CA2654283A1 - Methods of diagnosing and treating complications of pregnancy - Google Patents
Methods of diagnosing and treating complications of pregnancy Download PDFInfo
- Publication number
- CA2654283A1 CA2654283A1 CA002654283A CA2654283A CA2654283A1 CA 2654283 A1 CA2654283 A1 CA 2654283A1 CA 002654283 A CA002654283 A CA 002654283A CA 2654283 A CA2654283 A CA 2654283A CA 2654283 A1 CA2654283 A1 CA 2654283A1
- Authority
- CA
- Canada
- Prior art keywords
- soluble endoglin
- eclampsia
- endoglin
- subject
- tgf
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 230000035935 pregnancy Effects 0.000 title claims abstract description 229
- 238000000034 method Methods 0.000 title claims abstract description 164
- 108010036395 Endoglin Proteins 0.000 claims abstract description 637
- 102000012085 Endoglin Human genes 0.000 claims abstract description 634
- 201000011461 pre-eclampsia Diseases 0.000 claims abstract description 336
- 150000001875 compounds Chemical class 0.000 claims abstract description 160
- 206010020772 Hypertension Diseases 0.000 claims abstract description 142
- 208000002296 eclampsia Diseases 0.000 claims abstract description 126
- 230000004071 biological effect Effects 0.000 claims abstract description 82
- 230000014509 gene expression Effects 0.000 claims abstract description 75
- 108010023082 activin A Proteins 0.000 claims abstract description 35
- KAQKFAOMNZTLHT-OZUDYXHBSA-N prostaglandin I2 Chemical compound O1\C(=C/CCCC(O)=O)C[C@@H]2[C@@H](/C=C/[C@@H](O)CCCCC)[C@H](O)C[C@@H]21 KAQKFAOMNZTLHT-OZUDYXHBSA-N 0.000 claims abstract description 35
- 102100022544 Bone morphogenetic protein 7 Human genes 0.000 claims abstract description 34
- 102100024506 Bone morphogenetic protein 2 Human genes 0.000 claims abstract description 29
- 238000005259 measurement Methods 0.000 claims abstract description 27
- 101000862089 Clarkia lewisii Glucose-6-phosphate isomerase, cytosolic 1A Proteins 0.000 claims abstract description 25
- 101000762366 Homo sapiens Bone morphogenetic protein 2 Proteins 0.000 claims abstract description 21
- 101000899361 Homo sapiens Bone morphogenetic protein 7 Proteins 0.000 claims abstract description 20
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 136
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 129
- 229920001184 polypeptide Polymers 0.000 claims description 121
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 claims description 109
- 102100028452 Nitric oxide synthase, endothelial Human genes 0.000 claims description 91
- 230000027455 binding Effects 0.000 claims description 74
- 210000004027 cell Anatomy 0.000 claims description 63
- 210000002966 serum Anatomy 0.000 claims description 63
- 150000001413 amino acids Chemical class 0.000 claims description 58
- 239000012634 fragment Substances 0.000 claims description 58
- 230000001225 therapeutic effect Effects 0.000 claims description 51
- 210000002826 placenta Anatomy 0.000 claims description 48
- -1 sFlt-1 Proteins 0.000 claims description 44
- 230000001965 increasing effect Effects 0.000 claims description 42
- 239000000523 sample Substances 0.000 claims description 42
- 230000007423 decrease Effects 0.000 claims description 36
- 230000004913 activation Effects 0.000 claims description 35
- 239000003102 growth factor Substances 0.000 claims description 34
- 210000002381 plasma Anatomy 0.000 claims description 34
- 208000024891 symptom Diseases 0.000 claims description 34
- 210000004369 blood Anatomy 0.000 claims description 32
- 239000008280 blood Substances 0.000 claims description 32
- 210000002889 endothelial cell Anatomy 0.000 claims description 32
- 210000002700 urine Anatomy 0.000 claims description 30
- 206010070538 Gestational hypertension Diseases 0.000 claims description 29
- 239000000427 antigen Substances 0.000 claims description 25
- 108091007433 antigens Proteins 0.000 claims description 25
- 102000036639 antigens Human genes 0.000 claims description 25
- 238000012544 monitoring process Methods 0.000 claims description 25
- 230000004075 alteration Effects 0.000 claims description 24
- 101000881679 Homo sapiens Endoglin Proteins 0.000 claims description 23
- 102000048896 human ENG Human genes 0.000 claims description 22
- 239000011230 binding agent Substances 0.000 claims description 21
- 238000003745 diagnosis Methods 0.000 claims description 21
- 238000003776 cleavage reaction Methods 0.000 claims description 20
- 230000001419 dependent effect Effects 0.000 claims description 20
- 230000026731 phosphorylation Effects 0.000 claims description 20
- 238000006366 phosphorylation reaction Methods 0.000 claims description 20
- 239000013074 reference sample Substances 0.000 claims description 20
- 230000007017 scission Effects 0.000 claims description 20
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 19
- 230000030609 dephosphorylation Effects 0.000 claims description 18
- 238000006209 dephosphorylation reaction Methods 0.000 claims description 18
- 201000005624 HELLP Syndrome Diseases 0.000 claims description 16
- 210000003754 fetus Anatomy 0.000 claims description 16
- 108010049870 Bone Morphogenetic Protein 7 Proteins 0.000 claims description 14
- 101710143123 Mothers against decapentaplegic homolog 2 Proteins 0.000 claims description 14
- 102000035195 Peptidases Human genes 0.000 claims description 14
- 108091005804 Peptidases Proteins 0.000 claims description 14
- 210000001124 body fluid Anatomy 0.000 claims description 14
- 230000006872 improvement Effects 0.000 claims description 12
- 210000005059 placental tissue Anatomy 0.000 claims description 11
- 230000002255 enzymatic effect Effects 0.000 claims description 10
- 108010000684 Matrix Metalloproteinases Proteins 0.000 claims description 9
- 102000002274 Matrix Metalloproteinases Human genes 0.000 claims description 9
- 108010049931 Bone Morphogenetic Protein 2 Proteins 0.000 claims description 8
- 210000004381 amniotic fluid Anatomy 0.000 claims description 8
- 102000005600 Cathepsins Human genes 0.000 claims description 7
- 108010084457 Cathepsins Proteins 0.000 claims description 7
- 101000808011 Homo sapiens Vascular endothelial growth factor A Proteins 0.000 claims description 7
- 102000016387 Pancreatic elastase Human genes 0.000 claims description 7
- 108010067372 Pancreatic elastase Proteins 0.000 claims description 7
- 230000001684 chronic effect Effects 0.000 claims description 7
- 102000058223 human VEGFA Human genes 0.000 claims description 7
- 230000015556 catabolic process Effects 0.000 claims description 6
- 238000006731 degradation reaction Methods 0.000 claims description 6
- 241001494479 Pecora Species 0.000 claims description 5
- 238000010324 immunological assay Methods 0.000 claims description 5
- 241000283707 Capra Species 0.000 claims description 4
- 238000002965 ELISA Methods 0.000 claims description 4
- 239000003937 drug carrier Substances 0.000 claims description 3
- 210000000265 leukocyte Anatomy 0.000 claims description 3
- 210000001616 monocyte Anatomy 0.000 claims description 3
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 claims 6
- 102000057208 Smad2 Human genes 0.000 claims 5
- 101710090055 Nitric oxide synthase, endothelial Proteins 0.000 claims 3
- 108010075520 Nitric Oxide Synthase Type III Proteins 0.000 abstract description 90
- 102000008052 Nitric Oxide Synthase Type III Human genes 0.000 abstract 2
- 108090000623 proteins and genes Proteins 0.000 description 124
- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 description 104
- 102000009524 Vascular Endothelial Growth Factor A Human genes 0.000 description 104
- 102000004169 proteins and genes Human genes 0.000 description 104
- 235000018102 proteins Nutrition 0.000 description 98
- 101000595923 Homo sapiens Placenta growth factor Proteins 0.000 description 83
- 102100035194 Placenta growth factor Human genes 0.000 description 79
- 238000003556 assay Methods 0.000 description 65
- 150000007523 nucleic acids Chemical class 0.000 description 59
- 235000001014 amino acid Nutrition 0.000 description 58
- 229940024606 amino acid Drugs 0.000 description 58
- 102000039446 nucleic acids Human genes 0.000 description 54
- 108020004707 nucleic acids Proteins 0.000 description 54
- 101001001487 Homo sapiens Phosphatidylinositol-glycan biosynthesis class F protein Proteins 0.000 description 49
- MWUXSHHQAYIFBG-UHFFFAOYSA-N Nitric oxide Chemical compound O=[N] MWUXSHHQAYIFBG-UHFFFAOYSA-N 0.000 description 46
- 230000000694 effects Effects 0.000 description 45
- 230000001772 anti-angiogenic effect Effects 0.000 description 40
- 241000700159 Rattus Species 0.000 description 38
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 36
- 206010041092 Small for dates baby Diseases 0.000 description 34
- 238000001262 western blot Methods 0.000 description 32
- 230000033115 angiogenesis Effects 0.000 description 30
- 102000008299 Nitric Oxide Synthase Human genes 0.000 description 28
- 108010021487 Nitric Oxide Synthase Proteins 0.000 description 28
- 201000001474 proteinuria Diseases 0.000 description 28
- 238000002474 experimental method Methods 0.000 description 27
- 238000011282 treatment Methods 0.000 description 27
- DDRJAANPRJIHGJ-UHFFFAOYSA-N creatinine Chemical compound CN1CC(=O)NC1=N DDRJAANPRJIHGJ-UHFFFAOYSA-N 0.000 description 26
- 230000036772 blood pressure Effects 0.000 description 25
- 238000012384 transportation and delivery Methods 0.000 description 24
- 230000003247 decreasing effect Effects 0.000 description 22
- 102000005962 receptors Human genes 0.000 description 22
- 108020003175 receptors Proteins 0.000 description 22
- 230000015572 biosynthetic process Effects 0.000 description 21
- 210000004379 membrane Anatomy 0.000 description 21
- 239000012528 membrane Substances 0.000 description 21
- 239000000047 product Substances 0.000 description 21
- 239000002773 nucleotide Substances 0.000 description 20
- 108010076504 Protein Sorting Signals Proteins 0.000 description 19
- 239000000203 mixture Substances 0.000 description 19
- 125000003729 nucleotide group Chemical group 0.000 description 19
- 230000008774 maternal effect Effects 0.000 description 18
- 108020004999 messenger RNA Proteins 0.000 description 17
- 230000003169 placental effect Effects 0.000 description 17
- 210000002993 trophoblast Anatomy 0.000 description 17
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 16
- 102000014914 Carrier Proteins Human genes 0.000 description 16
- 108020004459 Small interfering RNA Proteins 0.000 description 16
- 108091008324 binding proteins Proteins 0.000 description 16
- 238000002405 diagnostic procedure Methods 0.000 description 16
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 15
- 239000003814 drug Substances 0.000 description 15
- 230000003511 endothelial effect Effects 0.000 description 15
- 238000005755 formation reaction Methods 0.000 description 15
- 239000004698 Polyethylene Substances 0.000 description 14
- 238000012228 RNA interference-mediated gene silencing Methods 0.000 description 14
- 239000002870 angiogenesis inducing agent Substances 0.000 description 14
- 230000000692 anti-sense effect Effects 0.000 description 14
- 238000010241 blood sampling Methods 0.000 description 14
- 230000008859 change Effects 0.000 description 14
- 238000011161 development Methods 0.000 description 14
- 230000018109 developmental process Effects 0.000 description 14
- 230000009368 gene silencing by RNA Effects 0.000 description 14
- 230000002028 premature Effects 0.000 description 14
- 201000005608 severe pre-eclampsia Diseases 0.000 description 14
- 241000701161 unidentified adenovirus Species 0.000 description 14
- 108010029485 Protein Isoforms Proteins 0.000 description 13
- 102000001708 Protein Isoforms Human genes 0.000 description 13
- 238000004458 analytical method Methods 0.000 description 13
- 229940109239 creatinine Drugs 0.000 description 13
- 201000010099 disease Diseases 0.000 description 13
- 230000003993 interaction Effects 0.000 description 13
- 230000011664 signaling Effects 0.000 description 13
- 238000008157 ELISA kit Methods 0.000 description 12
- 102000004190 Enzymes Human genes 0.000 description 12
- 108090000790 Enzymes Proteins 0.000 description 12
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 12
- 229940088598 enzyme Drugs 0.000 description 12
- 238000001114 immunoprecipitation Methods 0.000 description 12
- 239000003446 ligand Substances 0.000 description 12
- 108010047041 Complementarity Determining Regions Proteins 0.000 description 11
- 210000004185 liver Anatomy 0.000 description 11
- 239000001509 sodium citrate Substances 0.000 description 11
- 238000002560 therapeutic procedure Methods 0.000 description 11
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 11
- 229940038773 trisodium citrate Drugs 0.000 description 11
- 108060003951 Immunoglobulin Proteins 0.000 description 10
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 10
- 238000001514 detection method Methods 0.000 description 10
- 102000018358 immunoglobulin Human genes 0.000 description 10
- 239000003112 inhibitor Substances 0.000 description 10
- 238000012360 testing method Methods 0.000 description 10
- 210000001519 tissue Anatomy 0.000 description 10
- 230000024883 vasodilation Effects 0.000 description 10
- 108020004414 DNA Proteins 0.000 description 9
- 102100025751 Mothers against decapentaplegic homolog 2 Human genes 0.000 description 9
- 238000007792 addition Methods 0.000 description 9
- 230000002491 angiogenic effect Effects 0.000 description 9
- 229940079593 drug Drugs 0.000 description 9
- 230000001434 glomerular Effects 0.000 description 9
- 230000005764 inhibitory process Effects 0.000 description 9
- 210000004088 microvessel Anatomy 0.000 description 9
- 230000002265 prevention Effects 0.000 description 9
- 230000036266 weeks of gestation Effects 0.000 description 9
- 108091028043 Nucleic acid sequence Proteins 0.000 description 8
- 101100372762 Rattus norvegicus Flt1 gene Proteins 0.000 description 8
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 8
- 230000003527 anti-angiogenesis Effects 0.000 description 8
- 238000009396 hybridization Methods 0.000 description 8
- 238000003018 immunoassay Methods 0.000 description 8
- 238000001727 in vivo Methods 0.000 description 8
- 210000003734 kidney Anatomy 0.000 description 8
- 230000001404 mediated effect Effects 0.000 description 8
- 230000009257 reactivity Effects 0.000 description 8
- 230000002829 reductive effect Effects 0.000 description 8
- 230000001105 regulatory effect Effects 0.000 description 8
- 230000004044 response Effects 0.000 description 8
- 239000000126 substance Substances 0.000 description 8
- 238000006467 substitution reaction Methods 0.000 description 8
- 230000035488 systolic blood pressure Effects 0.000 description 8
- 230000002792 vascular Effects 0.000 description 8
- 239000003981 vehicle Substances 0.000 description 8
- 238000009007 Diagnostic Kit Methods 0.000 description 7
- ODKSFYDXXFIFQN-BYPYZUCNSA-N L-arginine Chemical compound OC(=O)[C@@H](N)CCCN=C(N)N ODKSFYDXXFIFQN-BYPYZUCNSA-N 0.000 description 7
- 229930064664 L-arginine Natural products 0.000 description 7
- 235000014852 L-arginine Nutrition 0.000 description 7
- 241000699666 Mus <mouse, genus> Species 0.000 description 7
- 108091034117 Oligonucleotide Proteins 0.000 description 7
- 208000002787 Pregnancy Complications Diseases 0.000 description 7
- 239000004365 Protease Substances 0.000 description 7
- 238000012217 deletion Methods 0.000 description 7
- 230000037430 deletion Effects 0.000 description 7
- 230000003205 diastolic effect Effects 0.000 description 7
- 238000009472 formulation Methods 0.000 description 7
- 238000000338 in vitro Methods 0.000 description 7
- 230000008506 pathogenesis Effects 0.000 description 7
- 229920001223 polyethylene glycol Polymers 0.000 description 7
- 238000003752 polymerase chain reaction Methods 0.000 description 7
- 230000035755 proliferation Effects 0.000 description 7
- 108010064377 prostacyclin synthetase Proteins 0.000 description 7
- 230000009467 reduction Effects 0.000 description 7
- 150000003839 salts Chemical class 0.000 description 7
- 239000000758 substrate Substances 0.000 description 7
- 230000003966 vascular damage Effects 0.000 description 7
- 102000000584 Calmodulin Human genes 0.000 description 6
- 108010041952 Calmodulin Proteins 0.000 description 6
- 208000001362 Fetal Growth Retardation Diseases 0.000 description 6
- 241001465754 Metazoa Species 0.000 description 6
- 238000000636 Northern blotting Methods 0.000 description 6
- 239000002202 Polyethylene glycol Substances 0.000 description 6
- 229920002684 Sepharose Polymers 0.000 description 6
- 239000003795 chemical substances by application Substances 0.000 description 6
- 238000003759 clinical diagnosis Methods 0.000 description 6
- 230000035487 diastolic blood pressure Effects 0.000 description 6
- 230000004069 differentiation Effects 0.000 description 6
- 230000001605 fetal effect Effects 0.000 description 6
- 208000030941 fetal growth restriction Diseases 0.000 description 6
- 238000004949 mass spectrometry Methods 0.000 description 6
- 108010082117 matrigel Proteins 0.000 description 6
- 238000013508 migration Methods 0.000 description 6
- 201000011460 mild pre-eclampsia Diseases 0.000 description 6
- 230000035772 mutation Effects 0.000 description 6
- 239000002953 phosphate buffered saline Substances 0.000 description 6
- 230000019491 signal transduction Effects 0.000 description 6
- 239000011780 sodium chloride Substances 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 238000010561 standard procedure Methods 0.000 description 6
- 208000011580 syndromic disease Diseases 0.000 description 6
- 238000003786 synthesis reaction Methods 0.000 description 6
- 238000005406 washing Methods 0.000 description 6
- 108010088751 Albumins Proteins 0.000 description 5
- 102000009027 Albumins Human genes 0.000 description 5
- COXVTLYNGOIATD-HVMBLDELSA-N CC1=C(C=CC(=C1)C1=CC(C)=C(C=C1)\N=N\C1=C(O)C2=C(N)C(=CC(=C2C=C1)S(O)(=O)=O)S(O)(=O)=O)\N=N\C1=CC=C2C(=CC(=C(N)C2=C1O)S(O)(=O)=O)S(O)(=O)=O Chemical compound CC1=C(C=CC(=C1)C1=CC(C)=C(C=C1)\N=N\C1=C(O)C2=C(N)C(=CC(=C2C=C1)S(O)(=O)=O)S(O)(=O)=O)\N=N\C1=CC=C2C(=CC(=C(N)C2=C1O)S(O)(=O)=O)S(O)(=O)=O COXVTLYNGOIATD-HVMBLDELSA-N 0.000 description 5
- 206010070531 Foetal growth restriction Diseases 0.000 description 5
- RHGKLRLOHDJJDR-BYPYZUCNSA-N L-citrulline Chemical compound NC(=O)NCCC[C@H]([NH3+])C([O-])=O RHGKLRLOHDJJDR-BYPYZUCNSA-N 0.000 description 5
- 241000124008 Mammalia Species 0.000 description 5
- 241000699670 Mus sp. Species 0.000 description 5
- 206010030113 Oedema Diseases 0.000 description 5
- 102000004316 Oxidoreductases Human genes 0.000 description 5
- 108090000854 Oxidoreductases Proteins 0.000 description 5
- 108010009583 Transforming Growth Factors Proteins 0.000 description 5
- 102000009618 Transforming Growth Factors Human genes 0.000 description 5
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 5
- 238000013459 approach Methods 0.000 description 5
- 239000000872 buffer Substances 0.000 description 5
- 230000004663 cell proliferation Effects 0.000 description 5
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 230000000295 complement effect Effects 0.000 description 5
- 230000000875 corresponding effect Effects 0.000 description 5
- 238000009826 distribution Methods 0.000 description 5
- 229960001123 epoprostenol Drugs 0.000 description 5
- 229960003699 evans blue Drugs 0.000 description 5
- 230000012010 growth Effects 0.000 description 5
- 230000036541 health Effects 0.000 description 5
- 238000001802 infusion Methods 0.000 description 5
- 150000002632 lipids Chemical class 0.000 description 5
- 238000004519 manufacturing process Methods 0.000 description 5
- 230000007246 mechanism Effects 0.000 description 5
- 230000005012 migration Effects 0.000 description 5
- 239000003226 mitogen Substances 0.000 description 5
- 230000004048 modification Effects 0.000 description 5
- 238000012986 modification Methods 0.000 description 5
- 210000004897 n-terminal region Anatomy 0.000 description 5
- 230000035699 permeability Effects 0.000 description 5
- 230000028742 placenta development Effects 0.000 description 5
- 238000012216 screening Methods 0.000 description 5
- 210000003462 vein Anatomy 0.000 description 5
- 239000004475 Arginine Substances 0.000 description 4
- 206010048962 Brain oedema Diseases 0.000 description 4
- 206010010904 Convulsion Diseases 0.000 description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- 206010018910 Haemolysis Diseases 0.000 description 4
- NTYJJOPFIAHURM-UHFFFAOYSA-N Histamine Chemical compound NCCC1=CN=CN1 NTYJJOPFIAHURM-UHFFFAOYSA-N 0.000 description 4
- 206010028851 Necrosis Diseases 0.000 description 4
- 239000002253 acid Substances 0.000 description 4
- 230000006907 apoptotic process Effects 0.000 description 4
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 4
- 235000009697 arginine Nutrition 0.000 description 4
- 230000000903 blocking effect Effects 0.000 description 4
- 208000006752 brain edema Diseases 0.000 description 4
- 210000004899 c-terminal region Anatomy 0.000 description 4
- 230000004087 circulation Effects 0.000 description 4
- 229960002173 citrulline Drugs 0.000 description 4
- 230000001086 cytosolic effect Effects 0.000 description 4
- 230000006378 damage Effects 0.000 description 4
- 238000013461 design Methods 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 231100000673 dose–response relationship Toxicity 0.000 description 4
- 210000003038 endothelium Anatomy 0.000 description 4
- 230000000004 hemodynamic effect Effects 0.000 description 4
- 230000008588 hemolysis Effects 0.000 description 4
- 239000000710 homodimer Substances 0.000 description 4
- 230000006698 induction Effects 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 230000002452 interceptive effect Effects 0.000 description 4
- 238000000670 ligand binding assay Methods 0.000 description 4
- 238000007726 management method Methods 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 238000002483 medication Methods 0.000 description 4
- 230000017074 necrotic cell death Effects 0.000 description 4
- 210000000056 organ Anatomy 0.000 description 4
- 230000003647 oxidation Effects 0.000 description 4
- 238000007254 oxidation reaction Methods 0.000 description 4
- 230000037361 pathway Effects 0.000 description 4
- 230000002093 peripheral effect Effects 0.000 description 4
- 239000000546 pharmaceutical excipient Substances 0.000 description 4
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 4
- 208000012113 pregnancy disease Diseases 0.000 description 4
- 230000001023 pro-angiogenic effect Effects 0.000 description 4
- YIBNHAJFJUQSRA-YNNPMVKQSA-N prostaglandin H2 Chemical compound C1[C@@H]2OO[C@H]1[C@H](/C=C/[C@@H](O)CCCCC)[C@H]2C\C=C/CCCC(O)=O YIBNHAJFJUQSRA-YNNPMVKQSA-N 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 238000012552 review Methods 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- 208000010110 spontaneous platelet aggregation Diseases 0.000 description 4
- 238000003860 storage Methods 0.000 description 4
- 230000002485 urinary effect Effects 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- FUOOLUPWFVMBKG-UHFFFAOYSA-N 2-Aminoisobutyric acid Chemical compound CC(C)(N)C(O)=O FUOOLUPWFVMBKG-UHFFFAOYSA-N 0.000 description 3
- 102000007469 Actins Human genes 0.000 description 3
- 108010085238 Actins Proteins 0.000 description 3
- 101100381481 Caenorhabditis elegans baz-2 gene Proteins 0.000 description 3
- 102000000844 Cell Surface Receptors Human genes 0.000 description 3
- 108010001857 Cell Surface Receptors Proteins 0.000 description 3
- 108091026890 Coding region Proteins 0.000 description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 3
- 206010048554 Endothelial dysfunction Diseases 0.000 description 3
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 3
- 108700024394 Exon Proteins 0.000 description 3
- 108060003393 Granulin Proteins 0.000 description 3
- 101000881680 Mus musculus Endoglin Proteins 0.000 description 3
- KCWZGJVSDFYRIX-YFKPBYRVSA-N N(gamma)-nitro-L-arginine methyl ester Chemical compound COC(=O)[C@@H](N)CCCN=C(N)N[N+]([O-])=O KCWZGJVSDFYRIX-YFKPBYRVSA-N 0.000 description 3
- RHGKLRLOHDJJDR-UHFFFAOYSA-N Ndelta-carbamoyl-DL-ornithine Natural products OC(=O)C(N)CCCNC(N)=O RHGKLRLOHDJJDR-UHFFFAOYSA-N 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 3
- 108090000417 Oxygenases Proteins 0.000 description 3
- 102000004020 Oxygenases Human genes 0.000 description 3
- 102000003923 Protein Kinase C Human genes 0.000 description 3
- 108090000315 Protein Kinase C Proteins 0.000 description 3
- 235000013290 Sagittaria latifolia Nutrition 0.000 description 3
- 229930006000 Sucrose Natural products 0.000 description 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 3
- 102100033663 Transforming growth factor beta receptor type 3 Human genes 0.000 description 3
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical class OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 239000004480 active ingredient Substances 0.000 description 3
- 239000000556 agonist Substances 0.000 description 3
- QWCKQJZIFLGMSD-UHFFFAOYSA-N alpha-aminobutyric acid Chemical compound CCC(N)C(O)=O QWCKQJZIFLGMSD-UHFFFAOYSA-N 0.000 description 3
- 230000002238 attenuated effect Effects 0.000 description 3
- 238000000211 autoradiogram Methods 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 108010079292 betaglycan Proteins 0.000 description 3
- 239000012472 biological sample Substances 0.000 description 3
- 210000004204 blood vessel Anatomy 0.000 description 3
- 210000000988 bone and bone Anatomy 0.000 description 3
- 201000011510 cancer Diseases 0.000 description 3
- 210000001736 capillary Anatomy 0.000 description 3
- 235000013477 citrulline Nutrition 0.000 description 3
- 235000015246 common arrowhead Nutrition 0.000 description 3
- 239000007857 degradation product Substances 0.000 description 3
- 239000003085 diluting agent Substances 0.000 description 3
- 230000008694 endothelial dysfunction Effects 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 230000013595 glycosylation Effects 0.000 description 3
- 238000006206 glycosylation reaction Methods 0.000 description 3
- 150000003278 haem Chemical group 0.000 description 3
- 238000003384 imaging method Methods 0.000 description 3
- 238000003364 immunohistochemistry Methods 0.000 description 3
- 230000001771 impaired effect Effects 0.000 description 3
- 230000008676 import Effects 0.000 description 3
- 238000003780 insertion Methods 0.000 description 3
- 230000037431 insertion Effects 0.000 description 3
- 238000001990 intravenous administration Methods 0.000 description 3
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 3
- 239000002502 liposome Substances 0.000 description 3
- 230000002297 mitogenic effect Effects 0.000 description 3
- 230000004899 motility Effects 0.000 description 3
- 230000004766 neurogenesis Effects 0.000 description 3
- 231100000252 nontoxic Toxicity 0.000 description 3
- 230000003000 nontoxic effect Effects 0.000 description 3
- 238000007911 parenteral administration Methods 0.000 description 3
- 125000001151 peptidyl group Chemical group 0.000 description 3
- 230000036470 plasma concentration Effects 0.000 description 3
- 229920000642 polymer Polymers 0.000 description 3
- 230000003389 potentiating effect Effects 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 230000006337 proteolytic cleavage Effects 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 238000003127 radioimmunoassay Methods 0.000 description 3
- 230000002441 reversible effect Effects 0.000 description 3
- 210000003296 saliva Anatomy 0.000 description 3
- 150000003384 small molecules Chemical class 0.000 description 3
- 239000005720 sucrose Substances 0.000 description 3
- 230000009885 systemic effect Effects 0.000 description 3
- 206010043554 thrombocytopenia Diseases 0.000 description 3
- 230000007838 tissue remodeling Effects 0.000 description 3
- 210000003606 umbilical vein Anatomy 0.000 description 3
- 210000003556 vascular endothelial cell Anatomy 0.000 description 3
- 230000006438 vascular health Effects 0.000 description 3
- 230000008728 vascular permeability Effects 0.000 description 3
- 230000006442 vascular tone Effects 0.000 description 3
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 2
- YKFCISHFRZHKHY-NGQGLHOPSA-N (2s)-2-amino-3-(3,4-dihydroxyphenyl)-2-methylpropanoic acid;trihydrate Chemical compound O.O.O.OC(=O)[C@](N)(C)CC1=CC=C(O)C(O)=C1.OC(=O)[C@](N)(C)CC1=CC=C(O)C(O)=C1 YKFCISHFRZHKHY-NGQGLHOPSA-N 0.000 description 2
- 238000010600 3H thymidine incorporation assay Methods 0.000 description 2
- WOVKYSAHUYNSMH-RRKCRQDMSA-N 5-bromodeoxyuridine Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(Br)=C1 WOVKYSAHUYNSMH-RRKCRQDMSA-N 0.000 description 2
- 108010059616 Activins Proteins 0.000 description 2
- 244000118350 Andrographis paniculata Species 0.000 description 2
- 102000008076 Angiogenic Proteins Human genes 0.000 description 2
- 108010074415 Angiogenic Proteins Proteins 0.000 description 2
- 102000004625 Aspartate Aminotransferases Human genes 0.000 description 2
- 108010003415 Aspartate Aminotransferases Proteins 0.000 description 2
- 101150061927 BMP2 gene Proteins 0.000 description 2
- 101800004538 Bradykinin Proteins 0.000 description 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 2
- 229920002134 Carboxymethyl cellulose Polymers 0.000 description 2
- PHEDXBVPIONUQT-UHFFFAOYSA-N Cocarcinogen A1 Natural products CCCCCCCCCCCCCC(=O)OC1C(C)C2(O)C3C=C(C)C(=O)C3(O)CC(CO)=CC2C2C1(OC(C)=O)C2(C)C PHEDXBVPIONUQT-UHFFFAOYSA-N 0.000 description 2
- 108091033380 Coding strand Proteins 0.000 description 2
- PMATZTZNYRCHOR-CGLBZJNRSA-N Cyclosporin A Chemical compound CC[C@@H]1NC(=O)[C@H]([C@H](O)[C@H](C)C\C=C\C)N(C)C(=O)[C@H](C(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)N(C)C(=O)CN(C)C1=O PMATZTZNYRCHOR-CGLBZJNRSA-N 0.000 description 2
- 108010036949 Cyclosporine Proteins 0.000 description 2
- 230000006820 DNA synthesis Effects 0.000 description 2
- 241000282326 Felis catus Species 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- QXZGBUJJYSLZLT-UHFFFAOYSA-N H-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-OH Natural products NC(N)=NCCCC(N)C(=O)N1CCCC1C(=O)N1C(C(=O)NCC(=O)NC(CC=2C=CC=CC=2)C(=O)NC(CO)C(=O)N2C(CCC2)C(=O)NC(CC=2C=CC=CC=2)C(=O)NC(CCCN=C(N)N)C(O)=O)CCC1 QXZGBUJJYSLZLT-UHFFFAOYSA-N 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 206010061218 Inflammation Diseases 0.000 description 2
- 102100026818 Inhibin beta E chain Human genes 0.000 description 2
- 102100035792 Kininogen-1 Human genes 0.000 description 2
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 2
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 2
- 229940124761 MMP inhibitor Drugs 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 108010076557 Matrix Metalloproteinase 14 Proteins 0.000 description 2
- 102100030216 Matrix metalloproteinase-14 Human genes 0.000 description 2
- 108010090054 Membrane Glycoproteins Proteins 0.000 description 2
- 102000012750 Membrane Glycoproteins Human genes 0.000 description 2
- SNIOPGDIGTZGOP-UHFFFAOYSA-N Nitroglycerin Chemical compound [O-][N+](=O)OCC(O[N+]([O-])=O)CO[N+]([O-])=O SNIOPGDIGTZGOP-UHFFFAOYSA-N 0.000 description 2
- 239000000006 Nitroglycerin Substances 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 101150053185 P450 gene Proteins 0.000 description 2
- 108091007960 PI3Ks Proteins 0.000 description 2
- 102000003993 Phosphatidylinositol 3-kinases Human genes 0.000 description 2
- 108090000430 Phosphatidylinositol 3-kinases Proteins 0.000 description 2
- 208000006399 Premature Obstetric Labor Diseases 0.000 description 2
- 239000012083 RIPA buffer Substances 0.000 description 2
- 101000852966 Rattus norvegicus Interleukin-1 receptor-like 1 Proteins 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 208000027418 Wounds and injury Diseases 0.000 description 2
- VVGPECAOVDZTLZ-UHFFFAOYSA-N [N]NC(N)=N Chemical compound [N]NC(N)=N VVGPECAOVDZTLZ-UHFFFAOYSA-N 0.000 description 2
- XJLXINKUBYWONI-DQQFMEOOSA-N [[(2r,3r,4r,5r)-5-(6-aminopurin-9-yl)-3-hydroxy-4-phosphonooxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2s,3r,4s,5s)-5-(3-carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl phosphate Chemical compound NC(=O)C1=CC=C[N+]([C@@H]2[C@H]([C@@H](O)[C@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-DQQFMEOOSA-N 0.000 description 2
- 230000005856 abnormality Effects 0.000 description 2
- 230000021736 acetylation Effects 0.000 description 2
- 238000006640 acetylation reaction Methods 0.000 description 2
- 150000007513 acids Chemical class 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 239000000488 activin Substances 0.000 description 2
- 230000001154 acute effect Effects 0.000 description 2
- 230000000996 additive effect Effects 0.000 description 2
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 description 2
- 230000002411 adverse Effects 0.000 description 2
- 238000001042 affinity chromatography Methods 0.000 description 2
- 238000000540 analysis of variance Methods 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 230000004872 arterial blood pressure Effects 0.000 description 2
- 238000003149 assay kit Methods 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 239000007640 basal medium Substances 0.000 description 2
- UCMIRNVEIXFBKS-UHFFFAOYSA-N beta-alanine Chemical compound NCCC(O)=O UCMIRNVEIXFBKS-UHFFFAOYSA-N 0.000 description 2
- 239000001045 blue dye Substances 0.000 description 2
- QXZGBUJJYSLZLT-FDISYFBBSA-N bradykinin Chemical compound NC(=N)NCCC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N1[C@H](C(=O)NCC(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CO)C(=O)N2[C@@H](CCC2)C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)CCC1 QXZGBUJJYSLZLT-FDISYFBBSA-N 0.000 description 2
- 210000004556 brain Anatomy 0.000 description 2
- 229910052791 calcium Inorganic materials 0.000 description 2
- 239000011575 calcium Substances 0.000 description 2
- 150000001720 carbohydrates Chemical group 0.000 description 2
- 239000001768 carboxy methyl cellulose Substances 0.000 description 2
- 235000010948 carboxy methyl cellulose Nutrition 0.000 description 2
- 239000008112 carboxymethyl-cellulose Substances 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 230000003197 catalytic effect Effects 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000000546 chi-square test Methods 0.000 description 2
- 229960001265 ciclosporin Drugs 0.000 description 2
- 238000002648 combination therapy Methods 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 229920001577 copolymer Polymers 0.000 description 2
- 230000002596 correlated effect Effects 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 238000004132 cross linking Methods 0.000 description 2
- 229930182912 cyclosporin Natural products 0.000 description 2
- XVOYSCVBGLVSOL-UHFFFAOYSA-N cysteic acid Chemical compound OC(=O)C(N)CS(O)(=O)=O XVOYSCVBGLVSOL-UHFFFAOYSA-N 0.000 description 2
- 230000000779 depleting effect Effects 0.000 description 2
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 2
- XEYBHCRIKKKOSS-UHFFFAOYSA-N disodium;azanylidyneoxidanium;iron(2+);pentacyanide Chemical compound [Na+].[Na+].[Fe+2].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].[O+]#N XEYBHCRIKKKOSS-UHFFFAOYSA-N 0.000 description 2
- 208000035475 disorder Diseases 0.000 description 2
- 230000003828 downregulation Effects 0.000 description 2
- 230000007783 downstream signaling Effects 0.000 description 2
- 230000004064 dysfunction Effects 0.000 description 2
- 150000002066 eicosanoids Chemical class 0.000 description 2
- 238000000635 electron micrograph Methods 0.000 description 2
- 239000003480 eluent Substances 0.000 description 2
- 239000000839 emulsion Substances 0.000 description 2
- DEFVIWRASFVYLL-UHFFFAOYSA-N ethylene glycol bis(2-aminoethyl)tetraacetic acid Chemical compound OC(=O)CN(CC(O)=O)CCOCCOCCN(CC(O)=O)CC(O)=O DEFVIWRASFVYLL-UHFFFAOYSA-N 0.000 description 2
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 2
- BTCSSZJGUNDROE-UHFFFAOYSA-N gamma-aminobutyric acid Chemical compound NCCCC(O)=O BTCSSZJGUNDROE-UHFFFAOYSA-N 0.000 description 2
- 239000007903 gelatin capsule Substances 0.000 description 2
- 230000007614 genetic variation Effects 0.000 description 2
- 229960003711 glyceryl trinitrate Drugs 0.000 description 2
- 239000011544 gradient gel Substances 0.000 description 2
- 229960001340 histamine Drugs 0.000 description 2
- 239000011539 homogenization buffer Substances 0.000 description 2
- 230000033444 hydroxylation Effects 0.000 description 2
- 238000005805 hydroxylation reaction Methods 0.000 description 2
- 238000003125 immunofluorescent labeling Methods 0.000 description 2
- 230000005847 immunogenicity Effects 0.000 description 2
- 238000002513 implantation Methods 0.000 description 2
- 238000010874 in vitro model Methods 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 230000004054 inflammatory process Effects 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 208000014674 injury Diseases 0.000 description 2
- 208000028867 ischemia Diseases 0.000 description 2
- 208000017169 kidney disease Diseases 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- JJTUDXZGHPGLLC-UHFFFAOYSA-N lactide Chemical compound CC1OC(=O)C(C)OC1=O JJTUDXZGHPGLLC-UHFFFAOYSA-N 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 210000004072 lung Anatomy 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 2
- 238000012423 maintenance Methods 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 230000003821 menstrual periods Effects 0.000 description 2
- 229910052751 metal Chemical class 0.000 description 2
- 239000002184 metal Chemical class 0.000 description 2
- 238000002493 microarray Methods 0.000 description 2
- 238000000386 microscopy Methods 0.000 description 2
- 238000010369 molecular cloning Methods 0.000 description 2
- DNIAPMSPPWPWGF-UHFFFAOYSA-N monopropylene glycol Natural products CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 2
- 230000000921 morphogenic effect Effects 0.000 description 2
- 210000003205 muscle Anatomy 0.000 description 2
- 230000007498 myristoylation Effects 0.000 description 2
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 2
- 239000002840 nitric oxide donor Substances 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 239000003921 oil Substances 0.000 description 2
- 235000019198 oils Nutrition 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 230000036961 partial effect Effects 0.000 description 2
- 230000036581 peripheral resistance Effects 0.000 description 2
- PHEDXBVPIONUQT-RGYGYFBISA-N phorbol 13-acetate 12-myristate Chemical compound C([C@]1(O)C(=O)C(C)=C[C@H]1[C@@]1(O)[C@H](C)[C@H]2OC(=O)CCCCCCCCCCCCC)C(CO)=C[C@H]1[C@H]1[C@]2(OC(C)=O)C1(C)C PHEDXBVPIONUQT-RGYGYFBISA-N 0.000 description 2
- 210000000557 podocyte Anatomy 0.000 description 2
- 238000006116 polymerization reaction Methods 0.000 description 2
- 102000054765 polymorphisms of proteins Human genes 0.000 description 2
- 230000012495 positive regulation of renal sodium excretion Effects 0.000 description 2
- 230000004481 post-translational protein modification Effects 0.000 description 2
- 230000032361 posttranscriptional gene silencing Effects 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 229940024999 proteolytic enzymes for treatment of wounds and ulcers Drugs 0.000 description 2
- 230000036593 pulmonary vascular resistance Effects 0.000 description 2
- 238000012207 quantitative assay Methods 0.000 description 2
- 230000004648 relaxation of smooth muscle Effects 0.000 description 2
- 230000008439 repair process Effects 0.000 description 2
- 230000000630 rising effect Effects 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- FSYKKLYZXJSNPZ-UHFFFAOYSA-N sarcosine Chemical compound C[NH2+]CC([O-])=O FSYKKLYZXJSNPZ-UHFFFAOYSA-N 0.000 description 2
- 238000007423 screening assay Methods 0.000 description 2
- 102000035025 signaling receptors Human genes 0.000 description 2
- 108091005475 signaling receptors Proteins 0.000 description 2
- 230000000391 smoking effect Effects 0.000 description 2
- 210000002460 smooth muscle Anatomy 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 229940083618 sodium nitroprusside Drugs 0.000 description 2
- 238000012453 sprague-dawley rat model Methods 0.000 description 2
- 238000007619 statistical method Methods 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- 230000019635 sulfation Effects 0.000 description 2
- 238000005670 sulfation reaction Methods 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 238000013268 sustained release Methods 0.000 description 2
- 239000012730 sustained-release form Substances 0.000 description 2
- 239000003826 tablet Substances 0.000 description 2
- ZFXYFBGIUFBOJW-UHFFFAOYSA-N theophylline Chemical compound O=C1N(C)C(=O)N(C)C2=C1NC=N2 ZFXYFBGIUFBOJW-UHFFFAOYSA-N 0.000 description 2
- 229940124597 therapeutic agent Drugs 0.000 description 2
- 238000013519 translation Methods 0.000 description 2
- 230000003827 upregulation Effects 0.000 description 2
- 230000001457 vasomotor Effects 0.000 description 2
- 230000037314 wound repair Effects 0.000 description 2
- GVJHHUAWPYXKBD-IEOSBIPESA-N α-tocopherol Chemical compound OC1=C(C)C(C)=C2O[C@@](CCC[C@H](C)CCC[C@H](C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-IEOSBIPESA-N 0.000 description 2
- SNICXCGAKADSCV-JTQLQIEISA-N (-)-Nicotine Chemical compound CN1CCC[C@H]1C1=CC=CN=C1 SNICXCGAKADSCV-JTQLQIEISA-N 0.000 description 1
- VEPOHXYIFQMVHW-PVJVQHJQSA-N (2r,3r)-2,3-dihydroxybutanedioic acid;(2s,3s)-3,4-dimethyl-2-phenylmorpholine Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O.O1CCN(C)[C@@H](C)[C@@H]1C1=CC=CC=C1 VEPOHXYIFQMVHW-PVJVQHJQSA-N 0.000 description 1
- GFUITADOEPNRML-SJORKVTESA-N (2r,3r)-3-(cyclopentylmethyl)-n-hydroxy-4-oxo-4-piperidin-1-yl-2-[(3,4,4-trimethyl-2,5-dioxoimidazolidin-1-yl)methyl]butanamide Chemical compound O=C1C(C)(C)N(C)C(=O)N1C[C@H](C(=O)NO)[C@H](C(=O)N1CCCCC1)CC1CCCC1 GFUITADOEPNRML-SJORKVTESA-N 0.000 description 1
- BVAUMRCGVHUWOZ-ZETCQYMHSA-N (2s)-2-(cyclohexylazaniumyl)propanoate Chemical compound OC(=O)[C@H](C)NC1CCCCC1 BVAUMRCGVHUWOZ-ZETCQYMHSA-N 0.000 description 1
- MRTPISKDZDHEQI-YFKPBYRVSA-N (2s)-2-(tert-butylamino)propanoic acid Chemical compound OC(=O)[C@H](C)NC(C)(C)C MRTPISKDZDHEQI-YFKPBYRVSA-N 0.000 description 1
- NPDBDJFLKKQMCM-SCSAIBSYSA-N (2s)-2-amino-3,3-dimethylbutanoic acid Chemical compound CC(C)(C)[C@H](N)C(O)=O NPDBDJFLKKQMCM-SCSAIBSYSA-N 0.000 description 1
- GTXSRFUZSLTDFX-HRCADAONSA-N (2s)-n-[(2s)-3,3-dimethyl-1-(methylamino)-1-oxobutan-2-yl]-4-methyl-2-[[(2s)-2-sulfanyl-4-(3,4,4-trimethyl-2,5-dioxoimidazolidin-1-yl)butanoyl]amino]pentanamide Chemical compound CNC(=O)[C@H](C(C)(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](S)CCN1C(=O)N(C)C(C)(C)C1=O GTXSRFUZSLTDFX-HRCADAONSA-N 0.000 description 1
- KPJZHOPZRAFDTN-ZRGWGRIASA-N (6aR,9R)-N-[(2S)-1-hydroxybutan-2-yl]-4,7-dimethyl-6,6a,8,9-tetrahydroindolo[4,3-fg]quinoline-9-carboxamide Chemical compound C1=CC(C=2[C@H](N(C)C[C@@H](C=2)C(=O)N[C@H](CO)CC)C2)=C3C2=CN(C)C3=C1 KPJZHOPZRAFDTN-ZRGWGRIASA-N 0.000 description 1
- TUSDEZXZIZRFGC-UHFFFAOYSA-N 1-O-galloyl-3,6-(R)-HHDP-beta-D-glucose Natural products OC1C(O2)COC(=O)C3=CC(O)=C(O)C(O)=C3C3=C(O)C(O)=C(O)C=C3C(=O)OC1C(O)C2OC(=O)C1=CC(O)=C(O)C(O)=C1 TUSDEZXZIZRFGC-UHFFFAOYSA-N 0.000 description 1
- OGNSCSPNOLGXSM-UHFFFAOYSA-N 2,4-diaminobutyric acid Chemical compound NCCC(N)C(O)=O OGNSCSPNOLGXSM-UHFFFAOYSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- SGUAFYQXFOLMHL-UHFFFAOYSA-N 2-hydroxy-5-{1-hydroxy-2-[(4-phenylbutan-2-yl)amino]ethyl}benzamide Chemical compound C=1C=C(O)C(C(N)=O)=CC=1C(O)CNC(C)CCC1=CC=CC=C1 SGUAFYQXFOLMHL-UHFFFAOYSA-N 0.000 description 1
- BMYNFMYTOJXKLE-UHFFFAOYSA-N 3-azaniumyl-2-hydroxypropanoate Chemical compound NCC(O)C(O)=O BMYNFMYTOJXKLE-UHFFFAOYSA-N 0.000 description 1
- NCGICGYLBXGBGN-UHFFFAOYSA-N 3-morpholin-4-yl-1-oxa-3-azonia-2-azanidacyclopent-3-en-5-imine;hydrochloride Chemical compound Cl.[N-]1OC(=N)C=[N+]1N1CCOCC1 NCGICGYLBXGBGN-UHFFFAOYSA-N 0.000 description 1
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- ODHCTXKNWHHXJC-VKHMYHEASA-N 5-oxo-L-proline Chemical compound OC(=O)[C@@H]1CCC(=O)N1 ODHCTXKNWHHXJC-VKHMYHEASA-N 0.000 description 1
- SLXKOJJOQWFEFD-UHFFFAOYSA-N 6-aminohexanoic acid Chemical compound NCCCCCC(O)=O SLXKOJJOQWFEFD-UHFFFAOYSA-N 0.000 description 1
- ZKRFOXLVOKTUTA-KQYNXXCUSA-N 9-(5-phosphoribofuranosyl)-6-mercaptopurine Chemical compound O[C@@H]1[C@H](O)[C@@H](COP(O)(O)=O)O[C@H]1N1C(NC=NC2=S)=C2N=C1 ZKRFOXLVOKTUTA-KQYNXXCUSA-N 0.000 description 1
- 230000005730 ADP ribosylation Effects 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 206010059245 Angiopathy Diseases 0.000 description 1
- 108010005853 Anti-Mullerian Hormone Proteins 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- BSYNRYMUTXBXSQ-UHFFFAOYSA-N Aspirin Chemical compound CC(=O)OC1=CC=CC=C1C(O)=O BSYNRYMUTXBXSQ-UHFFFAOYSA-N 0.000 description 1
- 241000972773 Aulopiformes Species 0.000 description 1
- 102100037135 BCL2/adenovirus E1B 19 kDa protein-interacting protein 2 Human genes 0.000 description 1
- 108091032955 Bacterial small RNA Proteins 0.000 description 1
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- 102000007350 Bone Morphogenetic Proteins Human genes 0.000 description 1
- 108010007726 Bone Morphogenetic Proteins Proteins 0.000 description 1
- 102100024505 Bone morphogenetic protein 4 Human genes 0.000 description 1
- 239000011547 Bouin solution Substances 0.000 description 1
- 206010055113 Breast cancer metastatic Diseases 0.000 description 1
- COVZYZSDYWQREU-UHFFFAOYSA-N Busulfan Chemical compound CS(=O)(=O)OCCCCOS(C)(=O)=O COVZYZSDYWQREU-UHFFFAOYSA-N 0.000 description 1
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 1
- 206010008111 Cerebral haemorrhage Diseases 0.000 description 1
- 206010008805 Chromosomal abnormalities Diseases 0.000 description 1
- 208000031404 Chromosome Aberrations Diseases 0.000 description 1
- 108060005980 Collagenase Proteins 0.000 description 1
- 102000029816 Collagenase Human genes 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 1
- 206010010071 Coma Diseases 0.000 description 1
- 108091035707 Consensus sequence Proteins 0.000 description 1
- 206010067477 Cytogenetic abnormality Diseases 0.000 description 1
- 101710112752 Cytotoxin Proteins 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 206010061818 Disease progression Diseases 0.000 description 1
- 206010058314 Dysplasia Diseases 0.000 description 1
- LVGKNOAMLMIIKO-UHFFFAOYSA-N Elaidinsaeure-aethylester Natural products CCCCCCCCC=CCCCCCCCC(=O)OCC LVGKNOAMLMIIKO-UHFFFAOYSA-N 0.000 description 1
- 102100040897 Embryonic growth/differentiation factor 1 Human genes 0.000 description 1
- 102100037241 Endoglin Human genes 0.000 description 1
- 102000010911 Enzyme Precursors Human genes 0.000 description 1
- 108010062466 Enzyme Precursors Proteins 0.000 description 1
- 206010015548 Euthanasia Diseases 0.000 description 1
- 239000001263 FEMA 3042 Substances 0.000 description 1
- 108091006020 Fc-tagged proteins Proteins 0.000 description 1
- 238000000729 Fisher's exact test Methods 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 108010026132 Gelatinases Proteins 0.000 description 1
- 102000013382 Gelatinases Human genes 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- 108010090296 Growth Differentiation Factor 1 Proteins 0.000 description 1
- 108010090293 Growth Differentiation Factor 3 Proteins 0.000 description 1
- 102100035364 Growth/differentiation factor 3 Human genes 0.000 description 1
- 108020005004 Guide RNA Proteins 0.000 description 1
- 229940121710 HMGCoA reductase inhibitor Drugs 0.000 description 1
- 206010019013 Haemorrhagic infarction Diseases 0.000 description 1
- 102100034051 Heat shock protein HSP 90-alpha Human genes 0.000 description 1
- 206010019837 Hepatocellular injury Diseases 0.000 description 1
- 108090000100 Hepatocyte Growth Factor Proteins 0.000 description 1
- 102100021866 Hepatocyte growth factor Human genes 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000740576 Homo sapiens BCL2/adenovirus E1B 19 kDa protein-interacting protein 2 Proteins 0.000 description 1
- 101000762379 Homo sapiens Bone morphogenetic protein 4 Proteins 0.000 description 1
- 101001016865 Homo sapiens Heat shock protein HSP 90-alpha Proteins 0.000 description 1
- 101000878605 Homo sapiens Low affinity immunoglobulin epsilon Fc receptor Proteins 0.000 description 1
- 101000990902 Homo sapiens Matrix metalloproteinase-9 Proteins 0.000 description 1
- 101000669513 Homo sapiens Metalloproteinase inhibitor 1 Proteins 0.000 description 1
- 102000003839 Human Proteins Human genes 0.000 description 1
- 108090000144 Human Proteins Proteins 0.000 description 1
- 102000008100 Human Serum Albumin Human genes 0.000 description 1
- 108091006905 Human Serum Albumin Proteins 0.000 description 1
- PMMYEEVYMWASQN-DMTCNVIQSA-N Hydroxyproline Chemical compound O[C@H]1CN[C@H](C(O)=O)C1 PMMYEEVYMWASQN-DMTCNVIQSA-N 0.000 description 1
- 206010020880 Hypertrophy Diseases 0.000 description 1
- 102000009786 Immunoglobulin Constant Regions Human genes 0.000 description 1
- 108010009817 Immunoglobulin Constant Regions Proteins 0.000 description 1
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 1
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 1
- 206010061216 Infarction Diseases 0.000 description 1
- 108010044467 Isoenzymes Proteins 0.000 description 1
- SNDPXSYFESPGGJ-BYPYZUCNSA-N L-2-aminopentanoic acid Chemical compound CCC[C@H](N)C(O)=O SNDPXSYFESPGGJ-BYPYZUCNSA-N 0.000 description 1
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- ZGUNAGUHMKGQNY-ZETCQYMHSA-N L-alpha-phenylglycine zwitterion Chemical compound OC(=O)[C@@H](N)C1=CC=CC=C1 ZGUNAGUHMKGQNY-ZETCQYMHSA-N 0.000 description 1
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- XIGSAGMEBXLVJJ-YFKPBYRVSA-N L-homocitrulline Chemical compound NC(=O)NCCCC[C@H]([NH3+])C([O-])=O XIGSAGMEBXLVJJ-YFKPBYRVSA-N 0.000 description 1
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 1
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 1
- SNDPXSYFESPGGJ-UHFFFAOYSA-N L-norVal-OH Natural products CCCC(N)C(O)=O SNDPXSYFESPGGJ-UHFFFAOYSA-N 0.000 description 1
- LRQKBLKVPFOOQJ-YFKPBYRVSA-N L-norleucine Chemical compound CCCC[C@H]([NH3+])C([O-])=O LRQKBLKVPFOOQJ-YFKPBYRVSA-N 0.000 description 1
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- 102400000401 Latency-associated peptide Human genes 0.000 description 1
- 101800001155 Latency-associated peptide Proteins 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- 102100038007 Low affinity immunoglobulin epsilon Fc receptor Human genes 0.000 description 1
- 206010067508 Low birth weight baby Diseases 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 208000000091 Maternal Death Diseases 0.000 description 1
- 102100030412 Matrix metalloproteinase-9 Human genes 0.000 description 1
- 102000005741 Metalloproteases Human genes 0.000 description 1
- 108010006035 Metalloproteases Proteins 0.000 description 1
- 102100039364 Metalloproteinase inhibitor 1 Human genes 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 206010027525 Microalbuminuria Diseases 0.000 description 1
- ZFMITUMMTDLWHR-UHFFFAOYSA-N Minoxidil Chemical compound NC1=[N+]([O-])C(N)=CC(N2CCCCC2)=N1 ZFMITUMMTDLWHR-UHFFFAOYSA-N 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 101100165560 Mus musculus Bmp7 gene Proteins 0.000 description 1
- 101500028702 Mus musculus Dll1-soluble form Proteins 0.000 description 1
- 101100175313 Mus musculus Gdf3 gene Proteins 0.000 description 1
- 101000808007 Mus musculus Vascular endothelial growth factor A Proteins 0.000 description 1
- FQWRAVYMZULPNK-UHFFFAOYSA-N N(5)-[(Z)-amino(hydroxyimino)methyl]ornithine Chemical compound OC(=O)C(N)CCCNC(N)=NO FQWRAVYMZULPNK-UHFFFAOYSA-N 0.000 description 1
- 102100031887 Nanos homolog 1 Human genes 0.000 description 1
- 101710196788 Nanos homolog 1 Proteins 0.000 description 1
- 102100031893 Nanos homolog 3 Human genes 0.000 description 1
- 101710196784 Nanos homolog 3 Proteins 0.000 description 1
- 102100022397 Nitric oxide synthase, brain Human genes 0.000 description 1
- 101710111444 Nitric oxide synthase, brain Proteins 0.000 description 1
- 102100029438 Nitric oxide synthase, inducible Human genes 0.000 description 1
- 101710089543 Nitric oxide synthase, inducible Proteins 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 108700020796 Oncogene Proteins 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- LRBQNJMCXXYXIU-PPKXGCFTSA-N Penta-digallate-beta-D-glucose Natural products OC1=C(O)C(O)=CC(C(=O)OC=2C(=C(O)C=C(C=2)C(=O)OC[C@@H]2[C@H]([C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)O2)OC(=O)C=2C=C(OC(=O)C=3C=C(O)C(O)=C(O)C=3)C(O)=C(O)C=2)O)=C1 LRBQNJMCXXYXIU-PPKXGCFTSA-N 0.000 description 1
- QGMRQYFBGABWDR-UHFFFAOYSA-M Pentobarbital sodium Chemical compound [Na+].CCCC(C)C1(CC)C(=O)NC(=O)[N-]C1=O QGMRQYFBGABWDR-UHFFFAOYSA-M 0.000 description 1
- 102000007079 Peptide Fragments Human genes 0.000 description 1
- 108010033276 Peptide Fragments Proteins 0.000 description 1
- 241000577979 Peromyscus spicilegus Species 0.000 description 1
- 241000009328 Perro Species 0.000 description 1
- 108091000080 Phosphotransferase Proteins 0.000 description 1
- 108010082093 Placenta Growth Factor Proteins 0.000 description 1
- 102100040681 Platelet-derived growth factor C Human genes 0.000 description 1
- 239000004372 Polyvinyl alcohol Substances 0.000 description 1
- 208000005347 Pregnancy-Induced Hypertension Diseases 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- 102000005569 Protein Phosphatase 1 Human genes 0.000 description 1
- 108010059000 Protein Phosphatase 1 Proteins 0.000 description 1
- 108010001267 Protein Subunits Proteins 0.000 description 1
- 102000004022 Protein-Tyrosine Kinases Human genes 0.000 description 1
- 108090000412 Protein-Tyrosine Kinases Proteins 0.000 description 1
- 206010037423 Pulmonary oedema Diseases 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 101000930457 Rattus norvegicus Albumin Proteins 0.000 description 1
- 102000004278 Receptor Protein-Tyrosine Kinases Human genes 0.000 description 1
- 108090000873 Receptor Protein-Tyrosine Kinases Proteins 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- AUNGANRZJHBGPY-SCRDCRAPSA-N Riboflavin Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-SCRDCRAPSA-N 0.000 description 1
- 108091006629 SLC13A2 Proteins 0.000 description 1
- 108010077895 Sarcosine Proteins 0.000 description 1
- 108091081021 Sense strand Proteins 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 108091027967 Small hairpin RNA Proteins 0.000 description 1
- 238000002105 Southern blotting Methods 0.000 description 1
- 208000005392 Spasm Diseases 0.000 description 1
- 235000021355 Stearic acid Nutrition 0.000 description 1
- 238000000692 Student's t-test Methods 0.000 description 1
- JXAGDPXECXQWBC-LJQANCHMSA-N Tanomastat Chemical compound C([C@H](C(=O)O)CC(=O)C=1C=CC(=CC=1)C=1C=CC(Cl)=CC=1)SC1=CC=CC=C1 JXAGDPXECXQWBC-LJQANCHMSA-N 0.000 description 1
- 206010043189 Telangiectasia Diseases 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- 102000003929 Transaminases Human genes 0.000 description 1
- 108090000340 Transaminases Proteins 0.000 description 1
- 101710195626 Transcriptional activator protein Proteins 0.000 description 1
- 108020004566 Transfer RNA Proteins 0.000 description 1
- 108700019146 Transgenes Proteins 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical class O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 1
- LEHOTFFKMJEONL-UHFFFAOYSA-N Uric Acid Chemical compound N1C(=O)NC(=O)C2=C1NC(=O)N2 LEHOTFFKMJEONL-UHFFFAOYSA-N 0.000 description 1
- TVWHNULVHGKJHS-UHFFFAOYSA-N Uric acid Natural products N1C(=O)NC(=O)C2NC(=O)NC21 TVWHNULVHGKJHS-UHFFFAOYSA-N 0.000 description 1
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 1
- 108010073925 Vascular Endothelial Growth Factor B Proteins 0.000 description 1
- 108010073923 Vascular Endothelial Growth Factor C Proteins 0.000 description 1
- 108010073919 Vascular Endothelial Growth Factor D Proteins 0.000 description 1
- 102000016548 Vascular Endothelial Growth Factor Receptor-1 Human genes 0.000 description 1
- 108010053096 Vascular Endothelial Growth Factor Receptor-1 Proteins 0.000 description 1
- 208000032594 Vascular Remodeling Diseases 0.000 description 1
- 102100038217 Vascular endothelial growth factor B Human genes 0.000 description 1
- 102100038232 Vascular endothelial growth factor C Human genes 0.000 description 1
- 102100038234 Vascular endothelial growth factor D Human genes 0.000 description 1
- 206010047141 Vasodilatation Diseases 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- FKAWLXNLHHIHLA-YCBIHMBMSA-N [(2r,3r,5r,7r,8s,9s)-2-[(1s,3s,4s,5r,6r,7e,9e,11e,13z)-14-cyano-3,5-dihydroxy-1-methoxy-4,6,8,9,13-pentamethyltetradeca-7,9,11,13-tetraenyl]-9-[(e)-3-[2-[(2s)-4-[[(2s,3s,4s)-4-(dimethylamino)-2,3-dihydroxy-5-methoxypentanoyl]amino]butan-2-yl]-1,3-oxazol-4 Chemical compound O1C([C@@H](C)CCNC(=O)[C@@H](O)[C@@H](O)[C@H](COC)N(C)C)=NC(\C=C\C[C@H]2[C@H]([C@H](O)C[C@]3(O2)C([C@@H](OP(O)(O)=O)[C@@H]([C@H](C[C@H](O)[C@H](C)[C@H](O)[C@H](C)\C=C(/C)\C(\C)=C\C=C\C(\C)=C/C#N)OC)O3)(C)C)C)=C1 FKAWLXNLHHIHLA-YCBIHMBMSA-N 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 229960001138 acetylsalicylic acid Drugs 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 230000010933 acylation Effects 0.000 description 1
- 238000005917 acylation reaction Methods 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 229960005305 adenosine Drugs 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 229940087168 alpha tocopherol Drugs 0.000 description 1
- VREFGVBLTWBCJP-UHFFFAOYSA-N alprazolam Chemical compound C12=CC(Cl)=CC=C2N2C(C)=NN=C2CN=C1C1=CC=CC=C1 VREFGVBLTWBCJP-UHFFFAOYSA-N 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 229960002684 aminocaproic acid Drugs 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 230000019552 anatomical structure morphogenesis Effects 0.000 description 1
- 239000004037 angiogenesis inhibitor Substances 0.000 description 1
- 230000008485 antagonism Effects 0.000 description 1
- 230000000181 anti-adherent effect Effects 0.000 description 1
- 230000001773 anti-convulsant effect Effects 0.000 description 1
- 239000000868 anti-mullerian hormone Substances 0.000 description 1
- 230000002785 anti-thrombosis Effects 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 239000001961 anticonvulsive agent Substances 0.000 description 1
- 229960003965 antiepileptics Drugs 0.000 description 1
- 239000002220 antihypertensive agent Substances 0.000 description 1
- 230000010516 arginylation Effects 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 229940009098 aspartate Drugs 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 238000002869 basic local alignment search tool Methods 0.000 description 1
- XFILPEOLDIKJHX-QYZOEREBSA-N batimastat Chemical compound C([C@@H](C(=O)NC)NC(=O)[C@H](CC(C)C)[C@H](CSC=1SC=CC=1)C(=O)NO)C1=CC=CC=C1 XFILPEOLDIKJHX-QYZOEREBSA-N 0.000 description 1
- 229950001858 batimastat Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid group Chemical group C(C1=CC=CC=C1)(=O)O WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 238000010876 biochemical test Methods 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 210000002459 blastocyst Anatomy 0.000 description 1
- 238000009530 blood pressure measurement Methods 0.000 description 1
- 238000006664 bond formation reaction Methods 0.000 description 1
- 229940112869 bone morphogenetic protein Drugs 0.000 description 1
- OZVBMTJYIDMWIL-AYFBDAFISA-N bromocriptine Chemical compound C1=CC(C=2[C@H](N(C)C[C@@H](C=2)C(=O)N[C@]2(C(=O)N3[C@H](C(N4CCC[C@H]4[C@]3(O)O2)=O)CC(C)C)C(C)C)C2)=C3C2=C(Br)NC3=C1 OZVBMTJYIDMWIL-AYFBDAFISA-N 0.000 description 1
- 229960002802 bromocriptine Drugs 0.000 description 1
- 239000003560 cancer drug Substances 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 210000001715 carotid artery Anatomy 0.000 description 1
- 210000000845 cartilage Anatomy 0.000 description 1
- 230000012292 cell migration Effects 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 229940083181 centrally acting adntiadrenergic agent methyldopa Drugs 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 239000007910 chewable tablet Substances 0.000 description 1
- ARUGKOZUKWAXDS-SEWALLKFSA-N cicaprost Chemical compound C1\C(=C/COCC(O)=O)C[C@@H]2[C@@H](C#C[C@@H](O)[C@@H](C)CC#CCC)[C@H](O)C[C@@H]21 ARUGKOZUKWAXDS-SEWALLKFSA-N 0.000 description 1
- 229950000634 cicaprost Drugs 0.000 description 1
- 210000002358 circulating endothelial cell Anatomy 0.000 description 1
- 230000015271 coagulation Effects 0.000 description 1
- 238000005345 coagulation Methods 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 230000001010 compromised effect Effects 0.000 description 1
- 230000008602 contraction Effects 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 210000005220 cytoplasmic tail Anatomy 0.000 description 1
- 231100000599 cytotoxic agent Toxicity 0.000 description 1
- 239000002619 cytotoxin Substances 0.000 description 1
- 210000003785 decidua Anatomy 0.000 description 1
- KWDSFGYQALRPMG-UHFFFAOYSA-N delta-N-Hydroxy-L-orginin Natural products OC(=O)C(N)CCCN(O)C(N)=N KWDSFGYQALRPMG-UHFFFAOYSA-N 0.000 description 1
- 230000017858 demethylation Effects 0.000 description 1
- 238000010520 demethylation reaction Methods 0.000 description 1
- 239000005547 deoxyribonucleotide Substances 0.000 description 1
- 125000002637 deoxyribonucleotide group Chemical group 0.000 description 1
- 230000003831 deregulation Effects 0.000 description 1
- 238000003795 desorption Methods 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- HPNMFZURTQLUMO-UHFFFAOYSA-N diethylamine Chemical compound CCNCC HPNMFZURTQLUMO-UHFFFAOYSA-N 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 238000006471 dimerization reaction Methods 0.000 description 1
- 230000005750 disease progression Effects 0.000 description 1
- PMMYEEVYMWASQN-UHFFFAOYSA-N dl-hydroxyproline Natural products OC1C[NH2+]C(C([O-])=O)C1 PMMYEEVYMWASQN-UHFFFAOYSA-N 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 238000009510 drug design Methods 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 238000001493 electron microscopy Methods 0.000 description 1
- 230000008753 endothelial function Effects 0.000 description 1
- 210000004954 endothelial membrane Anatomy 0.000 description 1
- 210000003989 endothelium vascular Anatomy 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- LVGKNOAMLMIIKO-QXMHVHEDSA-N ethyl oleate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC LVGKNOAMLMIIKO-QXMHVHEDSA-N 0.000 description 1
- 229940093471 ethyl oleate Drugs 0.000 description 1
- 239000005038 ethylene vinyl acetate Substances 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 238000010195 expression analysis Methods 0.000 description 1
- 230000008175 fetal development Effects 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 230000022244 formylation Effects 0.000 description 1
- 238000006170 formylation reaction Methods 0.000 description 1
- 238000002825 functional assay Methods 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 229960003692 gamma aminobutyric acid Drugs 0.000 description 1
- 230000006251 gamma-carboxylation Effects 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000011223 gene expression profiling Methods 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- 230000002518 glial effect Effects 0.000 description 1
- 210000005086 glomerual capillary Anatomy 0.000 description 1
- 238000007446 glucose tolerance test Methods 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 125000000291 glutamic acid group Chemical group N[C@@H](CCC(O)=O)C(=O)* 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 235000004554 glutamine Nutrition 0.000 description 1
- 210000002216 heart Anatomy 0.000 description 1
- 238000005534 hematocrit Methods 0.000 description 1
- 230000002008 hemorrhagic effect Effects 0.000 description 1
- 230000002440 hepatic effect Effects 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000036732 histological change Effects 0.000 description 1
- 230000003284 homeostatic effect Effects 0.000 description 1
- 230000009097 homeostatic mechanism Effects 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- ZUXNZUWOTSUBMN-UHFFFAOYSA-N hydralazine hydrochloride Chemical compound Cl.C1=CC=C2C(NN)=NN=CC2=C1 ZUXNZUWOTSUBMN-UHFFFAOYSA-N 0.000 description 1
- 229960005384 hydralazine hydrochloride Drugs 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000008172 hydrogenated vegetable oil Substances 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 239000002471 hydroxymethylglutaryl coenzyme A reductase inhibitor Substances 0.000 description 1
- 229960002591 hydroxyproline Drugs 0.000 description 1
- 230000001631 hypertensive effect Effects 0.000 description 1
- 229960002240 iloprost Drugs 0.000 description 1
- HIFJCPQKFCZDDL-ACWOEMLNSA-N iloprost Chemical compound C1\C(=C/CCCC(O)=O)C[C@@H]2[C@@H](/C=C/[C@@H](O)C(C)CC#CC)[C@H](O)C[C@@H]21 HIFJCPQKFCZDDL-ACWOEMLNSA-N 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 229940072221 immunoglobulins Drugs 0.000 description 1
- 238000005462 in vivo assay Methods 0.000 description 1
- 239000000411 inducer Substances 0.000 description 1
- 239000003701 inert diluent Substances 0.000 description 1
- 230000007574 infarction Effects 0.000 description 1
- 239000000893 inhibin Substances 0.000 description 1
- ZPNFWUPYTFPOJU-LPYSRVMUSA-N iniprol Chemical compound C([C@H]1C(=O)NCC(=O)NCC(=O)N[C@H]2CSSC[C@H]3C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@H](C(N[C@H](C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC=4C=CC=CC=4)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC=4C=CC=CC=4)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC2=O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC=2C=CC=CC=2)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H]2N(CCC2)C(=O)[C@@H](N)CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N2[C@@H](CCC2)C(=O)N2[C@@H](CCC2)C(=O)N[C@@H](CC=2C=CC(O)=CC=2)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N2[C@@H](CCC2)C(=O)N3)C(=O)NCC(=O)NCC(=O)N[C@@H](C)C(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@H](C(=O)N1)C(C)C)[C@@H](C)O)[C@@H](C)CC)=O)[C@@H](C)CC)C1=CC=C(O)C=C1 ZPNFWUPYTFPOJU-LPYSRVMUSA-N 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 230000026045 iodination Effects 0.000 description 1
- 238000006192 iodination reaction Methods 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 230000000302 ischemic effect Effects 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 238000006317 isomerization reaction Methods 0.000 description 1
- 230000006122 isoprenylation Effects 0.000 description 1
- 229960001632 labetalol Drugs 0.000 description 1
- 238000011545 laboratory measurement Methods 0.000 description 1
- 150000002611 lead compounds Chemical class 0.000 description 1
- 108020001756 ligand binding domains Proteins 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 238000011551 log transformation method Methods 0.000 description 1
- 238000007477 logistic regression Methods 0.000 description 1
- 208000018773 low birth weight Diseases 0.000 description 1
- 231100000533 low birth weight Toxicity 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 235000019359 magnesium stearate Nutrition 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 229960003390 magnesium sulfate Drugs 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 230000036244 malformation Effects 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- OCSMOTCMPXTDND-OUAUKWLOSA-N marimastat Chemical compound CNC(=O)[C@H](C(C)(C)C)NC(=O)[C@H](CC(C)C)[C@H](O)C(=O)NO OCSMOTCMPXTDND-OUAUKWLOSA-N 0.000 description 1
- 229950008959 marimastat Drugs 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000010197 meta-analysis Methods 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- MYWUZJCMWCOHBA-VIFPVBQESA-N methamphetamine Chemical compound CN[C@@H](C)CC1=CC=CC=C1 MYWUZJCMWCOHBA-VIFPVBQESA-N 0.000 description 1
- 125000001360 methionine group Chemical group N[C@@H](CCSC)C(=O)* 0.000 description 1
- 230000011987 methylation Effects 0.000 description 1
- 238000007069 methylation reaction Methods 0.000 description 1
- 229960001186 methysergide Drugs 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 150000007522 mineralic acids Chemical class 0.000 description 1
- 229960000841 minoxidil sulfate Drugs 0.000 description 1
- 229940126619 mouse monoclonal antibody Drugs 0.000 description 1
- 210000002464 muscle smooth vascular Anatomy 0.000 description 1
- BSIZUMJRKYHEBR-QGZVFWFLSA-N n-hydroxy-2(r)-[[(4-methoxyphenyl)sulfonyl](3-picolyl)amino]-3-methylbutanamide hydrochloride Chemical compound C1=CC(OC)=CC=C1S(=O)(=O)N([C@H](C(C)C)C(=O)NO)CC1=CC=CN=C1 BSIZUMJRKYHEBR-QGZVFWFLSA-N 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 230000001537 neural effect Effects 0.000 description 1
- 230000002182 neurohumoral effect Effects 0.000 description 1
- 230000000508 neurotrophic effect Effects 0.000 description 1
- SNICXCGAKADSCV-UHFFFAOYSA-N nicotine Natural products CN1CCCC1C1=CC=CN=C1 SNICXCGAKADSCV-UHFFFAOYSA-N 0.000 description 1
- 229960002715 nicotine Drugs 0.000 description 1
- HYIMSNHJOBLJNT-UHFFFAOYSA-N nifedipine Chemical compound COC(=O)C1=C(C)NC(C)=C(C(=O)OC)C1C1=CC=CC=C1[N+]([O-])=O HYIMSNHJOBLJNT-UHFFFAOYSA-N 0.000 description 1
- 229960001597 nifedipine Drugs 0.000 description 1
- 239000002687 nonaqueous vehicle Substances 0.000 description 1
- 239000000346 nonvolatile oil Substances 0.000 description 1
- 238000010606 normalization Methods 0.000 description 1
- 230000001019 normoglycemic effect Effects 0.000 description 1
- 125000003835 nucleoside group Chemical group 0.000 description 1
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 description 1
- OQCDKBAXFALNLD-UHFFFAOYSA-N octadecanoic acid Natural products CCCCCCCC(C)CCCCCCCCC(O)=O OQCDKBAXFALNLD-UHFFFAOYSA-N 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 230000002138 osteoinductive effect Effects 0.000 description 1
- 229940094443 oxytocics prostaglandins Drugs 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- IPCSVZSSVZVIGE-UHFFFAOYSA-N palmitic acid group Chemical group C(CCCCCCCCCCCCCCC)(=O)O IPCSVZSSVZVIGE-UHFFFAOYSA-N 0.000 description 1
- 230000026792 palmitoylation Effects 0.000 description 1
- 230000003076 paracrine Effects 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 230000007310 pathophysiology Effects 0.000 description 1
- 230000006320 pegylation Effects 0.000 description 1
- 229960002275 pentobarbital sodium Drugs 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 239000008194 pharmaceutical composition Substances 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- YXJYBPXSEKMEEJ-UHFFFAOYSA-N phosphoric acid;sulfuric acid Chemical compound OP(O)(O)=O.OS(O)(=O)=O YXJYBPXSEKMEEJ-UHFFFAOYSA-N 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 230000000865 phosphorylative effect Effects 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- OXNIZHLAWKMVMX-UHFFFAOYSA-N picric acid Chemical compound OC1=C([N+]([O-])=O)C=C([N+]([O-])=O)C=C1[N+]([O-])=O OXNIZHLAWKMVMX-UHFFFAOYSA-N 0.000 description 1
- 239000006187 pill Substances 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 108010017992 platelet-derived growth factor C Proteins 0.000 description 1
- 230000010287 polarization Effects 0.000 description 1
- 229920001200 poly(ethylene-vinyl acetate) Polymers 0.000 description 1
- 229920001515 polyalkylene glycol Polymers 0.000 description 1
- 102000040430 polynucleotide Human genes 0.000 description 1
- 108091033319 polynucleotide Proteins 0.000 description 1
- 239000002157 polynucleotide Substances 0.000 description 1
- 229920002503 polyoxyethylene-polyoxypropylene Polymers 0.000 description 1
- 229920002451 polyvinyl alcohol Polymers 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 230000029279 positive regulation of transcription, DNA-dependent Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 208000036335 preeclampsia/eclampsia 1 Diseases 0.000 description 1
- 230000013823 prenylation Effects 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 229950003608 prinomastat Drugs 0.000 description 1
- YKPYIPVDTNNYCN-INIZCTEOSA-N prinomastat Chemical compound ONC(=O)[C@H]1C(C)(C)SCCN1S(=O)(=O)C(C=C1)=CC=C1OC1=CC=NC=C1 YKPYIPVDTNNYCN-INIZCTEOSA-N 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- 238000011321 prophylaxis Methods 0.000 description 1
- 150000003180 prostaglandins Chemical class 0.000 description 1
- 230000009145 protein modification Effects 0.000 description 1
- 230000017854 proteolysis Effects 0.000 description 1
- 230000002797 proteolythic effect Effects 0.000 description 1
- 208000005333 pulmonary edema Diseases 0.000 description 1
- 230000035485 pulse pressure Effects 0.000 description 1
- 229940043131 pyroglutamate Drugs 0.000 description 1
- 230000006340 racemization Effects 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 230000000306 recurrent effect Effects 0.000 description 1
- 238000000611 regression analysis Methods 0.000 description 1
- 230000029865 regulation of blood pressure Effects 0.000 description 1
- 230000025053 regulation of cell proliferation Effects 0.000 description 1
- 230000026267 regulation of growth Effects 0.000 description 1
- 230000033904 relaxation of vascular smooth muscle Effects 0.000 description 1
- 230000008085 renal dysfunction Effects 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 210000001995 reticulocyte Anatomy 0.000 description 1
- 206010038796 reticulocytosis Diseases 0.000 description 1
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 1
- 238000007363 ring formation reaction Methods 0.000 description 1
- 229920002477 rna polymer Polymers 0.000 description 1
- 235000019515 salmon Nutrition 0.000 description 1
- 229940043230 sarcosine Drugs 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000002047 solid lipid nanoparticle Substances 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000012289 standard assay Methods 0.000 description 1
- 239000008117 stearic acid Substances 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 108091007196 stromelysin Proteins 0.000 description 1
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 1
- 239000007939 sustained release tablet Substances 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 230000005062 synaptic transmission Effects 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- 239000000454 talc Substances 0.000 description 1
- 229910052623 talc Inorganic materials 0.000 description 1
- 238000004885 tandem mass spectrometry Methods 0.000 description 1
- 235000015523 tannic acid Nutrition 0.000 description 1
- 229920002258 tannic acid Polymers 0.000 description 1
- LRBQNJMCXXYXIU-NRMVVENXSA-N tannic acid Chemical compound OC1=C(O)C(O)=CC(C(=O)OC=2C(=C(O)C=C(C=2)C(=O)OC[C@@H]2[C@H]([C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)O2)OC(=O)C=2C=C(OC(=O)C=3C=C(O)C(O)=C(O)C=3)C(O)=C(O)C=2)O)=C1 LRBQNJMCXXYXIU-NRMVVENXSA-N 0.000 description 1
- 229940033123 tannic acid Drugs 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 208000009056 telangiectasis Diseases 0.000 description 1
- 229960000278 theophylline Drugs 0.000 description 1
- 239000003767 thromboxane receptor stimulating agent Substances 0.000 description 1
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical class CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 229960000984 tocofersolan Drugs 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- FGMPLJWBKKVCDB-UHFFFAOYSA-N trans-L-hydroxy-proline Natural products ON1CCCC1C(O)=O FGMPLJWBKKVCDB-UHFFFAOYSA-N 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- YFDSDPIBEUFTMI-UHFFFAOYSA-N tribromoethanol Chemical compound OCC(Br)(Br)Br YFDSDPIBEUFTMI-UHFFFAOYSA-N 0.000 description 1
- 229950004616 tribromoethanol Drugs 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 238000010798 ubiquitination Methods 0.000 description 1
- 230000034512 ubiquitination Effects 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 241001515965 unidentified phage Species 0.000 description 1
- 229940116269 uric acid Drugs 0.000 description 1
- 208000019206 urinary tract infection Diseases 0.000 description 1
- 238000005353 urine analysis Methods 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- LSGOVYNHVSXFFJ-UHFFFAOYSA-N vanadate(3-) Chemical compound [O-][V]([O-])([O-])=O LSGOVYNHVSXFFJ-UHFFFAOYSA-N 0.000 description 1
- 208000019553 vascular disease Diseases 0.000 description 1
- 230000006492 vascular dysfunction Effects 0.000 description 1
- 230000001196 vasorelaxation Effects 0.000 description 1
- 230000002883 vasorelaxation effect Effects 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
- 229920003169 water-soluble polymer Polymers 0.000 description 1
- 230000004584 weight gain Effects 0.000 description 1
- 235000019786 weight gain Nutrition 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
- XOOUIPVCVHRTMJ-UHFFFAOYSA-L zinc stearate Chemical compound [Zn+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O XOOUIPVCVHRTMJ-UHFFFAOYSA-L 0.000 description 1
- 108010088577 zinc-binding protein Proteins 0.000 description 1
- 210000004340 zona pellucida Anatomy 0.000 description 1
- 239000002076 α-tocopherol Substances 0.000 description 1
- 235000004835 α-tocopherol Nutrition 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
- A61K38/4886—Metalloendopeptidases (3.4.24), e.g. collagenase
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
- A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
- A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
- A61K31/455—Nicotinic acids, e.g. niacin; Derivatives thereof, e.g. esters, amides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
- A61K31/52—Purines, e.g. adenine
- A61K31/522—Purines, e.g. adenine having oxo groups directly attached to the heterocyclic ring, e.g. hypoxanthine, guanine, acyclovir
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
- A61K38/1841—Transforming growth factor [TGF]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
- A61K38/1858—Platelet-derived growth factor [PDGF]
- A61K38/1866—Vascular endothelial growth factor [VEGF]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
- A61K38/1875—Bone morphogenic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
- A61K38/482—Serine endopeptidases (3.4.21)
- A61K38/486—Elastase (3.4.21.36 or 3.4.21.37)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/12—Antihypertensives
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/36—Gynecology or obstetrics
- G01N2800/368—Pregnancy complicated by disease or abnormalities of pregnancy, e.g. preeclampsia, preterm labour
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Engineering & Computer Science (AREA)
- Epidemiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Immunology (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Hematology (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Molecular Biology (AREA)
- Food Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Reproductive Health (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Cardiology (AREA)
- Diabetes (AREA)
- Heart & Thoracic Surgery (AREA)
- Vascular Medicine (AREA)
- Physics & Mathematics (AREA)
- Endocrinology (AREA)
- Biochemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
Abstract
Disclosed herein are methods for treating a pregnancy related hypertensive disorder, such as pre-eclampsia and eclampsia, using combinations of compounds that alter soluble endoglin, endothelial nitric oxide synthase, PGI2, TGF-.beta.l, TGF-.beta.3, activin A, BMP2, BMP7, and sFlt-1 expression levels or biological activity. Also disclosed are methods of diagnosing a pregnancy related hypertensive disorder, such as pre-eclampsia and eclampsia, that include the measurement of any one or more of the following: soluble endoglin, endothelial nitric oxide synthase, PGI2, TGF-.beta.l, TGF-.beta.3, activin A, BMP2, BMP7, and sFlt-1 expression levels or biological activity.
Description
METHODS OF DIAGNOSING AND TREATING
COMPLICATIONS OF PREGNANCY
Field of the Invention In general, this invention relates to the detection and treatment of subjects having a pregnancy related hypertensive disorder.
Background of the Invention = 10 Pre-eclampsia is a syndrome of hypertension, edema, and proteinuria that affects 5 to 10% of pregnancies and results in substantial maternal and fetal morbidity and mortality. Pre-eclampsia accounts for at least 200,000 maternal deaths worldwide per year. The symptoms of pre-eclampsia typically appear after the 200' week of pregnancy and are usually detected by routine measuring of the woman's blood pressure and urine.
However, these monitoring methods are ineffective for diagnosis of the syndrome at an early stage, which could reduce the risk to the subject or developing fetus, if an effective treatment were available.
Currently there are no known cures for pre-eclampsia. Pre-eclampsia can vary in severity from mild to life threatening. A mild form of pre-eclampsia can be treated with bed rest and frequent monitoring. For moderate to severe cases, hospitalization is recommended and blood pressure medication or anticonvulsant medications to prevent seizures are prescribed. If the condition becomes life threatening to the mother or the baby the pregnancy is terminated and the baby is delivered pre-term.
The proper development of the fetus and the placenta is mediated by several growth factors or angiogenic factors. One of these angiogenic factors is endoglin, also known as CD 105. Endoglin is a homodimeric cell membrane glycoprotein that is predominantly expressed on endothelial cells such as syncytiotrophoblasts, human unbilical vein endothelial cells (I-IWEC), and on vascular endothelial cells.
Endoglin shares sequence identity with betaglycan, a transforming growth factor (TGF)-(3 receptor (T13R) type III. Endoglin has been shown to be a regulatory component of the receptor complex, which modulates angiogenesis, proliferation, differentiation, and apoptosis. Endoglin also binds several other members of the TGF-P superfamily I
including activin-A, bone morphogenic protein (BMP)-2 and BMP-7. In particular, endoglin binds TGF-01 and TGF-03 with high affinity and forms heterotrimeric associations with the TGF-0 signaling receptors types I and II. Mutations in the coding region of the "endoglin gene are responsible for haemorrhagic telangiectasia type 1 (HHT1), a dominantly inherited vascular disorder characterized by multisystemic vascular dysplasia and recurrent hernorrhage. While endoglin immunoreactivity has been previously detected at increased levels in the plasma of patients with metastatic breast and colorectal cancer, its biochemical characteristics have not been determined and its exact functional role in the pathogenesis of cancer is unclear. Soluble endoglin production has not been reported to be associated with pre-eclampsia or normal pregnancy.
Several factors have been reported to have an association with fetal and placental development and, more specifically, with pre-eclampsia. They include vascular endothelial growth factor (VEGF), soluble Flt-1 receptor (sFlt-1), and placental growth factor (P1GF). VEGF is an endothelial cell-specific mitogen, an angiogenic inducer, and a mediator of vascular permeability. VEGF has also been shown to be important for glomerular capillary repair. VEGF binds as a homodimer to one of two homologous membrane-spanning tyrosine kinase receptors, the fms-like tyrosine kinase (Flt-1) and the kinase domain receptor (KDR), which are differentially expressed in endothelial cells obtained from many different tissues. FIt-1, but not KDR, is highly expressed by trophoblast cells which contribute to placental formation. PIGF is a VEGF
family member that'is also involved in placental development. P1GF is expressed by cytotrophoblasts and syncytiotrophoblasts and is capable of inducing proliferation, migration, and activation of endothelial cells. PIGF binds as a homodimer to the Fit-1 receptor, but not the KDR receptor. Both PIGF and VEGF contribute to the mitogenic activity and angiogenesis that are critical for the developing placenta.
sFlt-1, which lacks the transmembrane and cytoplasmic domains of the receptor, was recently identified in a cultured medium of human umbilical vein endothelial cells and in vivo expression was subsequently demonstrated in placental tissue. sFlt-1 binds to VEGF with a high affinity but does not stimulate mitogenesis of endothelial cells.
Careful regulation of angiogenic and mitogenic signaling pathways is critical for
COMPLICATIONS OF PREGNANCY
Field of the Invention In general, this invention relates to the detection and treatment of subjects having a pregnancy related hypertensive disorder.
Background of the Invention = 10 Pre-eclampsia is a syndrome of hypertension, edema, and proteinuria that affects 5 to 10% of pregnancies and results in substantial maternal and fetal morbidity and mortality. Pre-eclampsia accounts for at least 200,000 maternal deaths worldwide per year. The symptoms of pre-eclampsia typically appear after the 200' week of pregnancy and are usually detected by routine measuring of the woman's blood pressure and urine.
However, these monitoring methods are ineffective for diagnosis of the syndrome at an early stage, which could reduce the risk to the subject or developing fetus, if an effective treatment were available.
Currently there are no known cures for pre-eclampsia. Pre-eclampsia can vary in severity from mild to life threatening. A mild form of pre-eclampsia can be treated with bed rest and frequent monitoring. For moderate to severe cases, hospitalization is recommended and blood pressure medication or anticonvulsant medications to prevent seizures are prescribed. If the condition becomes life threatening to the mother or the baby the pregnancy is terminated and the baby is delivered pre-term.
The proper development of the fetus and the placenta is mediated by several growth factors or angiogenic factors. One of these angiogenic factors is endoglin, also known as CD 105. Endoglin is a homodimeric cell membrane glycoprotein that is predominantly expressed on endothelial cells such as syncytiotrophoblasts, human unbilical vein endothelial cells (I-IWEC), and on vascular endothelial cells.
Endoglin shares sequence identity with betaglycan, a transforming growth factor (TGF)-(3 receptor (T13R) type III. Endoglin has been shown to be a regulatory component of the receptor complex, which modulates angiogenesis, proliferation, differentiation, and apoptosis. Endoglin also binds several other members of the TGF-P superfamily I
including activin-A, bone morphogenic protein (BMP)-2 and BMP-7. In particular, endoglin binds TGF-01 and TGF-03 with high affinity and forms heterotrimeric associations with the TGF-0 signaling receptors types I and II. Mutations in the coding region of the "endoglin gene are responsible for haemorrhagic telangiectasia type 1 (HHT1), a dominantly inherited vascular disorder characterized by multisystemic vascular dysplasia and recurrent hernorrhage. While endoglin immunoreactivity has been previously detected at increased levels in the plasma of patients with metastatic breast and colorectal cancer, its biochemical characteristics have not been determined and its exact functional role in the pathogenesis of cancer is unclear. Soluble endoglin production has not been reported to be associated with pre-eclampsia or normal pregnancy.
Several factors have been reported to have an association with fetal and placental development and, more specifically, with pre-eclampsia. They include vascular endothelial growth factor (VEGF), soluble Flt-1 receptor (sFlt-1), and placental growth factor (P1GF). VEGF is an endothelial cell-specific mitogen, an angiogenic inducer, and a mediator of vascular permeability. VEGF has also been shown to be important for glomerular capillary repair. VEGF binds as a homodimer to one of two homologous membrane-spanning tyrosine kinase receptors, the fms-like tyrosine kinase (Flt-1) and the kinase domain receptor (KDR), which are differentially expressed in endothelial cells obtained from many different tissues. FIt-1, but not KDR, is highly expressed by trophoblast cells which contribute to placental formation. PIGF is a VEGF
family member that'is also involved in placental development. P1GF is expressed by cytotrophoblasts and syncytiotrophoblasts and is capable of inducing proliferation, migration, and activation of endothelial cells. PIGF binds as a homodimer to the Fit-1 receptor, but not the KDR receptor. Both PIGF and VEGF contribute to the mitogenic activity and angiogenesis that are critical for the developing placenta.
sFlt-1, which lacks the transmembrane and cytoplasmic domains of the receptor, was recently identified in a cultured medium of human umbilical vein endothelial cells and in vivo expression was subsequently demonstrated in placental tissue. sFlt-1 binds to VEGF with a high affinity but does not stimulate mitogenesis of endothelial cells.
Careful regulation of angiogenic and mitogenic signaling pathways is critical for
2
3 PCT/US2007/012787 maintaining appropriate proliferation, migration, and angiogenesis by trophoblast cells in the developing placenta.
There is a need for methods of accurately diagnosing subjects at risk for or having pre-eclampsia or eclampsia, particularly before the onset of the most severe symptoms.
A treatment is also needed.
Summary of the Invention We have discovered methods for diagnosing and treating pregnancy related hypertensive disorders, including pre-ecla.mpsia and eclampsia.
Using gene expression analysis, we have discovered that levels of soluble endoglin (sEng) are markedly elevated in placental tissue samples from pregnant women suffering from pregnancy complications associated with hypertension, including pre-eclampsia. Using western blotting, we have also discovered that soluble endoglin protein levels are elevated in blood serum samples taken from women with a pregnancy related hypertensive disorder, such as pre-eclampsia or eclampsia. Soluble endoglin may be formed by cleavage of the extracellular portion of the membrane bounds form by proteolytic enzymes. We have discovered that the soluble endoglin detected in these samples contains a minimum of the first 381 amino acids (excluding the leader peptide, 406 including the leader peptide) of the amino terminal portion of the full-length endoglin. Excess soluble endoglin in pre-eclampsia may be depleting the placenta of necessary amounts of these essential angiogenic and mitogenic factors by preventing binding of TGF-01 to TPRII on endothelial cells leading to decreased signaling, as described herein. We have also discovered that soluble endoglin interferes with TGF-0 I
signaling and endothelial nitric oxide synthase (eNOS) activation in endothelial cells, thereby disrupting key homeostatic mechariisms necessary for maintenance of vascular health. We demonstrate that soluble endoglin prevents binding of TGF-01 to T(3.RII on endothelial cells leading to decreased signaling. Since circulating TGF-01 is complexed with latency associated peptide and latent TGF-(31 binding protein, it cannot bind its receptors, unless activated. It is therefore likely that soluble endoglin only inhibits TGF-(31 effects locally where active TGF-Pl is generated. Taken together, these data suggest a crucial role for endoglin in linking TGF-0 receptor activation to nitric oxide (NO) synthesis. In addition, our functional studies suggest that soli.ible endoglin and sF]tl act in concert to induce vascular damage and HELLP syndrome by interfering with TGF- j31 and VEGF signaling respectively, likely via inhibition of the downstream activation of NOS.
In the present invention, compounds that bind to or neutralize soluble endoglin are used to reduce the elevated levels of soluble endoglin and to treat pregnancy complications associated with hypertension, including pre-eclampsia or eclampsia. For example, antibodies directed to soluble endoglin as well as RNA interference and antisense nucleobase oligomers directed to lowering the levels of biologically active soluble endoglin are also provided. The invention also features the use of any compound (e.g., polypeptide, small molecule, antibody, nucleic acid, and mimetic) that decreases soluble endoglin levels or biological activity or that increases the level or biological activity of a soluble endoglin binding protein (e.g., soluble endoglin binding protein (e.g., TGF-(31, TGF-(33, activin A, Bone Morphogenic Protein (BMP)-2 and BMP-7), NOS, and prostacyclin (PGI2) either alone or in combination with each other or with any compound that decreases the level of sFlt-1 or increases the level or activity of VEGF or PIGF (see for example, U.S. Patent Application Publication Numbers 20040126828, 20050025762, and 20050170444 and PCT Publication Numbers WO 2004/008946 and WO 2005/077007) to treat or prevent pregnancy related hypertensive disorders, such as pre-eclampsia or eclampsia in a subject. The invention also features methods for measuring levels of soluble endoglin, either alone or in combination with sFlt-1, VEGF, PIGF, TGF-{3, eNOS, or PGI2, as a detection tool for early diagnosis and management of a pregnancy related hypertensive disorder, including pre-eclampsia and eclampsia.
Accordingly, in a first aspect, the invention features a method of treating or preventing a pregnancy related hypertensive disorder in a subject, that includes administering to the subject (i) a compound capable of decreasing soluble endoglin expression levels or biological activity and (ii) a compound capable of decreasing sFlt-1 expression levels or biological activity, for a time and in an amount sufficient to treat or prevent the pregnancy related hypertensive disorder. Pregnancy related hypertensive disorder include, for example, pre-eclampsia, eclampsia, gestational hypertension, chronic hypertension, HELLP syndrome, and pregnancy with a small for gestational age
There is a need for methods of accurately diagnosing subjects at risk for or having pre-eclampsia or eclampsia, particularly before the onset of the most severe symptoms.
A treatment is also needed.
Summary of the Invention We have discovered methods for diagnosing and treating pregnancy related hypertensive disorders, including pre-ecla.mpsia and eclampsia.
Using gene expression analysis, we have discovered that levels of soluble endoglin (sEng) are markedly elevated in placental tissue samples from pregnant women suffering from pregnancy complications associated with hypertension, including pre-eclampsia. Using western blotting, we have also discovered that soluble endoglin protein levels are elevated in blood serum samples taken from women with a pregnancy related hypertensive disorder, such as pre-eclampsia or eclampsia. Soluble endoglin may be formed by cleavage of the extracellular portion of the membrane bounds form by proteolytic enzymes. We have discovered that the soluble endoglin detected in these samples contains a minimum of the first 381 amino acids (excluding the leader peptide, 406 including the leader peptide) of the amino terminal portion of the full-length endoglin. Excess soluble endoglin in pre-eclampsia may be depleting the placenta of necessary amounts of these essential angiogenic and mitogenic factors by preventing binding of TGF-01 to TPRII on endothelial cells leading to decreased signaling, as described herein. We have also discovered that soluble endoglin interferes with TGF-0 I
signaling and endothelial nitric oxide synthase (eNOS) activation in endothelial cells, thereby disrupting key homeostatic mechariisms necessary for maintenance of vascular health. We demonstrate that soluble endoglin prevents binding of TGF-01 to T(3.RII on endothelial cells leading to decreased signaling. Since circulating TGF-01 is complexed with latency associated peptide and latent TGF-(31 binding protein, it cannot bind its receptors, unless activated. It is therefore likely that soluble endoglin only inhibits TGF-(31 effects locally where active TGF-Pl is generated. Taken together, these data suggest a crucial role for endoglin in linking TGF-0 receptor activation to nitric oxide (NO) synthesis. In addition, our functional studies suggest that soli.ible endoglin and sF]tl act in concert to induce vascular damage and HELLP syndrome by interfering with TGF- j31 and VEGF signaling respectively, likely via inhibition of the downstream activation of NOS.
In the present invention, compounds that bind to or neutralize soluble endoglin are used to reduce the elevated levels of soluble endoglin and to treat pregnancy complications associated with hypertension, including pre-eclampsia or eclampsia. For example, antibodies directed to soluble endoglin as well as RNA interference and antisense nucleobase oligomers directed to lowering the levels of biologically active soluble endoglin are also provided. The invention also features the use of any compound (e.g., polypeptide, small molecule, antibody, nucleic acid, and mimetic) that decreases soluble endoglin levels or biological activity or that increases the level or biological activity of a soluble endoglin binding protein (e.g., soluble endoglin binding protein (e.g., TGF-(31, TGF-(33, activin A, Bone Morphogenic Protein (BMP)-2 and BMP-7), NOS, and prostacyclin (PGI2) either alone or in combination with each other or with any compound that decreases the level of sFlt-1 or increases the level or activity of VEGF or PIGF (see for example, U.S. Patent Application Publication Numbers 20040126828, 20050025762, and 20050170444 and PCT Publication Numbers WO 2004/008946 and WO 2005/077007) to treat or prevent pregnancy related hypertensive disorders, such as pre-eclampsia or eclampsia in a subject. The invention also features methods for measuring levels of soluble endoglin, either alone or in combination with sFlt-1, VEGF, PIGF, TGF-{3, eNOS, or PGI2, as a detection tool for early diagnosis and management of a pregnancy related hypertensive disorder, including pre-eclampsia and eclampsia.
Accordingly, in a first aspect, the invention features a method of treating or preventing a pregnancy related hypertensive disorder in a subject, that includes administering to the subject (i) a compound capable of decreasing soluble endoglin expression levels or biological activity and (ii) a compound capable of decreasing sFlt-1 expression levels or biological activity, for a time and in an amount sufficient to treat or prevent the pregnancy related hypertensive disorder. Pregnancy related hypertensive disorder include, for example, pre-eclampsia, eclampsia, gestational hypertension, chronic hypertension, HELLP syndrome, and pregnancy with a small for gestational age
4 (SGA) infant. Preferably, the pregnancy related hypertensive disorder is pre-eclampsia or eclampsia.
Assays for soluble endoglin or sFlt-1 expression levels or biological activity are known in the art. Preferred compounds wi11 decrease soluble endoglin or sFlt-1 expression levels or biological activity by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more. Non-limiting examples of compounds capable of decreasing soluble endoglin expression levels or biological activity include any compound that specifically binds soluble endoglin, for example, a purified soluble endoglin antibody, a soluble endoglin antigen-binding fragment, or a soluble endoglin binding protein (e.g., TGF-0l, TGF-(33, activin A, BMP-2 and BMP-7).
Additional examples of a compound capable of decreasing soluble endoglin expression levels or biological activity include any compound that inhibits a proteolytic enzyme (e.g., a matrix metalloproteinase (1VIIVIP), cathepsin, and elastase) or a compound that increases the level of a growth factor capable of binding to soluble endoglin. Growth factors such as TGF-(31, TGF-P3, activin A, BMP-2, BMP-7, or fragments thereof, are examples of compounds that increases the level of a growth factor capable of binding to soluble endoglin as are cyclosporine, alpha tocopherol, methysergide, bromocriptine, and aldomet.
Non-limiting examples of a compound capable of decreasing sFlt-1 expression levels or biological activity include a compound capable of specifically binding to sFlt-1, such as a purified sFIt-1 antibody or an sFlt-1 antigen-binding fragment;
compounds that increase the level of a growth factor capable of binding to sFlt-1, such as nicotine, theophylline, adenosine, nifedipine, minoxidil, and magnesium sulfate, VEGF
(e.g., VEGF121, VEGF165, or a modified form of VEGF), PIGF, or fragments thereof.
In preferred embodiments of the above method, a compound capable of decreasing soluble endoglin expression levels or biological activity or a compound capable of decreasing sFlt-1 expression levels or biological activity, or both, can also increase nitric oxide synthase (NOS) activity by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more. Assays for NOS activity are known in the art and described herein.
Assays for soluble endoglin or sFlt-1 expression levels or biological activity are known in the art. Preferred compounds wi11 decrease soluble endoglin or sFlt-1 expression levels or biological activity by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more. Non-limiting examples of compounds capable of decreasing soluble endoglin expression levels or biological activity include any compound that specifically binds soluble endoglin, for example, a purified soluble endoglin antibody, a soluble endoglin antigen-binding fragment, or a soluble endoglin binding protein (e.g., TGF-0l, TGF-(33, activin A, BMP-2 and BMP-7).
Additional examples of a compound capable of decreasing soluble endoglin expression levels or biological activity include any compound that inhibits a proteolytic enzyme (e.g., a matrix metalloproteinase (1VIIVIP), cathepsin, and elastase) or a compound that increases the level of a growth factor capable of binding to soluble endoglin. Growth factors such as TGF-(31, TGF-P3, activin A, BMP-2, BMP-7, or fragments thereof, are examples of compounds that increases the level of a growth factor capable of binding to soluble endoglin as are cyclosporine, alpha tocopherol, methysergide, bromocriptine, and aldomet.
Non-limiting examples of a compound capable of decreasing sFlt-1 expression levels or biological activity include a compound capable of specifically binding to sFlt-1, such as a purified sFIt-1 antibody or an sFlt-1 antigen-binding fragment;
compounds that increase the level of a growth factor capable of binding to sFlt-1, such as nicotine, theophylline, adenosine, nifedipine, minoxidil, and magnesium sulfate, VEGF
(e.g., VEGF121, VEGF165, or a modified form of VEGF), PIGF, or fragments thereof.
In preferred embodiments of the above method, a compound capable of decreasing soluble endoglin expression levels or biological activity or a compound capable of decreasing sFlt-1 expression levels or biological activity, or both, can also increase nitric oxide synthase (NOS) activity by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more. Assays for NOS activity are known in the art and described herein.
5 In a second aspect, the invention fetures a method of treating or preventing a pregnancy related hypertensive disorder in a subject, that includes the step of administering to the subject a compound capable of increasing the expression level or biological activity of NOS, for a time and in an amount sufficient to treat or prevent the pregnancy related hypertensive disorder in the subject. Desirably, the NOS is eNOS. In one embodiment, the compound is a compound that increases the phosphorylation of Ser 1177 of eNOS, such as VEGF (e.g., VEGF121, VEGF165, or a modified form of VEGF), or biologically active fragments thereof, or P1GF or biologically active fragments thereof.
In another embodiment, the compound is a compound that increases the dephosphorylation of Thr495 of eNOS, such as TGF-01 or TGF-P3, activin A, BMP-2, and BMP-7. In another embodiment, the compound is a compound that prevents a reduction in the levels of eNOS or increases the stability of eNOS.
Optionally, the method further includes administering to the subject a compound capable of reducing soluble endoglin expression levels or biological activity, wherein the administering is sufficient to treat or prevent the pregnancy related hypertensive disorder in the subject. Non-limiting examples of a compound capable of reducing soluble endoglin expression or biological activity include a purified antibody that specifically binds soluble endoglin or a soluble endoglin antigen-binding fragment or a compound that inhibits a proteolytic enzyme selected from the group consisting of a matrix metalloproteinase (MMP), cathepsin, and elastase, or growth factors such as TGF-(31, TGF-03, activin A, BMP-2, BMP-7, or fragments thereof. Exemplary antibodies that specifically bind soluble endoglin include antibodies that bind to a soluble endoglin polypeptide that includes an amino acid sequence selected from the group consisting of amino acids 26 to 437,40 to 406, or 26 to 587 of the human endoglin sequence shown in Figure 30B. Additional exemplary antibodies useful in the methods of the invention include an antibody that binds to an epitope on human endoglin that includes amino acids 40 to 86, 144 to 199, 206 to 222, 289 to 304, or 375 to 381 of the human endoglin sequence shown in Figure 30B.
In one embodimerit, the pregnancy related hypertensive disorder is characterized 'by elevated levels of sFlt-1 polypeptide as,compared to a normal reference.
Optionally, the method further includes administering to the subject a compound capable of reducing
In another embodiment, the compound is a compound that increases the dephosphorylation of Thr495 of eNOS, such as TGF-01 or TGF-P3, activin A, BMP-2, and BMP-7. In another embodiment, the compound is a compound that prevents a reduction in the levels of eNOS or increases the stability of eNOS.
Optionally, the method further includes administering to the subject a compound capable of reducing soluble endoglin expression levels or biological activity, wherein the administering is sufficient to treat or prevent the pregnancy related hypertensive disorder in the subject. Non-limiting examples of a compound capable of reducing soluble endoglin expression or biological activity include a purified antibody that specifically binds soluble endoglin or a soluble endoglin antigen-binding fragment or a compound that inhibits a proteolytic enzyme selected from the group consisting of a matrix metalloproteinase (MMP), cathepsin, and elastase, or growth factors such as TGF-(31, TGF-03, activin A, BMP-2, BMP-7, or fragments thereof. Exemplary antibodies that specifically bind soluble endoglin include antibodies that bind to a soluble endoglin polypeptide that includes an amino acid sequence selected from the group consisting of amino acids 26 to 437,40 to 406, or 26 to 587 of the human endoglin sequence shown in Figure 30B. Additional exemplary antibodies useful in the methods of the invention include an antibody that binds to an epitope on human endoglin that includes amino acids 40 to 86, 144 to 199, 206 to 222, 289 to 304, or 375 to 381 of the human endoglin sequence shown in Figure 30B.
In one embodimerit, the pregnancy related hypertensive disorder is characterized 'by elevated levels of sFlt-1 polypeptide as,compared to a normal reference.
Optionally, the method further includes administering to the subject a compound capable of reducing
6 sFlt-1 expression or biological activity, wherein the administering is sufficient to treat or prevent the pregnancy related hypertensive disorder in the subject. Non-limiting examples of a compound capable of reducing soluble sFlt-1 expression or biological activity include a purified antibody that specifically binds sFlt-I or a sFlt-1 antigen binding fragment, or a growth factor such as VEGF (e.g., VEGFi21, VEGF165, or a modified form of VEGF), PIGF, or fragments thereof that bind to sFlt-1.
For any of the above methods, the method can further include the step of administering to a subject an anti-hypertensive compound. In preferred embodiments of any of the above methods, the subject is a pregnant human, a post-partum human, or a non-human (e.g, a cow, a horse, a sheep, a pig, a goat, a dog, and a cat).
For any of the above methods, the method can further include the step of monitoring the pregnancy related hypertensive disorder in the subject, wherein the monitoring includes measuring the level of soluble endoglin polypeptide in a serum or plasma sample from the subject. If the absolute level of soluble endoglin is deteremined, a level of soluble endoglin polypeptide less than 25 ng/ml indicates an improvement in the pregnancy related hypertensive disorder. Alternatively or additionally, the soluble endoglin level can be measured on two or more occasions or compared to a positive reference sample (e.g., from a subject suffering from a pregnancy related hypertensive disorder), where a decrease in the soluble endoglin level either between measurements or as compared to the positive reference is an indicator of an improvement in the pregnancy related hypertensive disorder. The measuring of the soluble endoglin levels can include the use of an immunological assay. The soluble endoglin can include free, bound, or total soluble endoglin or the level of an endoglin polypeptide resulting from degradation or enzymatic cleavage. The monitoring methods can also include any of the metrics described herein for the diagnosis of pre-eclampsia or eclampsia. For example, the monitoring method can include measuring the levels of sFlt-1 and soluble endoglin in the first and second trimesters in a subject and calculating the delta value of sFltl x soluble endoglin (sEng) in each trimester using the following equation: [dproduct =(sFltl x sEng) in the second trimester -(sFltl x sEng) in the first trimester], where a value greater than 0, 1, 2, or more, including fractions thereof, (e.g., a positive value) is a diagnostic indicator of pre-eclampsia or eclampsia. A decrease in the value over time indicates an
For any of the above methods, the method can further include the step of administering to a subject an anti-hypertensive compound. In preferred embodiments of any of the above methods, the subject is a pregnant human, a post-partum human, or a non-human (e.g, a cow, a horse, a sheep, a pig, a goat, a dog, and a cat).
For any of the above methods, the method can further include the step of monitoring the pregnancy related hypertensive disorder in the subject, wherein the monitoring includes measuring the level of soluble endoglin polypeptide in a serum or plasma sample from the subject. If the absolute level of soluble endoglin is deteremined, a level of soluble endoglin polypeptide less than 25 ng/ml indicates an improvement in the pregnancy related hypertensive disorder. Alternatively or additionally, the soluble endoglin level can be measured on two or more occasions or compared to a positive reference sample (e.g., from a subject suffering from a pregnancy related hypertensive disorder), where a decrease in the soluble endoglin level either between measurements or as compared to the positive reference is an indicator of an improvement in the pregnancy related hypertensive disorder. The measuring of the soluble endoglin levels can include the use of an immunological assay. The soluble endoglin can include free, bound, or total soluble endoglin or the level of an endoglin polypeptide resulting from degradation or enzymatic cleavage. The monitoring methods can also include any of the metrics described herein for the diagnosis of pre-eclampsia or eclampsia. For example, the monitoring method can include measuring the levels of sFlt-1 and soluble endoglin in the first and second trimesters in a subject and calculating the delta value of sFltl x soluble endoglin (sEng) in each trimester using the following equation: [dproduct =(sFltl x sEng) in the second trimester -(sFltl x sEng) in the first trimester], where a value greater than 0, 1, 2, or more, including fractions thereof, (e.g., a positive value) is a diagnostic indicator of pre-eclampsia or eclampsia. A decrease in the value over time indicates an
7 improvement in the pregnancy related hypertensive disorder. In another example, the delta product for the sFlt-1 level (dsFlt-1) or the sEng level (dsEng) between the first and second trimesters is calculated, and a value greater than 0, 1, 2, or more, including fractions thereof, (e.g., a positive value) for (dsFlt-1) or (dsEng) is a diagnostic indicator of pre-eclampsia or eclampsia. A decrease in the value over time indicates an improvement in the pregnancy related hypertensive disorder.
For any of the monitoring methods, the method can be used to determine the therapeutic dosage of the compound. The method can also further include measuring the level of at least one of sFlt-1, VEGF, or PIGF polypeptide in a sample from the subject and a relationship between these levels can also, but need not, be calculated using a metric. Exemplary metrics include [(sFlt-1 + 0.25 soluble endoglin)/PIGF], [(soluble endoglin + sFl.t-1)/P1GF], [sFlt-I x soluble endoglin], and the dsFlt-1, dsEng, and [dproduct =(sFltl x sEng) in the second trimester -(sFltl x sEng) in the first trimester], as described above.
. In another aspect, the invention features an antibody or antigen-binding fragment thereof that specifically binds a soluble endoglin polypeptide, wherein the antibody binds to an epitope on human endoglin that includes amino acids 40 to 86, 144 to 199, 206 to 222, 289 to 304, or 375 to 381 of the human endoglin sequence shown in Figure 30B. In one embodiment, the antibody or antigen-binding fragment prevents binding of a growth factor (e.g., TGF-01, TGF-03, activin A, BMP-2, and BMP-7) to soluble endoglin.
The antibody can be any type of antibody or antibody fragment including a monoclonal antibody, chimeric antibody, humanized antibody, human antibody, an antibody lacks an Fc portion, is an F(ab')Z, an Fab, or an Fv structure. In one embodiment, the antibody or antigen-binding fragment thereof is present in a pharmaceuticaIly acceptable carrier.
In another aspect, the invention features a method of diagnosing a subject as having, or having a predisposition to, a pregnancy related hypertensive disorder, that includes measuring the level of a soluble endoglin polypeptide and at least one additional polypeptide selected from the group consisting of soluble endoglin binding proteins (e.g., TGF-01, TGF-03, activin A, BMP2, and BIv1P 7) and downstream mediators of soluble endoglin signaling (e.g., eNOS and PGI2) in a sample from the subject, wherein an
For any of the monitoring methods, the method can be used to determine the therapeutic dosage of the compound. The method can also further include measuring the level of at least one of sFlt-1, VEGF, or PIGF polypeptide in a sample from the subject and a relationship between these levels can also, but need not, be calculated using a metric. Exemplary metrics include [(sFlt-1 + 0.25 soluble endoglin)/PIGF], [(soluble endoglin + sFl.t-1)/P1GF], [sFlt-I x soluble endoglin], and the dsFlt-1, dsEng, and [dproduct =(sFltl x sEng) in the second trimester -(sFltl x sEng) in the first trimester], as described above.
. In another aspect, the invention features an antibody or antigen-binding fragment thereof that specifically binds a soluble endoglin polypeptide, wherein the antibody binds to an epitope on human endoglin that includes amino acids 40 to 86, 144 to 199, 206 to 222, 289 to 304, or 375 to 381 of the human endoglin sequence shown in Figure 30B. In one embodiment, the antibody or antigen-binding fragment prevents binding of a growth factor (e.g., TGF-01, TGF-03, activin A, BMP-2, and BMP-7) to soluble endoglin.
The antibody can be any type of antibody or antibody fragment including a monoclonal antibody, chimeric antibody, humanized antibody, human antibody, an antibody lacks an Fc portion, is an F(ab')Z, an Fab, or an Fv structure. In one embodiment, the antibody or antigen-binding fragment thereof is present in a pharmaceuticaIly acceptable carrier.
In another aspect, the invention features a method of diagnosing a subject as having, or having a predisposition to, a pregnancy related hypertensive disorder, that includes measuring the level of a soluble endoglin polypeptide and at least one additional polypeptide selected from the group consisting of soluble endoglin binding proteins (e.g., TGF-01, TGF-03, activin A, BMP2, and BIv1P 7) and downstream mediators of soluble endoglin signaling (e.g., eNOS and PGI2) in a sample from the subject, wherein an
8 increase in the soluble endoglin level and a decrease in the level of the at least one additional polypeptide as compared to a normal reference sample, standard, or level is a diagnostic indicator of a pregnancy related hypertensive disorder or a propensity to develop a pregnancy related hypertensive disorder.
In another aspect, the invention features a method of diagnosing a subject as having, or having a predisposition to, a pregnancy related hypertensive disorder, that includes measuring the level of a soluble endoglin polypeptide and an sFlt-1 polypeptide from the subject and calculating the relationship between the levels of soluble endoglin and sFlt-1 using a [soluble endoglin x sF1t-1] metric, wherein an increase in the metric in the subject sample relative to a normal reference sample, is a diagnostic indicator of a pregnancy related hypertensive disorder in the subject.
In another aspect, the invention features a method of diagnosing a subject as having, or having a predisposition to, a pregnancy related hypertensive disorder, that includes measuring the levels of sFlt-1 and soluble endoglin in the first and second trimesters in a subject and calculating the delta value of sFltl x soluble endoglin (sEng) in each trimester using the following equation: [dproduct =(sFltl x sEng) in the second trimester -(sFltl x sEng) in the first trimester], where a value greater than 0, 1, 2, or more, including fractions thereof (e.g., a positive value) is a diagnostic indicator of pre-eclampsia or eclampsia. A positive value can also be an indicator of pre-term pre-eclampsia. Such a measurement can be taken on numerous occasions during the first and second trimesters and the dproduct can be followed over time.
In another aspect, the invention features a method of diagnosing a subject as having, or having a predisposition to, a pregnancy related hypertensive disorder, that includes measuring the levels of sFlt-1 and soluble endoglin in the first and second trimesters in a subject and calculating the delta product for the sFlt-1 level (dsFlt-1) or the sEng level (dsEng) between the first and second trimesters, where a value greater than 0, 1, 2, or more, including fractions thereof, (e.g., a positive value) for (dsFlt-1) or (dsEng) is a diagnostic indicator of pre-eclampsia or eclampsia.
For any of the diagnostic methods of the invention, the measuring can include the use of an immunological assay, such as an ELISA. In one embodiment, the normal reference sample is a prior sample from the subject. In another embodiment, the metric
In another aspect, the invention features a method of diagnosing a subject as having, or having a predisposition to, a pregnancy related hypertensive disorder, that includes measuring the level of a soluble endoglin polypeptide and an sFlt-1 polypeptide from the subject and calculating the relationship between the levels of soluble endoglin and sFlt-1 using a [soluble endoglin x sF1t-1] metric, wherein an increase in the metric in the subject sample relative to a normal reference sample, is a diagnostic indicator of a pregnancy related hypertensive disorder in the subject.
In another aspect, the invention features a method of diagnosing a subject as having, or having a predisposition to, a pregnancy related hypertensive disorder, that includes measuring the levels of sFlt-1 and soluble endoglin in the first and second trimesters in a subject and calculating the delta value of sFltl x soluble endoglin (sEng) in each trimester using the following equation: [dproduct =(sFltl x sEng) in the second trimester -(sFltl x sEng) in the first trimester], where a value greater than 0, 1, 2, or more, including fractions thereof (e.g., a positive value) is a diagnostic indicator of pre-eclampsia or eclampsia. A positive value can also be an indicator of pre-term pre-eclampsia. Such a measurement can be taken on numerous occasions during the first and second trimesters and the dproduct can be followed over time.
In another aspect, the invention features a method of diagnosing a subject as having, or having a predisposition to, a pregnancy related hypertensive disorder, that includes measuring the levels of sFlt-1 and soluble endoglin in the first and second trimesters in a subject and calculating the delta product for the sFlt-1 level (dsFlt-1) or the sEng level (dsEng) between the first and second trimesters, where a value greater than 0, 1, 2, or more, including fractions thereof, (e.g., a positive value) for (dsFlt-1) or (dsEng) is a diagnostic indicator of pre-eclampsia or eclampsia.
For any of the diagnostic methods of the invention, the measuring can include the use of an immunological assay, such as an ELISA. In one embodiment, the normal reference sample is a prior sample from the subject. In another embodiment, the metric
9 also includes the body mass index of the mother or the gestational age of the fetus. The sample can be a bodily fluid (e.g., urine, amniotic fluid, blood, serum, and plasma), cell (e.g., an endothelial cell, a leukocyte, a monocyte, and a cell derived from the placenta), or a tissue (e.g., placental tissue) of the subject in which the soluble endoglin is normally detectable. The subject can be a non-pregnant human, a pregnant human, a post-partum human, or a non-human (e.g., a cow, a horse, a sheep, a pig, a goat, a dog, or a cat) and the method can be used to diagnose a pregnancy related hypertensive disorder or a propensity to develop a pregnancy related hypertensive disorder (e.g., at least four weeks prior to the onset of symptoms).
In yet another aspect, the invention features a kit for the diagnosis of a pregnancy related hypertensive disorder in a subject that includes (i) a soluble endoglin binding agent and (ii) at least one additional binding agent that binds to a polypeptide selected from the group consisting of TGF-(31, TGF-(33, eNOS, and PGI2 and (iii) instructions for the use of the binding agent of (i) and the at least one binding agent of (ii) for the diagnosis of a pregnancy related hypertensive disorder or a propensity to develop a pregnancy related hypertensive disorder. The binding agents can be an antibody, or antigen-binding fragment thereof, that specifically binds soluble endoglin or antibody, or antigen binding fragment thereof, that specifically binds TGF-0l, TGF-03, eNOS, or PGI2.
Optionally, the kit can also include a VEGF, sFlt-1, or PIGF binding molecule.
In another aspect, the invention features a method of identifying a compound that ameliorates a pregnancy related hypertensive disorder, that includes the following steps:
(a) contacting a cell with a soluble endoglin compound;
(b) determining the phosphorylation state of Thr495 of eNOS in the cell after contacting with the soluble endoglin compound;
(c) contacting the cell with a candidate compound;
(d) determining the phosphorylation state of Thr495 of eNOS in the cell after contacting the cell with the candidate compound; and (e) comparing the phosphorylation state determined in step (b) and step (d), wherein an increase in the dephosphorylation of Thr 495 of eNOS in step (d) as compared to step (b) identifies the candidate compound as a compound that ameliorates a pregnancy related hypertensive disorder.
In yet another aspect, the invention features a method of identifying a compound that ameliorates a pregnancy related hypertensive disorder, that includes the following steps:
(a) contacting a cell with a Smad2/3 dependent reporter construct and a soluble endoglin compound;
(b) determining the level of activation of the Smad2/3 reporter construct in the cell of step (a);
(c) contacting the cell of step (a) with a candidate compou.nd;
(d) determining the level of activation of the Smad2/3 reporter construct in the cell of step (c); and (e) comparing the level of activation of the Smad2/3 reporter construct determined in step (b) and step (d), wherein an increase in the level of activation of the Smad2/3 reporter construct in step (d) as compared to step (b) identifies the candidate compound as a compound that ameliorates a pregnancy related hypertensive disorder.
For any of the above aspects, the pregnancy related hypertensive disorder can be pre-eclampsia, eclampsia, gestational hypertension, chronic hypertension, HELLP
syndrome, and pregnancy with a SGA infant. In one embodiment, the pregnancy related hypertensive disorder is pre-eclampsia or eclampsia.
As described below, we have discovered that deregulation of both the soluble endoglin/TGF-R and the sFlt-1/VEGF/P1GF signaling pathways can act together to further the pathology of the pregnancy related hypertensive disorder.
Therefore, the invention also features combinations of the methods described herein with any of the therapeutic, diagnostic, or monitoring methods described in U.S. Patent Application Publication Numbers 20040126828, 20050025762, 20050170444, 2006/0067937, and 20070104707 and PCT Publication Numbers WO 2004/008946, WO 2005/077007, and WO 06/034507.
For the purpose of the present invention, the following abbreviations and terms are defined below.
By "alteration" is meant a change (increase or decrease). An alteration can include a change in the expression levels of a gene or polypeptide as detected by standard art known methods such as those described below. As used herein, an alteration includes a 10% change in expression levels, preferably a 25% change, more preferably a 40%, 50%, 60%, 70%, 80%, 90% or greater change in expression levels. "Alteration"
can also indicate a change (increase or decrease) in the biological activity of any of the polypeptides of the invention (e.g., soluble endoglin, sFlt-1, VEGF, PIGF, eNOS, or TGFR family member). As used herein, an alteration includes a 10% change in biological activity, preferably a 25% change, more preferably a 40%, 50%, 60%, 70%, 80%, 90% or greater change in biological activity. Examples of biological activity for soluble endoglin are angiogenesis and binding to substrates such as activin-A, BMP 2, BMP-7, TGF-(31 and TGF-(33. The biological activity of soluble endoglin can be measured by ligand binding assays, immunoassays, and angiogenesis assays that are standard in the art or are described herein. An example of such an assay is the in vitro matrigel endothelial tube formation assay in which antagonism of endoglin signaling led to massive loss of capillary formation (Li et al., Faseb Journal 14:55-64 (2000)).
Examples of biological activity for eNOS are known in the art and include catalyzing the formation of nitric oxide or "NO" from oxygen and arginine. Examples of biological activity for TGF-13 include regulation of growth, differentiation, motility, tissue remodeling, neurogenesis, wound repair, apoptosis, and angiogenesis in many cell types.
Such activities can be measured by assays known in the art or described herein. TGF-(3 also inhibits cell proliferation in many cell types and can stimulate the synthesis of matrix proteins. Other examples of biological activity for PIGF or VEGF include binding to receptors as measured by immunoassays, ligand binding assays or Scatchard plot analysis, and induction of cell proliferation or migration as measured by BrdU
labeling, cell counting experiments, or quantitative assays for DNA synthesis such as 3H-thymidine incorporation. Examples of biological activity for sFlt-l include binding to PIGF and VEGF as measured by immunoassays, ligand binding assays, or Scatchard plot analysis. Additional examples of assays for biological activity for each of the polypeptides are described herein.
By "antisense nucleobase oligomer" is meant a nucleobase oligomer, regardless of length, that is complementary to the coding strand or mRNA of an endoglin gene. By a "nucleobase oligomer" is meant a compound that includes a chain of at least eight nucleobases, preferably at least twelve, and most preferably at least sixteen bases, joined together by linkage groups. Included in this definition are natural and non-natural oligonucleotides, both modified and unmodified, as well as oligonucleotide mimetics such as Protein Nucleic Acids, locked nucleic acids, and arabinonucleic acids.
Numerous nucleobases and linkage groups may be employed in the nucleobase oligomers of the invention, including those described in U.S. Patent Publication Nos.
20030114412 (see for example paragraphs 27-45 of the publication) and 20030114407 (see for example paragraphs 35-52 of the publication), incorporated herein by reference. The nucleobase oligomer can also be targeted to the translational start and stop sites.
Preferably the antisense nucleobase oligomer comprises from about 8 to 30 nucleotides. The antisense nucleobase oligomer can also contain at least 40, 60, 85, 120, or more consecutive nucleotides that are complementary to endoglin mRNA or DNA, and may be as long as the full-length mRNA or gene.
By "binding" is meant a non-covalent or a covalent interaction, preferably non-covalent, that holds two molecules together. For example, two such molecules could be a ligand and its receptor, an enzyme and an inhibitor of that enzyme, an enzyme and its substrate, or an antibody and an antigen. Non-covalent interactions include, but are not limited to, hydrogen bonding, ionic interactions among charged groups, van der Waals interactions, and hydrophobic interactions among non-polar groups. One or more of these interactions can mediate the binding of two molecules to each other.
Binding may exhibit discriminatory properties such as specificity or selectivity.
By "body mass index" is meant a number, derived by using height and weight measurements, that gives a general indication of whether or not weight falls within a healthy range. The formula generally used to determine the body mass index is a person's weight in kilograms divided by a person's height in meters squared or weight (kg)/ (height (m))2.
By "compound" is meant any small molecule chemical compound (peptidyl or non-peptidyl), antibody, nucleic acid molecule, polypeptide, or fragments thereof.
Compounds particularly useful for the therapeutic methods of the invention can alter, preferably decrease, the levels or biological activity of soluble endoglin by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more.
By "chimeric antibody" is meant a polypeptide comprising at least the antigen-binding portion of an antibody molecule linked to at least part of another protein (typically an immunoglobulin constant domain).
By "double-stranded RNA (dsRNA)" is meant a ribonucleic acid molecule comprised of both a sense and an anti-sense strand. dsRNAs are typically used to mediate RNA interference.
By "endoglin" or "Eng," also known as CD105, is meant a mammalian growth factor that has endoglin biological activity (see Fonsatti et al., Oncogene 22:6557-6563, 2003; Fonsatti et al., Curr. Cancer Drug Targets 3:427-432, 2003; and Cheifetz et al:, J.
Biol. Chem. 267:19027-19030 (1992)) and is homologous to the protein defined by any of the following GenBank accession numbers: AAH29080 and NP 03195 8 (mouse);
AAS67893 (rat); NP_000109, P17813, VSP_004233, CAA80673 (pig); and CAA50891 and AAC63386 (human), or described in U.S.P.N. 6,562,957. Endoglin is a homodimeric cell membrane glycoprotein which is expressed at high levels in proliferating vascular endothelial cells and in the syncytiotrophoblasts from placentas.
There are two distinct isoforms of endoglin, L and S, which differ in their cytoplasmic tails by 47 amino acids. Both isoforms are included in the term endoglin as used herein.
Endoglin binds to TGF-(3 family members and, in the presence of TGF-0, endoglin can associate with the TGF-0 signaling receptors RI and RIl, and potentiate the response to the growth factors. Endoglin biological activities include binding to substrates such as TGF-(3 family members such as activin-A, BMP 2, BMP-7, TGF-01 and TGF- f33;
induction of angiogenesis, regulation of cell proliferation, attachment, migration, invasion; and activation of endothelial cells. Assays for endoglin biological activities are known in the art and include ligand binding assays or Scatchard plot analysis;
BrdU
labeling, cell counting experiments, or quantitative assays for DNA synthesis such as 3 H-thymidine incorporation used to measure cell proliferation; and angiogenesis assays such as those described herein or in McCarty et al., Intl. J. Oncol. 21:5-10, 2002;
Akhtar et al.
Clin. Chem. 49:32-40, 2003; and Yamashita et al, J. Biol. Chem. 269:1995-2001, 1994).
By "soluble endoglin polypeptide" or "sEng" is meant any circulating, non-membrane bound form of endoglin which includes at least a part of the extracellular portion of the endoglin protein and is substantially identical (e.g., 60%, 70%, 80%, 90%, 995%, 96%, 97%, 98%, 99%, or 100%) to the amino acid sequence encoding the extracellular portion of the endoglin protein (see FIGURES 1 and 2B). Soluble endoglin can result from the cleavage of the membrane bound form of endoglin by a proteolytic enzyme. One potential cleavage site is at amino acid 437 of human endoglin producing a soluble endoglin polypeptide that includes amino acids 1-437 of the endoglin polypeptide, including the peptide leader sequence, which is typically cleaved off in the ER (see FIGURES 3A and 3B), or a protein that is substantially identical to amino acids 1- 437 of the endoglin polypeptide. Additional forms of soluble endoglin contemplated by the invention include a protein substantially identical to amino acids 40 (glycine) to 406 (arginine) of the human endoglin shown in FIGURE 30B, substantially identical to amino acids 1 to 587 of human endoglin (the entire extracellular domain, including the peptide leader sequence, commercially available from R&D Systems, catalog number 1097-EN), substantially identical to amino acids 40 to 587 of human endoglin shown in FIGURE 30B (this is the entire extracellular domain with the peptide leader sequence excluded), any polypeptide that includes the peptides identified in bold and underlined in FIGURE 30B, and any polypeptide that includes the regions or domains of soluble endoglin that are required for binding to TGF-R or TGF-(3 receptors. It should be noted that the numbering of both endoglin and soluble endoglin depends on whether the leader peptide sequence is included. The numbering of endoglin shown in FIGURE 30B, starts at amino acid 26 (where the absent leader peptide sequence would be amino acids 1-25).
Soluble endoglin can also include circulating degradation products or fragments that result from enzymatic cleavage of endoglin and that maintain endoglin biological activity. Preferred soluble endoglin polypeptides have soluble endoglin biological activity such as binding to substrates such as TGF-P family members or TGF-(3 receptors, inhibiting the biological activity of TGF-(3 family members, or reversing or inhibiting angiogenesis by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more.
Examples of assays for measuring these activities are known in the art and described in U.S. Patent Application Publication Nos. 20060067937, 20050267021, and and PCT Publication No. WO 06/034507, incorporated herein by reference. For example, soluble endoglin biological activity can include the ability to reverse, reduce, or inhibit angiogenesis induced by TGF-0 or the ability to reverse activation of Smad 2/3 or Smad 2/3 dependent transcriptional activation. Soluble endoglin polypeptides may be isolated from a variety of sources, such as from mammalian tissue or cells (e.g., placental tissue or cells), or prepared by recombinant or synthetic methods. The term soluble endoglin also encompasses modifications to the polypeptide, fragments, derivatives, analogs, and variants of the endoglin polypeptide, examples of which are described below.
By "endoglin nucleic acid" is meant a nucleic acid that encodes any of the endoglin proteins described above. For example, the gene for human endoglin consists of 14 exons, where exon 1 encodes the signal peptide sequence, exons 2-12 encode the extracellular domain (includes exon 9a and 9b), exon 13 encodes the transmembrane domain, and exon 14 encodes C-terminal cytoplasmic domain (see FIGURES 1, 2A, and 2B). Desirably, the endoglin nucleic acid encodes any of the soluble endoglin polypeptides described above or is substantially identical (60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) to the nucleic acid sequence set forth in FIGURE 2A. It should be noted that the circulating protein is predicted to lack the peptide leader sequence (amino acids 1-25).
By "epitope" is meant a sequence of amino acids which, either as a result of linear structure or three dimensional conformation, forms the binding site for an antibody.
By "expression" is meant the detection of a gene or polypeptide by standard art known methods. For example, polypeptide expression is often detected by western blotting, DNA expression is often detected by Southern blotting or polymerase chain reaction (PCR), and RNA expression is often detected by northern blotting, PCR, or RNAse protection assays. Methods to measure protein expression levels generally include, but are not limited to: Western blot, immunoblot, enzyme-linked immunosorbant assay (ELISA), radioimmunoassay (RIA), immunoprecipitation, surface plasmon resonance, chemiluminescence, fluorescent polarization, phosphorescence, irnmunohistochemical analysis, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, microcytometry, microarray, microscopy, fluorescence activated cell sorting (FACS), and flow cytometry, as well as assays based on a property of the protein including but not limited to enzymatic activity or interaction with other protein partners. Exemplary assays are described in detail in U.S.
Patent Application Publication No. 2006/0067937 and PCT Publication No. WO 06/034507.
Any compound that decreases soluble endoglin levels by at least 10%, 20%, preferably 30%, more preferably at least 40% or 50%, and most preferably at least 60%, 70%, 80%, 90% or rnore is considered a therapeutic compound of the invention.
By "fragment" is meant a portion of a polypeptide or nucleic acid molecule.
This portion contains, preferably, at least 10%, 20%, 3 0%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A
fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1250, 1500, 1750, 1800 or more nucleotides or 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 640 amino acids or more. Exemplary fragments of soluble endoglin include from I to 437 amino acids (including the peptide leader sequence), 26 to 437 amino acids (excluding the leader sequence), from 40 to 406 amino acids, or from 1 to 587 amino acids, and from I to 1311, 10 to 1311, 80 to 1030, or I. to 1761 nucleotides.
By "gestational age" is meant a reference to the age of the fetus, counting from the first day of the mother's last menstrual period usually referred to in weeks.
By "gestational hypertension" is meant the development of high blood pressure without proteinuria after 20 weeks of pregnancy.
By a "history of pre-eclampsia or eclampsia" is meant a previous diagnosis of pre-eclampsia or eclampsia or pregnancy induced hypertension in the subject themselves or in a related family member.
By "homologous" is meant any gene or protein sequence that bears at least 30%
homology, more preferably 40%, 50%, 60%, 70%, 80 fo, and most preferably 90%
or more homology to a lanown gene or protein sequence over the length of the comparison sequence. A"homoiogous" protein can also have at least one biological activity of the comparison protein. In general, for proteins, the length of comparison sequences will be at least 10 amino acids, preferably 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 250, 300, 350, 400, 437, or at least 587 amino acids or more. For nucleic acids, the length of comparison sequences will generally be at least 25, 50, 100, 125, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, 1000, 1100, 1200, 1311, or at least 1761 nucleotides or more. "Homology" can also refer to a substantial similarity between an epitope used to generate antibodies and the protein or fragment thereof to which the antibodies are directed. In this case, homology refers to a similarity sufficient to elicit the production of antibodies that can specifically recognize the protein at issue.
By "humanized antibody" is meant an immunoglobulin amino acid sequence variant or fragment thereof that is capable of binding to a predetermined antigen.
Ordinarily, the antibody will contain both the light chain as well as at least the variable domain of a heavy chain. The antibody also may include the CH1, hinge, CH2, CH3, or CH4 regions of the heavy chain. The humanized antibody comprises a framework region (FR) having substantially the amino acid sequence of a human immunoglobulin and a complementarity determining region (CDR) having substantially the amino acid sequence of a non-human immunoglobulin (the "import" sequences).
Generally, a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains (Fab, Fab', F(ab')2, Fabc, Fv) in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. By "complementarity determining region (CDR)" is meant the three hypervariable sequences in the variable regions within each of the immunoglobulin light and heavy chains. By "framework region (FR)" is meant the sequences of amino acids located on either side of the three hypervariable sequences (CDR) of the immunoglobulin light and heavy chains.
The FR and CDR regions of the humanized antibody need not correspond precisely to the parental sequences, e.g., the import CDR or the consensus FR
may be mutagenized by substitution, insertion or deletion of at least one residue so that the CDR
or FR residue at that site does not correspond to either the consensus or the import antibody. Such mutations, however, will not be extensive. Usually, at least 75%, preferably 90%, and most preferably at least 95% of the humanized antibody residues will correspond to those of the parental FR and CDR sequences.
By "hybridize" is meant pair to form a double-stranded molecule between complementary polynucleotide sequences, or portions thereof, under various conditions of stringency. (See, e.g., Wahl and Berger Methods Enzymol. 152:399, 1987;
Kimmel, Methods Enzymol. 152:507, 1987.) For example, stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCI and 50 mM trisodium citrate, and most preferably less than about 250 mM NaCI and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and most preferably at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30 C, more preferably of at least about 37 C, and most preferably of at least about 42 C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art.
Various levels of stringency are accomplished by combining these various conditions as needed. In a preferred embodiment, hybridization will occur at 30 C in 750 mM
NaCI, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37 C in 500 mM NaCI, 50 mM trisodium citrate, 1% SDS, 35%
formamide, and 100 g/ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization will occur at 42 C in 250 mNi NaCl, 25 mM trisodium citrate, 1%
SDS, 50% formamide, and 200 gg/mi ssDNA. Useful var iations on these conditions will be readily apparent to those skilled in the art.
For most applications, washing steps that follow hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCI and 3 mM
trisodium citrate, and most preferably less than about 15 mM NaC1 and 1.5 mM trisodium citrate.
Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25 C, more preferably of at least about 42 C, and most preferably of at least about 68 C. In a preferred embodiment, wash steps will occur at 25 C in 30 mM
NaCI, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42 C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1 %
SDS. In a most preferred embodiment, wash steps will occur at 68 C in 15 mM NaCI, 1.5 mM
trisodium citrate, and 0.1 % SDS. Additional variations on these conditions will be.
readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975);
Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.
By "intrauterine growth retardation (IUGR)" is meant a syndrome resulting in a birth weight which is less that 10 percent of the predicted fetal weight for the gestational age of the fetus. The current World Health Organization criterion-for low birth weight is a weight less than 2,500 gm (51bs..8 oz.) or below the 10`h percentile for gestational age according to U.S. tables of birth weight for gestational age by race, parity, and infant sex (Zhang and Bowes, Obstet. G,ynecol. 86:200-208, 1995). These low birth weight babies are also referred to as "small for gestational age (SGA)". Pre-eclampsia is a condition known to be associated with IUGR or SGA.
By "metric" is meant a measure. A metric may be used, for example, to compare the levels of a polypeptide or nucleic acid molecule of interest. Exemplary metrics include, but are not limited to, mathematical formulas or algorithms, such as ratios. The metric to be used is that which best discriminates between levels of soluble endoglin, sFlt-1, VEGF, PIGF, or any combination thereof, in a subject having pregnancy related hypertensive disorder, such as pre-eclampsia or eclampsia, and a normal control subject.
Depending on the metric that is used the diagnostic indicator of pregnancy related hypertensive disorder may be significantly above or below a reference value (e.g., from a control subject not having a pregnancy related hypertensive disorder). Soluble endoglin level is determined by measuring the amount of free, bound (i.e., bound to growth factor), or total (free + bound) soluble endoglin. sFlt-1 level is measured by measuring the amount of free, bound (i.e., bound to growth factor), or total sFlt-1 (bound +
free). =
VEGF or PIGF levels are determined by measuring the amount of free PIGF or free VEGF (i.e., not bound to sFlt-1). One exemplary metric is [sFlt-l/(VEGF +
P1GF)], also referred to as the pre-eclampsia anti-angiogenic index (PAAI). Another example is the following soluble endoglin anti-angiogenic index: (sFlt-1 + 0.25(soluble endoglin polypeptide))/P1GF. Yet another exemplary metric is the following: (soluble endoglin +
sFlt-1)JPIGF. An increase in the value of either of these two exemplary metrics as compared to a normal reference is a diagnostic indicator of pre-eclampsia or eclampsia.
Another example includes the measurement of the levels of sFlt-1 and soluble endoglin in the first and second trimesters in a subject and calculating the delta value of sFltl x soluble endoglin (sEng) in each trimester using the following equation:
[dproduct =
(sFltl x sEng) in the second trimester -(sFltl x sEng) in the first trimester], where a value greater than 0, 1, 2, or more (e.g., a positive value) is a diagnostic indicator of pre-eclampsia=or eclampsia. Additional metrics include the dproduct of the sFlt-1 level (dsFlt-1) and the sEng level (dsEng) between the first and second trimesters, where a value greater than 0, 1, 2, or more (e.g., a positive value) for (dsFlt-I ) or (dsEng) is a diagnostic indicator of pre-eclampsia or eclampsia.
Any of the metrics of the invention can further include the BMI of the mother or gestational age of the infant, or parity. Any of the metrics can also include eNOS, TGF-(31 or (33 or PGI2 levels as well.
By "nitric oxide synthase" or "NOS" is meant an enzyme that catalyzes the formation of nitric oxide (NO) from oxygen and arginine. NOS is a complex enzyme containing several cofactors, a heme group which is part of the catalytic site, an N-terminal oxygenase domain, which belongs to the class of haem-thiolate proteins, and a C-terminal reductase domain which is homologous to NADPH:P450 reductase. NOS
produces NO by catalysing a five-electron oxidation of a guanidino nitrogen of L-arginine (L-Arg). Oxidation of L-Arg to L-citrulline occurs via two successive monooxygenation reactions producing N-hydroxy-L-arginine as an intermediate.
The interdomain linker between the oxygenase and reductase domains contains a CaM-binding sequence. NO functions at low concentrations as a signal in many diverse physiological processes such as blood pressure control, neurotransmission, learning and memory, and at high concentrations as a defensive cytotoxin.
In mammals, three distinct genes encode NOS isozymes: neuronal (nNOS or NOS-1), cytokine-inducible (iNOS orNOS-2) and endothelial (eNOS or NOS-3).
eNOS
is membrane associated and eNOS localization to endothelial membranes is mediated by cotranslational N-terminal myristoylation and post-translational palmitoylation. In preferred embodiments of the invention, the NOS is eNOS.
By "pre-eclampsia anti-angiogenesis index (PAAI)" is meant the ratio of sFlt-1/VEGF + PIGF used as an indicator of anti-angiogenic activity. A PAAI greater than
In yet another aspect, the invention features a kit for the diagnosis of a pregnancy related hypertensive disorder in a subject that includes (i) a soluble endoglin binding agent and (ii) at least one additional binding agent that binds to a polypeptide selected from the group consisting of TGF-(31, TGF-(33, eNOS, and PGI2 and (iii) instructions for the use of the binding agent of (i) and the at least one binding agent of (ii) for the diagnosis of a pregnancy related hypertensive disorder or a propensity to develop a pregnancy related hypertensive disorder. The binding agents can be an antibody, or antigen-binding fragment thereof, that specifically binds soluble endoglin or antibody, or antigen binding fragment thereof, that specifically binds TGF-0l, TGF-03, eNOS, or PGI2.
Optionally, the kit can also include a VEGF, sFlt-1, or PIGF binding molecule.
In another aspect, the invention features a method of identifying a compound that ameliorates a pregnancy related hypertensive disorder, that includes the following steps:
(a) contacting a cell with a soluble endoglin compound;
(b) determining the phosphorylation state of Thr495 of eNOS in the cell after contacting with the soluble endoglin compound;
(c) contacting the cell with a candidate compound;
(d) determining the phosphorylation state of Thr495 of eNOS in the cell after contacting the cell with the candidate compound; and (e) comparing the phosphorylation state determined in step (b) and step (d), wherein an increase in the dephosphorylation of Thr 495 of eNOS in step (d) as compared to step (b) identifies the candidate compound as a compound that ameliorates a pregnancy related hypertensive disorder.
In yet another aspect, the invention features a method of identifying a compound that ameliorates a pregnancy related hypertensive disorder, that includes the following steps:
(a) contacting a cell with a Smad2/3 dependent reporter construct and a soluble endoglin compound;
(b) determining the level of activation of the Smad2/3 reporter construct in the cell of step (a);
(c) contacting the cell of step (a) with a candidate compou.nd;
(d) determining the level of activation of the Smad2/3 reporter construct in the cell of step (c); and (e) comparing the level of activation of the Smad2/3 reporter construct determined in step (b) and step (d), wherein an increase in the level of activation of the Smad2/3 reporter construct in step (d) as compared to step (b) identifies the candidate compound as a compound that ameliorates a pregnancy related hypertensive disorder.
For any of the above aspects, the pregnancy related hypertensive disorder can be pre-eclampsia, eclampsia, gestational hypertension, chronic hypertension, HELLP
syndrome, and pregnancy with a SGA infant. In one embodiment, the pregnancy related hypertensive disorder is pre-eclampsia or eclampsia.
As described below, we have discovered that deregulation of both the soluble endoglin/TGF-R and the sFlt-1/VEGF/P1GF signaling pathways can act together to further the pathology of the pregnancy related hypertensive disorder.
Therefore, the invention also features combinations of the methods described herein with any of the therapeutic, diagnostic, or monitoring methods described in U.S. Patent Application Publication Numbers 20040126828, 20050025762, 20050170444, 2006/0067937, and 20070104707 and PCT Publication Numbers WO 2004/008946, WO 2005/077007, and WO 06/034507.
For the purpose of the present invention, the following abbreviations and terms are defined below.
By "alteration" is meant a change (increase or decrease). An alteration can include a change in the expression levels of a gene or polypeptide as detected by standard art known methods such as those described below. As used herein, an alteration includes a 10% change in expression levels, preferably a 25% change, more preferably a 40%, 50%, 60%, 70%, 80%, 90% or greater change in expression levels. "Alteration"
can also indicate a change (increase or decrease) in the biological activity of any of the polypeptides of the invention (e.g., soluble endoglin, sFlt-1, VEGF, PIGF, eNOS, or TGFR family member). As used herein, an alteration includes a 10% change in biological activity, preferably a 25% change, more preferably a 40%, 50%, 60%, 70%, 80%, 90% or greater change in biological activity. Examples of biological activity for soluble endoglin are angiogenesis and binding to substrates such as activin-A, BMP 2, BMP-7, TGF-(31 and TGF-(33. The biological activity of soluble endoglin can be measured by ligand binding assays, immunoassays, and angiogenesis assays that are standard in the art or are described herein. An example of such an assay is the in vitro matrigel endothelial tube formation assay in which antagonism of endoglin signaling led to massive loss of capillary formation (Li et al., Faseb Journal 14:55-64 (2000)).
Examples of biological activity for eNOS are known in the art and include catalyzing the formation of nitric oxide or "NO" from oxygen and arginine. Examples of biological activity for TGF-13 include regulation of growth, differentiation, motility, tissue remodeling, neurogenesis, wound repair, apoptosis, and angiogenesis in many cell types.
Such activities can be measured by assays known in the art or described herein. TGF-(3 also inhibits cell proliferation in many cell types and can stimulate the synthesis of matrix proteins. Other examples of biological activity for PIGF or VEGF include binding to receptors as measured by immunoassays, ligand binding assays or Scatchard plot analysis, and induction of cell proliferation or migration as measured by BrdU
labeling, cell counting experiments, or quantitative assays for DNA synthesis such as 3H-thymidine incorporation. Examples of biological activity for sFlt-l include binding to PIGF and VEGF as measured by immunoassays, ligand binding assays, or Scatchard plot analysis. Additional examples of assays for biological activity for each of the polypeptides are described herein.
By "antisense nucleobase oligomer" is meant a nucleobase oligomer, regardless of length, that is complementary to the coding strand or mRNA of an endoglin gene. By a "nucleobase oligomer" is meant a compound that includes a chain of at least eight nucleobases, preferably at least twelve, and most preferably at least sixteen bases, joined together by linkage groups. Included in this definition are natural and non-natural oligonucleotides, both modified and unmodified, as well as oligonucleotide mimetics such as Protein Nucleic Acids, locked nucleic acids, and arabinonucleic acids.
Numerous nucleobases and linkage groups may be employed in the nucleobase oligomers of the invention, including those described in U.S. Patent Publication Nos.
20030114412 (see for example paragraphs 27-45 of the publication) and 20030114407 (see for example paragraphs 35-52 of the publication), incorporated herein by reference. The nucleobase oligomer can also be targeted to the translational start and stop sites.
Preferably the antisense nucleobase oligomer comprises from about 8 to 30 nucleotides. The antisense nucleobase oligomer can also contain at least 40, 60, 85, 120, or more consecutive nucleotides that are complementary to endoglin mRNA or DNA, and may be as long as the full-length mRNA or gene.
By "binding" is meant a non-covalent or a covalent interaction, preferably non-covalent, that holds two molecules together. For example, two such molecules could be a ligand and its receptor, an enzyme and an inhibitor of that enzyme, an enzyme and its substrate, or an antibody and an antigen. Non-covalent interactions include, but are not limited to, hydrogen bonding, ionic interactions among charged groups, van der Waals interactions, and hydrophobic interactions among non-polar groups. One or more of these interactions can mediate the binding of two molecules to each other.
Binding may exhibit discriminatory properties such as specificity or selectivity.
By "body mass index" is meant a number, derived by using height and weight measurements, that gives a general indication of whether or not weight falls within a healthy range. The formula generally used to determine the body mass index is a person's weight in kilograms divided by a person's height in meters squared or weight (kg)/ (height (m))2.
By "compound" is meant any small molecule chemical compound (peptidyl or non-peptidyl), antibody, nucleic acid molecule, polypeptide, or fragments thereof.
Compounds particularly useful for the therapeutic methods of the invention can alter, preferably decrease, the levels or biological activity of soluble endoglin by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more.
By "chimeric antibody" is meant a polypeptide comprising at least the antigen-binding portion of an antibody molecule linked to at least part of another protein (typically an immunoglobulin constant domain).
By "double-stranded RNA (dsRNA)" is meant a ribonucleic acid molecule comprised of both a sense and an anti-sense strand. dsRNAs are typically used to mediate RNA interference.
By "endoglin" or "Eng," also known as CD105, is meant a mammalian growth factor that has endoglin biological activity (see Fonsatti et al., Oncogene 22:6557-6563, 2003; Fonsatti et al., Curr. Cancer Drug Targets 3:427-432, 2003; and Cheifetz et al:, J.
Biol. Chem. 267:19027-19030 (1992)) and is homologous to the protein defined by any of the following GenBank accession numbers: AAH29080 and NP 03195 8 (mouse);
AAS67893 (rat); NP_000109, P17813, VSP_004233, CAA80673 (pig); and CAA50891 and AAC63386 (human), or described in U.S.P.N. 6,562,957. Endoglin is a homodimeric cell membrane glycoprotein which is expressed at high levels in proliferating vascular endothelial cells and in the syncytiotrophoblasts from placentas.
There are two distinct isoforms of endoglin, L and S, which differ in their cytoplasmic tails by 47 amino acids. Both isoforms are included in the term endoglin as used herein.
Endoglin binds to TGF-(3 family members and, in the presence of TGF-0, endoglin can associate with the TGF-0 signaling receptors RI and RIl, and potentiate the response to the growth factors. Endoglin biological activities include binding to substrates such as TGF-(3 family members such as activin-A, BMP 2, BMP-7, TGF-01 and TGF- f33;
induction of angiogenesis, regulation of cell proliferation, attachment, migration, invasion; and activation of endothelial cells. Assays for endoglin biological activities are known in the art and include ligand binding assays or Scatchard plot analysis;
BrdU
labeling, cell counting experiments, or quantitative assays for DNA synthesis such as 3 H-thymidine incorporation used to measure cell proliferation; and angiogenesis assays such as those described herein or in McCarty et al., Intl. J. Oncol. 21:5-10, 2002;
Akhtar et al.
Clin. Chem. 49:32-40, 2003; and Yamashita et al, J. Biol. Chem. 269:1995-2001, 1994).
By "soluble endoglin polypeptide" or "sEng" is meant any circulating, non-membrane bound form of endoglin which includes at least a part of the extracellular portion of the endoglin protein and is substantially identical (e.g., 60%, 70%, 80%, 90%, 995%, 96%, 97%, 98%, 99%, or 100%) to the amino acid sequence encoding the extracellular portion of the endoglin protein (see FIGURES 1 and 2B). Soluble endoglin can result from the cleavage of the membrane bound form of endoglin by a proteolytic enzyme. One potential cleavage site is at amino acid 437 of human endoglin producing a soluble endoglin polypeptide that includes amino acids 1-437 of the endoglin polypeptide, including the peptide leader sequence, which is typically cleaved off in the ER (see FIGURES 3A and 3B), or a protein that is substantially identical to amino acids 1- 437 of the endoglin polypeptide. Additional forms of soluble endoglin contemplated by the invention include a protein substantially identical to amino acids 40 (glycine) to 406 (arginine) of the human endoglin shown in FIGURE 30B, substantially identical to amino acids 1 to 587 of human endoglin (the entire extracellular domain, including the peptide leader sequence, commercially available from R&D Systems, catalog number 1097-EN), substantially identical to amino acids 40 to 587 of human endoglin shown in FIGURE 30B (this is the entire extracellular domain with the peptide leader sequence excluded), any polypeptide that includes the peptides identified in bold and underlined in FIGURE 30B, and any polypeptide that includes the regions or domains of soluble endoglin that are required for binding to TGF-R or TGF-(3 receptors. It should be noted that the numbering of both endoglin and soluble endoglin depends on whether the leader peptide sequence is included. The numbering of endoglin shown in FIGURE 30B, starts at amino acid 26 (where the absent leader peptide sequence would be amino acids 1-25).
Soluble endoglin can also include circulating degradation products or fragments that result from enzymatic cleavage of endoglin and that maintain endoglin biological activity. Preferred soluble endoglin polypeptides have soluble endoglin biological activity such as binding to substrates such as TGF-P family members or TGF-(3 receptors, inhibiting the biological activity of TGF-(3 family members, or reversing or inhibiting angiogenesis by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more.
Examples of assays for measuring these activities are known in the art and described in U.S. Patent Application Publication Nos. 20060067937, 20050267021, and and PCT Publication No. WO 06/034507, incorporated herein by reference. For example, soluble endoglin biological activity can include the ability to reverse, reduce, or inhibit angiogenesis induced by TGF-0 or the ability to reverse activation of Smad 2/3 or Smad 2/3 dependent transcriptional activation. Soluble endoglin polypeptides may be isolated from a variety of sources, such as from mammalian tissue or cells (e.g., placental tissue or cells), or prepared by recombinant or synthetic methods. The term soluble endoglin also encompasses modifications to the polypeptide, fragments, derivatives, analogs, and variants of the endoglin polypeptide, examples of which are described below.
By "endoglin nucleic acid" is meant a nucleic acid that encodes any of the endoglin proteins described above. For example, the gene for human endoglin consists of 14 exons, where exon 1 encodes the signal peptide sequence, exons 2-12 encode the extracellular domain (includes exon 9a and 9b), exon 13 encodes the transmembrane domain, and exon 14 encodes C-terminal cytoplasmic domain (see FIGURES 1, 2A, and 2B). Desirably, the endoglin nucleic acid encodes any of the soluble endoglin polypeptides described above or is substantially identical (60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) to the nucleic acid sequence set forth in FIGURE 2A. It should be noted that the circulating protein is predicted to lack the peptide leader sequence (amino acids 1-25).
By "epitope" is meant a sequence of amino acids which, either as a result of linear structure or three dimensional conformation, forms the binding site for an antibody.
By "expression" is meant the detection of a gene or polypeptide by standard art known methods. For example, polypeptide expression is often detected by western blotting, DNA expression is often detected by Southern blotting or polymerase chain reaction (PCR), and RNA expression is often detected by northern blotting, PCR, or RNAse protection assays. Methods to measure protein expression levels generally include, but are not limited to: Western blot, immunoblot, enzyme-linked immunosorbant assay (ELISA), radioimmunoassay (RIA), immunoprecipitation, surface plasmon resonance, chemiluminescence, fluorescent polarization, phosphorescence, irnmunohistochemical analysis, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, microcytometry, microarray, microscopy, fluorescence activated cell sorting (FACS), and flow cytometry, as well as assays based on a property of the protein including but not limited to enzymatic activity or interaction with other protein partners. Exemplary assays are described in detail in U.S.
Patent Application Publication No. 2006/0067937 and PCT Publication No. WO 06/034507.
Any compound that decreases soluble endoglin levels by at least 10%, 20%, preferably 30%, more preferably at least 40% or 50%, and most preferably at least 60%, 70%, 80%, 90% or rnore is considered a therapeutic compound of the invention.
By "fragment" is meant a portion of a polypeptide or nucleic acid molecule.
This portion contains, preferably, at least 10%, 20%, 3 0%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A
fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1250, 1500, 1750, 1800 or more nucleotides or 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 640 amino acids or more. Exemplary fragments of soluble endoglin include from I to 437 amino acids (including the peptide leader sequence), 26 to 437 amino acids (excluding the leader sequence), from 40 to 406 amino acids, or from 1 to 587 amino acids, and from I to 1311, 10 to 1311, 80 to 1030, or I. to 1761 nucleotides.
By "gestational age" is meant a reference to the age of the fetus, counting from the first day of the mother's last menstrual period usually referred to in weeks.
By "gestational hypertension" is meant the development of high blood pressure without proteinuria after 20 weeks of pregnancy.
By a "history of pre-eclampsia or eclampsia" is meant a previous diagnosis of pre-eclampsia or eclampsia or pregnancy induced hypertension in the subject themselves or in a related family member.
By "homologous" is meant any gene or protein sequence that bears at least 30%
homology, more preferably 40%, 50%, 60%, 70%, 80 fo, and most preferably 90%
or more homology to a lanown gene or protein sequence over the length of the comparison sequence. A"homoiogous" protein can also have at least one biological activity of the comparison protein. In general, for proteins, the length of comparison sequences will be at least 10 amino acids, preferably 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 250, 300, 350, 400, 437, or at least 587 amino acids or more. For nucleic acids, the length of comparison sequences will generally be at least 25, 50, 100, 125, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, 1000, 1100, 1200, 1311, or at least 1761 nucleotides or more. "Homology" can also refer to a substantial similarity between an epitope used to generate antibodies and the protein or fragment thereof to which the antibodies are directed. In this case, homology refers to a similarity sufficient to elicit the production of antibodies that can specifically recognize the protein at issue.
By "humanized antibody" is meant an immunoglobulin amino acid sequence variant or fragment thereof that is capable of binding to a predetermined antigen.
Ordinarily, the antibody will contain both the light chain as well as at least the variable domain of a heavy chain. The antibody also may include the CH1, hinge, CH2, CH3, or CH4 regions of the heavy chain. The humanized antibody comprises a framework region (FR) having substantially the amino acid sequence of a human immunoglobulin and a complementarity determining region (CDR) having substantially the amino acid sequence of a non-human immunoglobulin (the "import" sequences).
Generally, a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains (Fab, Fab', F(ab')2, Fabc, Fv) in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. By "complementarity determining region (CDR)" is meant the three hypervariable sequences in the variable regions within each of the immunoglobulin light and heavy chains. By "framework region (FR)" is meant the sequences of amino acids located on either side of the three hypervariable sequences (CDR) of the immunoglobulin light and heavy chains.
The FR and CDR regions of the humanized antibody need not correspond precisely to the parental sequences, e.g., the import CDR or the consensus FR
may be mutagenized by substitution, insertion or deletion of at least one residue so that the CDR
or FR residue at that site does not correspond to either the consensus or the import antibody. Such mutations, however, will not be extensive. Usually, at least 75%, preferably 90%, and most preferably at least 95% of the humanized antibody residues will correspond to those of the parental FR and CDR sequences.
By "hybridize" is meant pair to form a double-stranded molecule between complementary polynucleotide sequences, or portions thereof, under various conditions of stringency. (See, e.g., Wahl and Berger Methods Enzymol. 152:399, 1987;
Kimmel, Methods Enzymol. 152:507, 1987.) For example, stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCI and 50 mM trisodium citrate, and most preferably less than about 250 mM NaCI and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and most preferably at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30 C, more preferably of at least about 37 C, and most preferably of at least about 42 C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art.
Various levels of stringency are accomplished by combining these various conditions as needed. In a preferred embodiment, hybridization will occur at 30 C in 750 mM
NaCI, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37 C in 500 mM NaCI, 50 mM trisodium citrate, 1% SDS, 35%
formamide, and 100 g/ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization will occur at 42 C in 250 mNi NaCl, 25 mM trisodium citrate, 1%
SDS, 50% formamide, and 200 gg/mi ssDNA. Useful var iations on these conditions will be readily apparent to those skilled in the art.
For most applications, washing steps that follow hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCI and 3 mM
trisodium citrate, and most preferably less than about 15 mM NaC1 and 1.5 mM trisodium citrate.
Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25 C, more preferably of at least about 42 C, and most preferably of at least about 68 C. In a preferred embodiment, wash steps will occur at 25 C in 30 mM
NaCI, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42 C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1 %
SDS. In a most preferred embodiment, wash steps will occur at 68 C in 15 mM NaCI, 1.5 mM
trisodium citrate, and 0.1 % SDS. Additional variations on these conditions will be.
readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975);
Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.
By "intrauterine growth retardation (IUGR)" is meant a syndrome resulting in a birth weight which is less that 10 percent of the predicted fetal weight for the gestational age of the fetus. The current World Health Organization criterion-for low birth weight is a weight less than 2,500 gm (51bs..8 oz.) or below the 10`h percentile for gestational age according to U.S. tables of birth weight for gestational age by race, parity, and infant sex (Zhang and Bowes, Obstet. G,ynecol. 86:200-208, 1995). These low birth weight babies are also referred to as "small for gestational age (SGA)". Pre-eclampsia is a condition known to be associated with IUGR or SGA.
By "metric" is meant a measure. A metric may be used, for example, to compare the levels of a polypeptide or nucleic acid molecule of interest. Exemplary metrics include, but are not limited to, mathematical formulas or algorithms, such as ratios. The metric to be used is that which best discriminates between levels of soluble endoglin, sFlt-1, VEGF, PIGF, or any combination thereof, in a subject having pregnancy related hypertensive disorder, such as pre-eclampsia or eclampsia, and a normal control subject.
Depending on the metric that is used the diagnostic indicator of pregnancy related hypertensive disorder may be significantly above or below a reference value (e.g., from a control subject not having a pregnancy related hypertensive disorder). Soluble endoglin level is determined by measuring the amount of free, bound (i.e., bound to growth factor), or total (free + bound) soluble endoglin. sFlt-1 level is measured by measuring the amount of free, bound (i.e., bound to growth factor), or total sFlt-1 (bound +
free). =
VEGF or PIGF levels are determined by measuring the amount of free PIGF or free VEGF (i.e., not bound to sFlt-1). One exemplary metric is [sFlt-l/(VEGF +
P1GF)], also referred to as the pre-eclampsia anti-angiogenic index (PAAI). Another example is the following soluble endoglin anti-angiogenic index: (sFlt-1 + 0.25(soluble endoglin polypeptide))/P1GF. Yet another exemplary metric is the following: (soluble endoglin +
sFlt-1)JPIGF. An increase in the value of either of these two exemplary metrics as compared to a normal reference is a diagnostic indicator of pre-eclampsia or eclampsia.
Another example includes the measurement of the levels of sFlt-1 and soluble endoglin in the first and second trimesters in a subject and calculating the delta value of sFltl x soluble endoglin (sEng) in each trimester using the following equation:
[dproduct =
(sFltl x sEng) in the second trimester -(sFltl x sEng) in the first trimester], where a value greater than 0, 1, 2, or more (e.g., a positive value) is a diagnostic indicator of pre-eclampsia=or eclampsia. Additional metrics include the dproduct of the sFlt-1 level (dsFlt-1) and the sEng level (dsEng) between the first and second trimesters, where a value greater than 0, 1, 2, or more (e.g., a positive value) for (dsFlt-I ) or (dsEng) is a diagnostic indicator of pre-eclampsia or eclampsia.
Any of the metrics of the invention can further include the BMI of the mother or gestational age of the infant, or parity. Any of the metrics can also include eNOS, TGF-(31 or (33 or PGI2 levels as well.
By "nitric oxide synthase" or "NOS" is meant an enzyme that catalyzes the formation of nitric oxide (NO) from oxygen and arginine. NOS is a complex enzyme containing several cofactors, a heme group which is part of the catalytic site, an N-terminal oxygenase domain, which belongs to the class of haem-thiolate proteins, and a C-terminal reductase domain which is homologous to NADPH:P450 reductase. NOS
produces NO by catalysing a five-electron oxidation of a guanidino nitrogen of L-arginine (L-Arg). Oxidation of L-Arg to L-citrulline occurs via two successive monooxygenation reactions producing N-hydroxy-L-arginine as an intermediate.
The interdomain linker between the oxygenase and reductase domains contains a CaM-binding sequence. NO functions at low concentrations as a signal in many diverse physiological processes such as blood pressure control, neurotransmission, learning and memory, and at high concentrations as a defensive cytotoxin.
In mammals, three distinct genes encode NOS isozymes: neuronal (nNOS or NOS-1), cytokine-inducible (iNOS orNOS-2) and endothelial (eNOS or NOS-3).
eNOS
is membrane associated and eNOS localization to endothelial membranes is mediated by cotranslational N-terminal myristoylation and post-translational palmitoylation. In preferred embodiments of the invention, the NOS is eNOS.
By "pre-eclampsia anti-angiogenesis index (PAAI)" is meant the ratio of sFlt-1/VEGF + PIGF used as an indicator of anti-angiogenic activity. A PAAI greater than
10, more preferably greater than 20, is indicative of a pregnancy related hypertensive disorder, such as pre-eclampsia or risk of pre-eclampsia.
By "soluble endoglin anti-angiogenic index" is meant the ratio of (sFlt-1 +
0.25 soluble endoglin)/PIGF. For example, a value of 75, or higher, preferably 100 or higher, or more preferably 200 or higher is indicative of a pregnancy complication associated with hypertension, such as pre-eclampsia or eclampsia.
By "operably linked" is meant that a gene and a regulatory sequence(s) are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequence(s).
By "pharmaceutically acceptable carrier ' is meant a carrier that is physiologically acceptable to the treated mammal while retaining the therapeutic properties of the compound with which it is administered. One exemplary pharmaceutically acceptable carrier substance is physiological saline. Other physiologically acceptable carriers and their formulations are known to one skilled in the art and described, for example, in Remington's Pharmaceutical Sciences, (20th edition), ed. A. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, PA.
By "placental growth factor (PIGF)" is meant a mammalian growth factor that is homologous to the protein defined by GenBank accession number P49763 and that has PIGF biological activity. P1GF is a glycosylated homodimer belonging to the VEGF
family and can be found in two distinct isoforms through alternative splicing mechanisms. PIGF is expressed by cyto- and syncytiotrophoblasts in the placenta and P1GF biological activities include induction of proliferation, migration, and activation of endothelial cells, particularly trophoblast cells.
By "polymorphism" is meant a genetic variation, mutation, deletion or addition in a soluble endoglin, sFlt-1, P1GF, or VEGF nucleic acid molecule that is indicative of a predisposition to develop pre-eclampsia or eclampsia. Such polymorphisms are known to the skilled artisan and are described, for example, by Raab et al. (Bzochem.
J. 339:579-588, 1999) and Parry et al. (Eur. Jlmmunogenet. 26:321-323, 1999). A
polymorphism may be present in the promoter sequence, an open reading frame, intronic sequence, or untranslated 3' region of a gene. Known examples of such polymorphisms in the endoglin gene include a 6 base insertion of GGGGGA in intron 7 at 26 bases beyond the 3' end of exon 7 (Ann. Neurol. 41:683-6, 1997).
By "pregnancy related hypertensive disorder" is meant any condition or disease or pregnancy that is associated with or characterized by an increase in blood pressure.
Included among these conditions are pre-eclampsia (including premature pre-eclampsia, severe pre-eclampsia), eclampsia, gestational hypertension, HELLP syndrome, (hemolysis, elevated liver enzymes, low platelets), abruption placenta, chronic hypertension, pregnancy with intra uterine growth restriction, and pregnancy with a small for gestational age (SGA) infant. It should be noted that although pregnancy with a SGA
infant is not often associated with hypertension, it is included in this definition.
By "pre-eclampsia" is meant the multi-system disorder that is characterized by hypertension with proteinuria or edema, or both, glomerular dysfunction, brain edema, liver edema, or coagulation abnormalities due to pregnancy or the influence of a recent pregnancy. All forms of pre-eclampsia, such as premature, mild, moderate, and severe pre-eclampsia are included in this definition. Pre-eclampsia generally occurs after the 20th week of gestation. Pre-eclampsia is generally defined as some combination of the following symptoms: (1) a systolic blood pressure (BP) >140 mmHg and a diastolic BP
>90 mmHg after 20 weeks gestation (generally measured on two occasions, 4-168 hours apart), (2) new onset proteinuria (l+ by dipstik on urinaiysis, > 300mg of protein in a 24-hour urine collection, or a single random urine sample having a protein/creatinine ratio >0.3), and (3) resolution of hypertension and proteinuria by 12 weeks postpartum.
Severe pre-eclampsia is generally defined as (1) a diastolic BP > 110 mmHg (generally measured on two occasions, 4-168 hours apart) or (2) proteinuria characterized by a measurement of 3.5 grams or more protein in a 24-hour urine collection or two random urine specimens with at least 3+ protein by dipstick. In pre-eclampsia, hypertension and proteinuria generally occur within seven days of each other. In severe pre-eclampsia, severe hypertension, severe proteinuria and HELLP syndrome (hemolysis, elevated liver enzymes, low platelets) or eclampsia can occur simultaneously or only one symptom at a time. HELLP syndrome is characterized by evidence of thrombocytopenia (<100000 cells/ l), increased LDH (>600 IU/L) and increased AST (>70 IU/L).
Occasionally, severe pre-eclampsia can lead to the development of seizures. This severe form of the syndrome is referred to as "eclampsia." Eclampsia can also include dysfunction or damage to several organs or tissues such as the liver (e.g., hepatocellular damage, periportal necrosis) and the central nervous system (e.g., cerebral edema and cerebral hemorrhage). The etiology of the seizures is thought to be secondary to the development of cerebral edema and focal spasm of small blood vessels in the kidney.
By "premature pre-eclampsia" is meant pre-eclampsia with onset of symptoms <37 weeks or <34 weeks.
By "prostacyclin" or "PGI2" is meant a member of the family of lipid molecules known as eicosanoids. It is produced in endothelial cells from prostaglandin H2 (PGH2) by the action of the enzyme prostacyclin synthase and is mainly synthesized by vascular endothelium and smooth muscle. PGI2 biological activity includes inhibition of platelet aggregation, relaxation of smooth muscle, reduction of systemic and pulmonary vascular resistance by direct vasodilation, and natriuresis in kidney.
By "protein" or "polypeptide" or "polypeptide fragment" is meant any chain of more than two amino acids, regardless of post-translational modification (e.g., glycosylation or phosphorylation), constituting all or part of a naturally occurring polypeptide or peptide, or constituting a non-naturally occurring polypeptide or peptide.
By "reference sample"'is meant any sample, standard, or level that is used for comparison purposes. A"nonnal reference sample" can be a prior sample taken from the same subject, a sample from a pregnant subject not having a pregnancy related hypertensive disorder, such as pre-eclampsia or eclampsia, a subject that is pregnant but the sample was taken early in pregnancy (e.g., in the first or second trimester or before the detection of a pregnancy related hypertensive disorder, such as pre-eclampsia or eclampsia), a subject that is pregnant and has no history of a pregnancy related hypertensive disorder, such as pre-eclampsia or eclampsia, a subject that is not pregnant, a sample of a purified reference polypeptide at a known normal concentration (i.e., not indicative of a pregnancy related hypertensive disorder, such as pre-eclampsia or eclampsia). By "reference standard or level" is meant a value or number derived from a reference sample. A normal reference standard or level can be a value or number derived from a normal subject that is matched to the sample subject by at least one of the following criteria: gestational age of the fetus, maternal age, matemal blood pressure prior to pregnancy, maternal blood pressure during pregnancy, BMI of the mother, weight of the fetus, prior diagnosis of pre-eclampsia or eclampsia, and a family history of pre-eclampsia or eclampsia. A "positive reference" sample, standard or value is a sample or value or number derived from a subject that is known to have a pregnancy related hypertensive disorder, such as pre-eclampsia or eclampsia, that is matched to the sample subject by at least one of the following criteria: gestational age of the fetus, maternal age, maternal blood pressure prior to pregnancy, maternal blood pressure during pregnancy, BMl of the mother, weight of the fetus, prior diagnosis of a pregnancy related hypertensive disorder, and a family history of a pregnancy related hypertensive disorder By "reduce or inhibit" is meant the ability to cause an overall decrease preferably of 20% or greater, more preferably of 40%, 50%, 60%, 70%, 80%, 90% or greater change in the level of protein or nucleic acid, detected by the aforementioned assays (see "expression"), as compared to an untreated sample By "sample" is meant a tissue biopsy, cell, bodily fluid (e.g., blood, serum, plasma, urine, saliva, amniotic fluid, or cerebrospinal fluid) or other specimen obtained from a subject. Desirably, the biological sample includes soluble endoglin nucleic acid molecules or polypeptides or both.
By "small interfering RNAs (siRNAs)" is meant an isolated dsRNA molecule, preferably greater than 10 nucleotides (nt) in length, more preferably greater than 15 nucleotides in length, and most preferably greater than 19 nucleotides in length that is used to identify the target gene or rnRNA to be degraded. A range of 19-25 nucleotides is the most preferred size for siRNAs. siRNAs can also include short hairpin RNAs in which both strands of an siRNA duplex are included within a single RNA
molecule.
siRNA includes any form of dsRNA (proteolytically cleaved products of larger dsRNA, partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA) as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and/or alteration of one or more nucleotides. Such alterations can include the addition of non-nucleotide material, such as to the end(s) of the 19, 20, 21, 22, 23, 24, or 25 nt RNA or internally (at one or more nucleotides of the RNA). In a preferred embodiment, the RNA molecules contain a 3' hydroxyl group.
Nucleotides in the RNA molecules of the present invention can also comprise non-standard nucleotides, including non-naturally occurring nucleotides or deoxyribonucleotides.
Collectively, all such altered RNAs are referred to as analogs of RNA. siRNAs of the present invention need only be sufficiently similar to natural RNA that it has the ability to mediate RNA
interference (RNAi). As used herein, RNAi refers to the ATP-dependent targeted cleavage and degradation of a specific mRNA molecule through the introduction of small interfering RNAs or dsRNAs into a cell or an organism. As used herein "mediate RNAi"
refers to the ability to distinguish or identify which RNAs are to be degraded.
By "soluble endoglin binding molecule" is meant a protein or small molecule compound that binds, preferably specifically binds, a soluble endoglin polypeptide. A
soluble endoglin binding molecule may be, for example, an antibody, antibody-related peptide, one or more CDR regions of a soluble endoglin binding antibody, or soluble endoglin interacting protein.
By "soluble Flt-1 (sFlt-1)" (also known as sVEGF-R1) is meant the soluble form of the Flt-1 receptor, that is homologous to the protein defined by GenBank accession number U01134, and that has sFlt-1 biological activity. The biological activity of an sFIt-1 polypeptide may be assayed using any standard method, for example, by assaying sFit-1 binding to VEGF. sFlt-1 lacks the transmembrane domain and the cytoplasmic tyrosine kinase domain of the Flt-1 receptor. sFlt-I can bind to VEGF and PIGF
with high affinity, but it cannot induce proliferation or angiogenesis and is therefore functionally different from the Flt-1 and KDR receptors. sFlt-1 was initially purified from human umbilical endothelial cells and later shown to be produced by trophoblast cells in vivo. As used herein, sFlt-1 includes any sFlt-1 family member or isoform. sFlt-1 can also mean degradation products or fragments that result from enzymatic cleavage of the Flt-1 receptor and that maintain sFlt-1 biological activity. In one example, specific metalloproteinases released from the placenta may cleave the extracellular domain of Flt-1 receptor to release the N-terminal portion of Flt-I into circulation.
By "specifically binds" is meant a compound or antibody which recognizes and binds a polypeptide of the invention but that does not substantially recognize and bind other molecules in a sample, for example, a biological sample, which naturally includes a polypeptide of the invention. In one example, an antibody that specifically binds soluble endoglin does not bind membrane bound endoglin. In another example, an antibody that specifically binds to soluble endoglin binds to an epitope within the extracellular domain of endoglin, particularly an epitope within amino acids 26 to 437 (excluding the peptide leader sequence), amino acids 40 to 406 of human endoglin (see FIGURE 30B), or amino acids 26 to 587 (excluding the peptide leader sequence); that may or may not be unique to soluble endoglin (e.g., in the three dimenstional structure of soluble endoglin). In another example, an antibody that specifically binds to soluble endoglin recognizes one or more of the amino acid sequences shown in bold and underlined in FIGURE 30B.
By `subject" is meant a mammal, including, but not limited to, a human or non-human mammal, such as a cow, a horse, a sheep, a pig, a goat, a dog, or a cat.
Included in this definition are pregnant, post-partum, and non-pregnant mammals.
By "substantially identical" is meant a nucleic acid or amino acid sequence that, when optimally aligned, for example using the methods described below, share at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with.a second nucleic acid or amino acid sequence, e.g., an endoglin or soluble endoglin sequence.
"Substantial identity" may be used to refer to various types and lengths of sequence, such as full-length sequence, epitopes or immunogenic peptides, functional domains, coding and/or regulatory sequences, exons,-introns, promoters,=and genomic sequences.
Percent identity between two polypeptides or nucleic acid sequences is determined in various ways that are within the skill in the art, for instance, using publicly available computer software such as Smith Waterman Alignment (Smith, T. F. and M. S. Waterman (1981) JMoI Biol 147:195-7);
"BestFit" (Smith and Waterman, Advances in Applied Mathematics, 482-489 (1981)) as incorporated into GeneMatcher P1usTm, Schwarz and Dayhof (1979) Atlas of Protein Sequence and Structure, Dayhof, M.O., Ed pp 353-358; BLAST program (Basic Local Alignment Search Tool; (Altschul, S. F., W. Gish, et al. (1990) J Mol Biol 215: 403-10), BLAST-2, BLAST-P, BLAST-N, BLAST-X, WU-BLAST-2, ALIGN, ALIGN-2, CLUSTAL, or Megalign (DNASTAR) software. In addition, those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the length of the sequences being compared. In general, for proteins, the length of comparison sequences will be at least 10 amino acids, preferably 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 250, 300, 350, 400, 437, or at least 587 amino acids or more. For nucleic acids, the length of comparison sequences will generally be at least 25, 50, 100, 125, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, 1000, 1100, 1200, 1311, or at least 1761 nucleotides or more. It is understood that for the purposes of determining sequence identity when comparing a DNA sequence to an RNA sequence, a thymine nucleotide is equivalent to a uracil nucleotide. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine;
aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
By "symptoms of pre-eclampsia" is meant any of the following: (1) a systolic blood pressure (BP) >140 mmHg and a diastolic BP >90 mmHg after 20 weeks gestation, (2) new onset proteinuria (1+ by dipstik on urinanaysis, >300mg of protein in a 24 hour urine collection, or random urine protein/creatinine ratio >0.3), and (3) resolution of hypertension and proteinuria by 12 weeks postpartum. The symptoms of pre-eclampsia can also include renal dysfunction and glomerular endotheliosis or hypertrophy, By "symptoms of eclampsia" is meant the development of any of the following symptoms due to pregnancy or the influence of a recent pregnancy: seizures, coma, thrombocytopenia, liver edema, pulmonary edema, and cerebral edema.
By "transforming growth factor (3 (TGF-0)" is meant a mammalian growth factor that has TGF-0 biological activity and is a member of a family of structurally related paracrine polypeptides found ubiquitously in vertebrates, and prototypic of a large family of metazoan growth, differentiation, and morphogenesis factors (see, for review, Massaque et al. Ann. Rev. Cell. Biol. 6:597-641 (1990); Massaque et al. Trends Cell Biol 4:172-178 (1994); Kingsley Gene Dev. 8:133-146 (1994); and Spom et al. J.
Cell. Biol.
119:1017-1021 (1992). As described in Kingsley, supra, the TGF-P superfamily has at least 25 members, and can be grouped into distinct sub-families with highly related sequences. The most obvious sub-families include the following: the TGF-P sub-family, which comprises at least four genes that are much more similar to TGF-Pl than to other members of the TGF-P superfamily; the bone morphogenetic proteins; the activin sub-family, comprising homo- or hetero-dimers or two sub-units, inhibin(3-A and inhibinp-B.
The decapentaplegic sub-family, which includes the mammalian factors BMP2 and BMP4, which can induce the formation of ectopic bone and cartilage when implanted under the skin or into muscles. The 60A sub-family, which includes a number of mammalian homologs, with osteoinductive activity, including BMP5-8. Other members of the TGF-(3 superfamily include the gross differentiation factor 1(GDF-1), GDF-3/VGR-2, dorsalin, nodal, mullerian-inhibiting substance (1VIIS), and glial-derived neurotrophic growth factor (GDNF). It is noted that the DPP and 60A sub-families are related more closely to one another than to other members of the TGF-P
superfamily, and have often been grouped together as part of a larger collection of molecules called DVR
(dpp and vgl related). Unless evidenced from the context in which it is used, the term TGF-0 as used throughout this specification wiIl be understood to generally refer to members of the TGF-0 superfamily as appropriate. (Massague et al., Annu. Rev.
Biochem. 67:753-91, 1998; Josso et al., Curr. Op. Gen. Dev., 7:371-377, 1997).
TGF-(3 functions to regulate growth, differentiation, motility, tissue remodeling, neurogenesis, would repair, apoptosis, and angiogenesis in many cell types. TGF-0 also inhibits cell proliferation in many cell types and can stimulate the synthesis of matrix proteins.
By "therapeutic amount" is meant an amount that when administered, either by administration directly to the patient or by an ex vivo approach, to a patient suffering from pre-eclampsia or eclampsia is sufficient to cause a qualitative or quantitative reduction in the symptoms of pre-eclampsia or eclampsia as described herein. A
"therapeutic amount" can also mean an amount that when administered, either by administration directly to the patient or by an ex vivo approach, to a patient suffering from pre-eclampsia or eclampsia is sufficient to cause a reduction in the expression levels of soluble endoglin or sFIt-1 or an increase in the expression levels of VEGF
or P1GF as measured by the assays described herein.
By "treating" is meant administering a compound or a pharmaceutical composition for therapeutic purposes. To "treat disease" or use for "therapeutic treatment" refers to administering treatment to a subject already suffering from a disease to improve the subject's condition. Preferably, the subject is diagnosed as suffering from a pregnancy complication associated with hypertension, such as pre-eclampsia or eclampsia, based on identification of any of the characteristic symptoms described below or the use of the diagnostic methods described herein. To "prevent disease"
refers to prophylactic treatment of a subject who is not yet ill, but who is susceptible to, or otherwise at risk of, developing a particular disease. Preferably a subject is determined to be at risk of developing pre-eclampsia or eclampsia using the diagnostic methods described herein. Thus, in the claims and embodiments, treating is the administration to a mammal either for therapeutic or prophylactic purposes.
By "trophoblast" is meant the mesectodermal cell layer covering the blastocyst that erodes the uterine-mucosa and through-which the embryo receives nourishment from the mother; the cells contribute to the formation of the placenta.
By "vascular endothelial growth factor (VEGF)" is meant a mammalian growth factor that is homologous to the growth factor defined in U.S. Patent Nos.
5,332,671;
5,240,848; 5,194,596; and Charnock-Jones et al. (Biol. Reproduction, 48: 1120-1128, 1993), and has VEGF biological activity. VEGF exists as a glycosylated homodimer and includes at least four different alternatively spliced isoforms. The biological activity of native VEGF includes the promotion of selective growth of vascular endothelial cells or umbilical vein endothelial cells and induction of angiogenesis. As used herein, VEGF
includes any VEGF family member or isoform (e.g., VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF189, VEGF 165, or VEGF 121). Preferably, VEGF is the VEGF121 or VEGF165 isoform (Tischer et al., J. Biol. Chem. 266, 11947-11954, 1991;
Neufed et al. Cancer Metastasis 15:153-158, 1996), which is described in U.S.
Patent Nos. 6,447,768; 5,219,739; and 5,194,596, hereby incorporated by reference.
Also included are mutant formsof VEGF such as the KDR-selective VEGF and Fit-selective VEGF described in Gille et al. (J. Biol. Chem. 276:3222-3230, 2001). As used herein VEGF also includes any modified forms of VEGF such as those described in LeCouter et al. (Science 299:890-893, 2003). Although human VEGF is preferred, the invention is not limited to human forms and can include other animal forms of VEGF (e.g.
mouse, rat, dog, or chicken).
By "vector" is meant a DNA molecule, usually derived from a plasmid or bacteriophage, into which fragments of DNA may be inserted or cloned. A
recombinant vector will contain one or more unique restriction sites, and may be capable of autonomous replication in a defined host or vehicle organism such that the cloned sequence is reproducible. A vector contains a promoter operably linked to a gene or coding region such that, upon transfection into a recipient cell, an RNA is expressed.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.
Brief Description of the Drawings FIGURE 1 is a schematic showing the endoglin protein. SP: signal peptide (also referred to as the peptide leader sequence), ZP: zona pellucida domain, CL:
potential cleavage site (amino acid 437) for the release of soluble endoglin, TM:
transmembrane domain, Cyto: cytoplasmic domain. Once the signal peptide is cleaved, the remaining mature protein starts at the glutamic acid residue at amino acid 26.
FIGURE 2A shows the predicted cDNA sequence (SEQ ID NO: 1) of soluble endoglin. FIGURE 2B shows the predicted amino acid sequence (SEQ ID NO: 2) of soluble endoglin which includes the signal peptide (amino acids 1-25). It should be noted that the sequence includes the leader peptide sequence that would normally be cleaved in the ER.
FIGURE 3 is a Northern blot showing endoglin mRNA levels in placentas from normal pregnancies (N), placentas from preterm pre-eclamptic pregnancies (p) and placentas from term pre-eclamptic pregnancies (P).
FIGURE 4 is a western blot showing endoglin protein levels in the placenta.
Samples are from two pre-eclamptic patients, p32 and p36, that presented to the Beth Israel Deaconess Medical Center in 2003 and maternal serum from a pregnant woman.
The Westem blot was probed using a N-terminal antibody obtained from Santa Cruz Biotechnology, I:nc., (Santa Cruz, CA) that shows both the I l OkD band in the placenta and a smaller 63 kD band that is present in the placenta and the-serum samples.
FIGURE 5 is a graph that shows the circulating concentrations of soluble endoglin in women with normal pregnancy, mild pre-eclampsia, severe pre-eclampsia and non-pre-eclamptic pregnancies complicated by pre-term delivery. All blood specimens were obtained within 24 hours prior to delivery. Soluble endoglin was measured using an ELISA kit from R & D Systems, MN (Cat # DNDGOO). These data show that soluble endoglin levels are significantly elevated in pre-eclamptic patients at the time of clinical disease.
FIGURE 6 is a graph showing the mean soluble endoglin concentration for the five different study groups of pregnant women throughout pregnancy during the various gestational age group-windows.
FIGURE 7 is a graph showing the mean sFltl concentrations for the five different study groups of pregnant women throughout pregnancy during the various gestational age group windows.
FIGURE 8 is a graph showing the mean PIGF concentrations for the five different study groups of pregnant women throughout pregnancy during the various gestational age group windows.
FIGURE 9 is a graph showing the values for the soluble endoglin anti-angiogenic index for pre-eclampsia anti-angiogenesis for samples taken prior to clinical symptoms.
FIGURE 10 is a graph showing the mean concentrations of soluble endoglin according to the number of weeks before clinical premature pre-eclampsia (PE
<37 weeks).
FIGURE 11 is a graph showing the soluble endoglin anti-angiogenic index values according to the number of weeks before clinical premature pre-eclampsia (PE
<37 weeks).
FIGURE 12 is a graph showing the alteration in soluble endoglin levels throughout pregnancy for term pre-eclampsia (PE>37 weeks) before and after symptoms.
FIGURE 13 is a graph showing the alteration in the soluble endoglin anti-angiogenic index levels throughout pregnancy for term pre-eclampsia (PE>37 weeks) before and after symptoms.
FIGURE 14 is a graph showing the soluble endoglin levels detected in women during gestational hypertension and before gestational hypertension (1-5 weeks preceding gestational hypertension (during weeks 33-36 of pregnancy)) and normotensive controls.
FIGURE 15 is a graph showing the soluble endoglin anti-angiogenic index levels in women during gestational hypertension and before gestational hypertension (1-5 weeks preceding gestational hypertension (during weeks 33-36 of pregnancy)) and normotensive controls.
- FIGURE 16 is a graph showing the soluble endoglin levels detected during the 33-36 week gestational windows in women with severe SGA, mild SGA, and normotensive controls.
FIGURE 17 is a graph showing the soluble endoglin anti-angiogenic index levels detected during the 33-36 week gestational windows in women with severe SGA, mild SGA, and normotensive controls.
FIGURE 18 is a graph showing the concentration of sFltl and soluble endoglin in the same pregnant patients plotted against each other.
FIGLIRE 19 shows photomicrographs of double immunofluorescence staining of endoglin (red) and smooth muscle actin (green) for pre-eclamptic placentas taken at 25.2 weeks. The antibody used to detect endoglin stains both full-length endoglin and the soluble endoglin. Control placentas for the appropriate gestational windows were derived from patients with pre-term labor.
FIGURE 20 shows photomicrographs of double immunofluorescence staining of endoglin (red) and smooth muscle actin (green) for pre-eclamptic placentas taken at 41.3 weeks. The antibody used to detect endoglin stains both full-length endoglin and the soluble endoglin. Control placentas for the appropriate gestational windows were derived from patients with pre-term labor.
FIGURE 21A shows an autaradiogram from immunoprecipitation and western blots experiments for endoglin using both pre-eclamptic placentas and serum.
FIGURE
21B shows an autoradiogram from immunoprecipitation and western blots experiments for endoglin using pre-eclamptic placentas. The three different N and P
samples represent individual patients. For both figures commercially available monoclonal antibodies were used for immunoprecipitation and polyclonal antibodies were used for the western blots. Both these antibodies were raised against the N-terminal region of the endoglin protein and detect both the full length and the truncated soluble endoglin protein.
FIGURE 22 is a graph showing the results of angiogenesis assays using HUVECs in growth factor reduced matrigels. Angiogenesis assays were performed in the presence of soluble endoglin or sFit1 or both and the endothelial tube lengths quantitated. C-represents control, E- represents 1 g/ml of soluble endoglin and S represents 1 g/ml of sFltl. E+S represent the combination of I g/ml of E + 1 g/ml of sFltl. Data represents a mean of three independent experiments.
FIGURE 23 is a graph showing the microvascular permeability in several organ beds assessed using Evans blue leakage in mice as described in the materials and methods. C- control (GFP), E-soluble endoglin, S-sFltl and S+E- sF1tI +
soluble endoglin. Data represents a mean of 4 independent experiments.
FIGURE 24 is a graph showing the percent change in rat renal microvessel diameter when subjected to microvascular reactivity experiments in the presence of TGF-p 1 (B 1) and TGF-(33 (133) from doses ranging from 200 pg/ml - 200 ng/ml.
These same experiments were repeated in the presence of soluble endoglin (E) at 1 g/ml.
These data presented are a mean of 4 independent experiments.
FIGURE 25 is a graph showing the percent change in the vascular diameter of renal microvessels in the presence of 1 ng/ml of VEGF (V), TGF-(31 (B 1) and the combination (V+B 1). Also shown is the effect of this combination in the presence of 1 g/ml each of sFltl (S) and soluble endoglin (E) (V+B I+S+E). The data represents a mean of 4 independent experiments.
FIGURE 26A is a photograph of a peripheral smear of blood samples taken at the time of sacrifice from pregnant rats injected with the combination of sFlt1 and a control adenoviruses (CMV). FIGURE 26B is a photograph of a peripheral smear of blood samples taken at the time of sacrifice from pregnant rats injected with the combination of sFlt and adenoviruses expressing soluble endoglin and demonstrates active hemolysis as evidenced by schistocyes and increased reticulocyte count. Arrowheads represent schistocyte.
FIGURES 27A-D are a series of photomicrographs showing the renal histology (1-I &E stain) of the various animal groups described in Table 8. FIGURE 27A
shows the renal histology for the control group with no evidence of glomerular endotheliosis.
FIGURE 27B shows the renal histology for the soluble endoglin injected group with no evidence of glomerular endotheliosis. FIGURE 27C shows the renal histology for sFltl injected rats showing moderate endotheliosis (shown by arrow head). FIGURE 27D
shows the renal histology for the soluble endoglin and sFltl injected rats showing extremely swollen glomeruli and severe glomerular endotheliosis with protein resorption droplets in the podocytes. All light micrographs were taken at 60X (original magnification).
FIGURE 28 is a graph showing the ELISA results for soluble endoglin (sEng) and sFltl in sera of patients with varying degrees of preeclampsia, control pregnancies and four non-pregnant healthy volunteers as described in Example 3. *P < 0.05 compared to pre-term controls and # P< 0.05 compared to severe preeclampsia.
FIGURE 29 is a graph showing ELISA results for soluble endoglin in a subset of pregnant patients (normal: n= 6; preeclampsia: n = 11) described in Example 3 with blood drawn pre- (0-12 hours) and post- (48 hours) delivery. * P < 0.05 as compared to T
= 0 samples.
FIGURE 30A is a western blot showing soluble endoglin after purification the serum of preeclamptic patients. Fractions 4 and 5 eluted from the 44G4-IgG
(anti-Eng) Sepharose were run on SDS-PAGE under reducing conditions and tested by Western blot using a polyclonal antibody to endoglin. The eluted fractions were subjected to mass spectrometry analysis (3 runs). FIGURE 30B shows the sequence of human endoglin (SEQ ID NO: 5). Peptides identified by mass spec are shown in bold and underlined.
The underlined amino acids represent the transmembrane domain of human cell surface endoglin. Note that the amino acid sequence numbering starts at 26 because amino acids 1-25 represents the leader peptide. Note that the sequence listed as SEQ ID
NO: 5 in the sequence listing begins at amino acid I so that amino acid 26 in the figure is amino acid 1 in the sequence listing, amino acid 658 in the figure is amino acid 633 in the sequence listing. The numbering of the amino acids is adjusted depending on the reference .
sequence (i.e., amino acids 26 to 658 for sequences referring to FIGURE 30B
are the same as amino acids 1 to 633 for sequences referring to SEQ ID NO: 5).
FIGURE 31 shows a series of photomicrographs showing soluble endoglin inhibits capillary formation and increases vascular permeability. Angiogenesis assays were performed using HUVEC in growth factor reduced MatrigelTm in the presence of 1 g of recombinant soluble endoglin, sFltl, or both, and endothelial tube lengths were quantified. A representative experiment (n = 4) is shown with tube lengths in pixels indicated below the panels.
FIGURE 32 is a series of graphs showing inhibition of TGF-(31-mediated vascular reactivity in mesenteric vessels by soluble endoglin. Microvascular reactivity of rat mesenteric microvessels was measured in the presence of TGF-(31 or TGF-(33 from 200 pg/ml to 200 ng/ml. The experiments were repeated in the presence of recombinant soluble endoglin at 1 g/ml. The mean f SE of 4 independent experiments is shown (upper panel). Also shown is the blocking effect of L-NAME on TGFP 1 at 1 ng/ml (lower panel).
FIGURE 33 is a series of photomicrographs showing glomerular endotheliosis in pregnant rats. Electron micrographs (EM) of glomeruli from a control pregnant rat (upper panel), soluble endoglin (sEng)-treated pregnant rat (middle panel) and the combination group - soluble endoglin (sEng)+sFltl (lower panel) are shown_ These photos were taken at 6200X (original magnification) for the upper and middle panel and 5000X (original magnification) for the lower panel. -FIGURES 34 A-H are a series of photomicrographs showing renal, placental and hepatic histological changes and peripheral blood smears in pregnant rats after soluble endoglin and sFltl treatment. Placental histology (H &E stain) ofcontrol.
(FIGURE
34A), sEng (FIGURE 34B), sFltl (FIGURE 34C) and sFltl+sEng (FIGURE 34D) groups. Both the soluble endoglin and sFltl treated animals show diffuse inflammation (arrow heads) at the maternal-fetal junction not seen in controls. There is hemorrhagic infarction and fibrinoid necrosis with lumen obstruction of a maternal vessel (arrow) in the decidua of the sFltl+sEng treated placenta (FIGURE 34D). Scale bar, 200 pm (FIGURE 34E-H). Liver histology in the control (FIGURE 34E), sEng (FIGURE
34F), sFltl (FIGt1RE 34G) and sFltl+sEng (FIGURE 34H) groups. Ischemic changes with multifocal necrosis (arrow head) are noted in the sFltl+sEng group (FIGURE
34H).
Control group and rats given sEng or sFltl showed no changes. Scale bar, 200 m.
FIGURES 35A-D are a series of graphs and autoradiograms showing recombinant sEng attenuates TGF-01 binding and activity and its effects on vasodilation via eNOS
activation. FIGURE 35A is a graph showing the microvascular responses of renal microvessels to I ng/ml of VEGF, TGF-p 1 and the combination. The effects of ng/ml each of sFltl and sEng on the combined response are shown. (n = 4). Also shown is the blocking effect of L-NAME on TGF(i1 and VEGF stimulated responses.
FIGURE
35B is a representative autoradiogram and graph of a dose-dependent increase in [1125 ]
TGF-01 binding to T(3RII on mouse endothelial cells. Treatment with 5 nM
recombinant soluble endoglin significantly reduced binding at 50 pM and 100 pM (*P < 0.05 vs.
untreated group). Competition with 40X excess cold TGF-(31 in cells treated with 100 pM [I125] TGF-J31 abolished receptor binding and served as background control.
FIGURE 35C is a graph showing significantly increased TGF-0-induced activation of the Smad 2/3-dependent CAGA-Luc reporter construct transfected in HUVECs and inhibition by treatment with sEng. (n = 3, **P < 0.01 vs. sEng untreated group).
FIGtTRE 35D is a representative western blots and graph (n = 4) showing significant dephosphorylation at eNOS Thr495 following treatment with TGF-(31 and attenuation by sEng (*P < 0.05 vs. untreated). Phosphorylation was unchanged at Serl 177 and total levels of eNOS remained constant throughout the experiments.
FIGURE 36 shows two western blots of rat plasma demonstrating expression of the recombinant sFlt1 and soluble endoglin. Upper panel: Plasma specimens from pregnant rats (at early third trimester) were used as described in Methods.
Lanes 1, 2 and 3 represent 200 pg, 500 pg and 2 ng of recombinant mouse Fltl -Fc protein used as a positive control. 20 l of plasma specimens from one control rat and two sFItl treated rats are shown. sFltl (53 kDa) band was detected in the sFltl treated rats.
Quantitation of the sFltl expression was performed using commercially available ELISA (Table 8).
Lower panel: Plasma specimens from pregnant rats were used (at early third trimester) to detect sEng expression. Lane 1 represents 500 pg of recombinant human soluble endoglin and lanes 2 and 3 represent 30 l of plasma from sEng treated and control rats respectively. The blot shows no soluble endoglin in control rats but robust expression of recombinant sEng in treated rats. Quantitation of soluble endoglin was performed using a commercially available ELISA (Table 8).
Figure 37 is a graph showing the distribution of delta sFltl and delta sEng (first trimester- second trimester values) in controls, all pre-eclampsia and in pre-eclampsia <37 weeks.
Figure 38 is a graph showing the distribution of sFlt x sEng product in the fsrst trimester (product 1), in the second trimester (product 2), delta product (product 1-product 2) in controls, all pre-eclampsia and in pre-eclampsia <37 weeks.
Figure 39 is a graph showing the risk of pre-eclampsia according to tertiles of delta product. The increase in risk of preterrn preeclampsia in the group whose delta product levels were greater than +1 [aOR 5.5, 95% Cl 1.4 - 22.4], compared to women whose delta product was less than -1 was statistically significant (P<0.05).
Figure 40A is a western blot showing endoglin is necessary for TGF-j31 induced dephosphorylation of eNOS at Thr495. Figure 40B is a graph showing the percent of eNOS Thr495 phosphorylation relative to total eNOS. The results show that the level of phosphorylated Thr495 decreases in the presence of TGF-01 in the presence of soluble endoglin but not in the absence of soluble endoglin.
Detailed Description We have discovered that soluble endoglin levels are elevated in blood serum samples taken from women with a pregnancy related hypertensive disorder, such as pre-eclampsia or eclampsia. Soluble endoglin may be formed by cleavage of the extracellular portion of the membrane bound form by proteolytic enzymes. The lack of detection of altemate splice variants in placenta and the partial peptide sequence of purified soluble endoglin as described herein suggest that it is an N-terminal cleavage product of full-length endoglin. Excess soluble endoglin may be depleting the placenta of necessary amounts of these essential angiogenic and mitogenic factors. We have discovered that excess circulating concentrations of soluble endoglin and sFltl in patients with preeclampsia contribute to the pathogenesis of pre-eclampsia and other pregnancy related hypertensive disorders. We have also discovered that soluble endoglin interferes with TGF-01 and TGF-j33 binding to its receptor leading to decreased signaling such as a reduction in eNOS activation in endothelial cells, thereby disrupting key homeostatic mechanisms necessary for maintenance of vascular health. These data suggest a crucial role for endoglin in linking TGF-(3 receptor activation to NO synthesis. In addition, we have discovered that soluble endoglin and sFltl act in concert to induce vascular damage and pregnancy related hypertensive disorders, such as pre-eclampsia or eclampisa, by interfering with TGF-P 1 and VEGF signaling respectively, likely via inhibition of the downstream activation of eNOS.
The present invention features the use of therapeutic agents that interfere with soluble endoglin binding to growth factors, agents that reduce soluble endoglin expression or biological activity, or agents that increase levels of growth factors, can be used to treat or prevent pregnancy related hypertensive disorders, such as pre-eclampsia or eclampsia in a subject. Such agents include, but are not limited to, antibodies that bind to soluble endoglin and inhibit soluble endoglin biological activity, oligonucleotides for antisense or RNAi that reduce levels of soluble endoglin, compounds that increase the levels of growth factors that bind to soluble endoglin, compounds that prevent the proteolytic cleavage of the membrane bound form of endoglin thereby preventing the release of soluble endoglin, and small molecules that bind soluble endoglin and block the growth factor binding site. Additionally or alternatively, the invention features the use of any compound (e.g., polypeptide, small molecule, antibody, nucleic acid, and nzimetic) that increases the level or biological activity of TGF-(3, eNOS, and PGI2 to treat or prevent pregnancy related hypertensive disorders, such as pre-eclampsia or eclampsia in a subject. Additionally, the invention features the use of any compound that decreases the level of sFlt-l or increases the level or activity of VEGF or PIGF (see for example, U.S.
Patent Application Publication Numbers 20040126828, 20050025762, and and PCT Publication Numbers WO 2004/008946 and WO 2005/077007) in combination with any of the therapeutic compounds described above to treat or prevent pregnancy related hypertensive disorders, such as pre-eclampsia or eclampsia, in a subject. In addition, the invention features the use of soluble endoglin, eNOS, TGF-(3, of PGI2, either alone or in combination, as a diagnostic marker of pregnancy related hypertensive disorders, including pre-eclampsia and eclampsia.
While the detailed description presented herein refers specifically to soluble endoglin, TGF-(31, eNOS, sFlt-1, VEGF, or PIGF, it will be clear to one skilled in the art that the detailed description can also apply to family members, isoforms, and/or variants of soluble endoglin, TGF-(3, eNOS, sFlt-1, VEGF, or PIGF.
Diagnostics We have discovered that soluble endoglin levels are elevated in blood serum samples taken from women with a pregnancy related hypertensive disorder, such as pre-eclampsia or eclampsia. Soluble endoglin starts rising 6-10 weeks before clinical symptoms of preeclampsia. Accordingly, a diagnostic test measuring soluble endoglin and sFltl, optionally in combination with free PIGF, in the serum will have enhanced sensitivity and specificity, and provide a powerful tool in the prevention of preeclampsia-induced mortality. The diagnostic test can also include measuring the levels of free VEGF; TGF-P family members, preferably TGF-(31, TGF- P3, free activin-A, BMP2, BMP7; NOS, preferably eNOS; or PGI2, either alone or in any combination thereof. An alteration in the levels of any of these proteins is diagnostic of a pregnancy related hypertensive disorder, such as pre-eclampsia or eclampsia. In one example, a decrease in the levels of free BMP2, BMP7, or activin A is diagnostic of a pregnancy related hypertensive disorder, such as pre-eclampsia or eclampsia.
While the methods described herein refer to pre-eclampsia and eclampsia specifically, it should be understood that the diagnostic and monitoring methods of the invention apply to any pregnancy related hypertensive disorder including, but not limited to, gestational hypertensiorn, pregnancy with a small for gestational age (SGA) infant, HELLP, chronic hypertension, pre-eclampsia (mild, moderate, and severe), and eclampsia.
Levels of soluble endoglin, either free, bound, or total levels, are measured in a subject sampl e and used as an indicator of pre-eclampsia, eclampsia, or the propensity to develop such conditions.
A subject having pre-eclampsia, eclampsia, or a predisposition to such conditions will show an increase in the expression of a soluble endoglin polypeptide. The soluble endoglin polypeptide can include full-length soluble endoglin, degradation products, alternatively spliced isoforms of soluble endoglin, enzymatic cleavage products of soluble endoglin, and the like. An antibody that specifically binds a soluble endoglin polypeptide may be used for the diagnosis of pre-eclampsia or eclampsia or to identify a subject at risk of developing such conditions. One example of an antibody useful in the methods of the invention is a monoclonal antibody against the N-terminal region of endoglin that is commercially available from Santa Cruz Biotechnology, Inc.
(cat # sc-20072). Additional examples include antibodies that specifically bind the extracellular domain of endoglin (e.g., amino acids 1 to 437 of endoglin, amino acids I to 587 of endoglin, or any of the amino acid sequences shown in bold and underlined in FIGURE
30B). A variety of protocols for measuring an alteration in the expression of such polypeptides are known, including immunological methods (such as ELISAs and RIAs), and provide a basis for diagnosing pre-eclampsia or eclampsia or a risk of developing such conditions.
Increased levels of soluble endoglin are a positive indicator of pre-eclampsia or eclampsia. For example, if the level of soluble endoglin is increased relative to a normal reference (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more), or increases over time in one or more samples from a subject, this is considered a positive indicator of pre-eclampsia or eclampsia. Additionally, any detectable alteration in levels of soluble endoglin, sFlt-1, VEGF, or PIGF relative to normal levels is indicative of eclampsia, pre-eclampsia, or the propensity to develop such conditions. Nonnally, circulating serum concentrations of soluble endoglin range from 2-7 ng/ml during the non-pregnant state and from 10-20 ng/ml during normal pregnancy. Elevated serum levels, greater than 15 ng/rnl, preferably greater than 20 ng/ml, and most preferably greater than 25 ng/ml or more, of soluble endoglin is considered a positive indicator of pre-eclampsia or eclampsia.
In one embodiment, the level of soluble endoglin is measured in combination with the level of sFlt-1, VEGF, or PIGF polypeptide or nucleic acid, or any combination thereof. Methods for the measurement of sFlt-1, VEGF, and PIGF are described in U.S.
Patent Application Publication Numbers 20040126828, 20050025762, and and PCT Publication Numbers WO 2004/008946 and WO 2005/077007, hereby incorporated by reference in their entirety. In additional preferred embodiments, the body mass index (BMI) and gestational age of the fetus is also measured and included the diagnostic metric.
In another embodiment, the level of TGF-(31, TGF-P3, or eNOS polypeptide or nucleic acid is measured in combination with the level of soluble endoglin, sFlt-1, VEGF, or PIGF polypeptide or nucleic acid. Antibodies useful for the measurement of TGF-(31 and P3 polypeptide levels are commercially available, for example, from Abcam, Abgent, BD Biosciences Pharmingen, Chemicon, GeneTex, and R&D Systems. The level of PGIZ
can also be used in combination with the level of any of the above polypeptides. PGI2 levels can be determined, for example, using the PGIa receptor as a binding molecule in any of the diagnostic assays described above, or using, for example, the urinary prostacyclin colorimetric ELISA kit (Assay Designs). Antibodies useful for the measurement of eNOS polypeptide levels are commercially available, for example, from Research Diagnostics Inc., Santa Cruz, Cayman Chemicals, and BD Biosciences.
In another embodiment, the biological activity of any one or more of TGF-(31, TGF-03, or eNOS polypeptide is measured in combination with the biological activity of soluble endoglin, sFlt-1, VEGF, or PIGF polypeptide and a decrease in the biological activity is a positive indicator of pre-eclampsia or eclampsia. The biological activity can be measured, for example using an assay for enzymatic activity or for the downstream signaling activity. In one example, the enzymatic activity of eNOS is determined by measuring citrulline conversion and a decrease in the enzymatic activity of eNOS is a positive indicator of pre-eclampsia or eclampsia.
In one embodiment, a metric incorporating soluble endoglin, sFlt-1, VEGF, or PIGF, or any combination therein, is used to determine whether a relationship between levels of at least two of the proteins is indicative of pre-eclampsia or eclampsia. In one example, the metric is a PAAI (sFlt-1/ VEGF + PIGF), which is used, in combination with soluble endoglin measurement, as an anti-angiogenic index that is diagnostic of pre-eclampsia, eclampsia, or the propensity to develop such conditions. If the level of soluble endoglin is increased relative to a reference sample (e.g., 1.5-fold, 2-fold, 3-fold, 4-fold, or even by as much as ] 0-fold or more), and the PAAI is greater than 10, more preferably greater than 20, then the subject is considered to have pre-eclampsia, eclampsia, or to be in imminent risk of developing the same. The PAAI (sFlt-1/
VEGF +
PIGF) ratio is merely one example of a useful metric that may be used as a diagnostic indicator. It is not intended to limit the invention. Virtually any metric that detects an alteration in the level of soluble endoglin, sFlt-1, PIGF, or VEGF, or any combination thereof, in a subject relative to a normal control may be used as a diagnostic indicator.
Another example is the following soluble endoglin anti-angiogenic index: (sFlt-1 +
0.25(soluble endoglin polypeptide))/P1GF. An increase in the value of the soluble endoglin metric over time or compared to a reference sample or value is a diagnostic indicator of pre-eclampsia or eclampsia. A soluble endoglin index above 100, preferably above 200 is a diagnostic indicator of pre-eclampsia or eclampsia. Additional examples include the following indexes: (soluble endoglin+ sFlt-1)/P1GF or sFlt-1 x soluble endoglin.
Another example includes the measurement of the levels of sFlt-1 and soluble endoglin in the first and second trimesters in a subject and calculating the delta value of sFlt1 x soluble endoglin (sEng) in each trimester using the following equation: [dproduct =(sFltl x sEng) in the second trimester - (sFltl x sEng) in the first trimester], where a value greater than 0, 1, 2, or more, including fractions thereof (e.g., a positive value) is a diagnostic indicator of pre-eclampsia or eclampsia. A positive value can also be an indicator of pre-term pre-eclampsia. Such a measurement can be taken on numerous occasions during the first and second trimesters and the dproduct can be followed over time. In addition, the dproduct of the sFlt-1 level (dsFIt-1) and the sEng level (dsEng) alone can also be calculated between the first and second trimesters, where a value greater than 0, 1, 2, or more, including fractions thereof (e.g., a positive value) for (dsFlt-1) or (dsEng) is a diagnostic indicator of pre-eclampsia or eclampsia.
In addition, the metric can further include the level of TGF-01, TGF-03, PGI2, or eNOS polypeptide. Any of the metrics can further include the BMI of the mother or the GA of the infant.
Standard methods may be used to measure levels of soluble endoglin, free VEGF, free P1GF, sFlt-1, TGF-p1, TGF-(33, PGI2, or eNOS polypeptide in any bodily fluid, including, but not limited to, urine, serum, plasma, saliva, amniotic fluid, or cerebrospinal fluid. Such methods include immunoassay, ELISA, western blotting using antibodies directed to soluble endoglin, free VEGF, free P1GF, sFlt-1, TGF-01, TGF-03, PGIZ, or eNOS polypeptide and quantitative enzyme immunoassay techniques such as those described in Ong et al. (Obstet. Gynecol. 98:608-611, 2001) and Su et al.
(Obstet.
Gynecol., 97:898-904, 2001). ELISA is the preferred method for measuring levels of soluble endoglin, VEGF, P1GF, sFlt-1, TGF-(31, TGF-(33, PGI2, or eNOS
polypeptide.
Preferably, soluble endoglin is measured alone or in combination with any one or more of the remaining polypeptides.
Oligonucleotides or longer fragments derived from an endoglin, sFlt-1, P1GF, or VEGF nucleic acid sequence may be used as a probe not only to monitor expression, but also to identify subjects having a genetic variation, mutation, or polymorphism in an endoglin, sFlt-1, PIGF, or VEGF nucleic acid molecule that are indicative of a predisposition to develop the pre-eclampsia or eclampsia. Such methods are described in detail in Abdalla et al., Hum. Mutat. 25:320-321 (2005), U.S. Patent Application Publication No. 2006/0067937 and PCT Publication No. WO 06/034507. Preferred oligonucleotides will hybridize at high stringency to the extracellular domain of endoglin or to any nucleic acid sequence encoding any of the peptides shown in bold and underlined in FIGRUE 30B.
The measurement of any of the nucleic acids or polypeptides described herein can occur on at least two different occasions and an alteration in the levels as compared to normal reference levels over time is used as an indicator of pre-eclampsia, eclampsia, or the propensity to develop such conditions.
In one example, the level of a soluble endoglin polypeptide or nucleic acid present in the bodily fluids of a subject having pre-eclampsia, eclampsia, or the propensity to develop such conditions may be increased by as little as 10%, 20%, 30%, or 40%, or by as much as 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more relative to levels in a normal control subject or relative to a previous sampling obtained from the same bodily fluids of the same subject. In another example, the level of a soluble endoglin polypeptide or nucleic acid in the bodily fluids of a subject having pre-eclampsia, eclampsia, or the propensity to develop such conditions may be altered by as little as 10%, 20%, 30%, or 40%, or by as much as 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% over time from one measurement to the next.
The level of sFlt-1, VEGF, or PIGF measured in combination with the level of soluble endoglin in the bodily fluids of a subject having pre-eclampsia, eclampsia, or the propensity to develop such conditions may be altered by as little as 10%, 20%, 30%, or 40%, or by as much as 50%, 60%, 70%, 80%, 90%, 95%, 96%, 9'7oJo, 98%, 99% or more relative to the level of sFlt-1, VEGF, or PIGF in a normal control. The level of sFlt-1, VEGF, or PIGF measured in combination with the level of soluble endoglin in the bodily fluids of a subject having pre-eclampsia, eclampsia, or the propensity to develop such.
conditions may be altered by as little as 10%, 20%, 30%, or 40%, or by as much as 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% over time from one measurement to the next.
In one embodiment, a subject sample of a bodily fluid (e.g., urine, plasma, serum, amniotic fluid, or cerebrospinal fluid) is collected earty in pregnancy prior to the onset of pre-eclampsia symptoms. In another example, the sample can be a tissue or cell collected early in pregnancy prior to the onset of pre-eclampsia symptoms. Non-limiting examples of tissues and cells include placental tissue, placental cells, circulating endothelial cells, and leukocytes such as monocytes. In humans, for example, maternal blood serum samples are collected from the antecubital vein of pregnant women during the first, second, or third trimesters of the pregnancy. Preferably, the assay is carried out during the first trimester, for example, at 4, 6, 8, 10, or 12 weeks, or any interval therein, or during the second trimester, for example at 14, 16, 18, 20, 22, or 24 weeks, or any interval therein. In one example, the assay is carried out between 13 and 16 weeks of pregnancy. Such assays may also be conducted at the end of the second trimester or the third trimester, for example at 26, 28, 30, 32,.34, 36, or 38 weeks, or any interval therein.
It is preferable that levels of soluble endoglin and/or any of the additional polypeptides described herein be measured twice during this period of time. For the diagnosis of post-partum pre-eclampsia or eclarnpsia, assays for soluble endoglin may be carried out postpartum. For the diagnosis of a predisposition to pre-eclampsia or eclampsia, the assay is carried out prior to the onset of pregnancy or prior to the development of symptoms of pre-eclampsia or eclampsia. In one example, for the monitoring and management of therapy, the assay is canied out during the pregnancy after the diagnosis of pre-eclampsia, and/or during therapy.
In one particular example, serial blood samples can be collected during pregnancy and the levels of soluble endoglin polypeptide and/or any of the additional polypeptides of the invention determined by ELISA. In another example, a sample is collected during the second trimester and early in the third trimester and in increase in the level of soluble endoglin of any of the other polypeptides of the invention from the first sampling to the next is indicative of pre-eclampsia or eclampsia, or the propensity to develop either.
The invention also include the measurement of any soluble endoglin binding protein (e.g., TGF-pl, TGF-(33, activin-A, BMP-2, and BMP-7) or downstream mediators of soluble endoglin signaling (e.g., PGIZ and eNOS) in a bodily fluid from a subject, preferably urine, and an alteration (e.g., increase or decrease) in the level of the soluble endoglin binding protein is indicative of pre-eclampsia or eclampsia. The methods and timing for measurement of soluble endoglin described herein can also be used for the measurement of any of the soluble endoglin binding protein, PGI2 or eNOS.
In veterinary practice, assays may be carried out at any time during the pregnancy, but are, preferably, carried out early in pregnancy, prior to the onset of pre-eclampsia symptoms. Given that the term of pregnancies varies widely between species, the timing of the assay vaill be determined by a veterinarian, but will generally correspond to the timing of assays during a human pregnancy.
The diagnostic methods described herein can be used individually or in combination with any other diagnostic method described herein or known in the art for a more accurate diagnosis of the presence of, severity of, or estimated time of onset of pre-eclampsia or eclampsia. In addition, the diagnostic methods described herein can be used in combination with any other diagnostic methods determined to be useful for the accurate diagnosis. of the presence of, severity of, or estimated time of onset of pre-eclampsia or eclampsia.
The diagnostic methods described herein can also be used to monitor and manage pre-eclampsia or eclampsia in a subject. In one example, a therapy is administered until the blood, plasma, or serum soluble endoglin level is less than 25 ng/ml or until the serum soluble endoglin levels (or soluble endoglin binding protein, PGIa, or eNOS
level) return to the baseline level-determined before onset ofpre-eclampsia or eclampsia. In another example, if a subject is determined to have an increased level of soluble endoglin relative to a normal control then the therapy can be administered until the serum P1GF
level rises to approximately 400 pg/mL or a return to baseline level prior to onset of pre-eclampsia or eclampsia. In this embodiment, the levels of soluble endoglin, sFlt-1, P1GF, VEGF, soluble endoglin binding protein, PGI2, eNOS or any and all of these, are measured repeatedly as a method of not only diagnosing disease but monitoring the treatment and management of the pre-eclampsia and eclampsia.
Diagnostic Kits The invention also provides for a diagnostic test kit. For example, a diagnostic test kit can include binding agents (e.g., polypeptides or antibodies) that specifically bind to soluble endoglin and means for detecting, and more preferably evaluating, binding between the binding agent and the soluble endoglin polypeptide. For detection, either the binding-agent or the soluble endoglin polypeptide is labeled, and either the binding agent or the soluble endoglin polypeptide is substrate-bound, such that soluble endoglin polypeptide-binding agent interaction can be established by determining the amount of label attached to the substrate following binding between the binding agent and the soluble endoglin polypeptide. A conventional ELISA is a common, art-known method for detecting antibody-substrate interaction and can be provided with the kit of the invention. Soluble endoglin polypeptides can be detected in virtually any bodily fluid including, but not limited to urine, serum, plasma, saliva, amniotic fluid, or cerebrospinal fluid. The invention also provides for a diagnostic test kit that includes a soluble endoglin nucleic acid that can be used to detect and determine levels of soluble endoglin nucleic acids. A kit that determines an alteration, for example, an increase, in the level of soluble endoglin polypeptide relative to a reference, such as the level present in a normal control, is useful as a diagnostic kit in the methods of the invention.
The diagnostic kits of the invention can include antibodies or nucleic acids for the detection of sFlt-1, VEGF, or P1GF polypeptides or nucleic acids as described U.S. Patent Application Publication Numbers 20040126828, 20050025762, and 20050170444 and PCT Publication Numbers WO 2004/008946 and WO 2005/077007.
In another embodiment, the kit can also include binding agents for the detection of soluble endoglin ligands including but not limited to TGF-(31, TGF-(33, or PGI2 or eNOS poIypeptides. Antibodies useful for the measurement of TGF-01 and (33 polypeptide levels are commercially available, for example, from Abcam, Abgent, BD
Biosciences Pharmingen, Chemicon, GeneTex, and R&D Systems. Antibodies useful for the measurement of eNOS polypeptide levels are commercially available, for example, from Research Diagnostics Inc., Santa Cruz, Cayman Chemicals, and BD
Biosciences.
Binding agents for the detection of PGI2 levels can also be included and include for example the PGIz receptor, or fragments thereof, as a binding molecule in any of the diagnostic assays described above, or using, for example, the urinary prostacyclin colorimetric ELISA kit (Assay Designs). A kit that determines an alteration, for example, a decrease, in the level of eNOS, TGF-(31 or (33 polypeptide or PGIa relative to a normal reference or standard or level, such as the level present in a normal control, is useful as a diagnostic kit in the methods of the invention. A kit that determines an alteration, for example, a decrease in the level of soluble endoglin or an increase in the level of a soluble endoglin binding protein (e.g., TGF-01, TGF-f33, activin A, BMP2, and BMP 7) or downstream mediators. of soluble endoglin signaling (e.g., eNOS and PGI2) relative to a positive reference standard or level is useful for monitoring the treatment of pre-eclampsia or eclampsia.
Desirably, the kit includes any of the components needed to perform any of the diagnostic methods described above. For example, the kit desirably includes a membrane, where the soluble endoglin binding agent or the agent that binds the soluble endoglin binding agent is immobilized on the membrane. The membrane can be supported on a dipstick structure where the sample is deposited on the membrane by placing the dipstick structure into the sample or the membrane can be supported in a lateral flow cassette where the sample is deposited on the membrane through an opening in the cassette.
The diagnostic kits also generally include a label or instructions for the intended use of the kit components and a reference sample or purified proteins to be used to establish a standard curve. In one example, the kit contains instructions for the use of the kit for the diagnosis of a pregnancy related hypertensive disorder, such as pre-eclampsia, eclampsia, or the propensity to develop pre-eclampsia or eclampsia. In yet another example, the kit contains instructions for the use of the kit to monitor therapeutic treatment or dosage regimens for the treatment of pre-eclampsia or eclampsia.
The diagnostic kit may also include a label or instructions for the use of the kit to determine the PAAI or soluble endoglin anti-angiogenesis index of the subject sample and to compare the PAAI or soluble endoglin anti-angiogenesis index to a reference sample value. It will be understood that the reference sample values will depend on the intended use of the kit. For example, the sample can be compared to a normal reference value, wherein an increase in the PAAI or soluble endoglin anti-angiogenesis index or in the soluble endoglin value is indicative of pre-eclampsia or eclampsia, or a predisposition to pre-eclampsia or eclampsia. Tn another example, a kit used for therapeutic monitoring can have a reference PAAI or soluble endoglin anti-angiogenesis index value or soluble endoglin value that is indicative of pre-eclampsia or eclampsia, wherein a decrease in the PAAI or soluble endoglin anti-angiogenesis index value or a decrease in the soluble endoglin value of the subject sample relative to the reference sample can be used to indicate therapeutic efficacy or effective dosages of therapeutic compounds. A
standard curve of levels of purified protein within the normal or positive reference range, depending on the use of the kit, can also be included.
Therapeutics The present invention features methods and compositions for treating or preventing pre-eclampsia or eclampsia in a subject. Given that levels of soluble endoglin are increased in subjects having pre-eclampsia, eclampsia, or having a predisposition to such conditions, any compound that decreases the expression levels and/or biological activity of a soluble endoglin polypeptide or nucleic acid molecule is useful in the methods of the invention. Such compounds include TGF-p1, TGF-(33, activin-A, BMP2, or BMP7, that can disrupt soluble endoglin binding to ligands; a purified antibody or antigen-binding fragment that specifically binds soluble endoglin; antisense nucleobase oligomers; and dsRNAs used to mediate RNA interference. Additional useful compounds include any compounds that can alter the biological activity of soluble endoglin, for example, as measured by an angiogenesis assay. Exemplary compounds and methods are described in detail below. These methods can also be combined with methods to decrease sFlt-1 levels or to increase VEGF or PIGF levels or decrease sFlt-1 levels as described in PCT Publication Number WO 2004/008946 and U.S. Patent Publication Nos. 20040126828 and 20050170444. In addition, any compound that increases the level or biological activity of TGF-01 or 3, eNOS, or PG12 are useful in the methods of the invention. Exemplary compounds and methods are described in detail below.
It should be noted that we have discovered that the soluble endoglin and sFlt-pathways may be functioning in a cooperative manner to further the pathogenesis of pre-eclampsia or eclampsia. Therefore, the invention includes any combination of any of the methods or compositioris described herein for the treatment or prevention of a pregnancy related hypertensive disorder. For example, a compound that targets the soluble endoglin pathway (e.g., downregulates soluble endoglin expression or biological activity or upregulates TGF-(3, eNOS, or PGI2 expression or biological activity) can be used in combination with a compound that targets the sFlt-1 pathway (e.g., downregulates sFlt-1 expression or biological activity or upregulates VEGF or PIGF expression of biological activity) for the treatment or prevention of a pregnancy related hypertensive disorder.
Therapeutics targeting the TGF=,8 signaling pathway TGF-0 is the prototype of a family of at least 25 growth factors which regulate growth, differentiation, motility, tissue remodeling, neurogenesis, wound repair, apoptosis, and angiogenesis in many cell types. TGF-(3 also inhibits cell proliferation in many cell types and can stimulate the synthesis of matrix proteins. Unless evidenced from the context in which it is used, the term TGF-0 as used throughout this specification will be understood to generally refer to any and all members of the TGF-0 superfamily as appropriate. Soluble endoglin binds several specific members of the TGF-0 family including TGF-01, TGF-03, activin, B1VIP-2 and BMP-7, and may serve to deplete the developing fetus or placenta of these necessary mitogenic and angiogenic factor. The present invention features methods of increasing the levels of these ligands to bind to soluble endoglin and to neutralize the effects of soluble endoglin.
Soluble endoglin ligands as therapeutic compounds In a preferred embodiment of the present invention, purified forms of any soluble endoglin ligand such as TGF-(3 family proteins, including but not limited to TGF-01, TGF-(33, activin-A, BMP2, and BMP7, are administered to the subject in order to treat or prevent pre-eclampsia or eclampsia.
Purified TGF-(3 family proteins include any protein with an amino acid sequence that is homologous, more desirably, substantially identical to the amino acid sequence of TGF-P l or TGF-(33, or any known TGF-(3 family member, that can induce angiogenesis.
Non-limiting examples include human TGF-01 (Cat #240-B-002) and human TGF-P3 (Cat #243-B3-002) from R & D Systems, MN. Preferred TGF-(3 family proteins useful in the methods of the invention will have the ability to bind to soluble endoglin (e.g., Barbaraet al, J. Biol. Chem. 274:584-94 (1999)).
Therapeutic compounds that inhibit proteolytic cleavage of endoglin We have identified a potential cleavage site in the extracellular domain of endoglin where a proteolytic enzyme could cleave the membrane bound form of endoglin, releasing the extracellular domain as a soluble form. Our sequence alignments of the cleavage site suggest that a matrix metalloproteinase (MMP) may be responsible for the cleavage and release of soluble endoglin. Alternatively, a cathepsin or an elastase may also be involved in the cleavage event. 1VIlVIPs are also known as collagenases, gelatinases, and stromelysins and there are currently 26 family members known (for a review see Whittaker and Ayscough, Cell Transmissions 17:1 (2001)). A
preferred NIlVIP
is MMP9, which is known to be up-regulated in placentas from pre-eclamptic patients (Lim et a1., Am. J Fathol. 151:1809-1818, 1997). The activity of TEvIPs is controlled through activation of pro-enzymes and inhibition by endogenous inhibitors such as the tissue inhibitors of inetalloproteinases (TIMPS). Inhibitors of MBsIPs are zinc binding proteins. There are 4 known endogenous inhibitors (TIMP 1-4), which are reviewed in Whittaker et al., supra. One preferred MMP inhibitor is the inhibitor of membrane type-lV1IvIl'1 that has been shown to cleave betaglycan, a molecule that shares similarity to enodglin (Velasco-Loyden et al., J. Biol. Chem. 279:7721-7733 (2004)). In addition, a variety of naturally-occurring and synthetic MMP inhibitors have been identified and are also reviewed in Whittaker et al., supra. Examples include antibodies directed to MMPs, and various compounds including marimastat, batimastat, CT1746, BAY 12-9566, Prinomastat, CGS-27023A, D9120, BMS275291 (Bristol Myers Squibb), and trocade, some of which are currently in clinical trials. Given the potential role of IVIIVI2's, cathepsins, or elastases in the release and up-regulation of soluble endoglin levels, the present invention also provides for the use of any compound, such as those described above, known to inhibit the activity of any MMP, cathepsin, or elastase involved in the cleavage and release of soluble endoglin, for the treatment or prevention of pre-eclampsia or eclampsia in a subject.
Therapeutic compounds that increase soluble endoglin bindingproteins The present invention provides for the use of any compound known to stimulate or increase blood serum levels of soluble endoglin binding proteins, including but not limited to TGF-(31, TGF-(33, activin-A, BMP2, and BMP7, for the treatment or prevention of pre-eclampsia in a subject. These compounds can be used alone or in combination with the purified proteins described above or any of the other methods used to increase TGF-0 family proteins protein levels described herein. In one example, cyclosporine is used at a dosage of 100-200 mg twice a day to stimulate TGF-P
production.
Therapeutic compounds that alter the anti-angiogenic activity of soluble endoglin Additional therapeutic compounds can be identified using angiogenesis assays.
For example, pre-eclamptic serum having elevated levels of soluble endoglin are added to a matrigel tube formation assay will induce an anti-angiogenic state. Test compounds can then be added to the assay and a reversion in the anti-angiogenic state by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more indicates that the compound can reduce the biological activity of soluble endoglin and is useful as a therapeutic compound.
Therapeutic compounds that increase the levels or biological activity ofNOS
NOS is a complex enzyme containing several cofactors, a heme group which is part of the catalytic site, an N-terminal oxygenase domain, which belongs to the class of haem-thiolate proteins, and a C-terminal reductase domain which is homologous to NADPH:P450 reductase. NOS produces NO by catalysing a five-electron oxidation of a guanidino nitrogen of L-arginine (L-Arg).
eNOS activation involves a coordinated increase in Serl 177 phosphorylation and Thr495 dephosphorylation. We have discovered that TGF-01 dephosphorylates eNOS
at Thr495, which is necessary to increase the Ca2+ sensitivity and enzyme activity and may work synergistically with VEGF, which activates eNOS by phosphorylating Serl 177.
Accordingly, any compound (e.g., polypeptide, nucleic acid molecule, small molecule compound, or antibody) that increases the level (e.g., by increasing stability, transcription or translation, or decreasing protein degradation) or biological activity of NOS, particularly eNOS, or any compound that prevents the downregulation of eNOS
activity is useful in the methods of the invention. Such compounds include purified NOS, preferably eNOS, or biologically active fragments thereof, nucleic acids encoding NOS, preferably eNOS, or biologically active fragments thereof, statins, vanadate, hepatocyte growth factor, phosphoinositide 3-kinase (P13K), Akt, VEGF, TGF-(3I, or any other compound that increases Ser1177 phosphorylation or Thr495 dephosphorylation or both. Nitric oxide is synthesized from L-arginine by nitric oxide synthase located in endothelial and other cells. Nitric oxide can also be generated by application of various nitric oxide donors such as sodium nitroprusside, nitroglycerin, SIN-1, isosorbid mononitrate, isosorbid dinitrate, and the like. Accordingly, compounds that increase (e.g., by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more) the level or biological activity of NOS can optionally be administered in combination with L-arginine ' or a nitric oxide donor (e.g., sodium nitroprusside, nitroglycerin, isosorbidmononitrate, and isosorbo dinitrate). NOS activity can be assayed by standard methods known in the art, including but not limited to the citrulline assay and other assays described in U.S.
Patent Application Publication No. 20050256199, the entire disclosure of which is herein incorporated by reference. The Thr495 residue of eNOS is located within the calmodulin (CaM)-binding domain of eNOS. Agonist-induced dephosphorylation of eNOS at Thr495 increases the binding of CaM to the enzyme (Fleming et al., Circ Res.
2001, 88:
E68-75), thereby increasing its calcium sensitivity and activation. In addition to TGF-Pl described herein, other agonists that have been shown to cause Thr495 dephosphorylation of eNOS including bradykinin, histamine and VEGF. Thr495 dephosphorylation can be enhanced by the protein kinase C (PKC) inhibitor Ro 31-8220 (Calbiochem) or after PKC
downregulation using phorbol 12-myristate 13-acetate (PMA) (Sigma Aldrich).
Moreover, agonist-induced dephosphorylation of Thr495 has been shown to be Ca2+/calmodulin-dependent and inhibitable by calyculin A (Sigma Aldrich), a protein phosphatase 1(PP1) inhibitor (Fleming I, et al. Cire Res. 2001, 88: E68-75).
Additional compounds that effect eNOS dephosphorylation at Thr495 include histamine and bradykinin (Sigma Aldrich).
Therapeutic compounds that increase the levels or biological activity of PGIZ
Prostacyclin is a member of the family of lipid molecules known as eicosanoids.
It is produced in endothelial cells from prostaglandin H2 (PGH2) by the action of the enzyme prostacyclin synthase. PG12 biological activity includes inhibition of platelet aggregation, relaxation of smooth muscle, reduction of systemic and pulmonary vascular resistance by direct vasodilation, and natriuresis in kidney.
PG12 is an anti-thrombotic factor that is stimulated by both VEGF and TGF-01.
PGI2 biological activity includes inhibition of platelet aggregation and relaxation of vascular smooth muscle and assays for PGIz biological activity include any platelet aggregation assay or other PGI2 assay known in the art such as those described in Jakubowski et al., Prostaglandins 47:404(1994). The invention features the use of any compound that increases (e.g., by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more) the level or activity of PGI2, as measured by standard assays known in the art including but not limited to PGIa mimetics, iloprost, cicaprost, and aspirin. Additional compounds are known in the art and examples are described in U.S.P.N.
5,910,482, the entire disclosure of which is herein incorporated by reference.
Purffied proteins For any of the purified proteins, or fragment thereof, the proteins are prepared using standard methods known in the art. Analogs or homologs of any of the therapeutic proteins described above are also included and can be constructed, for example, by making various substitutions of residues or sequences, deleting terminal or internal residues or sequences not needed for biological activity, or adding terminal or internal residues which may enhance biological activity. Amino acid substitutions, deletions, additions, or mutations can be made to improve expression, stability, or solubility of the protein in the various expression systems. Generally, substitutions are made conservatively and take into consideration the effect on biological activity.
Mutations, deletions, or additions in nucleotide sequences constructed for expression of analog proteins or fragments thereof must, of course, preserve the reading frame of the coding sequences and preferably will not create complementary regions that could hybridize to produce secondary mRNA structures such as loops or hairpins which would adversely affect translation of the mRNA.
Any of the therapeutic compounds of the invention (e.g., polypeptide, antibodies;
small molecule compounds) can also include any modified forms. Examples of post-.
translational modifications include but are not limited to phosphorylation, glycosylation, hydroxylation, sulfation, acetylation, isoprenylation, proline isomerization, subunit dimerization or multimerization, and cross-linking or attachment to any other proteins, or fragments thereof, or membrane components, or fragments thereof (e.g., cleavage of the protein from the membrane with a membrane lipid component attached).
Modifications that provide additional advantages such as increased affinity, decreased off-rate, solubility, stability and in vivo or in vitro circulating time of the polypeptide, or decreased immunogenicity and include, for example, acetylation, acylation, ADP-ribosylation, arnidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. (See, for instance, Creighton, "Proteins:
Structures and Molecular Properties," 2d Ed., W. H. Freeman and Co.,.N.Y., 1992;
"Postranslational Covalent Modification of Proteins," Johnson, ed., Academic Press, New York, 1983;
Seifter et al., Meth. Ena,y zol., 182:626-646, 1990; Rattan et al., Ann.
.NYAcad. Sci., 663:48-62, 1992) are also included. The peptidyl therapeutic compound of the invention can also include sequence variants of any of the compounds such as variants that include 1, 2, 3, 4, 5, greater than 5, or greater than 10 amino acid alterations such as substitutions, deletions, or insertions with respect to wild type sequence. Additionally, the therapeutic compound of the invention may contain one or more non-classical amino acids.
Non-classical amino acids include, but are not limited to, to the D-isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, omithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, P-alanine, fluoro-amino acids, designer amino acids such as (3-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs in general. Furthermore, the amino acid can be D (dextrorotary) or L
(levorotary).
Additional post-translational modifications encompassed by the invention include, for example, e.g., N-linked or 0-linked carbohydrate chains, processing of N-terminal or C-terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or 0-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue.
In addition, chemically modified derivatives of the therapeutic compounds described herein, which may provide additional advantages such as increased solubility, stability and circulating time of the polypeptide, or decreased immunogenicity (see U.S.
Pat. No. 4,179,337) are also included. The chemical moieties for derivitization may be selected from water soluble polymers such as, for example, polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like. The compound may be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.
The polymer may be of any molecular weight, and may be branched or unbranched. For polyethylene glycol, the preferred molecular weight is between about I
kDa and about 100 kDa (the term "about" indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing. Other sizes may be used, depending on the desired therapeutic profile (e_g., the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects of the polyethylene glycol to a therapeutic protein or analog).
As noted above, the polyethylene glycol may have a branched structure. Branched polyethylene glycols are described, for example, in U.S. Pat. No. 5,643,575; Morpurgo et al., Appl.
Biochem. Biotechnol. 56:59-72, (1996); Vorobjev et al., Nucleosides Nucleotides 18:2745-2750, (1999); and Caliceti et al., Bioconjug. Chem. 10:638-646, (1999), the disclosures of each of which are incorporated by reference.
Any of the therapeutic compounds of the present invention (e.g., polypeptide, antibodies, or small molecule compounds) may also be modified in a way to form a chimeric molecule comprising the therapeutic compound fused to another, heterologous polypeptide or amino acid sequence, such as an Fe sequence, a detectable label, or an additional therapeutic molecule. In one example, an anti-soluble endoglin antibody can be a peptide fused to an Fc fusion protein.
For any of the polypeptides, including antibodies, that are used in the methods of the invention, the nucleic acids encoding the polypeptides or antibodies, or fragments thereof, are also useful in the methods of the invention using standard techniques for gene therapy known in the art and described herein. The invention also includes mimetics, based on modeling the 3-dimensional structure of a polypeptide or peptide fragment and using rational drug design to provide potential inhibitor compounds with particular molecular shape, size and charge characteristics. Following identification of a therapeutic compound, suitable modeling techniques known in the art can be used to study the functional interactions and design mimetic compounds which contain functional groups arranged in such a manner that they could reproduced those interactions. The designing of mimetics to a known pharmaceutically active compound is a known approach to the development of pharmaceuticals based on a lead compound. This might be desirable where the active compound is difficult or expensive to synthesize or where it is unsuitable for a particular method of administration, e.g. peptides are not well suited as active agents for oral compositions as they tend to be quickly degraded by proteases in the alimentary canal. Mimetic design, synthesis and testing may be used to avoid randomly screening large number of molecules for a target property. The mimetic or mimetics can then be screened to see whether they reduce or inhibit soluble endoglin levels or biological activity and further optimization or modification can then be carried out to anive at one or more final mimetics for in vivo or clinical testing.
Therapeutic nucleic acids Recent work has shown that the delivery of nucleic acid (DNA or RNA) capable of expressing an endothelial cell mitogen such as VEGF to the site of a blood vessel injury will induce proliferation and reendothelialization of the injured vessel. While the present invention does not relate to blood vessel injury, these general techniques for the delivery of nucleic acid to endothelial cells can be used in the present invention for the delivery of nucleic acids encoding soluble endoglin binding proteins, such as TGF-01, TGF-03, activin-A, BMP2 and BMP7, or eNOS. The techniques can also be used for the delivery of nucleic acids encoding proteins, such as those described above, known to inhibit the activity of any AZNII', cathepsin, or elastase involved in the cleavage and release of soluble endoglin, for the treatment or prevention of pre-eclampsia or eclampsia in a subject. These general techniques are described in U.S. Patent Nos.
5,830,879 and 6,258,787 and are incorporated herein by reference.
In the present invention the nucleic acid may be any nucleic acid (DNA or RNA) including genomic DNA, eDNA, and mRNA, encoding a soluble endoglin binding proteins such as TGF-(31, TGF-03, activin-A, B114P2 and BMP7, or eNOS. The nucleic acids encoding the desired protein may be obtained using routine procedures in the art, e.g. recombinant DNA, PCR amplification.
Modes for delivering nucleic acids For any of the nucleic acid applications described herein, standard methods for administering nucleic acids can be used. Examples are described in U.S. Patent Application Publication No. 20060067937 and PCT Publication No. WO 06/034507.
Therapeutic nucleic acids that inhibit soluble endoglin expression The present invention also features the use of antisense nucleobase oligomers to downregulate expression of soluble endoglin mRNA directly. By binding to the complementary nucleic acid sequence (the sense or coding strand), antisense nucleobase oligomers are able to inhibit protein expression presumably through the enzymatic cleavage of the RNA strand by RNAse H. Preferably the antisense nucleobase oligomer is capable of reducing soluble endoglin protein expression in a cell that expresses increased levels of soluble endoglin. Preferably the decrease in soluble endoglin protein expression is at least 10% relative to cells treated with a control oligonucleotide, preferably 20% or greater, more preferably 40%, 50%, 60%, 70%, 80%, 90% or-greater.
Methods for selecting and preparing antisense nucleobase oligomers are well known in the art. For an example of the use of antisense nucleobase oligomers to downregulate VEGF expression see U.S. Patent No. 6,410,322, incorporated herein by reference.
Methods for assaying levels of protein expression are also well known in the art and include western blotting, immunoprecipitation, and ELISA.
The present invention also features the use of RNA interference (RNAi) to inhibit expression of soluble endoglin. RNA interference (RNAi) is a recently discovered mechanism of post-transcriptional gene silencing (PTGS) in which double-stranded RNA
(dsRNA) corresponding to a gene or mRNA of interest is introduced into an organism resulting in the degradation of the corresponding mRNA_ In the RNAi reaction, both the sense and anti-sense strands of a dsRNA molecule are processed into small RNA
fragments or segments ranging in length from 21 to 23 nucleotides (nt) and having 2-nucleotide 3' tails. Alternatively, synthetic dsRNAs, which are 21 to 23 nt in length and have 2-nucleotide 3' tails, can be synthesized, purified and used in the reaction. These 21 to 23 nt dsRNAs are known as "guide RNAs" or "short interfering RNAs"
(siRNAs).
The siRNA duplexes then bind to a nuclease complex composed of proteins that target and destroy endogenous mRNAs having homology to the siRNA within the complex. Although the identity of the proteins within the complex remains unclear, the function of the complex is to target the homologous mRNA molecule through base pairing interactions between one of the siRNA strands and the endogenous mRNA.
The mRNA is then cleaved approximately 12 nt from the 3' terminus of the siRNA and degraded. In this manner, specific genes can be targeted and degraded, thereby resulting in a loss of protein expression from the targeted gene. siRNAs can also be chemically synthesized or obtained from a company that chemically synthesizes siRNAs (e.g., Dharmacon Research Inc., Pharmacia, or ABI).
The specific requirements and modifications of dsRNA are described in PCT
Publication No. WO01/75164, and in U.S. Patent Application Publication No.
20060067937 and PCT Publication No. WO 06/034507, incorporated herein by reference.
Soluble endoglin based therapeutic compounds useful in earlypregnancy Inhibition of full-length endoglin signaling has been shown to enhance trophoblast invasiveness in villous explant cultures (Caniggia I et al, EndocrinoloSy, 1997, 138:4977-88). Soluble endoglin is therefore likely to enhance trophoblast invasiveness during early pregnancy. Accordingly, compositions that increase soluble endoglin levels early in pregnancy in a woman who does not have a pregnancy related hypertensive disorder or a predisposition to a pregnancy related hypertensive disorder may be beneficial for enhancing placentation. Examples of compositions that increase soluble endoglin levels include purified soluble endoglin polypeptides, soluble endoglin encoding nucleic acid molecules, and compounds or growth factors that increase the levels or biological activity of soluble endoglin.
Assays for gene and protein expression . The following methods can be used to evaluate protein or gene expression and determine efficacy for any of the above-mentioned methods for increasing soluble endoglin binding protein levels, or for decreasing soluble endoglin protein levels.
Blood serum from the subject is measured for levels of soluble endoglin, using methods such as ELISA, western blotting, or immunoassays using specific antibodies.
Blood serum from the subject can also be measured for levels of TGF-(31, TGF-(33, activin-A, BMP2, BMP7, or any protein ligand known to bind to soluble endoglin.
Methods used to measure serum levels of proteins include ELISA, western blotting, or immunoassays using specific antibodies. In addition, in vitro angiogenesis assays can be performed to determine if the subject's blood has converted from an anti-angiogenic state to a pro-angiogenic state. Such assays are described below in Example 4. A
result that is diagnostic of pre-eclampsia or eclampsia is considered an increase of at least 10%, 20%, preferably 30%, more preferably at least 40% or 50%, and most preferably at least 60%, 70%, 80%, 90% or more in the levels of soluble endoglin and a result indicating an improvement in the pre-eclampsia or eclampsia is a decrease of at least 10%, 20%, preferably 30%, more preferably at least 40% or 50%, and most preferably at least 60%, 70%, 80%, 90% or more in the levels of soluble endoglin. Alternatively or additionally, a result that is diagnostic of pre-eclampsia or eclampsia is considered a decrease of at least 10%, 20%, preferably 30%, more preferably at least 40% or 50%, and most preferably at least 60%, 70%, 80%, 90% or more in the levels of eNOS, PGI2, TGF-01, TGF-(33, activin-A, BMP2, BMP7, or any protein ligand known to bind to soluble endoglin and a result indicating an improvement in the pre-eclampsia or eclampsia is an increase of at least 10%, 20%, preferably 30%, more preferably at least 40% or 50%, and most preferably at least 60%, 70%, 80%, 90% or more in the levels of eNOS, PGTZ, TGF-P 1, TGF-(33, activin-A, BMP2, BMP7, or any protein ligand known to bind to soluble endoglin. A result indicating an improvement in the pre-eclampsia or eclampsia can also be considered conversion by at least 10%, preferably 20%, 30%, 40%, 50%, and most preferably at least 60%, 70%, 80%, 90% or more from an anti-angiogenic state to a pro-angiogenic state using the in vitro angiogenesis assay.
Blood serum or urine samples from the subject can also be measured for levels of nucleic acids or polypeptides encoding eNOS, TGF-f31, TGF-03, activin-A, BNIP2, BMP7, or soluble endoglin. There are several art-known methods to assay for gene expression. Some examples include the preparation of RNA from the blood samples of the subject and the use of the RNA for northern blotting, PCR based amplification, or RNAse protection assays. A positive result is considered an increase of at least 10%, 20%, preferably 30%, more preferably at least 40% or 50%, and most preferably at least 60%, 70%, 80%, 90% or more in the levels of soluble endoglin, TGF-0 1, TGF-03, activin-A, BMP2, BMP7 nucleic acids.
Therapeutic antibodies The elevated levels of soluble endoglin found in the serum samples taken from pregnant women suffering from.pre-eclampsia suggests that soluble endoglin is acting as a "physiologic sink" to bind to and deplete the trophoblast cells and maternal endothelial cells of functional growth factors required for the proper development and angiogenesis of the fetus or the placenta. The use of compounds, such as antibodies, to bind to soluble endoglin and neutralize the activity of soluble endoglin (e.g., binding to TGF-(31, TGF-f33, activin-A, BMP2, BMP7), may help prevent or treat pre-eclampsia or eclampsia, by producing an increase in free TGF-(31, TGF-(33, activin-A, BMP2, and BMP7.
Such an increase would allow for an increase in trophoblast proliferation, migration and angiogenesis required for placental development and fetal nourishment, and for systemic maternal endothelial cell health.
The present invention provides antibodies that specifically bind to soluble endoglin. Preferably, the antibodies bind to the extracellular domain of endoglin or to the ligand binding domain. The antibodies are used to neutralize the activity of soluble endoglin and the most effective mechanism is believed to be through direct blocking of the binding sites for TGF-(31, TGF-(33, activin-A, BMP2, or BMP7, however, other mechanisms cannot be ruled out. Preferred antibodies can bind to an epitope (either as a result of linear structure or three dimensional conformation) on human endoglin that includes any one or more of the peptide sequences indicated in bold and underlined in FIGRUE 30B (e.g., amino acids 40 to 86, 144 to 199, 206 to 222, 289 to 304, or 375 to 381) or to any of the preferred fragments of soluble endoglin (e.g., amino acids 1 to 437, 4 to 437, 40 to 406, or 1 to 587 of human endoglin). Methods for the preparation and use of antibodies for therapeutic purposes are described in several patents including U.S.
Patent Numbers 6,054,297; 5,821,337; 6,365,157; and 6,165,464; U.S. Patent Application Publication No. 2006/0067937; and PCT Publication No. WO 06/034507 and are incorporated herein by reference. Antibodies can be polyclonal or monoclonal;
monoclonal humanized antibodies are preferred. The present invention also includes the antibodies that bind to soluble endoglin, including but not limited to those that bind to any one or more of the peptide sequences indicated in bold and underlined in FIGRUE
30B or to any of the preferred fragments of soluble endoglin (e.g., amino acids I to 437, 4 to 437, 40 to 406, or I to 587 of human endoglin).
Therapeutic uses of antibodies When used in vivo for the treatment or prevention of pre-eclampsia or eclampsia, the antibodies of the subject invention are administered to the subject in therapeutically effective amounts. Preferably, the antibodies are administered parenterally or intravenously by continuous infusion. The dose and dosage regimen depends upon the severity of the disease, and the overall health of the subject. The amount of antibody administered is typically in the range of about 0.001 to about 10 mg/kg of subject weight, preferably 0.01 to about 5 mg/kg of subject weight.
For parenteral administration, the antibodies are formulated in a unit dosage injectable form (solution, suspension, emulsion) in association with a pharmaceutically, acceptable parenteral vehicle. Such vehicles are inherently nontoxic, and non-therapeutic. Examples of such vehicles are water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Nonaqueous vehicles such as fixed oils and ethyl oleate may also be used. Liposomes may be used as carriers. The vehicle may contain minor amounts of additives such as substances that enhance isotonicity and chemical stability, e.g., buffers and preservatives. The antibodies typically are formulated in such vehicles at concentrations of about 1 mg/ml to 10 mg/mI.
Combination therapies Optionally, a therapeutic may be administered in combination with any other standard pre-eclampsia or eclampsia therapy; such methods are known to the skilled artisan and include the methods described in U.S. Patent Application Publication Numbers 20040126828, 20050025762, 20050170444, 20060067937, and 20070104707 and PCT Publication Numbers WO 2004/008946, WO 2005/077007, and WO
06/034507.
Desirably, the invention features the use of a combination of any one or more of the therapeutic agents described herein. Given our discovery that soluble endoglin and sFlt-1 may act in concert to induce vascular damage and pregnancy related hypertensive disorders by interfering with TGF-Pl and VEGF signaling pathway respectively, possibly converging on the NOS signaling pathway, desirable therapeutic methods of the invention include the administration of a compound that decrease sFlt-1 levels or activity or increase VEGF or PIGF levels or activity in combination with a compound that decreases soluble endoglin levels or activity or increase TGF-P, NOS, or PG12 levels or activity. It will be understood by the skilled artisan that any combination of any of the agents can be used for this purpose. For example, an antibody that specifically binds to soluble endoglin can be administered in combination with VEGF. In another example, a compound that increases TGF-01 levels or activity can be administered in combination with a compound that increases VEGF or PIGF in order to target both the endoglin and the VEGF pathway. Alternatively, a combination of antibodies against both soluble endoglin and sFlt-1 may be used either directly or in an ex vivo approach (e.g., using a column that is lined with anti-soluble endoglin or sFlt-1 and circulating the patient's blood through the column). Any of these combinations can further include the administration of a compound that increases NOS levels or activity, preferably eNOS, in order to regulate the pathway downstream of the respective receptors.
In addition, the invention provides for the use of any chronic hypertension medications used in combination with any of the therapeutic methods described herein.
Medications used for the treatment of hypertension during pregnancy include methyldopa, hydralazine hydrochloride, or labetalol. For each of these medications, modes of administration and dosages are determined by the physician and by the manufacturer's instructions.
Dosages and Modes of Administration Preferably, the therapeutic is administered either directly or using an ex vivo approach during pregnancy for the treatment or prevention of pre-eclampsia or eclampsia or after pregnancy to treat post-partum pre-eclampsia or eclampsia. Techniques and dosages for administration vary depending on the type of compound (e.g., chemical compound, purified protein, antibody, antisense, RNAi, or nucleic acid vector) and are well known to those skilled in the art or are readily determined.
Therapeutic compounds of the present invention may be administered with a pharmaceutically acceptable diluent, carrier, or excipient, in unit dosage form.
Administration may be parenteral, intravenous, subcutaneous, oral or local by direct injection into the amniotic fluid. Intravenous delivery by continuous infusion is the preferred method for administering the therapeutic compounds of the present invention.
The therapeutic compound may be in form of a solution, a suspension, an emulsion, an infusion device, or a delivery device for implantation, or it may be presented as a dry powder to be reconstituted with water or another suitable vehicle before use.
The composition can be in the form of a pill, tablet; capsule, liquid, or sustained release tablet for oral administration; or a liquid for intravenous, subcutaneous or parenteral administration; or a polymer or other sustained release vehicle for local administration.
Methods well known in the- art for making formulations are found, for example, in "Remington: The Science and Practice of Pharmacy" (20th ed., ed. A.R. Gennaro AR., 2000, Lippincott Williams & Wilkins, Philadelphia, PA). Formulations for parenteral administration may, for example, contain excipients, sterile water, saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes. Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds. Nanoparticulate formulations (e.g., biodegradable nanoparticles, solid lipid nanoparticles, liposomes) may be used to control the biodistribution of the compounds. Other potentially useful parenteral delivery systems include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. The concentration of the compound in the formulation varies depending upon a number of factors, including the.dosage of the drug to be administered, and the route of administration.
The compound may be optionally administered as a pharmaceutically acceptable salt, such as non-toxic acid addition salts or metal complexes that are commonly used in the pharmaceutical industry. Examples of acid addition salts include organic acids such as acetic, lactic, pamoic, =maleic; citric, malic, ascorbic, succinic, benzoic, palmitic, suberic, salicylic, tartaric, methanesulfonic, toluenesulfonic, or trifluoroacetic acids or the like; polymeric acids such as tannic acid, carboxymethyl cellulose, or the like; and inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid phosphoric acid, or the like. Metal complexes include zinc, iron, and the like.
Formulations for oral use include tablets containing the active ingredient(s) in a mixture with non-toxic pharmaceutically acceptable excipients. These excipients may be, for example, inert diluents or fillers (e.g., sucrose and sorbitol), lubricating agents, glidants, and anti-adhesives (e.g., magnesium stearate, zinc stearate, stearic acid, silicas, hydrogenated vegetable oils, or talc).
Formulations for oral use may also be provided as chewable tablets, or as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium.
The dosage and the timing of administering the compound depends on various clinical factors including the overall health of the subject and the severity of the symptoms of pre-eclampsia. In general, once pre-eclampsia or a predisposition to pre-eclampsia is detected, continuous infusion of the purified protein is used to treat or prevent further progression of the condition. Treatment can be continued for a period of time ranging from 1 to 100 days, more preferably 1 to 60 days, and most preferably I to days, or until the completion of pregnancy. Dosages vary depending on each compound and the severity of the condition and are titrated to achieve a steady-state 20 blood serum concentration ranging from 10 to 20 ng/ml soluble endoglin;
and/or 1 to 500 pg/niL free VEGF or free P1GF, or both, preferably 1 to 100 pg/xnL, more preferably 5 to 50 pg/mL and most preferably 5 to 10 pg/mL VEGF or PIGF, or 1-5 ng of sFlt-1.
The diagnostic methods described herein can be used to monitor the pre-eclarnpsia or eclampsia during therapy or to determine the dosages of therapeutic compounds. In one example, a therapeutic compound is administered and the PAAI
is determined during the course of therapy. If the PAAI is less than 20, preferably less than 10, then the therapeutic dosage is considered to be an effective dosage. In another example, a therapeutic compound is administered and the soluble endoglin anti-angiogenic index is determined during the course of therapy. If the soluble endoglin anti-angiogenic index is less than 200, preferably less than 100, then the therapeutic dosage is considered to be an effective dosage.
Subject monitoring The disease state or treatment of a subject having pre-eclampsia, eclampsia, or a predisposition to such a condition can be monitored using the diagnostic methods, kits, and compositions of the invention. For example, the expression of a soluble endoglin polypeptide present in a bodily fluid, such as blood, serum, urine, plasma, amniotic fluid, or CSF, can be monitored. The soluble endoglin monitoring can be combined with methods for monitoring the expression of an sFlt-1, VEGF, or PIGF, TGF-0, or eNOS
polypeptide or nucleic acid, or PGI2. Such monitoring may be useful, for example, in assessing the efficacy of a particular drug in a subject or in assessing disease progression.
Therapeutics that decrease the expression or biological activity of a soluble endoglin nucleic acid molecule or polypeptide are taken as particularly useful in the invention.
Screening Assays As discussed above, the level of a soluble endoglin nucleic acid or polypeptide is increased in a subject having pre-eclampsia, eclampsia, or a predisposition to such conditions. Based on these discoveries, compositions of the invention are useful for the high-throughput low-cost screening of candidate compounds to identify those that modulate the expression of a soluble endoglin polypeptide or nucleic acid molecule whose expression is altered in a subject having a pre-eclampsia or eclampsia.
Any number of methods are available for carrying out screening assays to identify new candidate compounds that alter the expression of a soluble endoglin nucleic acid molecule. Examples are described in detail in U.S. Patent Application Publication No.
20060067937 and PCT Publication No. WO 06/034507.
In one working example, candidate compounds may be screened for those that specifically bind to a soluble endoglin polypeptide. The efficacy of such a candidate compound is dependent upon its ability to interact with such a polypeptide or a functional equivalent thereof. Such an interaction can be readily assayed using any number of standard binding techniques and functional assays such as immunoassays or affinity chromatography based assays(e.g., those described in Ausubel et al., supra).
In one embodiment, a soluble endoglin polypeptide is immobilized and compounds are tested for the ability to bind to the immobilized soluble endoglin using standard affinity chromatography based assays. Compounds that bind to the immobilized soluble endoglin can then be eluted and purified and tested further for its ability to bind to soluble endoglin both in vivo and in vitro or its ability to inhibit the biological activity of soluble endoglin.
In another example, a candidate compound is tested for its ability to decrease the biological activity of a soluble endoglin polypeptide by decreasing binding of a soluble endoglin polypeptide and a growth factor, such as TGF-P l, TGF-(33, activin-A, and BMP-7. These assays can be performed in vivo or in vitro and the biological activity of the soluble endoglin polypeptide can be assayed using any of the assays for any of the soluble endoglin activities known in the art or described herein. For example, cells can be incubated with a Smad2/3-dependent reporter construct. If desired, the cells can also be incubated in the presence of TGF-0 to enhance the signal on the Smad2/3 dependent reporter construct. The cells can then be incubated in the presence of soluble endoglin which will reduce or inhibit TGF-0-induced activation of the Smad2/3 dependent reporter construct. Candidate compounds can be added to the cell and any compound that results in an increase of TGF-0-induced activation of the Smad2/3 dependent reporter in the soluble endoglin treated cells as compared to cells not treated with the compound, is considered a compound that may be useful for the treatment of pre-eclampsia or eclampsia.
In another example, the TGF- j3-induced dephosphorylation of eNOS at Thr495 can also be used as an assay for changes in soluble endoglin biological activity. In this example, cells are incubated in the presence of soluble endoglin, which as shown in the experiments described below, inhibits the TGF-(31 dephosphorylation of Thr495 of eNOS. Candidate compounds are then added to the cells and the phosphorylation state of Thr495 is determined. Any compound that results in an increase of TGF-p-induced activation of Thr495 dephosphorylation in the soluble endoglin treated cells as compared to cells not treated with the compound, is considered a compound that may be useful for the treatment of pre-eclampsia or eclampsia.
Examples The following examples are intended to illustrate the invention. They are not meant to limit the invention in any way.
Example 1. Increased levels of endoglin mRNA and protein in pregnant women with pre-eclampsia.
In an attempt to identify novel secreted factors playing a pathologic role in pre-eclampsia, we performed gene expression profiling of placental tissue from 17 pregnant women with pre-eclampsia and 13 normal pregnant women using Affymetrix U95A
microarray chips. We found that the gene for endoglin was upregulated in women with pre-eclampsia.
In order to confirm the upregulation of endoglin in pre-eclampsia, we performed Northern blots to analyze the placental endoglin mRNA levels (FIGURE 3) and western blot analysis to measure serum protein levels of endoglin (FIGURE 4) in pre-eclamptic pregnant women as compared with normotensive pregnant women. Pre-eclampsia was defined as (1) a systolic blood pressure (BP) >140 mmHg and a diastolic BP >90 mmHg after 20 weeks gestation, (2) new onset proteinuria (1+ by dipstik on urinanalysis, >300mg of protein in a 24 hour urine collection, or random urine protein/creatinine ratio >0.3, and (3) resolution of hypertension and proteinuria by 12 weeks postpartum.
Patients with underlying hypertension, proteinuria, or renal disease were excluded.
Patients were divided into mild and severe pre-eclampsia based on the presence or absence of nephrotic range proteinuria (>3g of protein on a 24 hour urine collection or urine protein/creatinine ratio greater than 3.0). The mean urine protein/creatinine ratios in the mild pre-eclampsia group were 0.94 +/- 0.2 and in the severe pre-eclampsia group were 7.8 +/- 2.1. The mean gestational ages of the various groups were as follows:
normal 38.8 +/-0.2 weeks, mild pre-eclampsia 34 +/- 1.2 weeks, severe pre-eclampsia 31.3 +1-0.6 weeks, and pre-term. 29.5 +/- 2.0 weeks. Placental samples were obtained immediately after delivery. Four random samples were taken from each placenta, placed in RNAlater stabilization solution (Ambion, Austin, TX) and stored at -70 C.
RNA
isolation was performed using Qiagen RNAeasy Maxi Kit (Qiagen, Valencia, CA).
Northern blots probed with a 400 base pair probe in the coding region of endoglin (Unigene Hs.76753) corresponding to the N-terminal region (gene bank #BC014271) and an 18S probe as a normalization control showed an increase in placental endoglin mRNA
(see Knebelmann et al., Cancer Res. 58:226-231 (1998)). Western blots probed with an antibody to the amino terminus of endoglin showed an increase in both placental and maternal serum levels of endoglin protein in pre-eclamptic pregnant women as compared to normotensive pregnant women.
Example 2. Demonstration of a soluble endoglin polypeptide in the placentas and serum of pre-eclamptic patients.
The western blot analysis used to measure the levels of endoglin protein in placentas and serum from pre-eclamptic women suggested the presence of a smaller protein (approximately 63-65 kDa), that was present in the placenta and serum of pre-eclamptic pregnant women (FIGURES 4 and 30A). We have demonstrated that this smaller fragment is the extracellular domain of endoglin. This truncated version is likely to be shed from the placental syncitiotrophoblasts and endothelial cells and circulated in excess quantities in patients with pre-eclampsia. This soluble form of endoglin may be acting as an anti-angiogenic agent by binding to circulating ligands that are necessary for normal vascular health.
The predicted length of the soluble form of the protein is approximately 437 amino acids (including the peptide leader sequence, 412 amino acids without the leader sequence). sEng was purified from the serum of preeclamptic patients.
Fractions 4 and 5 eluted from the 44G4-IgG (anti-Eng) Sepharose, were run on SDS-PAGE under reducing conditions and tested by Western blot using a polyclonal antibody to Eng. The eluted fractions were subjected to mass spectrometry analysis (3 runs) and the peptides identified are shown in (FIGURE 30B). The purification and analysis by mass spectrometry revealed several Eng-specific peptides ranging from Gly4O to Arg406 indicating a soluble form (soluble endoglin) corresponding to the N-terminal region of the full-length protein bold on the sequence of human endoglin.
Example 3. Circulating concentrations of soluble endoglin in women with normal versus pre-eclamptic pregnancies.
In order to compare the levels of circulating, soluble endoglin from the serum of normal, mildly pre-eclamptic, or severely pre-eclamptic women, we performed ELISA
analysis on blood samples taken from these women. All the patients for this study were recruited at the Beth Israel Deaconess Medical Center after obtaining appropriate IRB-approved consents. Pre-eclampsia was defined as (1) Systolic BP >140 and diastolic BP
>90 after 20 weeks gestation in a previously normotensive patient, (2) new onset proteinuria (1+ by dipstick on urinanalysis or > 300 mg of protein in a 24 hr urine collection or random urine protein/creatinine ratio >0.3), and (3) resolution of hypertension and proteinuria by 12 weeks postparlum. Patients with baseline hypertension, proteinuria, or renal disease were excluded. For the purposes of this study, patients were divided into mild and severe pre-eclampsia based on the absence or presence of nephrotic-range proteinuria (> 3 g of protein on a 24 hour urine collection or urine protein to creatinine ratio greater than 3.0). HELLP syndrome was defined when patients had evidence of thrombocytopenia (<100000 cel.ls/ l), increased LDH
(>600 IU/L) and increased AST (>70 IU/L). Healthy pregnant women were included as controls. 8 patients with pre-term deliveries for other medical reasons were included as additional controls. Placental samples were obtained immediately after delivery. Serum was collected from pregnant patients at the time of delivery (0-12 hours prior to delivery of the placenta) after obtaining informed consent. These experiments were approved by ' the Institutional Review Board at the Beth Israel Deaconess Medical Center.
Using the serum specimens from patients described in Table 1, we measured the circulating concentrations of soluble endoglin in the various groups of pre-eclamptic patients and control pregnant patients. When pre-eclamptic patients were further sub-divided into those with and without HELLP, sEng concentrations were three-, five- and ten-fold higher in mild, severe and HELLP syndrome preeclamptics, respectively, compared to gestational age-matched pre-term controls (FIGURE 28).
Concentrations of sEng in pregnant patients correlated with those of sFltl (R2 = 0.56), except in the HELLP
group where sEng was higher than sFltl. In a subset of patients, blood samples obtained 48 hours after placental delivery showed a 70% reduction in mean sEng circulating levels in preeclamptic and normal pregnant patients (FIGURE 29).
Table 1: Clinical characteristics and circulating soluble endoglin in the various patient groups Mild pre- Severe pre- Severe pre-Normal eclampsia eclampsia, no eclampsia with Pre-term (n=30) (n=l 1) HELLP (n=17) HELLP (n=11) (n=8) Maternal age (yrs) 32.43 33.18 29.5 33.73 31.88 Gestational age (wks) 38.65 3].91* 29.06* 26.52* 30.99*
Primiparous (%) 43.3 63.6 47.1 90.9 62.5 Systolic blood pressure (mmHg) 122 157* 170* 166* 123 Diastolic blood pressure (mmHg) 72 99* 104* 103* 77 Proteinuria (g protein/g creatinine) 0.37 2.5* 8.64* 5.16* 0.6 Uric acid (mg/dl) 5.27 6.24 7.29* 6.31 7.35 Hematocrit (%) 35.5 33.6 33.7 33.5 34.3 Platelet count 238 230 249 69.4* 229 Creatinine (mg/dl) 0.55 0.62 0.62 0.64 0.67 Soluble endoglin in (ng/ml) 18.73 36.12* 52.55** 99.83*** 10.9 *P <0.05, **P<0.005 The average serum concentrations of soluble endoglin was at least two fold higher in mild pre-eclampsia and 3-4 fold higher in patients with severe pre-eclampsia. In pre-eclamptic patients complicated with the HELLP syndrome, the concentration of soluble endoglin was at least 5-10 fold higher than gestational age matched control specimens.
Additionally, the levels of soluble endoglin in pregnant patients correlate with the levels of sFlt-1 (FIGURE 18). The R2 value for correlation was 0.6. (Note that the circulating concentrations of sFlt-1 reported here are at least 4-5 fold higher than previously published (Maynard et al., supra). This is due-to a difference in the sensitivity of a new ELISA kit from R&D systems which lacks urea in the assay diluent and therefore gives consistently higher values than previously published.) In other words, patients with the highest levels of soluble endoglin also had the highest circulating levels of sFltl. The origin of soluble endoglin is most likely the syncitiotrophoblast of the placenta as evidenced by the enhanced staining seen on our placental immunohistochemistry (FIGURES 19 and 20). These figures show that endoglin protein is expressed by the syncitiotrophoblasts and is vastly upregulated in pre-eclampsia. Our western blot data (FIGLTRES 21A and 21B) and the lack of detectable alternative splice variants by northern blot supports the notion that soluble endoglin is likely a shed form of the extracellular domain of the membrane endoglin protein. It is approximately 65 kDA in size and is produced at elevated levels in pre-eclamptic placentas and it circulates in higher amounts in pre-eclamptic sera. This protein was present at much lower levels in the sera of normal pregnant women and barely detectable in non-pregnant women.
Soluble endoglin expression in pre-eclamptic placenta was four-fold higher than in normal pregnancy (n=l-/group, P<0.01). Quantitation of sEng/Eng in these specimens showed no significant difference between normal (0_43) and preeclamptic (0.56) placentae (n = 10/group, P = 0.4), suggesting that sEng is derived from the full-length protein and that both Eng and sEng are similarly increased in preeclampsia.
The following methods were used for some of the experiments described in this example.
Immunohistochemistry Immunohistochemistry on placental samples for endoglin and a-Smooth muscle actin (SMA) was done as reported by (Leach et al., Lancet 360:1215-1219 (2002)).
Briefly, the frozen placenta section obtained from patients without preeclampsia (n=10) and with preeclampsia (n=10) slides were incubated with a serum-free protein blocking solution (DAKO) for 30 minutes at room temperature and then with the primary antibody at room temperature (mouse monoclonal anti-Endoglin: 1:50 dilution; DAKO) for hours. The slides were then washed with phosphate buffered saline for 10 minutes. The secondary antibody, Rhodamine conjugated sheep anti-mouse IgG, 1:200 dilution (Biomeda) was applied for 1 hour. Sections were again washed with phosphate buffered saline and subsequently incubated with a 1:400 dilution of FITC-conjugated mouse anti-human SMA (Dako) for 30 minutes at room temperature. Immunoreactivity of Endoglin was reviewed using a SPOT advanced imaging system (RT SLIDER Diagnostic Instruments, Inc) by a pathologist who was blinded to the clinical diagnosis.
ELI,SA and Western blots ELISA was performed using a commercially available ELISA kit from R & D
Systems, MN (for example, Cat # DNDG00) and as previously described (Maynard et al, J. Clin. Invest. 111:649-658, 2003). Western blots were performed essentially as described previously (Maynard et al, supra, and Kuo et al. Proc. Natl. Acad.
,Sci.
98:4605-4610 (2001))).
Immunoprecipitation (IP) experiments IP followed by western blots were used to identify and characterize soluble endoglin in the placental tissue and serum specimens from patients with pre-eclampsia.
Human placental tissue was washed with cold PBS and lysed in homogenization buffer [10 mM Tris-HCI, pH 7.4; 15 mM NaCl; 60 mM.KCI; 1 mM EDTA; 0.1 mM EGTA;
0.5% Nonidet P-40; 5% sucrose; protease mixture from Roche (Indianapolis, IN)]
for 10 minutes. Placental lysates were then subjected to immunoprecipitation with an anti-human monoclonal mouse endoglin antibody (mAb P4A4, Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Immunoaffinity columns were prepared by the directional coupling of 3-5 mg of the purified antibody to 2m1 protein A-Sepharose using an immunopure IgG orientation kit (Pierce Chemical Co., Rockford, Illinois, USA) according to the manufacturer's instructions. Columns were then washed extensively with RIPA buffer containing protease mixture, and bound proteins were eluted with 0.1 mol/L glycine-HC1 buffer, pH 2.8. The eluent was collected in 0.5-m1 fractions containing 1 mol/L Tris-HCI buffer. Protein-containing fractions were pooled and concentrated 9- to 10-fold with CENTRICON Centrifugal Concentrator (Millipore Corp., Bedford, Massachusetts, USA). The immunoprecipitated samples were separated on a 4-12% gradient gel (Invitrogen) and proteins were transferred to polyvinylidene difluoride (PVDF) membranes. Endoglin protein was detected by western blots using polyclonal anti-human rabbit endoglin primary antibody (H-300, Santa Cruz Biotechnology, Inc., Santa Cruz, CA).
Purification of soluble endoglin and analysis by mass spectrometry Serum (10 ml) from preeclamptic patients was sequentially applied onto CM Aff -gel blue and protein A Sepharose (Bio-Rad) columns to remove albumin and immunoglobulins, respectively. The flow through was slowly applied to a 2.5 ml column of mAb 44G4 IgG to human Eng, conjugated to Sepharose (Gougos et al., Int, Immuno.
4:83-92, (1992)). Bound fractions were eluted with 0.02 M diethylamine pH 11.4 and immediately neutralized with I M Tris pH 7.8. Fractions 4 and 5 with elevated absorbance at 280 nm were pooled, reduced with 10 mM DTT for 1 h at 57 C and alkylated with 0.055 M iodoacetomide. The samples were then completely digested with trypsin (1:100). The lyophilized sample was resuspended in 0.1% tri-fluoroacetic acid and injected in a CapLC (Waters) HPLC instrument. Peptides were separated using a 75 pm Nano Series column (LC Packings) and analyzed using a Qstar XL MS/MS
system.
The data was searched using the Mascot search engine (Matrix Science) against the human protein database, NCBInr.
Example 4. Model assay for angiogenesis An endothelial tube assay can be used an in vitro model of angiogenesis.
Growth factor reduced Matrigel (7 mg/mL, Collaborative Biomedical Products, Bedford, MA) is placed in wells (100 pl/well) of a pre-chilled 48-well cell culture plate and is incubated at 37 C for 25-30 minutes to allow polymerization. Human umbilical vein endothelial cells (30,000 + in 300 l of endothelial basal medium with no serum, Clonetics, Walkersville, MD) at passages 3-5 are treated with 10% patient serum, plated onto the Matrigel coated wells, and are incubated at 37 C for 12-16 hours. Tube formation is then assessed through an inverted phase contrast microscope at 4X (Nikon Corporation, Tokyo, Japan) and is analyzed (tube area and total length) using the Simple PCI imaging analysis software.
Example S. Soluble endoglin protein levels as a diagnostic indicator of pre-eclampsia and eclampsia in women (Romero Study).
This study was designed to evaluate whether soluble endoglin is altered during clinical pre-eclampsia and whether it can be used to predict pre-eclampsia and eclampsia in women.
This study was done under collaboration with Dr. Roberto Romero, at the Wayne State University/NICHD Perinatology Branch, Detroit, MI. A retrospective longitudinal case-control study was conducted using a banked biological sample database as previously described in Chaiworapongsa et al. (The Journal of Matemal-Fetal and Neonatal Medicine, January 2005, 17 (1):3-18). All women were enrolled in the prenatal clinic at the Sotero del Rio Hospital, Santiago, Chile, and followed until delivery.
Prenatal visits were scheduled at 4-week intervals in the first and second trimester, and every two weeks in the third trimester until delivery. Plasma samples were selected from each patient only once for each of the following six intervals: (1) 7-16 weeks, (2) 16-24 weeks, (3) 24-28 weeks, (4) 28-32 weeks, (5) 32-37 weeks, and (6) >37 weeks of gestation. For each pre-eclamptic case, one control was selected by matching for gestational age (+/- 2 weeks) at the time of clinical diagnosis of pre-eclampsia. The clinical criteria for the diagnosis of,pre-eclampsia were the same as previously described in Chaiworapongsa et al, supra.
Measurement of Plasma Endoglin Levels The plasma samples stored at -70 C were thawed and plasma soluble endoglin levels were measured in one batch using the commercially available ELISA kits from R&D systems, Minneapolis, MN.(Catalog # DNDG00).
Stastistical Analysis Analysis of covariance was used to assess the difference in plasma concentrations of soluble endoglin between patients destined to develop pre-eclampsia and in normal pregnancy after adjusting for gestational age at blood sampling and intervals of sample storage. Chi-square or Fisher's exact tests were employed for comparisons of proportions. The statistics package used was SPSS V.12 (SPSS Inc., Chicago, IL).
Significance was assumed for a p value of less than 0.05.
Results The clinical characteristics of the study population are described in Table 2.
The group with pre-eclampsia included more nulliparous women and delivered earlier than the control group. Importantly, the birth weights of the fetuses were smaller in the pre-eclamptic group and there were a higher proportion of women carrying small-for-gestational-age (SGA) infants.
Table 2. Clinical characteristics of the study population Normal Pre-eclampsia p pregnancy n = 44 n=44 Age (y) 29t6 26:L 6 0.04*
N=ulliparity 11 30 <0.001 (25%) (68.2%) *
Smoking 10 1 0.007*
(22.7%) (2.3%) GA at delivery (weeks) 39.7=1: 1.1 36.9-+2.7 <0.00 1 *
Birthweight (grams) 3,372 383 2,710 766 <0.001 *
Birthweight <10`'' percentile 0 16 <0.001 (36.4 %) *
Value expressed as mean sd or number (percent) GA: gestational age The clinical characteristics of patients with pre-eclampsia are described in Table 3. Thirty-two (72%) of the patients had severe pre-eclampsia, while 10 patients had severe early-onset pre-eclampsia defined as onset <34 weeks.
Table 3. Clinical characteristics of patients with pre-eclampsia Blood pressure (mmHg) Systolic 155 15 Diastolic 100 f 8 Mean arterial pressure 118 9 Proteinuria (dipstick) 3 0.8 Aspartate aminotransferase" (SGOT) (U/L) 29 31 Platelet counta (x 103) ( /L) 206 t 59 Severe pre-eclampsia 32 (72.7%) GA at pre-eclampsia diagnosed <34 weeks 10 (22.7%) GA at pre-eclampsia diagnosed >37 weeks 27 (61.4%) Value expressed as mean sd or number (percent) C (n =26); a (n=42) The serum soluble endoglin levels in the controls and the pre-eclamptic women measured in the 6 gestational age windows are shown in Table 4. Amongst the pre-eclamptics, their specimens were divided into two groups - clinical pre-eclampsia (samples taken at the time of symptoms of pre-eclampsia) and preclinical pre-eclampsia (samples taken prior to clinical symptoms). The data shows that at mid-pregnancy (24-28 weeks of gestation), serum soluble endoglin concentrations start rising in women destined t`o develop pre-eclampsia and become at least 3 fold higher than conttols by 28-32 weeks of gestation. Blood samples taken from women with clinical pre-eclampsia show a very dramatic (nearly 10-15 fold) elevation when compared to gestational age matched controls.
Table 4. Plasma soluble endoglin concentrations in normal pregnancy and pre-eclampsia Normal p Pre-clinical p Clinical samples pregnancy samples Pre-eclampsia Pre-eclampsia 151 blood sampling (7.1-16 weeks) -'"
Soluble Endoglin (ng/mi) 3.89 t.928 0.9 3.96 :~ 1.28 Gestational age (weeks) 12.3 f 2.2 0.2 11.6 2.4 Range 8.4-15.9 7.7 - 15.1 n=37 n=34 2nd blood sampling (16.1-24 weeks) Soluble Endoglin (ng/ml) 3.36 4:1.11 0.1 3.79 + 1.37 Gestational age (weeks) = 19.4 f 1.7 0.06 20.2 t 2.1 Range 16.3 -23.4 16.7 -24.0 n=44 n=36 3'a blood sampling(24.1-28 weeks) Soluble Endoglin (ng/ml) 3.18 -L .729 0.009* 5.27 t 4.12 Gestational age (weeks) 25.9 f 1.3 0.2 26.4 +1.1 Range 24.1 -28.0 24.6 -28.0 n=38 n=29 4s` blood sampling (28.1-32 weeks) Soluble Endoglin (ng/ml) 3.7 zE 1.1 <0.001 * 10.2 :E9.8 0.01 * 96.1 t 25.7 0.05 Gestationat age (weeks) 29.9 f 1.1 1.0 30.2 ;1z 1.0 1.0 30.4 t 1.4 1.0 Range 28.3-32.0 28.7 -32.0 29.4 -31.4 n=42 n=33 n=2a 5s' blood sampling (32.1-36.9 weeks 5.79 2.42 0.003* 10.5116,59 <0.001 43.14 f 25.6 <0.001 *
Soluble Endoglin (ng/ml) 34.7 f 1.3 1.0 34.8 t 1.5 1.0 34.5 ~ 1.2 1.0 Gestational age (weeks) 32.4 -36.6 32.6 -36.7 32.6 -36.6 Range n=37 n=20 n=13 6'h blood samnling (?=37weeks) Soluble Endoglin (ng/ml) 8.9 zi: 4.5 -- 15.23 ~ 10.61 0.006*
Gestational age (weeks) 39.4 t 1.0 38.8 + 1.1 0.05 Range 37.0 - 40.7 37.6 - 41.4 n=27 n=27 pa :compared between samples at clinical manifestation of pre-eclarnpsia and normal pregnancy Value expressed as mean sd 2 pre-eclamptic patients had no blood samples available at clinical manifestation To examine the relationship between plasma soluble endoglin concentrations and the interval to clinical diagnosis of pre-eclampsia, plasma samples of pre-eclamptic patients at different gestational ages were stratified according to the interval from blood sampling to clinical diagnosis into five groups: (1) at clinical diagnosis, (2) 2-5.9 weeks before clinical manifestation, (3) 6-10.9 weeks before clinical.manifestation, (4) 11-15.9 weeks before clinical manifestation, and (5) 16-25 weeks before clinical manifestation.
The data shown in Table 5 demonstrates that the plasma soluble endoglin levels start going up at 6-10.9 weeks before onset of symptoms in pre-eclamptics and are at least 3 fold higher at 2-5.9 weeks before symptoms in women destined to develop pre-eclampsia.
Table 5. Plasma soluble endoglin concentrations in normal and pre-eclamptic pregnant women.
Blood sampling Normal pregnancy Pre-eclampsia p At clinical manifestation Soluble Endoglin (ng/ml) 7.63 ~ 4.22 27.72 + 26.20 <0.001 *
Gestational age (weeks) 37.2 ~ 3.0 37.1:h 2.7 0.9 Range 28.9 -40.7 29.4 -- 41.4 n=42 n=42 S
2-5.9 weeks before clinical manifestation 4.67 2.32 15.07 + 10.15 <0.001*
Soluble Endoglin (ng/ml) 31.6 =L 3.8 32.8 f 2.8 0.2 Gestational age (weeks) 24.1 -36.3 27.1- 36.7 Range n=27 n=27 3.8 + 1.1 Interval before clinical manifestation (weeks) 6-10.9 weeks before clinical manifestation 3.61 -+1.05 5.89 3.07 <0.001*
Soluble Endoglin (ng/ml) 28.5 +2.9 28.5 =L 2.9 0.9 Gestational age (weeks) 19.7-32.6 19.6 -34.4 Range n=37 n=37 8.3 -+1.4 Interval before clinical manifestation (weeks)
By "soluble endoglin anti-angiogenic index" is meant the ratio of (sFlt-1 +
0.25 soluble endoglin)/PIGF. For example, a value of 75, or higher, preferably 100 or higher, or more preferably 200 or higher is indicative of a pregnancy complication associated with hypertension, such as pre-eclampsia or eclampsia.
By "operably linked" is meant that a gene and a regulatory sequence(s) are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequence(s).
By "pharmaceutically acceptable carrier ' is meant a carrier that is physiologically acceptable to the treated mammal while retaining the therapeutic properties of the compound with which it is administered. One exemplary pharmaceutically acceptable carrier substance is physiological saline. Other physiologically acceptable carriers and their formulations are known to one skilled in the art and described, for example, in Remington's Pharmaceutical Sciences, (20th edition), ed. A. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, PA.
By "placental growth factor (PIGF)" is meant a mammalian growth factor that is homologous to the protein defined by GenBank accession number P49763 and that has PIGF biological activity. P1GF is a glycosylated homodimer belonging to the VEGF
family and can be found in two distinct isoforms through alternative splicing mechanisms. PIGF is expressed by cyto- and syncytiotrophoblasts in the placenta and P1GF biological activities include induction of proliferation, migration, and activation of endothelial cells, particularly trophoblast cells.
By "polymorphism" is meant a genetic variation, mutation, deletion or addition in a soluble endoglin, sFlt-1, P1GF, or VEGF nucleic acid molecule that is indicative of a predisposition to develop pre-eclampsia or eclampsia. Such polymorphisms are known to the skilled artisan and are described, for example, by Raab et al. (Bzochem.
J. 339:579-588, 1999) and Parry et al. (Eur. Jlmmunogenet. 26:321-323, 1999). A
polymorphism may be present in the promoter sequence, an open reading frame, intronic sequence, or untranslated 3' region of a gene. Known examples of such polymorphisms in the endoglin gene include a 6 base insertion of GGGGGA in intron 7 at 26 bases beyond the 3' end of exon 7 (Ann. Neurol. 41:683-6, 1997).
By "pregnancy related hypertensive disorder" is meant any condition or disease or pregnancy that is associated with or characterized by an increase in blood pressure.
Included among these conditions are pre-eclampsia (including premature pre-eclampsia, severe pre-eclampsia), eclampsia, gestational hypertension, HELLP syndrome, (hemolysis, elevated liver enzymes, low platelets), abruption placenta, chronic hypertension, pregnancy with intra uterine growth restriction, and pregnancy with a small for gestational age (SGA) infant. It should be noted that although pregnancy with a SGA
infant is not often associated with hypertension, it is included in this definition.
By "pre-eclampsia" is meant the multi-system disorder that is characterized by hypertension with proteinuria or edema, or both, glomerular dysfunction, brain edema, liver edema, or coagulation abnormalities due to pregnancy or the influence of a recent pregnancy. All forms of pre-eclampsia, such as premature, mild, moderate, and severe pre-eclampsia are included in this definition. Pre-eclampsia generally occurs after the 20th week of gestation. Pre-eclampsia is generally defined as some combination of the following symptoms: (1) a systolic blood pressure (BP) >140 mmHg and a diastolic BP
>90 mmHg after 20 weeks gestation (generally measured on two occasions, 4-168 hours apart), (2) new onset proteinuria (l+ by dipstik on urinaiysis, > 300mg of protein in a 24-hour urine collection, or a single random urine sample having a protein/creatinine ratio >0.3), and (3) resolution of hypertension and proteinuria by 12 weeks postpartum.
Severe pre-eclampsia is generally defined as (1) a diastolic BP > 110 mmHg (generally measured on two occasions, 4-168 hours apart) or (2) proteinuria characterized by a measurement of 3.5 grams or more protein in a 24-hour urine collection or two random urine specimens with at least 3+ protein by dipstick. In pre-eclampsia, hypertension and proteinuria generally occur within seven days of each other. In severe pre-eclampsia, severe hypertension, severe proteinuria and HELLP syndrome (hemolysis, elevated liver enzymes, low platelets) or eclampsia can occur simultaneously or only one symptom at a time. HELLP syndrome is characterized by evidence of thrombocytopenia (<100000 cells/ l), increased LDH (>600 IU/L) and increased AST (>70 IU/L).
Occasionally, severe pre-eclampsia can lead to the development of seizures. This severe form of the syndrome is referred to as "eclampsia." Eclampsia can also include dysfunction or damage to several organs or tissues such as the liver (e.g., hepatocellular damage, periportal necrosis) and the central nervous system (e.g., cerebral edema and cerebral hemorrhage). The etiology of the seizures is thought to be secondary to the development of cerebral edema and focal spasm of small blood vessels in the kidney.
By "premature pre-eclampsia" is meant pre-eclampsia with onset of symptoms <37 weeks or <34 weeks.
By "prostacyclin" or "PGI2" is meant a member of the family of lipid molecules known as eicosanoids. It is produced in endothelial cells from prostaglandin H2 (PGH2) by the action of the enzyme prostacyclin synthase and is mainly synthesized by vascular endothelium and smooth muscle. PGI2 biological activity includes inhibition of platelet aggregation, relaxation of smooth muscle, reduction of systemic and pulmonary vascular resistance by direct vasodilation, and natriuresis in kidney.
By "protein" or "polypeptide" or "polypeptide fragment" is meant any chain of more than two amino acids, regardless of post-translational modification (e.g., glycosylation or phosphorylation), constituting all or part of a naturally occurring polypeptide or peptide, or constituting a non-naturally occurring polypeptide or peptide.
By "reference sample"'is meant any sample, standard, or level that is used for comparison purposes. A"nonnal reference sample" can be a prior sample taken from the same subject, a sample from a pregnant subject not having a pregnancy related hypertensive disorder, such as pre-eclampsia or eclampsia, a subject that is pregnant but the sample was taken early in pregnancy (e.g., in the first or second trimester or before the detection of a pregnancy related hypertensive disorder, such as pre-eclampsia or eclampsia), a subject that is pregnant and has no history of a pregnancy related hypertensive disorder, such as pre-eclampsia or eclampsia, a subject that is not pregnant, a sample of a purified reference polypeptide at a known normal concentration (i.e., not indicative of a pregnancy related hypertensive disorder, such as pre-eclampsia or eclampsia). By "reference standard or level" is meant a value or number derived from a reference sample. A normal reference standard or level can be a value or number derived from a normal subject that is matched to the sample subject by at least one of the following criteria: gestational age of the fetus, maternal age, matemal blood pressure prior to pregnancy, maternal blood pressure during pregnancy, BMI of the mother, weight of the fetus, prior diagnosis of pre-eclampsia or eclampsia, and a family history of pre-eclampsia or eclampsia. A "positive reference" sample, standard or value is a sample or value or number derived from a subject that is known to have a pregnancy related hypertensive disorder, such as pre-eclampsia or eclampsia, that is matched to the sample subject by at least one of the following criteria: gestational age of the fetus, maternal age, maternal blood pressure prior to pregnancy, maternal blood pressure during pregnancy, BMl of the mother, weight of the fetus, prior diagnosis of a pregnancy related hypertensive disorder, and a family history of a pregnancy related hypertensive disorder By "reduce or inhibit" is meant the ability to cause an overall decrease preferably of 20% or greater, more preferably of 40%, 50%, 60%, 70%, 80%, 90% or greater change in the level of protein or nucleic acid, detected by the aforementioned assays (see "expression"), as compared to an untreated sample By "sample" is meant a tissue biopsy, cell, bodily fluid (e.g., blood, serum, plasma, urine, saliva, amniotic fluid, or cerebrospinal fluid) or other specimen obtained from a subject. Desirably, the biological sample includes soluble endoglin nucleic acid molecules or polypeptides or both.
By "small interfering RNAs (siRNAs)" is meant an isolated dsRNA molecule, preferably greater than 10 nucleotides (nt) in length, more preferably greater than 15 nucleotides in length, and most preferably greater than 19 nucleotides in length that is used to identify the target gene or rnRNA to be degraded. A range of 19-25 nucleotides is the most preferred size for siRNAs. siRNAs can also include short hairpin RNAs in which both strands of an siRNA duplex are included within a single RNA
molecule.
siRNA includes any form of dsRNA (proteolytically cleaved products of larger dsRNA, partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA) as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and/or alteration of one or more nucleotides. Such alterations can include the addition of non-nucleotide material, such as to the end(s) of the 19, 20, 21, 22, 23, 24, or 25 nt RNA or internally (at one or more nucleotides of the RNA). In a preferred embodiment, the RNA molecules contain a 3' hydroxyl group.
Nucleotides in the RNA molecules of the present invention can also comprise non-standard nucleotides, including non-naturally occurring nucleotides or deoxyribonucleotides.
Collectively, all such altered RNAs are referred to as analogs of RNA. siRNAs of the present invention need only be sufficiently similar to natural RNA that it has the ability to mediate RNA
interference (RNAi). As used herein, RNAi refers to the ATP-dependent targeted cleavage and degradation of a specific mRNA molecule through the introduction of small interfering RNAs or dsRNAs into a cell or an organism. As used herein "mediate RNAi"
refers to the ability to distinguish or identify which RNAs are to be degraded.
By "soluble endoglin binding molecule" is meant a protein or small molecule compound that binds, preferably specifically binds, a soluble endoglin polypeptide. A
soluble endoglin binding molecule may be, for example, an antibody, antibody-related peptide, one or more CDR regions of a soluble endoglin binding antibody, or soluble endoglin interacting protein.
By "soluble Flt-1 (sFlt-1)" (also known as sVEGF-R1) is meant the soluble form of the Flt-1 receptor, that is homologous to the protein defined by GenBank accession number U01134, and that has sFlt-1 biological activity. The biological activity of an sFIt-1 polypeptide may be assayed using any standard method, for example, by assaying sFit-1 binding to VEGF. sFlt-1 lacks the transmembrane domain and the cytoplasmic tyrosine kinase domain of the Flt-1 receptor. sFlt-I can bind to VEGF and PIGF
with high affinity, but it cannot induce proliferation or angiogenesis and is therefore functionally different from the Flt-1 and KDR receptors. sFlt-1 was initially purified from human umbilical endothelial cells and later shown to be produced by trophoblast cells in vivo. As used herein, sFlt-1 includes any sFlt-1 family member or isoform. sFlt-1 can also mean degradation products or fragments that result from enzymatic cleavage of the Flt-1 receptor and that maintain sFlt-1 biological activity. In one example, specific metalloproteinases released from the placenta may cleave the extracellular domain of Flt-1 receptor to release the N-terminal portion of Flt-I into circulation.
By "specifically binds" is meant a compound or antibody which recognizes and binds a polypeptide of the invention but that does not substantially recognize and bind other molecules in a sample, for example, a biological sample, which naturally includes a polypeptide of the invention. In one example, an antibody that specifically binds soluble endoglin does not bind membrane bound endoglin. In another example, an antibody that specifically binds to soluble endoglin binds to an epitope within the extracellular domain of endoglin, particularly an epitope within amino acids 26 to 437 (excluding the peptide leader sequence), amino acids 40 to 406 of human endoglin (see FIGURE 30B), or amino acids 26 to 587 (excluding the peptide leader sequence); that may or may not be unique to soluble endoglin (e.g., in the three dimenstional structure of soluble endoglin). In another example, an antibody that specifically binds to soluble endoglin recognizes one or more of the amino acid sequences shown in bold and underlined in FIGURE 30B.
By `subject" is meant a mammal, including, but not limited to, a human or non-human mammal, such as a cow, a horse, a sheep, a pig, a goat, a dog, or a cat.
Included in this definition are pregnant, post-partum, and non-pregnant mammals.
By "substantially identical" is meant a nucleic acid or amino acid sequence that, when optimally aligned, for example using the methods described below, share at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with.a second nucleic acid or amino acid sequence, e.g., an endoglin or soluble endoglin sequence.
"Substantial identity" may be used to refer to various types and lengths of sequence, such as full-length sequence, epitopes or immunogenic peptides, functional domains, coding and/or regulatory sequences, exons,-introns, promoters,=and genomic sequences.
Percent identity between two polypeptides or nucleic acid sequences is determined in various ways that are within the skill in the art, for instance, using publicly available computer software such as Smith Waterman Alignment (Smith, T. F. and M. S. Waterman (1981) JMoI Biol 147:195-7);
"BestFit" (Smith and Waterman, Advances in Applied Mathematics, 482-489 (1981)) as incorporated into GeneMatcher P1usTm, Schwarz and Dayhof (1979) Atlas of Protein Sequence and Structure, Dayhof, M.O., Ed pp 353-358; BLAST program (Basic Local Alignment Search Tool; (Altschul, S. F., W. Gish, et al. (1990) J Mol Biol 215: 403-10), BLAST-2, BLAST-P, BLAST-N, BLAST-X, WU-BLAST-2, ALIGN, ALIGN-2, CLUSTAL, or Megalign (DNASTAR) software. In addition, those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the length of the sequences being compared. In general, for proteins, the length of comparison sequences will be at least 10 amino acids, preferably 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 250, 300, 350, 400, 437, or at least 587 amino acids or more. For nucleic acids, the length of comparison sequences will generally be at least 25, 50, 100, 125, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, 1000, 1100, 1200, 1311, or at least 1761 nucleotides or more. It is understood that for the purposes of determining sequence identity when comparing a DNA sequence to an RNA sequence, a thymine nucleotide is equivalent to a uracil nucleotide. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine;
aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
By "symptoms of pre-eclampsia" is meant any of the following: (1) a systolic blood pressure (BP) >140 mmHg and a diastolic BP >90 mmHg after 20 weeks gestation, (2) new onset proteinuria (1+ by dipstik on urinanaysis, >300mg of protein in a 24 hour urine collection, or random urine protein/creatinine ratio >0.3), and (3) resolution of hypertension and proteinuria by 12 weeks postpartum. The symptoms of pre-eclampsia can also include renal dysfunction and glomerular endotheliosis or hypertrophy, By "symptoms of eclampsia" is meant the development of any of the following symptoms due to pregnancy or the influence of a recent pregnancy: seizures, coma, thrombocytopenia, liver edema, pulmonary edema, and cerebral edema.
By "transforming growth factor (3 (TGF-0)" is meant a mammalian growth factor that has TGF-0 biological activity and is a member of a family of structurally related paracrine polypeptides found ubiquitously in vertebrates, and prototypic of a large family of metazoan growth, differentiation, and morphogenesis factors (see, for review, Massaque et al. Ann. Rev. Cell. Biol. 6:597-641 (1990); Massaque et al. Trends Cell Biol 4:172-178 (1994); Kingsley Gene Dev. 8:133-146 (1994); and Spom et al. J.
Cell. Biol.
119:1017-1021 (1992). As described in Kingsley, supra, the TGF-P superfamily has at least 25 members, and can be grouped into distinct sub-families with highly related sequences. The most obvious sub-families include the following: the TGF-P sub-family, which comprises at least four genes that are much more similar to TGF-Pl than to other members of the TGF-P superfamily; the bone morphogenetic proteins; the activin sub-family, comprising homo- or hetero-dimers or two sub-units, inhibin(3-A and inhibinp-B.
The decapentaplegic sub-family, which includes the mammalian factors BMP2 and BMP4, which can induce the formation of ectopic bone and cartilage when implanted under the skin or into muscles. The 60A sub-family, which includes a number of mammalian homologs, with osteoinductive activity, including BMP5-8. Other members of the TGF-(3 superfamily include the gross differentiation factor 1(GDF-1), GDF-3/VGR-2, dorsalin, nodal, mullerian-inhibiting substance (1VIIS), and glial-derived neurotrophic growth factor (GDNF). It is noted that the DPP and 60A sub-families are related more closely to one another than to other members of the TGF-P
superfamily, and have often been grouped together as part of a larger collection of molecules called DVR
(dpp and vgl related). Unless evidenced from the context in which it is used, the term TGF-0 as used throughout this specification wiIl be understood to generally refer to members of the TGF-0 superfamily as appropriate. (Massague et al., Annu. Rev.
Biochem. 67:753-91, 1998; Josso et al., Curr. Op. Gen. Dev., 7:371-377, 1997).
TGF-(3 functions to regulate growth, differentiation, motility, tissue remodeling, neurogenesis, would repair, apoptosis, and angiogenesis in many cell types. TGF-0 also inhibits cell proliferation in many cell types and can stimulate the synthesis of matrix proteins.
By "therapeutic amount" is meant an amount that when administered, either by administration directly to the patient or by an ex vivo approach, to a patient suffering from pre-eclampsia or eclampsia is sufficient to cause a qualitative or quantitative reduction in the symptoms of pre-eclampsia or eclampsia as described herein. A
"therapeutic amount" can also mean an amount that when administered, either by administration directly to the patient or by an ex vivo approach, to a patient suffering from pre-eclampsia or eclampsia is sufficient to cause a reduction in the expression levels of soluble endoglin or sFIt-1 or an increase in the expression levels of VEGF
or P1GF as measured by the assays described herein.
By "treating" is meant administering a compound or a pharmaceutical composition for therapeutic purposes. To "treat disease" or use for "therapeutic treatment" refers to administering treatment to a subject already suffering from a disease to improve the subject's condition. Preferably, the subject is diagnosed as suffering from a pregnancy complication associated with hypertension, such as pre-eclampsia or eclampsia, based on identification of any of the characteristic symptoms described below or the use of the diagnostic methods described herein. To "prevent disease"
refers to prophylactic treatment of a subject who is not yet ill, but who is susceptible to, or otherwise at risk of, developing a particular disease. Preferably a subject is determined to be at risk of developing pre-eclampsia or eclampsia using the diagnostic methods described herein. Thus, in the claims and embodiments, treating is the administration to a mammal either for therapeutic or prophylactic purposes.
By "trophoblast" is meant the mesectodermal cell layer covering the blastocyst that erodes the uterine-mucosa and through-which the embryo receives nourishment from the mother; the cells contribute to the formation of the placenta.
By "vascular endothelial growth factor (VEGF)" is meant a mammalian growth factor that is homologous to the growth factor defined in U.S. Patent Nos.
5,332,671;
5,240,848; 5,194,596; and Charnock-Jones et al. (Biol. Reproduction, 48: 1120-1128, 1993), and has VEGF biological activity. VEGF exists as a glycosylated homodimer and includes at least four different alternatively spliced isoforms. The biological activity of native VEGF includes the promotion of selective growth of vascular endothelial cells or umbilical vein endothelial cells and induction of angiogenesis. As used herein, VEGF
includes any VEGF family member or isoform (e.g., VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF189, VEGF 165, or VEGF 121). Preferably, VEGF is the VEGF121 or VEGF165 isoform (Tischer et al., J. Biol. Chem. 266, 11947-11954, 1991;
Neufed et al. Cancer Metastasis 15:153-158, 1996), which is described in U.S.
Patent Nos. 6,447,768; 5,219,739; and 5,194,596, hereby incorporated by reference.
Also included are mutant formsof VEGF such as the KDR-selective VEGF and Fit-selective VEGF described in Gille et al. (J. Biol. Chem. 276:3222-3230, 2001). As used herein VEGF also includes any modified forms of VEGF such as those described in LeCouter et al. (Science 299:890-893, 2003). Although human VEGF is preferred, the invention is not limited to human forms and can include other animal forms of VEGF (e.g.
mouse, rat, dog, or chicken).
By "vector" is meant a DNA molecule, usually derived from a plasmid or bacteriophage, into which fragments of DNA may be inserted or cloned. A
recombinant vector will contain one or more unique restriction sites, and may be capable of autonomous replication in a defined host or vehicle organism such that the cloned sequence is reproducible. A vector contains a promoter operably linked to a gene or coding region such that, upon transfection into a recipient cell, an RNA is expressed.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.
Brief Description of the Drawings FIGURE 1 is a schematic showing the endoglin protein. SP: signal peptide (also referred to as the peptide leader sequence), ZP: zona pellucida domain, CL:
potential cleavage site (amino acid 437) for the release of soluble endoglin, TM:
transmembrane domain, Cyto: cytoplasmic domain. Once the signal peptide is cleaved, the remaining mature protein starts at the glutamic acid residue at amino acid 26.
FIGURE 2A shows the predicted cDNA sequence (SEQ ID NO: 1) of soluble endoglin. FIGURE 2B shows the predicted amino acid sequence (SEQ ID NO: 2) of soluble endoglin which includes the signal peptide (amino acids 1-25). It should be noted that the sequence includes the leader peptide sequence that would normally be cleaved in the ER.
FIGURE 3 is a Northern blot showing endoglin mRNA levels in placentas from normal pregnancies (N), placentas from preterm pre-eclamptic pregnancies (p) and placentas from term pre-eclamptic pregnancies (P).
FIGURE 4 is a western blot showing endoglin protein levels in the placenta.
Samples are from two pre-eclamptic patients, p32 and p36, that presented to the Beth Israel Deaconess Medical Center in 2003 and maternal serum from a pregnant woman.
The Westem blot was probed using a N-terminal antibody obtained from Santa Cruz Biotechnology, I:nc., (Santa Cruz, CA) that shows both the I l OkD band in the placenta and a smaller 63 kD band that is present in the placenta and the-serum samples.
FIGURE 5 is a graph that shows the circulating concentrations of soluble endoglin in women with normal pregnancy, mild pre-eclampsia, severe pre-eclampsia and non-pre-eclamptic pregnancies complicated by pre-term delivery. All blood specimens were obtained within 24 hours prior to delivery. Soluble endoglin was measured using an ELISA kit from R & D Systems, MN (Cat # DNDGOO). These data show that soluble endoglin levels are significantly elevated in pre-eclamptic patients at the time of clinical disease.
FIGURE 6 is a graph showing the mean soluble endoglin concentration for the five different study groups of pregnant women throughout pregnancy during the various gestational age group-windows.
FIGURE 7 is a graph showing the mean sFltl concentrations for the five different study groups of pregnant women throughout pregnancy during the various gestational age group windows.
FIGURE 8 is a graph showing the mean PIGF concentrations for the five different study groups of pregnant women throughout pregnancy during the various gestational age group windows.
FIGURE 9 is a graph showing the values for the soluble endoglin anti-angiogenic index for pre-eclampsia anti-angiogenesis for samples taken prior to clinical symptoms.
FIGURE 10 is a graph showing the mean concentrations of soluble endoglin according to the number of weeks before clinical premature pre-eclampsia (PE
<37 weeks).
FIGURE 11 is a graph showing the soluble endoglin anti-angiogenic index values according to the number of weeks before clinical premature pre-eclampsia (PE
<37 weeks).
FIGURE 12 is a graph showing the alteration in soluble endoglin levels throughout pregnancy for term pre-eclampsia (PE>37 weeks) before and after symptoms.
FIGURE 13 is a graph showing the alteration in the soluble endoglin anti-angiogenic index levels throughout pregnancy for term pre-eclampsia (PE>37 weeks) before and after symptoms.
FIGURE 14 is a graph showing the soluble endoglin levels detected in women during gestational hypertension and before gestational hypertension (1-5 weeks preceding gestational hypertension (during weeks 33-36 of pregnancy)) and normotensive controls.
FIGURE 15 is a graph showing the soluble endoglin anti-angiogenic index levels in women during gestational hypertension and before gestational hypertension (1-5 weeks preceding gestational hypertension (during weeks 33-36 of pregnancy)) and normotensive controls.
- FIGURE 16 is a graph showing the soluble endoglin levels detected during the 33-36 week gestational windows in women with severe SGA, mild SGA, and normotensive controls.
FIGURE 17 is a graph showing the soluble endoglin anti-angiogenic index levels detected during the 33-36 week gestational windows in women with severe SGA, mild SGA, and normotensive controls.
FIGURE 18 is a graph showing the concentration of sFltl and soluble endoglin in the same pregnant patients plotted against each other.
FIGLIRE 19 shows photomicrographs of double immunofluorescence staining of endoglin (red) and smooth muscle actin (green) for pre-eclamptic placentas taken at 25.2 weeks. The antibody used to detect endoglin stains both full-length endoglin and the soluble endoglin. Control placentas for the appropriate gestational windows were derived from patients with pre-term labor.
FIGURE 20 shows photomicrographs of double immunofluorescence staining of endoglin (red) and smooth muscle actin (green) for pre-eclamptic placentas taken at 41.3 weeks. The antibody used to detect endoglin stains both full-length endoglin and the soluble endoglin. Control placentas for the appropriate gestational windows were derived from patients with pre-term labor.
FIGURE 21A shows an autaradiogram from immunoprecipitation and western blots experiments for endoglin using both pre-eclamptic placentas and serum.
FIGURE
21B shows an autoradiogram from immunoprecipitation and western blots experiments for endoglin using pre-eclamptic placentas. The three different N and P
samples represent individual patients. For both figures commercially available monoclonal antibodies were used for immunoprecipitation and polyclonal antibodies were used for the western blots. Both these antibodies were raised against the N-terminal region of the endoglin protein and detect both the full length and the truncated soluble endoglin protein.
FIGURE 22 is a graph showing the results of angiogenesis assays using HUVECs in growth factor reduced matrigels. Angiogenesis assays were performed in the presence of soluble endoglin or sFit1 or both and the endothelial tube lengths quantitated. C-represents control, E- represents 1 g/ml of soluble endoglin and S represents 1 g/ml of sFltl. E+S represent the combination of I g/ml of E + 1 g/ml of sFltl. Data represents a mean of three independent experiments.
FIGURE 23 is a graph showing the microvascular permeability in several organ beds assessed using Evans blue leakage in mice as described in the materials and methods. C- control (GFP), E-soluble endoglin, S-sFltl and S+E- sF1tI +
soluble endoglin. Data represents a mean of 4 independent experiments.
FIGURE 24 is a graph showing the percent change in rat renal microvessel diameter when subjected to microvascular reactivity experiments in the presence of TGF-p 1 (B 1) and TGF-(33 (133) from doses ranging from 200 pg/ml - 200 ng/ml.
These same experiments were repeated in the presence of soluble endoglin (E) at 1 g/ml.
These data presented are a mean of 4 independent experiments.
FIGURE 25 is a graph showing the percent change in the vascular diameter of renal microvessels in the presence of 1 ng/ml of VEGF (V), TGF-(31 (B 1) and the combination (V+B 1). Also shown is the effect of this combination in the presence of 1 g/ml each of sFltl (S) and soluble endoglin (E) (V+B I+S+E). The data represents a mean of 4 independent experiments.
FIGURE 26A is a photograph of a peripheral smear of blood samples taken at the time of sacrifice from pregnant rats injected with the combination of sFlt1 and a control adenoviruses (CMV). FIGURE 26B is a photograph of a peripheral smear of blood samples taken at the time of sacrifice from pregnant rats injected with the combination of sFlt and adenoviruses expressing soluble endoglin and demonstrates active hemolysis as evidenced by schistocyes and increased reticulocyte count. Arrowheads represent schistocyte.
FIGURES 27A-D are a series of photomicrographs showing the renal histology (1-I &E stain) of the various animal groups described in Table 8. FIGURE 27A
shows the renal histology for the control group with no evidence of glomerular endotheliosis.
FIGURE 27B shows the renal histology for the soluble endoglin injected group with no evidence of glomerular endotheliosis. FIGURE 27C shows the renal histology for sFltl injected rats showing moderate endotheliosis (shown by arrow head). FIGURE 27D
shows the renal histology for the soluble endoglin and sFltl injected rats showing extremely swollen glomeruli and severe glomerular endotheliosis with protein resorption droplets in the podocytes. All light micrographs were taken at 60X (original magnification).
FIGURE 28 is a graph showing the ELISA results for soluble endoglin (sEng) and sFltl in sera of patients with varying degrees of preeclampsia, control pregnancies and four non-pregnant healthy volunteers as described in Example 3. *P < 0.05 compared to pre-term controls and # P< 0.05 compared to severe preeclampsia.
FIGURE 29 is a graph showing ELISA results for soluble endoglin in a subset of pregnant patients (normal: n= 6; preeclampsia: n = 11) described in Example 3 with blood drawn pre- (0-12 hours) and post- (48 hours) delivery. * P < 0.05 as compared to T
= 0 samples.
FIGURE 30A is a western blot showing soluble endoglin after purification the serum of preeclamptic patients. Fractions 4 and 5 eluted from the 44G4-IgG
(anti-Eng) Sepharose were run on SDS-PAGE under reducing conditions and tested by Western blot using a polyclonal antibody to endoglin. The eluted fractions were subjected to mass spectrometry analysis (3 runs). FIGURE 30B shows the sequence of human endoglin (SEQ ID NO: 5). Peptides identified by mass spec are shown in bold and underlined.
The underlined amino acids represent the transmembrane domain of human cell surface endoglin. Note that the amino acid sequence numbering starts at 26 because amino acids 1-25 represents the leader peptide. Note that the sequence listed as SEQ ID
NO: 5 in the sequence listing begins at amino acid I so that amino acid 26 in the figure is amino acid 1 in the sequence listing, amino acid 658 in the figure is amino acid 633 in the sequence listing. The numbering of the amino acids is adjusted depending on the reference .
sequence (i.e., amino acids 26 to 658 for sequences referring to FIGURE 30B
are the same as amino acids 1 to 633 for sequences referring to SEQ ID NO: 5).
FIGURE 31 shows a series of photomicrographs showing soluble endoglin inhibits capillary formation and increases vascular permeability. Angiogenesis assays were performed using HUVEC in growth factor reduced MatrigelTm in the presence of 1 g of recombinant soluble endoglin, sFltl, or both, and endothelial tube lengths were quantified. A representative experiment (n = 4) is shown with tube lengths in pixels indicated below the panels.
FIGURE 32 is a series of graphs showing inhibition of TGF-(31-mediated vascular reactivity in mesenteric vessels by soluble endoglin. Microvascular reactivity of rat mesenteric microvessels was measured in the presence of TGF-(31 or TGF-(33 from 200 pg/ml to 200 ng/ml. The experiments were repeated in the presence of recombinant soluble endoglin at 1 g/ml. The mean f SE of 4 independent experiments is shown (upper panel). Also shown is the blocking effect of L-NAME on TGFP 1 at 1 ng/ml (lower panel).
FIGURE 33 is a series of photomicrographs showing glomerular endotheliosis in pregnant rats. Electron micrographs (EM) of glomeruli from a control pregnant rat (upper panel), soluble endoglin (sEng)-treated pregnant rat (middle panel) and the combination group - soluble endoglin (sEng)+sFltl (lower panel) are shown_ These photos were taken at 6200X (original magnification) for the upper and middle panel and 5000X (original magnification) for the lower panel. -FIGURES 34 A-H are a series of photomicrographs showing renal, placental and hepatic histological changes and peripheral blood smears in pregnant rats after soluble endoglin and sFltl treatment. Placental histology (H &E stain) ofcontrol.
(FIGURE
34A), sEng (FIGURE 34B), sFltl (FIGURE 34C) and sFltl+sEng (FIGURE 34D) groups. Both the soluble endoglin and sFltl treated animals show diffuse inflammation (arrow heads) at the maternal-fetal junction not seen in controls. There is hemorrhagic infarction and fibrinoid necrosis with lumen obstruction of a maternal vessel (arrow) in the decidua of the sFltl+sEng treated placenta (FIGURE 34D). Scale bar, 200 pm (FIGURE 34E-H). Liver histology in the control (FIGURE 34E), sEng (FIGURE
34F), sFltl (FIGt1RE 34G) and sFltl+sEng (FIGURE 34H) groups. Ischemic changes with multifocal necrosis (arrow head) are noted in the sFltl+sEng group (FIGURE
34H).
Control group and rats given sEng or sFltl showed no changes. Scale bar, 200 m.
FIGURES 35A-D are a series of graphs and autoradiograms showing recombinant sEng attenuates TGF-01 binding and activity and its effects on vasodilation via eNOS
activation. FIGURE 35A is a graph showing the microvascular responses of renal microvessels to I ng/ml of VEGF, TGF-p 1 and the combination. The effects of ng/ml each of sFltl and sEng on the combined response are shown. (n = 4). Also shown is the blocking effect of L-NAME on TGF(i1 and VEGF stimulated responses.
FIGURE
35B is a representative autoradiogram and graph of a dose-dependent increase in [1125 ]
TGF-01 binding to T(3RII on mouse endothelial cells. Treatment with 5 nM
recombinant soluble endoglin significantly reduced binding at 50 pM and 100 pM (*P < 0.05 vs.
untreated group). Competition with 40X excess cold TGF-(31 in cells treated with 100 pM [I125] TGF-J31 abolished receptor binding and served as background control.
FIGURE 35C is a graph showing significantly increased TGF-0-induced activation of the Smad 2/3-dependent CAGA-Luc reporter construct transfected in HUVECs and inhibition by treatment with sEng. (n = 3, **P < 0.01 vs. sEng untreated group).
FIGtTRE 35D is a representative western blots and graph (n = 4) showing significant dephosphorylation at eNOS Thr495 following treatment with TGF-(31 and attenuation by sEng (*P < 0.05 vs. untreated). Phosphorylation was unchanged at Serl 177 and total levels of eNOS remained constant throughout the experiments.
FIGURE 36 shows two western blots of rat plasma demonstrating expression of the recombinant sFlt1 and soluble endoglin. Upper panel: Plasma specimens from pregnant rats (at early third trimester) were used as described in Methods.
Lanes 1, 2 and 3 represent 200 pg, 500 pg and 2 ng of recombinant mouse Fltl -Fc protein used as a positive control. 20 l of plasma specimens from one control rat and two sFItl treated rats are shown. sFltl (53 kDa) band was detected in the sFltl treated rats.
Quantitation of the sFltl expression was performed using commercially available ELISA (Table 8).
Lower panel: Plasma specimens from pregnant rats were used (at early third trimester) to detect sEng expression. Lane 1 represents 500 pg of recombinant human soluble endoglin and lanes 2 and 3 represent 30 l of plasma from sEng treated and control rats respectively. The blot shows no soluble endoglin in control rats but robust expression of recombinant sEng in treated rats. Quantitation of soluble endoglin was performed using a commercially available ELISA (Table 8).
Figure 37 is a graph showing the distribution of delta sFltl and delta sEng (first trimester- second trimester values) in controls, all pre-eclampsia and in pre-eclampsia <37 weeks.
Figure 38 is a graph showing the distribution of sFlt x sEng product in the fsrst trimester (product 1), in the second trimester (product 2), delta product (product 1-product 2) in controls, all pre-eclampsia and in pre-eclampsia <37 weeks.
Figure 39 is a graph showing the risk of pre-eclampsia according to tertiles of delta product. The increase in risk of preterrn preeclampsia in the group whose delta product levels were greater than +1 [aOR 5.5, 95% Cl 1.4 - 22.4], compared to women whose delta product was less than -1 was statistically significant (P<0.05).
Figure 40A is a western blot showing endoglin is necessary for TGF-j31 induced dephosphorylation of eNOS at Thr495. Figure 40B is a graph showing the percent of eNOS Thr495 phosphorylation relative to total eNOS. The results show that the level of phosphorylated Thr495 decreases in the presence of TGF-01 in the presence of soluble endoglin but not in the absence of soluble endoglin.
Detailed Description We have discovered that soluble endoglin levels are elevated in blood serum samples taken from women with a pregnancy related hypertensive disorder, such as pre-eclampsia or eclampsia. Soluble endoglin may be formed by cleavage of the extracellular portion of the membrane bound form by proteolytic enzymes. The lack of detection of altemate splice variants in placenta and the partial peptide sequence of purified soluble endoglin as described herein suggest that it is an N-terminal cleavage product of full-length endoglin. Excess soluble endoglin may be depleting the placenta of necessary amounts of these essential angiogenic and mitogenic factors. We have discovered that excess circulating concentrations of soluble endoglin and sFltl in patients with preeclampsia contribute to the pathogenesis of pre-eclampsia and other pregnancy related hypertensive disorders. We have also discovered that soluble endoglin interferes with TGF-01 and TGF-j33 binding to its receptor leading to decreased signaling such as a reduction in eNOS activation in endothelial cells, thereby disrupting key homeostatic mechanisms necessary for maintenance of vascular health. These data suggest a crucial role for endoglin in linking TGF-(3 receptor activation to NO synthesis. In addition, we have discovered that soluble endoglin and sFltl act in concert to induce vascular damage and pregnancy related hypertensive disorders, such as pre-eclampsia or eclampisa, by interfering with TGF-P 1 and VEGF signaling respectively, likely via inhibition of the downstream activation of eNOS.
The present invention features the use of therapeutic agents that interfere with soluble endoglin binding to growth factors, agents that reduce soluble endoglin expression or biological activity, or agents that increase levels of growth factors, can be used to treat or prevent pregnancy related hypertensive disorders, such as pre-eclampsia or eclampsia in a subject. Such agents include, but are not limited to, antibodies that bind to soluble endoglin and inhibit soluble endoglin biological activity, oligonucleotides for antisense or RNAi that reduce levels of soluble endoglin, compounds that increase the levels of growth factors that bind to soluble endoglin, compounds that prevent the proteolytic cleavage of the membrane bound form of endoglin thereby preventing the release of soluble endoglin, and small molecules that bind soluble endoglin and block the growth factor binding site. Additionally or alternatively, the invention features the use of any compound (e.g., polypeptide, small molecule, antibody, nucleic acid, and nzimetic) that increases the level or biological activity of TGF-(3, eNOS, and PGI2 to treat or prevent pregnancy related hypertensive disorders, such as pre-eclampsia or eclampsia in a subject. Additionally, the invention features the use of any compound that decreases the level of sFlt-l or increases the level or activity of VEGF or PIGF (see for example, U.S.
Patent Application Publication Numbers 20040126828, 20050025762, and and PCT Publication Numbers WO 2004/008946 and WO 2005/077007) in combination with any of the therapeutic compounds described above to treat or prevent pregnancy related hypertensive disorders, such as pre-eclampsia or eclampsia, in a subject. In addition, the invention features the use of soluble endoglin, eNOS, TGF-(3, of PGI2, either alone or in combination, as a diagnostic marker of pregnancy related hypertensive disorders, including pre-eclampsia and eclampsia.
While the detailed description presented herein refers specifically to soluble endoglin, TGF-(31, eNOS, sFlt-1, VEGF, or PIGF, it will be clear to one skilled in the art that the detailed description can also apply to family members, isoforms, and/or variants of soluble endoglin, TGF-(3, eNOS, sFlt-1, VEGF, or PIGF.
Diagnostics We have discovered that soluble endoglin levels are elevated in blood serum samples taken from women with a pregnancy related hypertensive disorder, such as pre-eclampsia or eclampsia. Soluble endoglin starts rising 6-10 weeks before clinical symptoms of preeclampsia. Accordingly, a diagnostic test measuring soluble endoglin and sFltl, optionally in combination with free PIGF, in the serum will have enhanced sensitivity and specificity, and provide a powerful tool in the prevention of preeclampsia-induced mortality. The diagnostic test can also include measuring the levels of free VEGF; TGF-P family members, preferably TGF-(31, TGF- P3, free activin-A, BMP2, BMP7; NOS, preferably eNOS; or PGI2, either alone or in any combination thereof. An alteration in the levels of any of these proteins is diagnostic of a pregnancy related hypertensive disorder, such as pre-eclampsia or eclampsia. In one example, a decrease in the levels of free BMP2, BMP7, or activin A is diagnostic of a pregnancy related hypertensive disorder, such as pre-eclampsia or eclampsia.
While the methods described herein refer to pre-eclampsia and eclampsia specifically, it should be understood that the diagnostic and monitoring methods of the invention apply to any pregnancy related hypertensive disorder including, but not limited to, gestational hypertensiorn, pregnancy with a small for gestational age (SGA) infant, HELLP, chronic hypertension, pre-eclampsia (mild, moderate, and severe), and eclampsia.
Levels of soluble endoglin, either free, bound, or total levels, are measured in a subject sampl e and used as an indicator of pre-eclampsia, eclampsia, or the propensity to develop such conditions.
A subject having pre-eclampsia, eclampsia, or a predisposition to such conditions will show an increase in the expression of a soluble endoglin polypeptide. The soluble endoglin polypeptide can include full-length soluble endoglin, degradation products, alternatively spliced isoforms of soluble endoglin, enzymatic cleavage products of soluble endoglin, and the like. An antibody that specifically binds a soluble endoglin polypeptide may be used for the diagnosis of pre-eclampsia or eclampsia or to identify a subject at risk of developing such conditions. One example of an antibody useful in the methods of the invention is a monoclonal antibody against the N-terminal region of endoglin that is commercially available from Santa Cruz Biotechnology, Inc.
(cat # sc-20072). Additional examples include antibodies that specifically bind the extracellular domain of endoglin (e.g., amino acids 1 to 437 of endoglin, amino acids I to 587 of endoglin, or any of the amino acid sequences shown in bold and underlined in FIGURE
30B). A variety of protocols for measuring an alteration in the expression of such polypeptides are known, including immunological methods (such as ELISAs and RIAs), and provide a basis for diagnosing pre-eclampsia or eclampsia or a risk of developing such conditions.
Increased levels of soluble endoglin are a positive indicator of pre-eclampsia or eclampsia. For example, if the level of soluble endoglin is increased relative to a normal reference (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more), or increases over time in one or more samples from a subject, this is considered a positive indicator of pre-eclampsia or eclampsia. Additionally, any detectable alteration in levels of soluble endoglin, sFlt-1, VEGF, or PIGF relative to normal levels is indicative of eclampsia, pre-eclampsia, or the propensity to develop such conditions. Nonnally, circulating serum concentrations of soluble endoglin range from 2-7 ng/ml during the non-pregnant state and from 10-20 ng/ml during normal pregnancy. Elevated serum levels, greater than 15 ng/rnl, preferably greater than 20 ng/ml, and most preferably greater than 25 ng/ml or more, of soluble endoglin is considered a positive indicator of pre-eclampsia or eclampsia.
In one embodiment, the level of soluble endoglin is measured in combination with the level of sFlt-1, VEGF, or PIGF polypeptide or nucleic acid, or any combination thereof. Methods for the measurement of sFlt-1, VEGF, and PIGF are described in U.S.
Patent Application Publication Numbers 20040126828, 20050025762, and and PCT Publication Numbers WO 2004/008946 and WO 2005/077007, hereby incorporated by reference in their entirety. In additional preferred embodiments, the body mass index (BMI) and gestational age of the fetus is also measured and included the diagnostic metric.
In another embodiment, the level of TGF-(31, TGF-P3, or eNOS polypeptide or nucleic acid is measured in combination with the level of soluble endoglin, sFlt-1, VEGF, or PIGF polypeptide or nucleic acid. Antibodies useful for the measurement of TGF-(31 and P3 polypeptide levels are commercially available, for example, from Abcam, Abgent, BD Biosciences Pharmingen, Chemicon, GeneTex, and R&D Systems. The level of PGIZ
can also be used in combination with the level of any of the above polypeptides. PGI2 levels can be determined, for example, using the PGIa receptor as a binding molecule in any of the diagnostic assays described above, or using, for example, the urinary prostacyclin colorimetric ELISA kit (Assay Designs). Antibodies useful for the measurement of eNOS polypeptide levels are commercially available, for example, from Research Diagnostics Inc., Santa Cruz, Cayman Chemicals, and BD Biosciences.
In another embodiment, the biological activity of any one or more of TGF-(31, TGF-03, or eNOS polypeptide is measured in combination with the biological activity of soluble endoglin, sFlt-1, VEGF, or PIGF polypeptide and a decrease in the biological activity is a positive indicator of pre-eclampsia or eclampsia. The biological activity can be measured, for example using an assay for enzymatic activity or for the downstream signaling activity. In one example, the enzymatic activity of eNOS is determined by measuring citrulline conversion and a decrease in the enzymatic activity of eNOS is a positive indicator of pre-eclampsia or eclampsia.
In one embodiment, a metric incorporating soluble endoglin, sFlt-1, VEGF, or PIGF, or any combination therein, is used to determine whether a relationship between levels of at least two of the proteins is indicative of pre-eclampsia or eclampsia. In one example, the metric is a PAAI (sFlt-1/ VEGF + PIGF), which is used, in combination with soluble endoglin measurement, as an anti-angiogenic index that is diagnostic of pre-eclampsia, eclampsia, or the propensity to develop such conditions. If the level of soluble endoglin is increased relative to a reference sample (e.g., 1.5-fold, 2-fold, 3-fold, 4-fold, or even by as much as ] 0-fold or more), and the PAAI is greater than 10, more preferably greater than 20, then the subject is considered to have pre-eclampsia, eclampsia, or to be in imminent risk of developing the same. The PAAI (sFlt-1/
VEGF +
PIGF) ratio is merely one example of a useful metric that may be used as a diagnostic indicator. It is not intended to limit the invention. Virtually any metric that detects an alteration in the level of soluble endoglin, sFlt-1, PIGF, or VEGF, or any combination thereof, in a subject relative to a normal control may be used as a diagnostic indicator.
Another example is the following soluble endoglin anti-angiogenic index: (sFlt-1 +
0.25(soluble endoglin polypeptide))/P1GF. An increase in the value of the soluble endoglin metric over time or compared to a reference sample or value is a diagnostic indicator of pre-eclampsia or eclampsia. A soluble endoglin index above 100, preferably above 200 is a diagnostic indicator of pre-eclampsia or eclampsia. Additional examples include the following indexes: (soluble endoglin+ sFlt-1)/P1GF or sFlt-1 x soluble endoglin.
Another example includes the measurement of the levels of sFlt-1 and soluble endoglin in the first and second trimesters in a subject and calculating the delta value of sFlt1 x soluble endoglin (sEng) in each trimester using the following equation: [dproduct =(sFltl x sEng) in the second trimester - (sFltl x sEng) in the first trimester], where a value greater than 0, 1, 2, or more, including fractions thereof (e.g., a positive value) is a diagnostic indicator of pre-eclampsia or eclampsia. A positive value can also be an indicator of pre-term pre-eclampsia. Such a measurement can be taken on numerous occasions during the first and second trimesters and the dproduct can be followed over time. In addition, the dproduct of the sFlt-1 level (dsFIt-1) and the sEng level (dsEng) alone can also be calculated between the first and second trimesters, where a value greater than 0, 1, 2, or more, including fractions thereof (e.g., a positive value) for (dsFlt-1) or (dsEng) is a diagnostic indicator of pre-eclampsia or eclampsia.
In addition, the metric can further include the level of TGF-01, TGF-03, PGI2, or eNOS polypeptide. Any of the metrics can further include the BMI of the mother or the GA of the infant.
Standard methods may be used to measure levels of soluble endoglin, free VEGF, free P1GF, sFlt-1, TGF-p1, TGF-(33, PGI2, or eNOS polypeptide in any bodily fluid, including, but not limited to, urine, serum, plasma, saliva, amniotic fluid, or cerebrospinal fluid. Such methods include immunoassay, ELISA, western blotting using antibodies directed to soluble endoglin, free VEGF, free P1GF, sFlt-1, TGF-01, TGF-03, PGIZ, or eNOS polypeptide and quantitative enzyme immunoassay techniques such as those described in Ong et al. (Obstet. Gynecol. 98:608-611, 2001) and Su et al.
(Obstet.
Gynecol., 97:898-904, 2001). ELISA is the preferred method for measuring levels of soluble endoglin, VEGF, P1GF, sFlt-1, TGF-(31, TGF-(33, PGI2, or eNOS
polypeptide.
Preferably, soluble endoglin is measured alone or in combination with any one or more of the remaining polypeptides.
Oligonucleotides or longer fragments derived from an endoglin, sFlt-1, P1GF, or VEGF nucleic acid sequence may be used as a probe not only to monitor expression, but also to identify subjects having a genetic variation, mutation, or polymorphism in an endoglin, sFlt-1, PIGF, or VEGF nucleic acid molecule that are indicative of a predisposition to develop the pre-eclampsia or eclampsia. Such methods are described in detail in Abdalla et al., Hum. Mutat. 25:320-321 (2005), U.S. Patent Application Publication No. 2006/0067937 and PCT Publication No. WO 06/034507. Preferred oligonucleotides will hybridize at high stringency to the extracellular domain of endoglin or to any nucleic acid sequence encoding any of the peptides shown in bold and underlined in FIGRUE 30B.
The measurement of any of the nucleic acids or polypeptides described herein can occur on at least two different occasions and an alteration in the levels as compared to normal reference levels over time is used as an indicator of pre-eclampsia, eclampsia, or the propensity to develop such conditions.
In one example, the level of a soluble endoglin polypeptide or nucleic acid present in the bodily fluids of a subject having pre-eclampsia, eclampsia, or the propensity to develop such conditions may be increased by as little as 10%, 20%, 30%, or 40%, or by as much as 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more relative to levels in a normal control subject or relative to a previous sampling obtained from the same bodily fluids of the same subject. In another example, the level of a soluble endoglin polypeptide or nucleic acid in the bodily fluids of a subject having pre-eclampsia, eclampsia, or the propensity to develop such conditions may be altered by as little as 10%, 20%, 30%, or 40%, or by as much as 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% over time from one measurement to the next.
The level of sFlt-1, VEGF, or PIGF measured in combination with the level of soluble endoglin in the bodily fluids of a subject having pre-eclampsia, eclampsia, or the propensity to develop such conditions may be altered by as little as 10%, 20%, 30%, or 40%, or by as much as 50%, 60%, 70%, 80%, 90%, 95%, 96%, 9'7oJo, 98%, 99% or more relative to the level of sFlt-1, VEGF, or PIGF in a normal control. The level of sFlt-1, VEGF, or PIGF measured in combination with the level of soluble endoglin in the bodily fluids of a subject having pre-eclampsia, eclampsia, or the propensity to develop such.
conditions may be altered by as little as 10%, 20%, 30%, or 40%, or by as much as 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% over time from one measurement to the next.
In one embodiment, a subject sample of a bodily fluid (e.g., urine, plasma, serum, amniotic fluid, or cerebrospinal fluid) is collected earty in pregnancy prior to the onset of pre-eclampsia symptoms. In another example, the sample can be a tissue or cell collected early in pregnancy prior to the onset of pre-eclampsia symptoms. Non-limiting examples of tissues and cells include placental tissue, placental cells, circulating endothelial cells, and leukocytes such as monocytes. In humans, for example, maternal blood serum samples are collected from the antecubital vein of pregnant women during the first, second, or third trimesters of the pregnancy. Preferably, the assay is carried out during the first trimester, for example, at 4, 6, 8, 10, or 12 weeks, or any interval therein, or during the second trimester, for example at 14, 16, 18, 20, 22, or 24 weeks, or any interval therein. In one example, the assay is carried out between 13 and 16 weeks of pregnancy. Such assays may also be conducted at the end of the second trimester or the third trimester, for example at 26, 28, 30, 32,.34, 36, or 38 weeks, or any interval therein.
It is preferable that levels of soluble endoglin and/or any of the additional polypeptides described herein be measured twice during this period of time. For the diagnosis of post-partum pre-eclampsia or eclarnpsia, assays for soluble endoglin may be carried out postpartum. For the diagnosis of a predisposition to pre-eclampsia or eclampsia, the assay is carried out prior to the onset of pregnancy or prior to the development of symptoms of pre-eclampsia or eclampsia. In one example, for the monitoring and management of therapy, the assay is canied out during the pregnancy after the diagnosis of pre-eclampsia, and/or during therapy.
In one particular example, serial blood samples can be collected during pregnancy and the levels of soluble endoglin polypeptide and/or any of the additional polypeptides of the invention determined by ELISA. In another example, a sample is collected during the second trimester and early in the third trimester and in increase in the level of soluble endoglin of any of the other polypeptides of the invention from the first sampling to the next is indicative of pre-eclampsia or eclampsia, or the propensity to develop either.
The invention also include the measurement of any soluble endoglin binding protein (e.g., TGF-pl, TGF-(33, activin-A, BMP-2, and BMP-7) or downstream mediators of soluble endoglin signaling (e.g., PGIZ and eNOS) in a bodily fluid from a subject, preferably urine, and an alteration (e.g., increase or decrease) in the level of the soluble endoglin binding protein is indicative of pre-eclampsia or eclampsia. The methods and timing for measurement of soluble endoglin described herein can also be used for the measurement of any of the soluble endoglin binding protein, PGI2 or eNOS.
In veterinary practice, assays may be carried out at any time during the pregnancy, but are, preferably, carried out early in pregnancy, prior to the onset of pre-eclampsia symptoms. Given that the term of pregnancies varies widely between species, the timing of the assay vaill be determined by a veterinarian, but will generally correspond to the timing of assays during a human pregnancy.
The diagnostic methods described herein can be used individually or in combination with any other diagnostic method described herein or known in the art for a more accurate diagnosis of the presence of, severity of, or estimated time of onset of pre-eclampsia or eclampsia. In addition, the diagnostic methods described herein can be used in combination with any other diagnostic methods determined to be useful for the accurate diagnosis. of the presence of, severity of, or estimated time of onset of pre-eclampsia or eclampsia.
The diagnostic methods described herein can also be used to monitor and manage pre-eclampsia or eclampsia in a subject. In one example, a therapy is administered until the blood, plasma, or serum soluble endoglin level is less than 25 ng/ml or until the serum soluble endoglin levels (or soluble endoglin binding protein, PGIa, or eNOS
level) return to the baseline level-determined before onset ofpre-eclampsia or eclampsia. In another example, if a subject is determined to have an increased level of soluble endoglin relative to a normal control then the therapy can be administered until the serum P1GF
level rises to approximately 400 pg/mL or a return to baseline level prior to onset of pre-eclampsia or eclampsia. In this embodiment, the levels of soluble endoglin, sFlt-1, P1GF, VEGF, soluble endoglin binding protein, PGI2, eNOS or any and all of these, are measured repeatedly as a method of not only diagnosing disease but monitoring the treatment and management of the pre-eclampsia and eclampsia.
Diagnostic Kits The invention also provides for a diagnostic test kit. For example, a diagnostic test kit can include binding agents (e.g., polypeptides or antibodies) that specifically bind to soluble endoglin and means for detecting, and more preferably evaluating, binding between the binding agent and the soluble endoglin polypeptide. For detection, either the binding-agent or the soluble endoglin polypeptide is labeled, and either the binding agent or the soluble endoglin polypeptide is substrate-bound, such that soluble endoglin polypeptide-binding agent interaction can be established by determining the amount of label attached to the substrate following binding between the binding agent and the soluble endoglin polypeptide. A conventional ELISA is a common, art-known method for detecting antibody-substrate interaction and can be provided with the kit of the invention. Soluble endoglin polypeptides can be detected in virtually any bodily fluid including, but not limited to urine, serum, plasma, saliva, amniotic fluid, or cerebrospinal fluid. The invention also provides for a diagnostic test kit that includes a soluble endoglin nucleic acid that can be used to detect and determine levels of soluble endoglin nucleic acids. A kit that determines an alteration, for example, an increase, in the level of soluble endoglin polypeptide relative to a reference, such as the level present in a normal control, is useful as a diagnostic kit in the methods of the invention.
The diagnostic kits of the invention can include antibodies or nucleic acids for the detection of sFlt-1, VEGF, or P1GF polypeptides or nucleic acids as described U.S. Patent Application Publication Numbers 20040126828, 20050025762, and 20050170444 and PCT Publication Numbers WO 2004/008946 and WO 2005/077007.
In another embodiment, the kit can also include binding agents for the detection of soluble endoglin ligands including but not limited to TGF-(31, TGF-(33, or PGI2 or eNOS poIypeptides. Antibodies useful for the measurement of TGF-01 and (33 polypeptide levels are commercially available, for example, from Abcam, Abgent, BD
Biosciences Pharmingen, Chemicon, GeneTex, and R&D Systems. Antibodies useful for the measurement of eNOS polypeptide levels are commercially available, for example, from Research Diagnostics Inc., Santa Cruz, Cayman Chemicals, and BD
Biosciences.
Binding agents for the detection of PGI2 levels can also be included and include for example the PGIz receptor, or fragments thereof, as a binding molecule in any of the diagnostic assays described above, or using, for example, the urinary prostacyclin colorimetric ELISA kit (Assay Designs). A kit that determines an alteration, for example, a decrease, in the level of eNOS, TGF-(31 or (33 polypeptide or PGIa relative to a normal reference or standard or level, such as the level present in a normal control, is useful as a diagnostic kit in the methods of the invention. A kit that determines an alteration, for example, a decrease in the level of soluble endoglin or an increase in the level of a soluble endoglin binding protein (e.g., TGF-01, TGF-f33, activin A, BMP2, and BMP 7) or downstream mediators. of soluble endoglin signaling (e.g., eNOS and PGI2) relative to a positive reference standard or level is useful for monitoring the treatment of pre-eclampsia or eclampsia.
Desirably, the kit includes any of the components needed to perform any of the diagnostic methods described above. For example, the kit desirably includes a membrane, where the soluble endoglin binding agent or the agent that binds the soluble endoglin binding agent is immobilized on the membrane. The membrane can be supported on a dipstick structure where the sample is deposited on the membrane by placing the dipstick structure into the sample or the membrane can be supported in a lateral flow cassette where the sample is deposited on the membrane through an opening in the cassette.
The diagnostic kits also generally include a label or instructions for the intended use of the kit components and a reference sample or purified proteins to be used to establish a standard curve. In one example, the kit contains instructions for the use of the kit for the diagnosis of a pregnancy related hypertensive disorder, such as pre-eclampsia, eclampsia, or the propensity to develop pre-eclampsia or eclampsia. In yet another example, the kit contains instructions for the use of the kit to monitor therapeutic treatment or dosage regimens for the treatment of pre-eclampsia or eclampsia.
The diagnostic kit may also include a label or instructions for the use of the kit to determine the PAAI or soluble endoglin anti-angiogenesis index of the subject sample and to compare the PAAI or soluble endoglin anti-angiogenesis index to a reference sample value. It will be understood that the reference sample values will depend on the intended use of the kit. For example, the sample can be compared to a normal reference value, wherein an increase in the PAAI or soluble endoglin anti-angiogenesis index or in the soluble endoglin value is indicative of pre-eclampsia or eclampsia, or a predisposition to pre-eclampsia or eclampsia. Tn another example, a kit used for therapeutic monitoring can have a reference PAAI or soluble endoglin anti-angiogenesis index value or soluble endoglin value that is indicative of pre-eclampsia or eclampsia, wherein a decrease in the PAAI or soluble endoglin anti-angiogenesis index value or a decrease in the soluble endoglin value of the subject sample relative to the reference sample can be used to indicate therapeutic efficacy or effective dosages of therapeutic compounds. A
standard curve of levels of purified protein within the normal or positive reference range, depending on the use of the kit, can also be included.
Therapeutics The present invention features methods and compositions for treating or preventing pre-eclampsia or eclampsia in a subject. Given that levels of soluble endoglin are increased in subjects having pre-eclampsia, eclampsia, or having a predisposition to such conditions, any compound that decreases the expression levels and/or biological activity of a soluble endoglin polypeptide or nucleic acid molecule is useful in the methods of the invention. Such compounds include TGF-p1, TGF-(33, activin-A, BMP2, or BMP7, that can disrupt soluble endoglin binding to ligands; a purified antibody or antigen-binding fragment that specifically binds soluble endoglin; antisense nucleobase oligomers; and dsRNAs used to mediate RNA interference. Additional useful compounds include any compounds that can alter the biological activity of soluble endoglin, for example, as measured by an angiogenesis assay. Exemplary compounds and methods are described in detail below. These methods can also be combined with methods to decrease sFlt-1 levels or to increase VEGF or PIGF levels or decrease sFlt-1 levels as described in PCT Publication Number WO 2004/008946 and U.S. Patent Publication Nos. 20040126828 and 20050170444. In addition, any compound that increases the level or biological activity of TGF-01 or 3, eNOS, or PG12 are useful in the methods of the invention. Exemplary compounds and methods are described in detail below.
It should be noted that we have discovered that the soluble endoglin and sFlt-pathways may be functioning in a cooperative manner to further the pathogenesis of pre-eclampsia or eclampsia. Therefore, the invention includes any combination of any of the methods or compositioris described herein for the treatment or prevention of a pregnancy related hypertensive disorder. For example, a compound that targets the soluble endoglin pathway (e.g., downregulates soluble endoglin expression or biological activity or upregulates TGF-(3, eNOS, or PGI2 expression or biological activity) can be used in combination with a compound that targets the sFlt-1 pathway (e.g., downregulates sFlt-1 expression or biological activity or upregulates VEGF or PIGF expression of biological activity) for the treatment or prevention of a pregnancy related hypertensive disorder.
Therapeutics targeting the TGF=,8 signaling pathway TGF-0 is the prototype of a family of at least 25 growth factors which regulate growth, differentiation, motility, tissue remodeling, neurogenesis, wound repair, apoptosis, and angiogenesis in many cell types. TGF-(3 also inhibits cell proliferation in many cell types and can stimulate the synthesis of matrix proteins. Unless evidenced from the context in which it is used, the term TGF-0 as used throughout this specification will be understood to generally refer to any and all members of the TGF-0 superfamily as appropriate. Soluble endoglin binds several specific members of the TGF-0 family including TGF-01, TGF-03, activin, B1VIP-2 and BMP-7, and may serve to deplete the developing fetus or placenta of these necessary mitogenic and angiogenic factor. The present invention features methods of increasing the levels of these ligands to bind to soluble endoglin and to neutralize the effects of soluble endoglin.
Soluble endoglin ligands as therapeutic compounds In a preferred embodiment of the present invention, purified forms of any soluble endoglin ligand such as TGF-(3 family proteins, including but not limited to TGF-01, TGF-(33, activin-A, BMP2, and BMP7, are administered to the subject in order to treat or prevent pre-eclampsia or eclampsia.
Purified TGF-(3 family proteins include any protein with an amino acid sequence that is homologous, more desirably, substantially identical to the amino acid sequence of TGF-P l or TGF-(33, or any known TGF-(3 family member, that can induce angiogenesis.
Non-limiting examples include human TGF-01 (Cat #240-B-002) and human TGF-P3 (Cat #243-B3-002) from R & D Systems, MN. Preferred TGF-(3 family proteins useful in the methods of the invention will have the ability to bind to soluble endoglin (e.g., Barbaraet al, J. Biol. Chem. 274:584-94 (1999)).
Therapeutic compounds that inhibit proteolytic cleavage of endoglin We have identified a potential cleavage site in the extracellular domain of endoglin where a proteolytic enzyme could cleave the membrane bound form of endoglin, releasing the extracellular domain as a soluble form. Our sequence alignments of the cleavage site suggest that a matrix metalloproteinase (MMP) may be responsible for the cleavage and release of soluble endoglin. Alternatively, a cathepsin or an elastase may also be involved in the cleavage event. 1VIlVIPs are also known as collagenases, gelatinases, and stromelysins and there are currently 26 family members known (for a review see Whittaker and Ayscough, Cell Transmissions 17:1 (2001)). A
preferred NIlVIP
is MMP9, which is known to be up-regulated in placentas from pre-eclamptic patients (Lim et a1., Am. J Fathol. 151:1809-1818, 1997). The activity of TEvIPs is controlled through activation of pro-enzymes and inhibition by endogenous inhibitors such as the tissue inhibitors of inetalloproteinases (TIMPS). Inhibitors of MBsIPs are zinc binding proteins. There are 4 known endogenous inhibitors (TIMP 1-4), which are reviewed in Whittaker et al., supra. One preferred MMP inhibitor is the inhibitor of membrane type-lV1IvIl'1 that has been shown to cleave betaglycan, a molecule that shares similarity to enodglin (Velasco-Loyden et al., J. Biol. Chem. 279:7721-7733 (2004)). In addition, a variety of naturally-occurring and synthetic MMP inhibitors have been identified and are also reviewed in Whittaker et al., supra. Examples include antibodies directed to MMPs, and various compounds including marimastat, batimastat, CT1746, BAY 12-9566, Prinomastat, CGS-27023A, D9120, BMS275291 (Bristol Myers Squibb), and trocade, some of which are currently in clinical trials. Given the potential role of IVIIVI2's, cathepsins, or elastases in the release and up-regulation of soluble endoglin levels, the present invention also provides for the use of any compound, such as those described above, known to inhibit the activity of any MMP, cathepsin, or elastase involved in the cleavage and release of soluble endoglin, for the treatment or prevention of pre-eclampsia or eclampsia in a subject.
Therapeutic compounds that increase soluble endoglin bindingproteins The present invention provides for the use of any compound known to stimulate or increase blood serum levels of soluble endoglin binding proteins, including but not limited to TGF-(31, TGF-(33, activin-A, BMP2, and BMP7, for the treatment or prevention of pre-eclampsia in a subject. These compounds can be used alone or in combination with the purified proteins described above or any of the other methods used to increase TGF-0 family proteins protein levels described herein. In one example, cyclosporine is used at a dosage of 100-200 mg twice a day to stimulate TGF-P
production.
Therapeutic compounds that alter the anti-angiogenic activity of soluble endoglin Additional therapeutic compounds can be identified using angiogenesis assays.
For example, pre-eclamptic serum having elevated levels of soluble endoglin are added to a matrigel tube formation assay will induce an anti-angiogenic state. Test compounds can then be added to the assay and a reversion in the anti-angiogenic state by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more indicates that the compound can reduce the biological activity of soluble endoglin and is useful as a therapeutic compound.
Therapeutic compounds that increase the levels or biological activity ofNOS
NOS is a complex enzyme containing several cofactors, a heme group which is part of the catalytic site, an N-terminal oxygenase domain, which belongs to the class of haem-thiolate proteins, and a C-terminal reductase domain which is homologous to NADPH:P450 reductase. NOS produces NO by catalysing a five-electron oxidation of a guanidino nitrogen of L-arginine (L-Arg).
eNOS activation involves a coordinated increase in Serl 177 phosphorylation and Thr495 dephosphorylation. We have discovered that TGF-01 dephosphorylates eNOS
at Thr495, which is necessary to increase the Ca2+ sensitivity and enzyme activity and may work synergistically with VEGF, which activates eNOS by phosphorylating Serl 177.
Accordingly, any compound (e.g., polypeptide, nucleic acid molecule, small molecule compound, or antibody) that increases the level (e.g., by increasing stability, transcription or translation, or decreasing protein degradation) or biological activity of NOS, particularly eNOS, or any compound that prevents the downregulation of eNOS
activity is useful in the methods of the invention. Such compounds include purified NOS, preferably eNOS, or biologically active fragments thereof, nucleic acids encoding NOS, preferably eNOS, or biologically active fragments thereof, statins, vanadate, hepatocyte growth factor, phosphoinositide 3-kinase (P13K), Akt, VEGF, TGF-(3I, or any other compound that increases Ser1177 phosphorylation or Thr495 dephosphorylation or both. Nitric oxide is synthesized from L-arginine by nitric oxide synthase located in endothelial and other cells. Nitric oxide can also be generated by application of various nitric oxide donors such as sodium nitroprusside, nitroglycerin, SIN-1, isosorbid mononitrate, isosorbid dinitrate, and the like. Accordingly, compounds that increase (e.g., by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more) the level or biological activity of NOS can optionally be administered in combination with L-arginine ' or a nitric oxide donor (e.g., sodium nitroprusside, nitroglycerin, isosorbidmononitrate, and isosorbo dinitrate). NOS activity can be assayed by standard methods known in the art, including but not limited to the citrulline assay and other assays described in U.S.
Patent Application Publication No. 20050256199, the entire disclosure of which is herein incorporated by reference. The Thr495 residue of eNOS is located within the calmodulin (CaM)-binding domain of eNOS. Agonist-induced dephosphorylation of eNOS at Thr495 increases the binding of CaM to the enzyme (Fleming et al., Circ Res.
2001, 88:
E68-75), thereby increasing its calcium sensitivity and activation. In addition to TGF-Pl described herein, other agonists that have been shown to cause Thr495 dephosphorylation of eNOS including bradykinin, histamine and VEGF. Thr495 dephosphorylation can be enhanced by the protein kinase C (PKC) inhibitor Ro 31-8220 (Calbiochem) or after PKC
downregulation using phorbol 12-myristate 13-acetate (PMA) (Sigma Aldrich).
Moreover, agonist-induced dephosphorylation of Thr495 has been shown to be Ca2+/calmodulin-dependent and inhibitable by calyculin A (Sigma Aldrich), a protein phosphatase 1(PP1) inhibitor (Fleming I, et al. Cire Res. 2001, 88: E68-75).
Additional compounds that effect eNOS dephosphorylation at Thr495 include histamine and bradykinin (Sigma Aldrich).
Therapeutic compounds that increase the levels or biological activity of PGIZ
Prostacyclin is a member of the family of lipid molecules known as eicosanoids.
It is produced in endothelial cells from prostaglandin H2 (PGH2) by the action of the enzyme prostacyclin synthase. PG12 biological activity includes inhibition of platelet aggregation, relaxation of smooth muscle, reduction of systemic and pulmonary vascular resistance by direct vasodilation, and natriuresis in kidney.
PG12 is an anti-thrombotic factor that is stimulated by both VEGF and TGF-01.
PGI2 biological activity includes inhibition of platelet aggregation and relaxation of vascular smooth muscle and assays for PGIz biological activity include any platelet aggregation assay or other PGI2 assay known in the art such as those described in Jakubowski et al., Prostaglandins 47:404(1994). The invention features the use of any compound that increases (e.g., by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more) the level or activity of PGI2, as measured by standard assays known in the art including but not limited to PGIa mimetics, iloprost, cicaprost, and aspirin. Additional compounds are known in the art and examples are described in U.S.P.N.
5,910,482, the entire disclosure of which is herein incorporated by reference.
Purffied proteins For any of the purified proteins, or fragment thereof, the proteins are prepared using standard methods known in the art. Analogs or homologs of any of the therapeutic proteins described above are also included and can be constructed, for example, by making various substitutions of residues or sequences, deleting terminal or internal residues or sequences not needed for biological activity, or adding terminal or internal residues which may enhance biological activity. Amino acid substitutions, deletions, additions, or mutations can be made to improve expression, stability, or solubility of the protein in the various expression systems. Generally, substitutions are made conservatively and take into consideration the effect on biological activity.
Mutations, deletions, or additions in nucleotide sequences constructed for expression of analog proteins or fragments thereof must, of course, preserve the reading frame of the coding sequences and preferably will not create complementary regions that could hybridize to produce secondary mRNA structures such as loops or hairpins which would adversely affect translation of the mRNA.
Any of the therapeutic compounds of the invention (e.g., polypeptide, antibodies;
small molecule compounds) can also include any modified forms. Examples of post-.
translational modifications include but are not limited to phosphorylation, glycosylation, hydroxylation, sulfation, acetylation, isoprenylation, proline isomerization, subunit dimerization or multimerization, and cross-linking or attachment to any other proteins, or fragments thereof, or membrane components, or fragments thereof (e.g., cleavage of the protein from the membrane with a membrane lipid component attached).
Modifications that provide additional advantages such as increased affinity, decreased off-rate, solubility, stability and in vivo or in vitro circulating time of the polypeptide, or decreased immunogenicity and include, for example, acetylation, acylation, ADP-ribosylation, arnidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. (See, for instance, Creighton, "Proteins:
Structures and Molecular Properties," 2d Ed., W. H. Freeman and Co.,.N.Y., 1992;
"Postranslational Covalent Modification of Proteins," Johnson, ed., Academic Press, New York, 1983;
Seifter et al., Meth. Ena,y zol., 182:626-646, 1990; Rattan et al., Ann.
.NYAcad. Sci., 663:48-62, 1992) are also included. The peptidyl therapeutic compound of the invention can also include sequence variants of any of the compounds such as variants that include 1, 2, 3, 4, 5, greater than 5, or greater than 10 amino acid alterations such as substitutions, deletions, or insertions with respect to wild type sequence. Additionally, the therapeutic compound of the invention may contain one or more non-classical amino acids.
Non-classical amino acids include, but are not limited to, to the D-isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, omithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, P-alanine, fluoro-amino acids, designer amino acids such as (3-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs in general. Furthermore, the amino acid can be D (dextrorotary) or L
(levorotary).
Additional post-translational modifications encompassed by the invention include, for example, e.g., N-linked or 0-linked carbohydrate chains, processing of N-terminal or C-terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or 0-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue.
In addition, chemically modified derivatives of the therapeutic compounds described herein, which may provide additional advantages such as increased solubility, stability and circulating time of the polypeptide, or decreased immunogenicity (see U.S.
Pat. No. 4,179,337) are also included. The chemical moieties for derivitization may be selected from water soluble polymers such as, for example, polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like. The compound may be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.
The polymer may be of any molecular weight, and may be branched or unbranched. For polyethylene glycol, the preferred molecular weight is between about I
kDa and about 100 kDa (the term "about" indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing. Other sizes may be used, depending on the desired therapeutic profile (e_g., the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects of the polyethylene glycol to a therapeutic protein or analog).
As noted above, the polyethylene glycol may have a branched structure. Branched polyethylene glycols are described, for example, in U.S. Pat. No. 5,643,575; Morpurgo et al., Appl.
Biochem. Biotechnol. 56:59-72, (1996); Vorobjev et al., Nucleosides Nucleotides 18:2745-2750, (1999); and Caliceti et al., Bioconjug. Chem. 10:638-646, (1999), the disclosures of each of which are incorporated by reference.
Any of the therapeutic compounds of the present invention (e.g., polypeptide, antibodies, or small molecule compounds) may also be modified in a way to form a chimeric molecule comprising the therapeutic compound fused to another, heterologous polypeptide or amino acid sequence, such as an Fe sequence, a detectable label, or an additional therapeutic molecule. In one example, an anti-soluble endoglin antibody can be a peptide fused to an Fc fusion protein.
For any of the polypeptides, including antibodies, that are used in the methods of the invention, the nucleic acids encoding the polypeptides or antibodies, or fragments thereof, are also useful in the methods of the invention using standard techniques for gene therapy known in the art and described herein. The invention also includes mimetics, based on modeling the 3-dimensional structure of a polypeptide or peptide fragment and using rational drug design to provide potential inhibitor compounds with particular molecular shape, size and charge characteristics. Following identification of a therapeutic compound, suitable modeling techniques known in the art can be used to study the functional interactions and design mimetic compounds which contain functional groups arranged in such a manner that they could reproduced those interactions. The designing of mimetics to a known pharmaceutically active compound is a known approach to the development of pharmaceuticals based on a lead compound. This might be desirable where the active compound is difficult or expensive to synthesize or where it is unsuitable for a particular method of administration, e.g. peptides are not well suited as active agents for oral compositions as they tend to be quickly degraded by proteases in the alimentary canal. Mimetic design, synthesis and testing may be used to avoid randomly screening large number of molecules for a target property. The mimetic or mimetics can then be screened to see whether they reduce or inhibit soluble endoglin levels or biological activity and further optimization or modification can then be carried out to anive at one or more final mimetics for in vivo or clinical testing.
Therapeutic nucleic acids Recent work has shown that the delivery of nucleic acid (DNA or RNA) capable of expressing an endothelial cell mitogen such as VEGF to the site of a blood vessel injury will induce proliferation and reendothelialization of the injured vessel. While the present invention does not relate to blood vessel injury, these general techniques for the delivery of nucleic acid to endothelial cells can be used in the present invention for the delivery of nucleic acids encoding soluble endoglin binding proteins, such as TGF-01, TGF-03, activin-A, BMP2 and BMP7, or eNOS. The techniques can also be used for the delivery of nucleic acids encoding proteins, such as those described above, known to inhibit the activity of any AZNII', cathepsin, or elastase involved in the cleavage and release of soluble endoglin, for the treatment or prevention of pre-eclampsia or eclampsia in a subject. These general techniques are described in U.S. Patent Nos.
5,830,879 and 6,258,787 and are incorporated herein by reference.
In the present invention the nucleic acid may be any nucleic acid (DNA or RNA) including genomic DNA, eDNA, and mRNA, encoding a soluble endoglin binding proteins such as TGF-(31, TGF-03, activin-A, B114P2 and BMP7, or eNOS. The nucleic acids encoding the desired protein may be obtained using routine procedures in the art, e.g. recombinant DNA, PCR amplification.
Modes for delivering nucleic acids For any of the nucleic acid applications described herein, standard methods for administering nucleic acids can be used. Examples are described in U.S. Patent Application Publication No. 20060067937 and PCT Publication No. WO 06/034507.
Therapeutic nucleic acids that inhibit soluble endoglin expression The present invention also features the use of antisense nucleobase oligomers to downregulate expression of soluble endoglin mRNA directly. By binding to the complementary nucleic acid sequence (the sense or coding strand), antisense nucleobase oligomers are able to inhibit protein expression presumably through the enzymatic cleavage of the RNA strand by RNAse H. Preferably the antisense nucleobase oligomer is capable of reducing soluble endoglin protein expression in a cell that expresses increased levels of soluble endoglin. Preferably the decrease in soluble endoglin protein expression is at least 10% relative to cells treated with a control oligonucleotide, preferably 20% or greater, more preferably 40%, 50%, 60%, 70%, 80%, 90% or-greater.
Methods for selecting and preparing antisense nucleobase oligomers are well known in the art. For an example of the use of antisense nucleobase oligomers to downregulate VEGF expression see U.S. Patent No. 6,410,322, incorporated herein by reference.
Methods for assaying levels of protein expression are also well known in the art and include western blotting, immunoprecipitation, and ELISA.
The present invention also features the use of RNA interference (RNAi) to inhibit expression of soluble endoglin. RNA interference (RNAi) is a recently discovered mechanism of post-transcriptional gene silencing (PTGS) in which double-stranded RNA
(dsRNA) corresponding to a gene or mRNA of interest is introduced into an organism resulting in the degradation of the corresponding mRNA_ In the RNAi reaction, both the sense and anti-sense strands of a dsRNA molecule are processed into small RNA
fragments or segments ranging in length from 21 to 23 nucleotides (nt) and having 2-nucleotide 3' tails. Alternatively, synthetic dsRNAs, which are 21 to 23 nt in length and have 2-nucleotide 3' tails, can be synthesized, purified and used in the reaction. These 21 to 23 nt dsRNAs are known as "guide RNAs" or "short interfering RNAs"
(siRNAs).
The siRNA duplexes then bind to a nuclease complex composed of proteins that target and destroy endogenous mRNAs having homology to the siRNA within the complex. Although the identity of the proteins within the complex remains unclear, the function of the complex is to target the homologous mRNA molecule through base pairing interactions between one of the siRNA strands and the endogenous mRNA.
The mRNA is then cleaved approximately 12 nt from the 3' terminus of the siRNA and degraded. In this manner, specific genes can be targeted and degraded, thereby resulting in a loss of protein expression from the targeted gene. siRNAs can also be chemically synthesized or obtained from a company that chemically synthesizes siRNAs (e.g., Dharmacon Research Inc., Pharmacia, or ABI).
The specific requirements and modifications of dsRNA are described in PCT
Publication No. WO01/75164, and in U.S. Patent Application Publication No.
20060067937 and PCT Publication No. WO 06/034507, incorporated herein by reference.
Soluble endoglin based therapeutic compounds useful in earlypregnancy Inhibition of full-length endoglin signaling has been shown to enhance trophoblast invasiveness in villous explant cultures (Caniggia I et al, EndocrinoloSy, 1997, 138:4977-88). Soluble endoglin is therefore likely to enhance trophoblast invasiveness during early pregnancy. Accordingly, compositions that increase soluble endoglin levels early in pregnancy in a woman who does not have a pregnancy related hypertensive disorder or a predisposition to a pregnancy related hypertensive disorder may be beneficial for enhancing placentation. Examples of compositions that increase soluble endoglin levels include purified soluble endoglin polypeptides, soluble endoglin encoding nucleic acid molecules, and compounds or growth factors that increase the levels or biological activity of soluble endoglin.
Assays for gene and protein expression . The following methods can be used to evaluate protein or gene expression and determine efficacy for any of the above-mentioned methods for increasing soluble endoglin binding protein levels, or for decreasing soluble endoglin protein levels.
Blood serum from the subject is measured for levels of soluble endoglin, using methods such as ELISA, western blotting, or immunoassays using specific antibodies.
Blood serum from the subject can also be measured for levels of TGF-(31, TGF-(33, activin-A, BMP2, BMP7, or any protein ligand known to bind to soluble endoglin.
Methods used to measure serum levels of proteins include ELISA, western blotting, or immunoassays using specific antibodies. In addition, in vitro angiogenesis assays can be performed to determine if the subject's blood has converted from an anti-angiogenic state to a pro-angiogenic state. Such assays are described below in Example 4. A
result that is diagnostic of pre-eclampsia or eclampsia is considered an increase of at least 10%, 20%, preferably 30%, more preferably at least 40% or 50%, and most preferably at least 60%, 70%, 80%, 90% or more in the levels of soluble endoglin and a result indicating an improvement in the pre-eclampsia or eclampsia is a decrease of at least 10%, 20%, preferably 30%, more preferably at least 40% or 50%, and most preferably at least 60%, 70%, 80%, 90% or more in the levels of soluble endoglin. Alternatively or additionally, a result that is diagnostic of pre-eclampsia or eclampsia is considered a decrease of at least 10%, 20%, preferably 30%, more preferably at least 40% or 50%, and most preferably at least 60%, 70%, 80%, 90% or more in the levels of eNOS, PGI2, TGF-01, TGF-(33, activin-A, BMP2, BMP7, or any protein ligand known to bind to soluble endoglin and a result indicating an improvement in the pre-eclampsia or eclampsia is an increase of at least 10%, 20%, preferably 30%, more preferably at least 40% or 50%, and most preferably at least 60%, 70%, 80%, 90% or more in the levels of eNOS, PGTZ, TGF-P 1, TGF-(33, activin-A, BMP2, BMP7, or any protein ligand known to bind to soluble endoglin. A result indicating an improvement in the pre-eclampsia or eclampsia can also be considered conversion by at least 10%, preferably 20%, 30%, 40%, 50%, and most preferably at least 60%, 70%, 80%, 90% or more from an anti-angiogenic state to a pro-angiogenic state using the in vitro angiogenesis assay.
Blood serum or urine samples from the subject can also be measured for levels of nucleic acids or polypeptides encoding eNOS, TGF-f31, TGF-03, activin-A, BNIP2, BMP7, or soluble endoglin. There are several art-known methods to assay for gene expression. Some examples include the preparation of RNA from the blood samples of the subject and the use of the RNA for northern blotting, PCR based amplification, or RNAse protection assays. A positive result is considered an increase of at least 10%, 20%, preferably 30%, more preferably at least 40% or 50%, and most preferably at least 60%, 70%, 80%, 90% or more in the levels of soluble endoglin, TGF-0 1, TGF-03, activin-A, BMP2, BMP7 nucleic acids.
Therapeutic antibodies The elevated levels of soluble endoglin found in the serum samples taken from pregnant women suffering from.pre-eclampsia suggests that soluble endoglin is acting as a "physiologic sink" to bind to and deplete the trophoblast cells and maternal endothelial cells of functional growth factors required for the proper development and angiogenesis of the fetus or the placenta. The use of compounds, such as antibodies, to bind to soluble endoglin and neutralize the activity of soluble endoglin (e.g., binding to TGF-(31, TGF-f33, activin-A, BMP2, BMP7), may help prevent or treat pre-eclampsia or eclampsia, by producing an increase in free TGF-(31, TGF-(33, activin-A, BMP2, and BMP7.
Such an increase would allow for an increase in trophoblast proliferation, migration and angiogenesis required for placental development and fetal nourishment, and for systemic maternal endothelial cell health.
The present invention provides antibodies that specifically bind to soluble endoglin. Preferably, the antibodies bind to the extracellular domain of endoglin or to the ligand binding domain. The antibodies are used to neutralize the activity of soluble endoglin and the most effective mechanism is believed to be through direct blocking of the binding sites for TGF-(31, TGF-(33, activin-A, BMP2, or BMP7, however, other mechanisms cannot be ruled out. Preferred antibodies can bind to an epitope (either as a result of linear structure or three dimensional conformation) on human endoglin that includes any one or more of the peptide sequences indicated in bold and underlined in FIGRUE 30B (e.g., amino acids 40 to 86, 144 to 199, 206 to 222, 289 to 304, or 375 to 381) or to any of the preferred fragments of soluble endoglin (e.g., amino acids 1 to 437, 4 to 437, 40 to 406, or 1 to 587 of human endoglin). Methods for the preparation and use of antibodies for therapeutic purposes are described in several patents including U.S.
Patent Numbers 6,054,297; 5,821,337; 6,365,157; and 6,165,464; U.S. Patent Application Publication No. 2006/0067937; and PCT Publication No. WO 06/034507 and are incorporated herein by reference. Antibodies can be polyclonal or monoclonal;
monoclonal humanized antibodies are preferred. The present invention also includes the antibodies that bind to soluble endoglin, including but not limited to those that bind to any one or more of the peptide sequences indicated in bold and underlined in FIGRUE
30B or to any of the preferred fragments of soluble endoglin (e.g., amino acids I to 437, 4 to 437, 40 to 406, or I to 587 of human endoglin).
Therapeutic uses of antibodies When used in vivo for the treatment or prevention of pre-eclampsia or eclampsia, the antibodies of the subject invention are administered to the subject in therapeutically effective amounts. Preferably, the antibodies are administered parenterally or intravenously by continuous infusion. The dose and dosage regimen depends upon the severity of the disease, and the overall health of the subject. The amount of antibody administered is typically in the range of about 0.001 to about 10 mg/kg of subject weight, preferably 0.01 to about 5 mg/kg of subject weight.
For parenteral administration, the antibodies are formulated in a unit dosage injectable form (solution, suspension, emulsion) in association with a pharmaceutically, acceptable parenteral vehicle. Such vehicles are inherently nontoxic, and non-therapeutic. Examples of such vehicles are water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Nonaqueous vehicles such as fixed oils and ethyl oleate may also be used. Liposomes may be used as carriers. The vehicle may contain minor amounts of additives such as substances that enhance isotonicity and chemical stability, e.g., buffers and preservatives. The antibodies typically are formulated in such vehicles at concentrations of about 1 mg/ml to 10 mg/mI.
Combination therapies Optionally, a therapeutic may be administered in combination with any other standard pre-eclampsia or eclampsia therapy; such methods are known to the skilled artisan and include the methods described in U.S. Patent Application Publication Numbers 20040126828, 20050025762, 20050170444, 20060067937, and 20070104707 and PCT Publication Numbers WO 2004/008946, WO 2005/077007, and WO
06/034507.
Desirably, the invention features the use of a combination of any one or more of the therapeutic agents described herein. Given our discovery that soluble endoglin and sFlt-1 may act in concert to induce vascular damage and pregnancy related hypertensive disorders by interfering with TGF-Pl and VEGF signaling pathway respectively, possibly converging on the NOS signaling pathway, desirable therapeutic methods of the invention include the administration of a compound that decrease sFlt-1 levels or activity or increase VEGF or PIGF levels or activity in combination with a compound that decreases soluble endoglin levels or activity or increase TGF-P, NOS, or PG12 levels or activity. It will be understood by the skilled artisan that any combination of any of the agents can be used for this purpose. For example, an antibody that specifically binds to soluble endoglin can be administered in combination with VEGF. In another example, a compound that increases TGF-01 levels or activity can be administered in combination with a compound that increases VEGF or PIGF in order to target both the endoglin and the VEGF pathway. Alternatively, a combination of antibodies against both soluble endoglin and sFlt-1 may be used either directly or in an ex vivo approach (e.g., using a column that is lined with anti-soluble endoglin or sFlt-1 and circulating the patient's blood through the column). Any of these combinations can further include the administration of a compound that increases NOS levels or activity, preferably eNOS, in order to regulate the pathway downstream of the respective receptors.
In addition, the invention provides for the use of any chronic hypertension medications used in combination with any of the therapeutic methods described herein.
Medications used for the treatment of hypertension during pregnancy include methyldopa, hydralazine hydrochloride, or labetalol. For each of these medications, modes of administration and dosages are determined by the physician and by the manufacturer's instructions.
Dosages and Modes of Administration Preferably, the therapeutic is administered either directly or using an ex vivo approach during pregnancy for the treatment or prevention of pre-eclampsia or eclampsia or after pregnancy to treat post-partum pre-eclampsia or eclampsia. Techniques and dosages for administration vary depending on the type of compound (e.g., chemical compound, purified protein, antibody, antisense, RNAi, or nucleic acid vector) and are well known to those skilled in the art or are readily determined.
Therapeutic compounds of the present invention may be administered with a pharmaceutically acceptable diluent, carrier, or excipient, in unit dosage form.
Administration may be parenteral, intravenous, subcutaneous, oral or local by direct injection into the amniotic fluid. Intravenous delivery by continuous infusion is the preferred method for administering the therapeutic compounds of the present invention.
The therapeutic compound may be in form of a solution, a suspension, an emulsion, an infusion device, or a delivery device for implantation, or it may be presented as a dry powder to be reconstituted with water or another suitable vehicle before use.
The composition can be in the form of a pill, tablet; capsule, liquid, or sustained release tablet for oral administration; or a liquid for intravenous, subcutaneous or parenteral administration; or a polymer or other sustained release vehicle for local administration.
Methods well known in the- art for making formulations are found, for example, in "Remington: The Science and Practice of Pharmacy" (20th ed., ed. A.R. Gennaro AR., 2000, Lippincott Williams & Wilkins, Philadelphia, PA). Formulations for parenteral administration may, for example, contain excipients, sterile water, saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes. Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds. Nanoparticulate formulations (e.g., biodegradable nanoparticles, solid lipid nanoparticles, liposomes) may be used to control the biodistribution of the compounds. Other potentially useful parenteral delivery systems include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. The concentration of the compound in the formulation varies depending upon a number of factors, including the.dosage of the drug to be administered, and the route of administration.
The compound may be optionally administered as a pharmaceutically acceptable salt, such as non-toxic acid addition salts or metal complexes that are commonly used in the pharmaceutical industry. Examples of acid addition salts include organic acids such as acetic, lactic, pamoic, =maleic; citric, malic, ascorbic, succinic, benzoic, palmitic, suberic, salicylic, tartaric, methanesulfonic, toluenesulfonic, or trifluoroacetic acids or the like; polymeric acids such as tannic acid, carboxymethyl cellulose, or the like; and inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid phosphoric acid, or the like. Metal complexes include zinc, iron, and the like.
Formulations for oral use include tablets containing the active ingredient(s) in a mixture with non-toxic pharmaceutically acceptable excipients. These excipients may be, for example, inert diluents or fillers (e.g., sucrose and sorbitol), lubricating agents, glidants, and anti-adhesives (e.g., magnesium stearate, zinc stearate, stearic acid, silicas, hydrogenated vegetable oils, or talc).
Formulations for oral use may also be provided as chewable tablets, or as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium.
The dosage and the timing of administering the compound depends on various clinical factors including the overall health of the subject and the severity of the symptoms of pre-eclampsia. In general, once pre-eclampsia or a predisposition to pre-eclampsia is detected, continuous infusion of the purified protein is used to treat or prevent further progression of the condition. Treatment can be continued for a period of time ranging from 1 to 100 days, more preferably 1 to 60 days, and most preferably I to days, or until the completion of pregnancy. Dosages vary depending on each compound and the severity of the condition and are titrated to achieve a steady-state 20 blood serum concentration ranging from 10 to 20 ng/ml soluble endoglin;
and/or 1 to 500 pg/niL free VEGF or free P1GF, or both, preferably 1 to 100 pg/xnL, more preferably 5 to 50 pg/mL and most preferably 5 to 10 pg/mL VEGF or PIGF, or 1-5 ng of sFlt-1.
The diagnostic methods described herein can be used to monitor the pre-eclarnpsia or eclampsia during therapy or to determine the dosages of therapeutic compounds. In one example, a therapeutic compound is administered and the PAAI
is determined during the course of therapy. If the PAAI is less than 20, preferably less than 10, then the therapeutic dosage is considered to be an effective dosage. In another example, a therapeutic compound is administered and the soluble endoglin anti-angiogenic index is determined during the course of therapy. If the soluble endoglin anti-angiogenic index is less than 200, preferably less than 100, then the therapeutic dosage is considered to be an effective dosage.
Subject monitoring The disease state or treatment of a subject having pre-eclampsia, eclampsia, or a predisposition to such a condition can be monitored using the diagnostic methods, kits, and compositions of the invention. For example, the expression of a soluble endoglin polypeptide present in a bodily fluid, such as blood, serum, urine, plasma, amniotic fluid, or CSF, can be monitored. The soluble endoglin monitoring can be combined with methods for monitoring the expression of an sFlt-1, VEGF, or PIGF, TGF-0, or eNOS
polypeptide or nucleic acid, or PGI2. Such monitoring may be useful, for example, in assessing the efficacy of a particular drug in a subject or in assessing disease progression.
Therapeutics that decrease the expression or biological activity of a soluble endoglin nucleic acid molecule or polypeptide are taken as particularly useful in the invention.
Screening Assays As discussed above, the level of a soluble endoglin nucleic acid or polypeptide is increased in a subject having pre-eclampsia, eclampsia, or a predisposition to such conditions. Based on these discoveries, compositions of the invention are useful for the high-throughput low-cost screening of candidate compounds to identify those that modulate the expression of a soluble endoglin polypeptide or nucleic acid molecule whose expression is altered in a subject having a pre-eclampsia or eclampsia.
Any number of methods are available for carrying out screening assays to identify new candidate compounds that alter the expression of a soluble endoglin nucleic acid molecule. Examples are described in detail in U.S. Patent Application Publication No.
20060067937 and PCT Publication No. WO 06/034507.
In one working example, candidate compounds may be screened for those that specifically bind to a soluble endoglin polypeptide. The efficacy of such a candidate compound is dependent upon its ability to interact with such a polypeptide or a functional equivalent thereof. Such an interaction can be readily assayed using any number of standard binding techniques and functional assays such as immunoassays or affinity chromatography based assays(e.g., those described in Ausubel et al., supra).
In one embodiment, a soluble endoglin polypeptide is immobilized and compounds are tested for the ability to bind to the immobilized soluble endoglin using standard affinity chromatography based assays. Compounds that bind to the immobilized soluble endoglin can then be eluted and purified and tested further for its ability to bind to soluble endoglin both in vivo and in vitro or its ability to inhibit the biological activity of soluble endoglin.
In another example, a candidate compound is tested for its ability to decrease the biological activity of a soluble endoglin polypeptide by decreasing binding of a soluble endoglin polypeptide and a growth factor, such as TGF-P l, TGF-(33, activin-A, and BMP-7. These assays can be performed in vivo or in vitro and the biological activity of the soluble endoglin polypeptide can be assayed using any of the assays for any of the soluble endoglin activities known in the art or described herein. For example, cells can be incubated with a Smad2/3-dependent reporter construct. If desired, the cells can also be incubated in the presence of TGF-0 to enhance the signal on the Smad2/3 dependent reporter construct. The cells can then be incubated in the presence of soluble endoglin which will reduce or inhibit TGF-0-induced activation of the Smad2/3 dependent reporter construct. Candidate compounds can be added to the cell and any compound that results in an increase of TGF-0-induced activation of the Smad2/3 dependent reporter in the soluble endoglin treated cells as compared to cells not treated with the compound, is considered a compound that may be useful for the treatment of pre-eclampsia or eclampsia.
In another example, the TGF- j3-induced dephosphorylation of eNOS at Thr495 can also be used as an assay for changes in soluble endoglin biological activity. In this example, cells are incubated in the presence of soluble endoglin, which as shown in the experiments described below, inhibits the TGF-(31 dephosphorylation of Thr495 of eNOS. Candidate compounds are then added to the cells and the phosphorylation state of Thr495 is determined. Any compound that results in an increase of TGF-p-induced activation of Thr495 dephosphorylation in the soluble endoglin treated cells as compared to cells not treated with the compound, is considered a compound that may be useful for the treatment of pre-eclampsia or eclampsia.
Examples The following examples are intended to illustrate the invention. They are not meant to limit the invention in any way.
Example 1. Increased levels of endoglin mRNA and protein in pregnant women with pre-eclampsia.
In an attempt to identify novel secreted factors playing a pathologic role in pre-eclampsia, we performed gene expression profiling of placental tissue from 17 pregnant women with pre-eclampsia and 13 normal pregnant women using Affymetrix U95A
microarray chips. We found that the gene for endoglin was upregulated in women with pre-eclampsia.
In order to confirm the upregulation of endoglin in pre-eclampsia, we performed Northern blots to analyze the placental endoglin mRNA levels (FIGURE 3) and western blot analysis to measure serum protein levels of endoglin (FIGURE 4) in pre-eclamptic pregnant women as compared with normotensive pregnant women. Pre-eclampsia was defined as (1) a systolic blood pressure (BP) >140 mmHg and a diastolic BP >90 mmHg after 20 weeks gestation, (2) new onset proteinuria (1+ by dipstik on urinanalysis, >300mg of protein in a 24 hour urine collection, or random urine protein/creatinine ratio >0.3, and (3) resolution of hypertension and proteinuria by 12 weeks postpartum.
Patients with underlying hypertension, proteinuria, or renal disease were excluded.
Patients were divided into mild and severe pre-eclampsia based on the presence or absence of nephrotic range proteinuria (>3g of protein on a 24 hour urine collection or urine protein/creatinine ratio greater than 3.0). The mean urine protein/creatinine ratios in the mild pre-eclampsia group were 0.94 +/- 0.2 and in the severe pre-eclampsia group were 7.8 +/- 2.1. The mean gestational ages of the various groups were as follows:
normal 38.8 +/-0.2 weeks, mild pre-eclampsia 34 +/- 1.2 weeks, severe pre-eclampsia 31.3 +1-0.6 weeks, and pre-term. 29.5 +/- 2.0 weeks. Placental samples were obtained immediately after delivery. Four random samples were taken from each placenta, placed in RNAlater stabilization solution (Ambion, Austin, TX) and stored at -70 C.
RNA
isolation was performed using Qiagen RNAeasy Maxi Kit (Qiagen, Valencia, CA).
Northern blots probed with a 400 base pair probe in the coding region of endoglin (Unigene Hs.76753) corresponding to the N-terminal region (gene bank #BC014271) and an 18S probe as a normalization control showed an increase in placental endoglin mRNA
(see Knebelmann et al., Cancer Res. 58:226-231 (1998)). Western blots probed with an antibody to the amino terminus of endoglin showed an increase in both placental and maternal serum levels of endoglin protein in pre-eclamptic pregnant women as compared to normotensive pregnant women.
Example 2. Demonstration of a soluble endoglin polypeptide in the placentas and serum of pre-eclamptic patients.
The western blot analysis used to measure the levels of endoglin protein in placentas and serum from pre-eclamptic women suggested the presence of a smaller protein (approximately 63-65 kDa), that was present in the placenta and serum of pre-eclamptic pregnant women (FIGURES 4 and 30A). We have demonstrated that this smaller fragment is the extracellular domain of endoglin. This truncated version is likely to be shed from the placental syncitiotrophoblasts and endothelial cells and circulated in excess quantities in patients with pre-eclampsia. This soluble form of endoglin may be acting as an anti-angiogenic agent by binding to circulating ligands that are necessary for normal vascular health.
The predicted length of the soluble form of the protein is approximately 437 amino acids (including the peptide leader sequence, 412 amino acids without the leader sequence). sEng was purified from the serum of preeclamptic patients.
Fractions 4 and 5 eluted from the 44G4-IgG (anti-Eng) Sepharose, were run on SDS-PAGE under reducing conditions and tested by Western blot using a polyclonal antibody to Eng. The eluted fractions were subjected to mass spectrometry analysis (3 runs) and the peptides identified are shown in (FIGURE 30B). The purification and analysis by mass spectrometry revealed several Eng-specific peptides ranging from Gly4O to Arg406 indicating a soluble form (soluble endoglin) corresponding to the N-terminal region of the full-length protein bold on the sequence of human endoglin.
Example 3. Circulating concentrations of soluble endoglin in women with normal versus pre-eclamptic pregnancies.
In order to compare the levels of circulating, soluble endoglin from the serum of normal, mildly pre-eclamptic, or severely pre-eclamptic women, we performed ELISA
analysis on blood samples taken from these women. All the patients for this study were recruited at the Beth Israel Deaconess Medical Center after obtaining appropriate IRB-approved consents. Pre-eclampsia was defined as (1) Systolic BP >140 and diastolic BP
>90 after 20 weeks gestation in a previously normotensive patient, (2) new onset proteinuria (1+ by dipstick on urinanalysis or > 300 mg of protein in a 24 hr urine collection or random urine protein/creatinine ratio >0.3), and (3) resolution of hypertension and proteinuria by 12 weeks postparlum. Patients with baseline hypertension, proteinuria, or renal disease were excluded. For the purposes of this study, patients were divided into mild and severe pre-eclampsia based on the absence or presence of nephrotic-range proteinuria (> 3 g of protein on a 24 hour urine collection or urine protein to creatinine ratio greater than 3.0). HELLP syndrome was defined when patients had evidence of thrombocytopenia (<100000 cel.ls/ l), increased LDH
(>600 IU/L) and increased AST (>70 IU/L). Healthy pregnant women were included as controls. 8 patients with pre-term deliveries for other medical reasons were included as additional controls. Placental samples were obtained immediately after delivery. Serum was collected from pregnant patients at the time of delivery (0-12 hours prior to delivery of the placenta) after obtaining informed consent. These experiments were approved by ' the Institutional Review Board at the Beth Israel Deaconess Medical Center.
Using the serum specimens from patients described in Table 1, we measured the circulating concentrations of soluble endoglin in the various groups of pre-eclamptic patients and control pregnant patients. When pre-eclamptic patients were further sub-divided into those with and without HELLP, sEng concentrations were three-, five- and ten-fold higher in mild, severe and HELLP syndrome preeclamptics, respectively, compared to gestational age-matched pre-term controls (FIGURE 28).
Concentrations of sEng in pregnant patients correlated with those of sFltl (R2 = 0.56), except in the HELLP
group where sEng was higher than sFltl. In a subset of patients, blood samples obtained 48 hours after placental delivery showed a 70% reduction in mean sEng circulating levels in preeclamptic and normal pregnant patients (FIGURE 29).
Table 1: Clinical characteristics and circulating soluble endoglin in the various patient groups Mild pre- Severe pre- Severe pre-Normal eclampsia eclampsia, no eclampsia with Pre-term (n=30) (n=l 1) HELLP (n=17) HELLP (n=11) (n=8) Maternal age (yrs) 32.43 33.18 29.5 33.73 31.88 Gestational age (wks) 38.65 3].91* 29.06* 26.52* 30.99*
Primiparous (%) 43.3 63.6 47.1 90.9 62.5 Systolic blood pressure (mmHg) 122 157* 170* 166* 123 Diastolic blood pressure (mmHg) 72 99* 104* 103* 77 Proteinuria (g protein/g creatinine) 0.37 2.5* 8.64* 5.16* 0.6 Uric acid (mg/dl) 5.27 6.24 7.29* 6.31 7.35 Hematocrit (%) 35.5 33.6 33.7 33.5 34.3 Platelet count 238 230 249 69.4* 229 Creatinine (mg/dl) 0.55 0.62 0.62 0.64 0.67 Soluble endoglin in (ng/ml) 18.73 36.12* 52.55** 99.83*** 10.9 *P <0.05, **P<0.005 The average serum concentrations of soluble endoglin was at least two fold higher in mild pre-eclampsia and 3-4 fold higher in patients with severe pre-eclampsia. In pre-eclamptic patients complicated with the HELLP syndrome, the concentration of soluble endoglin was at least 5-10 fold higher than gestational age matched control specimens.
Additionally, the levels of soluble endoglin in pregnant patients correlate with the levels of sFlt-1 (FIGURE 18). The R2 value for correlation was 0.6. (Note that the circulating concentrations of sFlt-1 reported here are at least 4-5 fold higher than previously published (Maynard et al., supra). This is due-to a difference in the sensitivity of a new ELISA kit from R&D systems which lacks urea in the assay diluent and therefore gives consistently higher values than previously published.) In other words, patients with the highest levels of soluble endoglin also had the highest circulating levels of sFltl. The origin of soluble endoglin is most likely the syncitiotrophoblast of the placenta as evidenced by the enhanced staining seen on our placental immunohistochemistry (FIGURES 19 and 20). These figures show that endoglin protein is expressed by the syncitiotrophoblasts and is vastly upregulated in pre-eclampsia. Our western blot data (FIGLTRES 21A and 21B) and the lack of detectable alternative splice variants by northern blot supports the notion that soluble endoglin is likely a shed form of the extracellular domain of the membrane endoglin protein. It is approximately 65 kDA in size and is produced at elevated levels in pre-eclamptic placentas and it circulates in higher amounts in pre-eclamptic sera. This protein was present at much lower levels in the sera of normal pregnant women and barely detectable in non-pregnant women.
Soluble endoglin expression in pre-eclamptic placenta was four-fold higher than in normal pregnancy (n=l-/group, P<0.01). Quantitation of sEng/Eng in these specimens showed no significant difference between normal (0_43) and preeclamptic (0.56) placentae (n = 10/group, P = 0.4), suggesting that sEng is derived from the full-length protein and that both Eng and sEng are similarly increased in preeclampsia.
The following methods were used for some of the experiments described in this example.
Immunohistochemistry Immunohistochemistry on placental samples for endoglin and a-Smooth muscle actin (SMA) was done as reported by (Leach et al., Lancet 360:1215-1219 (2002)).
Briefly, the frozen placenta section obtained from patients without preeclampsia (n=10) and with preeclampsia (n=10) slides were incubated with a serum-free protein blocking solution (DAKO) for 30 minutes at room temperature and then with the primary antibody at room temperature (mouse monoclonal anti-Endoglin: 1:50 dilution; DAKO) for hours. The slides were then washed with phosphate buffered saline for 10 minutes. The secondary antibody, Rhodamine conjugated sheep anti-mouse IgG, 1:200 dilution (Biomeda) was applied for 1 hour. Sections were again washed with phosphate buffered saline and subsequently incubated with a 1:400 dilution of FITC-conjugated mouse anti-human SMA (Dako) for 30 minutes at room temperature. Immunoreactivity of Endoglin was reviewed using a SPOT advanced imaging system (RT SLIDER Diagnostic Instruments, Inc) by a pathologist who was blinded to the clinical diagnosis.
ELI,SA and Western blots ELISA was performed using a commercially available ELISA kit from R & D
Systems, MN (for example, Cat # DNDG00) and as previously described (Maynard et al, J. Clin. Invest. 111:649-658, 2003). Western blots were performed essentially as described previously (Maynard et al, supra, and Kuo et al. Proc. Natl. Acad.
,Sci.
98:4605-4610 (2001))).
Immunoprecipitation (IP) experiments IP followed by western blots were used to identify and characterize soluble endoglin in the placental tissue and serum specimens from patients with pre-eclampsia.
Human placental tissue was washed with cold PBS and lysed in homogenization buffer [10 mM Tris-HCI, pH 7.4; 15 mM NaCl; 60 mM.KCI; 1 mM EDTA; 0.1 mM EGTA;
0.5% Nonidet P-40; 5% sucrose; protease mixture from Roche (Indianapolis, IN)]
for 10 minutes. Placental lysates were then subjected to immunoprecipitation with an anti-human monoclonal mouse endoglin antibody (mAb P4A4, Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Immunoaffinity columns were prepared by the directional coupling of 3-5 mg of the purified antibody to 2m1 protein A-Sepharose using an immunopure IgG orientation kit (Pierce Chemical Co., Rockford, Illinois, USA) according to the manufacturer's instructions. Columns were then washed extensively with RIPA buffer containing protease mixture, and bound proteins were eluted with 0.1 mol/L glycine-HC1 buffer, pH 2.8. The eluent was collected in 0.5-m1 fractions containing 1 mol/L Tris-HCI buffer. Protein-containing fractions were pooled and concentrated 9- to 10-fold with CENTRICON Centrifugal Concentrator (Millipore Corp., Bedford, Massachusetts, USA). The immunoprecipitated samples were separated on a 4-12% gradient gel (Invitrogen) and proteins were transferred to polyvinylidene difluoride (PVDF) membranes. Endoglin protein was detected by western blots using polyclonal anti-human rabbit endoglin primary antibody (H-300, Santa Cruz Biotechnology, Inc., Santa Cruz, CA).
Purification of soluble endoglin and analysis by mass spectrometry Serum (10 ml) from preeclamptic patients was sequentially applied onto CM Aff -gel blue and protein A Sepharose (Bio-Rad) columns to remove albumin and immunoglobulins, respectively. The flow through was slowly applied to a 2.5 ml column of mAb 44G4 IgG to human Eng, conjugated to Sepharose (Gougos et al., Int, Immuno.
4:83-92, (1992)). Bound fractions were eluted with 0.02 M diethylamine pH 11.4 and immediately neutralized with I M Tris pH 7.8. Fractions 4 and 5 with elevated absorbance at 280 nm were pooled, reduced with 10 mM DTT for 1 h at 57 C and alkylated with 0.055 M iodoacetomide. The samples were then completely digested with trypsin (1:100). The lyophilized sample was resuspended in 0.1% tri-fluoroacetic acid and injected in a CapLC (Waters) HPLC instrument. Peptides were separated using a 75 pm Nano Series column (LC Packings) and analyzed using a Qstar XL MS/MS
system.
The data was searched using the Mascot search engine (Matrix Science) against the human protein database, NCBInr.
Example 4. Model assay for angiogenesis An endothelial tube assay can be used an in vitro model of angiogenesis.
Growth factor reduced Matrigel (7 mg/mL, Collaborative Biomedical Products, Bedford, MA) is placed in wells (100 pl/well) of a pre-chilled 48-well cell culture plate and is incubated at 37 C for 25-30 minutes to allow polymerization. Human umbilical vein endothelial cells (30,000 + in 300 l of endothelial basal medium with no serum, Clonetics, Walkersville, MD) at passages 3-5 are treated with 10% patient serum, plated onto the Matrigel coated wells, and are incubated at 37 C for 12-16 hours. Tube formation is then assessed through an inverted phase contrast microscope at 4X (Nikon Corporation, Tokyo, Japan) and is analyzed (tube area and total length) using the Simple PCI imaging analysis software.
Example S. Soluble endoglin protein levels as a diagnostic indicator of pre-eclampsia and eclampsia in women (Romero Study).
This study was designed to evaluate whether soluble endoglin is altered during clinical pre-eclampsia and whether it can be used to predict pre-eclampsia and eclampsia in women.
This study was done under collaboration with Dr. Roberto Romero, at the Wayne State University/NICHD Perinatology Branch, Detroit, MI. A retrospective longitudinal case-control study was conducted using a banked biological sample database as previously described in Chaiworapongsa et al. (The Journal of Matemal-Fetal and Neonatal Medicine, January 2005, 17 (1):3-18). All women were enrolled in the prenatal clinic at the Sotero del Rio Hospital, Santiago, Chile, and followed until delivery.
Prenatal visits were scheduled at 4-week intervals in the first and second trimester, and every two weeks in the third trimester until delivery. Plasma samples were selected from each patient only once for each of the following six intervals: (1) 7-16 weeks, (2) 16-24 weeks, (3) 24-28 weeks, (4) 28-32 weeks, (5) 32-37 weeks, and (6) >37 weeks of gestation. For each pre-eclamptic case, one control was selected by matching for gestational age (+/- 2 weeks) at the time of clinical diagnosis of pre-eclampsia. The clinical criteria for the diagnosis of,pre-eclampsia were the same as previously described in Chaiworapongsa et al, supra.
Measurement of Plasma Endoglin Levels The plasma samples stored at -70 C were thawed and plasma soluble endoglin levels were measured in one batch using the commercially available ELISA kits from R&D systems, Minneapolis, MN.(Catalog # DNDG00).
Stastistical Analysis Analysis of covariance was used to assess the difference in plasma concentrations of soluble endoglin between patients destined to develop pre-eclampsia and in normal pregnancy after adjusting for gestational age at blood sampling and intervals of sample storage. Chi-square or Fisher's exact tests were employed for comparisons of proportions. The statistics package used was SPSS V.12 (SPSS Inc., Chicago, IL).
Significance was assumed for a p value of less than 0.05.
Results The clinical characteristics of the study population are described in Table 2.
The group with pre-eclampsia included more nulliparous women and delivered earlier than the control group. Importantly, the birth weights of the fetuses were smaller in the pre-eclamptic group and there were a higher proportion of women carrying small-for-gestational-age (SGA) infants.
Table 2. Clinical characteristics of the study population Normal Pre-eclampsia p pregnancy n = 44 n=44 Age (y) 29t6 26:L 6 0.04*
N=ulliparity 11 30 <0.001 (25%) (68.2%) *
Smoking 10 1 0.007*
(22.7%) (2.3%) GA at delivery (weeks) 39.7=1: 1.1 36.9-+2.7 <0.00 1 *
Birthweight (grams) 3,372 383 2,710 766 <0.001 *
Birthweight <10`'' percentile 0 16 <0.001 (36.4 %) *
Value expressed as mean sd or number (percent) GA: gestational age The clinical characteristics of patients with pre-eclampsia are described in Table 3. Thirty-two (72%) of the patients had severe pre-eclampsia, while 10 patients had severe early-onset pre-eclampsia defined as onset <34 weeks.
Table 3. Clinical characteristics of patients with pre-eclampsia Blood pressure (mmHg) Systolic 155 15 Diastolic 100 f 8 Mean arterial pressure 118 9 Proteinuria (dipstick) 3 0.8 Aspartate aminotransferase" (SGOT) (U/L) 29 31 Platelet counta (x 103) ( /L) 206 t 59 Severe pre-eclampsia 32 (72.7%) GA at pre-eclampsia diagnosed <34 weeks 10 (22.7%) GA at pre-eclampsia diagnosed >37 weeks 27 (61.4%) Value expressed as mean sd or number (percent) C (n =26); a (n=42) The serum soluble endoglin levels in the controls and the pre-eclamptic women measured in the 6 gestational age windows are shown in Table 4. Amongst the pre-eclamptics, their specimens were divided into two groups - clinical pre-eclampsia (samples taken at the time of symptoms of pre-eclampsia) and preclinical pre-eclampsia (samples taken prior to clinical symptoms). The data shows that at mid-pregnancy (24-28 weeks of gestation), serum soluble endoglin concentrations start rising in women destined t`o develop pre-eclampsia and become at least 3 fold higher than conttols by 28-32 weeks of gestation. Blood samples taken from women with clinical pre-eclampsia show a very dramatic (nearly 10-15 fold) elevation when compared to gestational age matched controls.
Table 4. Plasma soluble endoglin concentrations in normal pregnancy and pre-eclampsia Normal p Pre-clinical p Clinical samples pregnancy samples Pre-eclampsia Pre-eclampsia 151 blood sampling (7.1-16 weeks) -'"
Soluble Endoglin (ng/mi) 3.89 t.928 0.9 3.96 :~ 1.28 Gestational age (weeks) 12.3 f 2.2 0.2 11.6 2.4 Range 8.4-15.9 7.7 - 15.1 n=37 n=34 2nd blood sampling (16.1-24 weeks) Soluble Endoglin (ng/ml) 3.36 4:1.11 0.1 3.79 + 1.37 Gestational age (weeks) = 19.4 f 1.7 0.06 20.2 t 2.1 Range 16.3 -23.4 16.7 -24.0 n=44 n=36 3'a blood sampling(24.1-28 weeks) Soluble Endoglin (ng/ml) 3.18 -L .729 0.009* 5.27 t 4.12 Gestational age (weeks) 25.9 f 1.3 0.2 26.4 +1.1 Range 24.1 -28.0 24.6 -28.0 n=38 n=29 4s` blood sampling (28.1-32 weeks) Soluble Endoglin (ng/ml) 3.7 zE 1.1 <0.001 * 10.2 :E9.8 0.01 * 96.1 t 25.7 0.05 Gestationat age (weeks) 29.9 f 1.1 1.0 30.2 ;1z 1.0 1.0 30.4 t 1.4 1.0 Range 28.3-32.0 28.7 -32.0 29.4 -31.4 n=42 n=33 n=2a 5s' blood sampling (32.1-36.9 weeks 5.79 2.42 0.003* 10.5116,59 <0.001 43.14 f 25.6 <0.001 *
Soluble Endoglin (ng/ml) 34.7 f 1.3 1.0 34.8 t 1.5 1.0 34.5 ~ 1.2 1.0 Gestational age (weeks) 32.4 -36.6 32.6 -36.7 32.6 -36.6 Range n=37 n=20 n=13 6'h blood samnling (?=37weeks) Soluble Endoglin (ng/ml) 8.9 zi: 4.5 -- 15.23 ~ 10.61 0.006*
Gestational age (weeks) 39.4 t 1.0 38.8 + 1.1 0.05 Range 37.0 - 40.7 37.6 - 41.4 n=27 n=27 pa :compared between samples at clinical manifestation of pre-eclarnpsia and normal pregnancy Value expressed as mean sd 2 pre-eclamptic patients had no blood samples available at clinical manifestation To examine the relationship between plasma soluble endoglin concentrations and the interval to clinical diagnosis of pre-eclampsia, plasma samples of pre-eclamptic patients at different gestational ages were stratified according to the interval from blood sampling to clinical diagnosis into five groups: (1) at clinical diagnosis, (2) 2-5.9 weeks before clinical manifestation, (3) 6-10.9 weeks before clinical.manifestation, (4) 11-15.9 weeks before clinical manifestation, and (5) 16-25 weeks before clinical manifestation.
The data shown in Table 5 demonstrates that the plasma soluble endoglin levels start going up at 6-10.9 weeks before onset of symptoms in pre-eclamptics and are at least 3 fold higher at 2-5.9 weeks before symptoms in women destined to develop pre-eclampsia.
Table 5. Plasma soluble endoglin concentrations in normal and pre-eclamptic pregnant women.
Blood sampling Normal pregnancy Pre-eclampsia p At clinical manifestation Soluble Endoglin (ng/ml) 7.63 ~ 4.22 27.72 + 26.20 <0.001 *
Gestational age (weeks) 37.2 ~ 3.0 37.1:h 2.7 0.9 Range 28.9 -40.7 29.4 -- 41.4 n=42 n=42 S
2-5.9 weeks before clinical manifestation 4.67 2.32 15.07 + 10.15 <0.001*
Soluble Endoglin (ng/ml) 31.6 =L 3.8 32.8 f 2.8 0.2 Gestational age (weeks) 24.1 -36.3 27.1- 36.7 Range n=27 n=27 3.8 + 1.1 Interval before clinical manifestation (weeks) 6-10.9 weeks before clinical manifestation 3.61 -+1.05 5.89 3.07 <0.001*
Soluble Endoglin (ng/ml) 28.5 +2.9 28.5 =L 2.9 0.9 Gestational age (weeks) 19.7-32.6 19.6 -34.4 Range n=37 n=37 8.3 -+1.4 Interval before clinical manifestation (weeks)
11-15.9 weeks before clinical manifestation 3.35 +0.77 3.57 ~ 0.92 0.5"
Soluble Endoglin (ng/ml) 24.5 d: 3.1 24.2 ~ 3.3 0.8 Gestational age (weeks) 17.6- 27.9 17.7 - 28.0 Range n=19 n=19 13.2 t 1.3 Interval before clinical manifestation (weeks) 16-25 weeks before clinical manifestation 3.44 ~ 1.07 3.69 =L 1.18 0.3 Soluble Endoglin (ng/ml) 17.6 3.5 16.5 :b 4.5 0.2 Gestational age (weeks) 9.1- 23.4 8.0 -22.7 Range n=42 n=42 Interval before clinical manifestation 20.6 :h 3.6 (weeks) Value expressed as mean sd 2 pre-eclamptic patients had no blood samples available at clinical manifestation To examine the diagnostic potential of plasma soluble endoglin concentrations to identify those destined to develop pre-eclampsia, patients were stratified into early onset pre-eclampsia (PE<34 weeks) and late onset pre-eclampsia (PE>34 weeks). For patients with early-onset pre-eclampsia, the mean plasma soluble endoglin levels was significantly higher in pre-eclampsia (before clinical diagnosis) than in normal pregnancy starting around 16-24 weeks of gestation (Table 6) with very dramatic differences in 24-28 week and 28-32 week gestational windows. In contrast, for patients with late-onset pre-eclampsia, plasma soluble endoglin concentrations in pre-clinical pre-eclampsia was significantly higher than in nonnal pregnancy only at 28-32 weeks with very dramatic differences at 32-36 week of gestation (Table 7).
Table 6. Plasma soluble endoglin concentrations in normal pregnant women and patients who developed clinical Pre-eclam sia at 34 weeks of gestation or less.
Normal p Pre-olinical samples p Clinical samples pregnancy Pre-eclampsia pre-colampsia l" blood samplin (7.1-16 weeks) =
Soluble Endoglin (ng/ml) 3.89 t.928 0.7 3.81 * 1.11 Gestational age (weeks) 12.3 :L 2.2 0.4 11.6 f 2.6 Range 8.4-15.9 8.0 -15.1 n=37 n=8 2ad blood sampling (16.1-24 weeks) Soluble Endoglin (ng/ml) 3.36 f 1.11 0.02* 4.60 d:1.72 Gestational age (weeks) 19.4 f 1.7 0.7 19.8 f 2.9 Range 16.3 -23.4 17.3 -23.9 n=44 n=7 3'd blood sampling (24.1-28 weeks) Soluble Endoglin (ng/ml) 3.189 f.729 <01001 10.22 :E 6.17 Gestational age (weeks) 25.9 f 1.3 0.03* 26.8 t 0.6 Range 24. l -28.0 26.0 -27.3 n=38 n=6 4N blood sampling (28.1-32 weeks) = Soluble Endoglin (ng/ml) 3.70 t 1.10 0.01* 17.66 8.9 0.008* 96.10 t 25.76 0.05 Gestational age (weeks) 29.9 :k 1.1 1.0 29.7 t 1.1 1.0 30.4 :1: 1.4 1.0 Range 28.3-32.0 28.7-31.3 29.4-31.4 n=42 n=6 n=2 5ei blood sampling (32.1-36.9 weeks) Soluble Endoglin (ng/ml) 5.79 t 2.42 53.38 zk 32.09 0.001 *
Gestational age (weeks) 34.7 :~ 1.3 33.5 f 0.5 <0.001 *
Range 32.4 -36.6 32.6 -34.0 n=37 n=6 p :compared between samples at clinical manifestation of pre-eclampsia and normal pregnancy Value expressed as mean sd 6:2 pre-eclamptic patients had no blood samples available at clinical manifestation Table 7. Plasma soluble endoglin concentrations in normal pregnant women and pre-eclamptics (34 weeks of gestation) Normal p Pre-clinical samples p Clinical pp, pregnancy Pre-eclampsia samples Pre-eclampsia )A1 blood sam hnp,(7.l-16 weeks) Soluble Endoglin (ng/ml) 3.89 ,928 0.9 4.01f 1.35 Gestational age (weeks) 12.3 t 2.2 0.2 1 S.6 f 2.4 Range 8.4-15.9 7.7 - 15.1 n=37 n=26 2"d blood samplina (16.1-24 weeks) Soluble Endoglin (ng/ml) 3.36 f 1.11 0.4 3.59 t 1.23 Gestational age (weeks) 19.4 t 1.7 0.04* 20.3 t 1.9 Range 16.3 -23.4 16.7 -24.0 n=44 n=29 3'd blood samulinp (24.1-28 weeks) Soluble Endoglin (ng/ml) 3.18 t.729 0.1 3.98 ~z 2.13 Gestational age (weeks) 25.9 11.3 0.4 ' 26.3 d: 1.1 Range 24.1 -28.0 24.6 -28.0 n=38 n=23 4'h blood sampling (28.1-32 weeks) Soluble Endoglin (ng/ml) 3.70 t 1.10 0.001* 8.57 f 9.45 Gestational age (weeks) 29.9 1.1 0.2 30.3 t 1.0 Range 28.3-32.0 28.7 -32.0 n=42 n=27 5'h blood sampling (32.1-36.9 weeks) Soluble Endoglin (ng/ml) 5.79 :h 2.42 <0.001 * 10.51 f 6.59 <0,001 34,36 f 16.30 <0.001 *
Gestational age (weeks) 34.7 f 1.3 1.0 34.8 t 1.5 0.9 35.4 f 0.9 0.7 Range 32.4 -36.6 32.6 -36.7 34.3 -36.6 n=37 n=20 n=7 6 i blood samplinp (>=37weeks) Soluble Endoglin (ng/mi) 8.98 t 45.12 -- 15.23 10.61 0.006*
Gestational age (weeks) 39.411.0 38.8 1.1 0.05 Range 37.0 - 40.7 37.6 - 41.4 n=27 n=27 p:compared between samples at clinical manifestation of pre-eclampsia and normal pregnancy Value expressed as mean. sd ~
Summary The results of these experiments demonstrate that women with clinical pre-eclarnpsia have very high levels of circulating soluble endoglin when compared to gestational age matched controls. The results also demonstrate that women destined to develop pre-eclampsia (pre-clinical pre-eclampsia) have higher plasma soluble endoglin levels than those who are predicted to have a normal pregnancy. The increase in soluble endoglin levels is detectable at least 6-10 weeks prior to onset of clinical symptoms.
Finally, these results demonstrate that both early onset and late onset pre-eclampsia have elevated circulating soluble endoglin concentrations, but the alterations are more dramatic in the early onset pre-eclampsia.
Example 6. Soluble endoglin protein levels as a diagnostic indicator of pre-eclampsia and eclampsia in women (CPEP Study).
As described above, we have discovered that soluble endoglin, a cell surface receptor for the pro-angiogenic protein TGF-(3 and expressed on endothelium and syncytiotrophoblast, is upregulated in pre-eclarnptic placentas. In the experiments described above, we have shown that in pre-eclampsia excess soluble endoglin is released from the placenta into the circulation through shedding of the extracellular domain;
soluble endoglin may then synergize with sFltl, an anti-angiogenic factor which binds placental growth factor (PIGF) and VEGF, to cause endothelial dysfunction. To test this hypothesis, we compared serum concentrations of soluble endoglin, sFltl, and free PIGF
throughout pregnancy in women who developed pre-eclampsia and in those women with other pregnancy complication such as gestational hypertension (GH) and pregnancies complicated by small-for-gestational (SGA) infants to those of women with nonnotensive control pregnancies. This study was done in collaboration with the Dr.
Richard Levine at the NIH.
There were two principal objectives of this study. The first objective was to determine whether, in comparison with normotensive controls, elevated serum concentrations of soluble endoglin, sFltl, and reduced levels of P1GF can be detected before the onset of pre-eclampsia and other gestational disorders such as gestational hypertension or pregnancies complicated by small-for-gestational (SGA) infants. The second objective was to describe the time course of maternal serum concentrations of soluble endoglin, sFlt-1, and free PIGF with respect to gestational age in women with pre-eclampsia, gestational hypertension, or SGA with separate examination of specimens obtained before and after onset of clinical symptoms, and in normotensive controls.
Methods Clinical information This study was a case control study of pregnancy complications (premature pre-eclampsia, term pre-eclampsia, gestational hypertension, pregnancies with SGA
infants, normotensive control pregnancies) nested within the cohort of 4,589 healthy nulliparous women who participated in the Calcium for Pre-eclampsia Prevention trial (CPEP). 120 random cases were selected from each of the study groups. The study methods were identical to the nested case control study recently performed for pre-eclampsia (Levine et al, N. Eng. J. Med. 2004, 350:672-83). From each woman blood specimens were obtained before study enrollment (13-21 wks), at 26-29 weeks, at 36 weeks, and on suspicion of hypertension or proteinuria. All serum specimens collected at any time during pregnancy before onset of labor and delivery were eligible for the study. Cases included 120 women who developed term pre-eclampsia, gestational hypertension, or SGA and who delivered a liveborn or stillborn male baby without known major structural or chromosomal abnormalities, and from whom a baseline serum specimen was obtained.
For premature pre-eclampsia, defined as (PE<37 weeks) all 72 patients from the CPEP
cohort were studied. The clinical criterion for the diagnosis of pre-eclampsia is described in Levine et al., (2004), surpa. All cases of gestational hypertension were required to have a normal urine protein measurement within the interval from 1 day prior to onset of gestational hypertension through 7 days following. SGA was defined as <10th and <5th (severe SGA) percentile, using Zhang & Bowes' tables of birthweight for gestational age, specific for race, nulliparity, and infant gender. Controls were randomly selected from women without pre-eclampsia or gestational hypertension or SGA who delivered a liveborn or stillborn baby without known major structural malformations or chromosomal anomalies and matched, one control to one case, by the clinical center, gestational age at collection of the first serum specimen I wk), by freezer storage time (zL 1 year), and by number of freeze-thaws. A total of 1674 serum speciinens were studied.
Matching by gestational age was done to control for gestational age-related differences in levels of sFlt-1, VEGF, and P1GF. Matching for freezer storage time was done to minimize differences due to possible degradation during freezer storage. Matching by clinical center was done to control for the fact that pre-eclampsia rates differed significantly between centers, perhaps due to differences in the pathophysiology of the disease. In addition, the centers may have used slightly different procedures for collecting, preparing, and storing specimens. Matching by number of thaws was also performed to ensure that cases and controls will have been subjected equally to freeze thaw degradation.
ELISA measurements ELISA for the various angiogenic markers were performed at the Karumanchi laboratory by a single research assistant that was blinded to the clinical outcomes.
Commercially available ELISA kits for soluble endoglin (DNDGOO), sFItl (DVR 100), PIGF (DPGOO) were obtained from R&D systems, (Minneapolis, MN).
Statistical analysis T-test was used for the comparison of the various measurements after logarithm tic transformation to determine significance. P<0.05 was considered as statistically significant.
Results The mean soluble endoglin (FIGURE 6), sFltl (FIGURE 7) and P1GF (FIGURE
8) concentrations for the five different study groups of pregnant women throughout pregnancy during the various gestational age group windows= as described in the methods are shown in FIGURES 6-8. For the pre-eclampsia groups and gestational hypertensive groups, specimens taken after onset of clinical symptoms are not shown here.
Compared with gestational age-matched control specimens, soluble endoglin and sFitl increased and free P1GF decreased beginning 9-11 weeks before preterm pre-eclampsia, reaching levels 5-fold (46.4 vs 9.8 ng/ml, P<.0001) and 3-fold higher (6356 vs 2316 pg/ml, P<.0001) and 4-fold lower (144 vs 546 pg/ml, P<.0001), respectively, after pre-eclampsia onset. For term pre-eclampsia, soluble endoglin increased beginning 12-14 weeks, free PIGF decreased beginning 9-11 weeks, and sFltl increased <5 weeks'before pre-eclampsia onset. Serum concentrations of sFlt1 and free PIGF did not differ significantly between pregnancies with SGA or average for gestation age/large for gestation age (AGA/LGA) infants from 10-42 weeks of gestation. Serum soluble endoglin was modestly increased in SGA pregnancies beginning at 17-20 weeks (7.2 vs 5.8 ng/ml, P=.03), attaining concentrations of 15.7 and 43.7 ng/ml at 37-42 weeks for mild and severe SGA, respectively, as compared with 12.9 ng/ml in AGA/LGA pregnancies (severe SGA vs AGA/LGA, P=.002). In the gestational hypertension study, compared with GA-matched control specimens, modest increases in soluble endoglin were apparent <1-5 weeks before gestational hypertension, reaching levels 2-fold higher for soluble endoglin (29.7 vs 12.5 ng/ml, P=.002) after onset of gestational hypertension.
The adjusted odds ratio for subsequent preterm PE for specimens obtained at 21-32 weeks which were in the highest quartile of control soluble endoglin concentrations (>7.2 ng/ml), as compared to all other quartiles, was 9.8 (95% CI 4.5-21.5).
The soluble endoglin anti-angiogenic index for pre-eclampsia was defined as (sFltl + 0.25 soluble endoglin)/P1GF. The index was calculated throughout the various gestational age groups for the five different study groups. The soluble endoglin anti-angiogenic index for pre-eclampsia anti-angiogenesis for samples taken prior to clinical symptoms=is shown in FIGURE 9. Elevated values for the soluble endoglin anti-angiogenic index were noted as early as 17-20 weeks of pregnancies and seemed to get more dramatic with advancing gestation in severe pre-mature pre-eclampsia. In term pre-eclampsia, SGA and GH, there was a modest elevation during the end of pregnancy (33-36 weeks) when compared to control women.
FIGURES 10 and 11 depict the mean concentrations of soluble endoglin (FIGURE 10) and soluble endoglin anti-angiogenic index (FIGURE 11) according to the number of weeks before clinical premature pre-eclampsia (PE <37 weeks). Even as early 9-11 weeks prior to the onset of premature pre-eclampsia, there was a 2-3 fold elevation in soluble endoglin and soluble endoglin anti-angiogenic index in women destined to develop pre-eclampsia with dramatic elevations (>5 fold) in 1-5 weeks preceding clinical symptoms.
FIGURES 12 and 13 show the alteration in soluble endoglin (FIGURE 12) and the soluble endoglin anti-angiogenic index (FIGURE 13) throughout pregnancy for term pre-eclampsia (PE>37 weeks) before and after symptoms. Elevation in soluble endoglin and the soluble endoglin anti-angiogenic index are noted starting at 33-36 weeks of pregnancy reaching on average 2-fold higher levels at the time of clinical pre-eclampsia.
FIGURES 14 and 15 show a modest elevation in soluble endoglin (FIGURE 14) and the soluble endoglin anti-angiogenic index (FIGURE 15) detected in women during gestational hypertension, and 1-5 weeks preceding gestational hypertension (during 33-36 week of pregnancy) when compared to normotensive controls.
FIGURES 16 and 17 show a modest elevations in soluble endoglin (FIGURE 16) and the soluble endoglin anti-angiogenic index (FIGURE 17) detected during the week gestational windows in women with severe SGA and not in all women with SGA
when compared to control pregnancies.
Summary The results of this study show that the soluble endoglin levels and soluble endoglin anti-angiogenic index levels, when measured prior to 33 weeks of pregnancy, was dramatically elevated in women destined to develop premature pre-eclampsia and in women with clinical premature pre-eclampsia (PE <37 weeks) when compared to normal control pregnancy. Therefore, soluble endoglin levels and soluble endoglin anti-angiogenic index levels (prior to 33 weeks) can not only be used for the diagnosis of premature pre-eclampsia, but also for the prediction of pre-eclampsia. It appears that elevations in soluble endoglin levels and soluble endoglin anti-angiogenic index levels start as early as 10-12 weeks prior to symptoms of pre-eclampsia.
The soluble endoglin levels and soluble endoglin anti-angiogenic index levels were also significantly elevated in term pre-eclampsia (PE >37 weeks) and modestly elevated in gestational hypertension and severe SGA when measured late in pregnancy (33-36 week gestational windows). Therefore, soluble endoglin levels and soluble endoglin anti-angiogenic index levels can also be used to identify other pregnancy complications such as SGA and gestation hypertension when measured after 33 weeks of pregnancy.
Example 7. Involvement of soluble endoglin in the pathogenesis of pre-eclampsia.
We have shown that endoglin, a cell surface receptor for the pro-angiogenic protein TGF-0 and expressed on endothelium and syncytiotrophoblast, is upregulated in pre-eclamptic placentas. We have also shown that in pre-eclampsia, excess soluble endoglin is released from the placenta into the circulation through shedding of the extracellular domain. The experiments described below were designed to test the hypothesis that soluble endoglin may synergize with sFltl, an anti-angiogenic factor which binds placental growth factor (PIGF) and VEGF, to cause endothelial dysfunction.
Materials and Methods Reagents Recombinant Human endoglin, human sFltl, mouse endoglin, mouse sFltl, human TGF-pl, human TGF-P3, mouse VEGF were obtained from R&D systems (Minneapolis, MN). Mouse monoclonal antibody (catalog # sc 20072) and polyclonal antibody (sc 20632) against the N-terminal region of human endoglin was obtained from Santa Cruz Biotechnology, Inc. ELISA kits for human sFlt1, mouse sFltl and human soluble endoglin were obtained from R&D systems, MN.
Generation ofadenoviruses Adenoviruses against sFltl and control adenovirus (CMV) have been previously described (Maynard et al, J. Clin. Invest. 111: 649:658 (2003)) and were generated at the Harvard Medical Core facility in collaboration with Dr. Richard Mulligan. To create the soluble endoglin adenovirus, we used the Adeasy Kit (Stratagene). Briefly, human soluble endoglin (encoding the entire extracellular region of the endoglin protein) was PCR amplified using human cDNA full length endoglin clone (Invitrogen, CA) as the template and the following oligonucleotides as primers: forward 5'-ACG AAG CTT
GAA ACA GTC CAT TGT GAC CTT-3' (SEQ ID NO: 3) and reverse 5'TTA GAT
ATC TGG CCT TTG CTT GTG CAA CC-3' (SEQ ID NO: 4). Amplified PCR
fragments were initially subcloned into pSecTag2-B (Invitrogen, CA) and the DNA
sequence was confirmed. A mammalian expression construct encoding His-tagged human soluble endoglin was PCR amplified using pSecTag2 B-soluble endoglin as the template and subcloned into pShuttle-CMV vector (Stratagene; Kpnl and Scal sites), an adenovirus transfer vector, for adenovirus generation. Adenovirus expressing soluble endoglin (sE) was -then. generated using the standard protocol per manufacturer instructions and confirmed for expression by western blotting. The confirmed clone was then amplified on 293 cells and purified on a CsC12 density gradient as previously described (Kuo et al, Proc. Natl. Acad. Sci. USA 98:4605-4610 (2001)). The final products were titered by an optical absorbance method (Sweeney et al, Virology, 2002, 295:284-288). The titer is expressed as plaque forming units (pfu)/mL based on a formula derived from previous virus preps that were titered using the standard plaque dilution based titration assay kit (BD Biosciences Clontech, Palo Alto, CA, Cat. No.
K1653-1) and the optical absorbance method.
Western blots Western blots were used for checking the expression of adenoviral-infected transgenes in the rat plasma as described elsewhere (Maynard et al, supra).
Imrrzunoprecipitation (IP) experiments IP followed by western blots were used to identify and characterize soluble endoglin in the placental tissue and serum specimens from patients with pre-eclampsia.
Human placental tissue was washed with cold PBS and lysed in homogenization buffer [10 mM Tris-HCI, pH 7.4; 15 mM NaCI; 60 mM KCI; 1 mM EDTA; 0.1 mM EGTA;
0.5% Nonidet P-40; 5% sucrose; protease mixture from Roche (Indianapolis, IN)]
for 10 minutes. Placental lysates were then subjected to immunoprecipitation with an anti-human monoclonal mouse endoglin antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Immunoaffinity columns were prepared by the directional coupling of mg of the purified antibody to 2ml protein A-Sepharose using an immunopure IgG
orientation kit (Pierce Chemical Co., Rockford, Illinois, USA) according to the manufacturer's instructions. Columns were then washed extensively with RIPA
buffer containing protease mixture, and bound proteins were eluted with 0.1 mol/L
glycine-HC1 buffer, pH 2.8. The eluent was collected in 0.5-mi fractions containing 1 mol/L Tris-HCl buffer. Protein-containing fractions were pooled and concentrated 9- to 10-fold with CENTRICON Centrifugal Concentrator (Millipore Corp., Bedford, Massachusetts, USA).
The immunoprecipitated samples were separated on a 4-12% gradient gel (Invitrogen) and proteins were transferred to polyvinylidene difluoride (PVDF) membranes.
Endoglin protein was detected by western blots using rabbit polyclonal antibody to human endoglin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).
Endothelial tube assay Growth factor reduced matrigel (7mg/mL, Collaborative Biomedical Products, Bedford, MA) was placed in wells (1001/well) of a pre-chilled 48-well cell culture plate and incubated at 37 C for 30 minutes to allow polymerization. HUVEC cells (30,000 +
in 300 l of endothelial basal medium with no serum, Clonetics, Walkersville, MD) were treated with various combinations of recombinant protein (soluble endoglin, sFltl, or both) and plated onto the Matrigel coated wells, and incubated at 37 C for 12-16 hours.
Tube formation was then assessed through an inverted phase contrast microscope at 4X
(Nikon Corporation, Tokyo, Japan) and quantitatively analyzed (tube area and total length) using the Simple PCI imaging analysis software.
Microvascular permeability experiments Balb-C mice were injected through the retro-orbital venous plexus with lx 108 pfu of adenovirus expressing GFP or soluble endoglin or sFitl or combinations and microvascular permeability assay was performed 48 hours later. Mice were anesthetized by IP injection of 0.5 ml Avertin. 100 ml of 1% Evans blue dye (in PBS) was injected into the tail vein. 40 minutes later, mice were perfused via heart puncture with PBS
containing 2 mM EDTA for 20 minutes. Organs (brain, lung, liver, kidney) were harvested and incubated in fornnamide for 3 days to elute Evans blue dye. OD
of formamide solution was measured using 620 nm wave length.
Renal microvascular reactivity experiments Microvascular reactivity experiments were done as described previously (Maynard et al., supra) using rat renal microvessels (70-170 }tm internal diameter). In all experimental groups, the relaxation responses of kidney microvessels were examined after pre-contraction of the microvessels with U46619 (thromboxane agonist) to 40-60%
of their baseline diameter at a distending pressure of 40 mmHg. Once the steady-state tone was reached, the responses to various reagents such as TGF-(31 or TGF-03 or VEGF
were examined in a standardized order. All drugs were applied extraluminally.
Animal models Both pregnant and non-pregnant Sprague-Dawley rats were injected with 2 x 109 pfu of adenoviruses (Ad CMV or Ad sFItl or Ad sE or Ad sFltl +Ad sE) by tail vein injections. Pregnant rats were injected at day 8-9 of pregnancy (early second trimester) and blood pressure measured at day 16-17 of pregnancy (early third trimester).
Blood pressures were measured in the rats after anesthesia with pentobarbital sodium (60 mg/kg, i.p.). The carotid artery was isolated and cannulated with a 3-Fr high-fidelity microtip catheter connected to a pressure transducer (Millar Instruments, Houston, TX).
Blood pressure was recorded and averaged over a 10-minute period. Blood, tissue and urine samples were then obtained before euthanasia. Plasma levels were measured on the day of blood pressure measurement (day 8 after injection of the adenoviruses), recognizing that 7-10 days after adenoviral injection corresponds to the peak level of expression of these proteins. Circulating sFlt-I and soluble endoglin levels were confirmed initially by western blotting and then quantified using commercially available murine ELISA kits (R & D Systems, Minneapolis, MN). Urinary albumin was measured both by both standard dipstick and quantified by competitive enzyme-linked immunoassay using a commercially available rat albumin ELISA kit (Nephrat kit, Exocell Inc, Philadelphia, PA). Urinary creatinine was measured by a picric acid colorimetric procedure kit (Metra creatinine assay kit, Quidel Corp, San Diego, CA).
AST and LDH were measured using the commercially available kits (Thermo Electron, Louisville, CO). Platelet counts from rat blood were measured using an automated hemocytometer (Hemavet 850, Drew Scientific Inc, Oxford, CT). A peripheral smear of the blood with Wright's stain was performed for t.he detection of schistocytes in circulating blood. After the measurement of blood pressure and collection of specimens, the rats were sacrificed and organs harvested for histology. The litter was counted and individual placentas and fetuses weighed. Harvested kidneys were placed in Bouin's solution, paraffin embedded, sectioned and stained with H&E, PAS or Masson's trichrome stain.
Statistical comparisons Results are presented as mean standard error of mean (SEM) and comparisons between multiple groups were made by analysis of variance using ANOVA.
Significant differences are reported when p< 0.05.
Results Soluble endoglin is an anti-angiogenic molecule and induces vascular dysfunction We used an in vitro model of angiogenesis,to understand the function of the.
soluble endoglin. Soluble endoglin modestly inhibits endothelial tube fonnation, that is further enhanced by the presence of sFltl (FIGURE 22 and FIGURE 31). In pre-eclampsia, it has been reported that in addition to endothelial dysfunction, there is also enhanced microvascular permeability as evidenced by edema and enhanced leakage of Evan's blue bound albumin extracellularly. In order to see if soluble endoglin induces microvascular leak, we used mice treated for 48 hours with soluble endoglin and sFlt adenoviruses. A combination of soluble endoglin and sFltl induced a dramatic increase in albumin leakage in the lungs, liver and the kidney and a modest leakage in the brain as demonstrated using Evan's blue assay (FIGURE 23). Soluble endoglin alone induced a modest leakage in the liver. Importantly, the combination of soluble endoglin and sFlt-1 showed an additive effect in the liver, indicating that these soluble receptors may act in concert to disrupt endothelial integrity and induce significant vascular damage and leak.
These data suggest that soluble endoglin and sFltl combination are potent anti-angiogenic molecules and can induce significant vascular leakage.
To assess the hemodynamic effects of soluble endoglin, a series of microvascular reactivity experiments in rat renal microvessels were performed. We studied first the effects of TGF-0 1 and TGF-(33 -two known ligands of endoglin. Both TGF-(31 and TGF-(33 induced a dose-dependent increase in vascular diameter. Both TGF-(31 and (33 induced a dose-dependent increase in arterial diameter, whereas, TGF-(32, which is not a ligand for endoglin, failed to produce any significant vasodilation. (<2% at 0.1 and 1 g/ml). Importantly in the presence of excess soluble endoglin, the effect of both the TGF-(is were significantly attenuated (FIGURE 24). This acute effect of TGF-01 and TGF-03 isoforms on vascular tone was also seen in mesenteric vessels (FIGURE
32).
Finally, the combination of VEGF and TGF-01 induced vasodilation which was blocked by excess soluble endoglin and sFltl (FIGURE 25). This suggests that the sFltl and soluble endoglin may oppose the physiological vasodilation induced by angiogenic growth factors such as VEGF and TGF-(31 and induce hypertension.
In vivo effects of soluble endoglin and sFltl In order to assess the vascular effects of soluble endoglin and sFltl, we resorted to adenoviral expression system in pregnant rats. Adenovirus encoding a control gene (CMV) or soluble endoglin or sFltl or sFltl + soluble endoglin were injected by tail vein on day 8 of pregnancy in Sprague Dawley rats. On day 17, animals were examined for pre-eclampsia phenotype. Table 8 includes the hemodynamic and biochemical data.
Table 8. Hemodynamic and biochemical data for adenovirus treated rat animal models.
Groups N MAP in Urine Platelet LDH AST Fetal mm Hg Alb/creat count x U/L U/L weight (g) /m 1000/pl Control 6 83 5 186 94 1,098 +_ 75 156 + 54 + 4 2.1 +_ 0.5 (CMV) 32 sFltl 6 117 + 7* 2,295 867* 1,131 + 91 172 + 94 +_ 4* 1.75 + 0.4 SEng 6 104+6* 432+249 1,195+78 188--- 110+13* 1.6+0.4 sFltl + 6 121 + 9* 9,029 4043* 615 + 67* 1,952 + 210 + 92* 0.75 + 0.3*
sEng - 784* -Data are presented as mean s.e.m. MAP- mean arterial pressure (diastolic pressure +
1/3 pulse pressure); Fetal weight is the average weight of the litter for each group in grams; Alb/Creat - Albumin/creatinine ratios; LDH- Lactate dehyrogenase; AST-Aspartate Aminotransferase.
*P<0.05 when compared to control group.
Expression of sFltl and sEng were first confirmed in rat plasma by.Western blots (FIGURE 36) and circulating concentration quantified using commercially available ELISA kits.
The mean plasma concentrations of sFltl in the control, sEng, sFltl and sFltl+
sEng groups were 0.64 ng/ml, 0.66 ng/ml, 249 ng/ml, and 204 ng/ml respectively. The concentrations of sEng in these four groups were 0.39 ng/ml, 129 ng/ml, 0.37 ng/ml, and 123 ng/ml, respectively.
Soluble endoglin alone induced a mild hypertension. sFltl induced both hypertension and proteinuria, as previously reported. Fetal growth restriction was observed in litters born to the sFltl+sEng group, probably related to the placental vascular ischemia and damage. Importantly, the combination of sFlt'l and soluble endoglin induced severe hypertension, nephrotic range proteinuria, growth restriction of the fetuses and biochemical evidence of the development of the HELLP syndrome (elevated LDH, elevated AST and decreasing platelet counts) (Table 8).
Evidence of hemolysis in the soluble endoglin+sFltl group was confirmed by peripheral smear which revealed schistocytes and reticulocytosis (FIGURES 26A-B). Finally, renal histology also revealed focal endotheliosis in the soluble endoglin group and a severe glomerular endotheliosis in the soluble endoglin+sFltl group (FIGURES 27A-27D, and 33).
Note that in FIGURE 33, the control group is within normal limits. Note open capillary loops with fenestrated endothelium. The soluble endoglin panel shows endothelial swelling with loss of fenestrae and partial luminal occlusion. Note a red blood cell squeezing through the compromised lumen. While light microscopy of the kidneys of soluble endoglin treated rats was not striking for significant endotheliosis, electron microscopy revealed focal endotheliosis. Importantly, the animals that received both soluble endoglin and sFlt-1 had severe glomerular endotheliosis. The combination therapy group (lower panel) shows massive endocapillary occlusion with swollen endothelial cells.
Note the relative preservation of podocyte foot processes (shown as arrows) despite severe proteinuria. Extensive vascular damage of the placenta including infarction at the maternal-fetal junction was observed in the sFltl+sEng group, but not in control rats or in those treated with either agent alone (FIGURES 34A-H). Diffuse inflammation in the giant cell layer (corresponding to human invasive trophoblasts) was noted in the sFitl and sEng groups, and was higher in the combined group. Liver histology revealed signs of ischemia and areas of necrosis in the sFitl+sEng group, similar to those seen in patients with the HELLP syndrome (FIGURES 34A-H). Signs of severe maternal vascular damage were also seen when sFltl+sEng were injected to non-pregnant rats, suggesting that the observed phenotype in pregnant rats was due to a direct effect on the maternal vessels and did not require the placenta.
Summary These results demonstrate that soluble endoglin is up-regulated in pre-eclamptic placentas and is present at extremely high levels in patients with pre-eclampsia. The highest levels of soluble endoglin were present in patients with HELLP
syndrome, one of the most severe forms of pre-eclampsia. These results also demonstrate that soluble endoglin levels correlated with the elevated sFltl in pregnant patients and was higher in those patients in whom there is a higher circulating sFltl levels. In addition, the results indicate that soluble endoglin is an anti-angiogenic molecule and disrupts endothelial function in multiple endothelial assays such as angiogenesis assays, microvascular permeability assays, and microvascular reactivity experiments. Importantly, soluble endoglin can amplify the toxic consequence of sFltl in these in vitro endothelial assays.
Further, in in vivo assays, adenoviral expression of soluble endoglin induces mild hypertension without any significant proteinuria. However, in the presence of sFltl, soluble endoglin induces significant vascular damage as evidenced by the presence of severe hypertension, proteinuria, glomerular endotheliosis, development of the HELLP
syndrome and fetal growth restriction.
The mechanism of soluble endoglin release is likely proteolytic cleavage of the extracellular region of the endoglin molecule. Specific proteases that are up-regulated in the pre-eclamptic tissue may serve as candidate molecules. One example would be the membrane type matrix metalloproteinase-1 (MT1-MMP) that has been shown to cleave betaglycan, a molecule that shares similarity to endoglin (Velasco-Loyden G et al, J.
Biol. Chem. 279:7721-33 (2004)). Therefore, inhibitors of such proteases can serve as valuable targets for the treatment of pre-eclampsia.
Example S. Soluble endoglin inhibits TGF-(31 and TGF-P3 mediated NOS-dependent vasodilation.
eNOS is a Ca2+/calmodulin-regulated nitric oxide (NO) synthase that can be activated by fluid shear stress and neurohumoral stimuli. Endothelium-derived NO is a very potent vasorelaxant contributing to systemic blood pressure regulation, vascular permeability, and angiogenesis. In fact, the effects of VEGF ori angiogenesis and vascular tone are partly mediated by activation of eNOS, through increased eNOS/Hsp90 association and Akt-dependent eNOS phosphorylation at Ser1177. Our recent demonstration that increased placenta-derived sFltl in sera of preeclamptic patients is anti-angiogenic and induces hypertension may in fact reflect impaired VEGF-dependent eNOS activation (Maynard et al., supra). More recently, dephosphorylation of eNOS
Thr495 has been shown to precede Ser1177 phosphorylation and these coordinated events determine eNOS activity in endothelial cells (Fleming et al., Cir. Res.
88:E68-75 (2001)). Given the known effect of VEGF on reducing vascular reactivity via eNOS
activation and the recent demonstration that endoglin modulates eNOS-dependent vasomotor activity (Toporsian et al., Circ. Res. 96:684-692 (2005)), we assessed the hemodynamic effects of TGF-(3 isoforms and soluble endoglin in isolated rat renal microvessels. As described in Example 7 and in FIGURES 24 and 25, both TGF-(3]
and -03 induced a dose-dependent increase in arterial diameter which was significantly attenuated by soluble endoglin. This acute effect of TGF-f31 and -P3 isoforms on vascular tone has not been previously recognized and was also seen in mesenteric vessels (FIGURE 32). VEGF and TGF-01 had additive effects on vasodilation, which were blocked by sEng+sFltl at concentrations noted in patients with preeclampsia (FIGURE
and 35A). L-NAME blocked the vasodilation mediated by TGF-(31 and VEGF
25 indicating a NOS dependent response (FIGURE 35A). These data suggest that circulating sFItl and sEng may oppose the physiological NO-dependent vasodilatation elicited by these angiogenic growth factors, contributirng to the development of hypertension seen in preeclampsia.
Example 9. Soluble endoglin inhibits TGF-P1 binding and signaling in endothelial cells.
Given *that endoglin is a co-receptor for TGF-0 1 and -03 isoforms, we hypothesized that soluble endoglin acts by interfering with cell surface receptor binding.
Pre-incubating radio-labeled TGF-01 with recombinant soluble endoglin significantly reduced its binding to TGF-0 receptor type II (T(3RII) at both 50 and 100 pM
(FIGURE
35B). Thus soluble endoglin competes for TGF-0 1 binding to its receptors on endothelial cells. To test whether this leads to impaired signaling, the activity of a CAGA-Luc reporter construct was assessed in human endothelial cells. TGF-P 1 induced the activation of the Smad 2/3-dependent CAGA-Luc reporter and this response was abolished by treatment with soluble endoglin (FIGURE 35C).
Example 10. Soluble endoglin blocks TGF-P1 mediated eNOS activation.
Given our findings that TGF-(31 induces a NOS-dependent vasorelaxation in both renal and mesenteric resistance vessels, we explored its immediate effects on eNOS
activation. While TGF-0 I had no effect on eNOS Ser1177 phosphorylation, it induced a significant dephosphorylation at Thr495 (FIGURE 35D) suggesting that TGF-0 regulates the phosphorylation status of a key residue in eNOS activation. This effect was significantly attenuated by soluble endoglin (FIGURE 35D).
Taken together, the results in Examples 8-10 demonstrate that soluble endoglin interferes with TGF-0 receptor binding and downstream signaling in endothelial cells and attenuates eNOS activation. Soluble endoglin and sFlt-1 may be working in concert to inhibit endothelial dependent NO activation and vasomotor effects by both the VEGF and the TGF-(3 signaling pathways.
Example 11. Sequential changes in angiogenic factos can identify women at risk for pre-eclampsia or eclampsia.
In the examples described above, we have shown that both sFltl and soluble endoglin (sEng) are intimately related to the pathogenesis of preeclampsia. In the example described below we measured the concentrations of sFlt 1, and sEng in paired serum specimens collected in first and second trimesters from women followed prospectively during pregnancy and whose pregnancy outcomes were characterized in detail in order to determine if sequential changes of these markers between first and second trimesters are associated with the development of pre-eclampsia.
Materials and Methods Study Population We performed a prospective, nested case-control study of women who enrolled in the Massachusetts General Hospital Obstetrical Maternal Study (MOMS) whose methods have been described previously (Thadhani et al., Obstet. Gynecol. 97:515-20 (2001) and Wolf et al., Obstet. Gynecol. 98:757-62 (2001)). In brief, the MOMS cohort was established in 1998 for the prospective study of early gestational risk factors for adverse outcomes that occur later in pregnancy. Women who received prenatal care at Massachusetts General Hospital and affiliated health centers were eligible for inclusion in the cohort. For the current study, consecutive women with singleton gestations between June 1, 2001, and May 1, 2003, who enrolled in the MOMS cohort at or before 12 weeks of gestation and who delivered after 20 weeks were eligible for inclusion.
Cases (n=39) were defined as those with blood collections in the first and second trimester who subsequently developed pre-eclampsia, and controls (n=147) were consecutive contemporaneous women enrolled in the same cohort who delivered at term (>37 weeks) and remained normotensive, normoglycemic, and without evidence of proteinuria throughout pregnancy. Cases and controls were matched by age (f 2 years) and body mass index (+ I kg/m2) given potential for confounding by these exposures (Thadhani et al., Obstet. Gynecol. 94:543-50 (1999)). All subjects provided written informed consent, and this study was approved by the Institutional Review Board of the Massachusetts General Hospital.
Primary Exposures Blood samples were collected at the first prenatal visit (11-13 wks) and again in the second trimester (17-20 wks) in all women. Following collection, samples were stored at -80C for future analysis. The primary exposures were serum sFltl and sEng that were measured using commercial ELISA kits (R&D systems, Minneapolis, MN) (Maynard et al., supra and Venkatesha et al., Nat Med. 12:642-9 (2006)). The intraassay precision coefficients of variation for sFltl and sEng were 3.5 and 3.2%
respectively.
The interassay precision coefficients of variation for sFltl and endoglin were 8.1 and 9.5% respectively. All samples were run in duplicate, and if more than 10%
variation existed between duplicates, the assay was repeated, and averages were reported. All assays were performed by someone who was blinded to case status. Samples were randomly ordered for analysis.
Covariates and Confounders The electronic medical record (EMR), which is the medical record used at the Massachusetts General Hospital, provides clinical and demographic data that prospectively details the events of pregnancy through the early postpartum period.
Specific information obtained from the EMR collected at baseline (first prenatal visit) and at all subsequent prenatal visits included age, race, height, weight, smoking status, gestational age estimated from the last menstrual period and verified by ultrasound, blood pressure, and the results of urine analysis and fetal gestational age estimation. All pregnancy outcome information is also entered in the EMR including results of glucose tolerance tests and other routinely measured laboratory values, and delivery characteristics such as birth weight, route of delivery, and diagnosis of preeclampsia.
Primary Outcomes All pregnancy outcomes were verified by detailed examination of medical records, including prenatal flow sheets and laboratory measurements. At each prenatal visit blood pressure was obtained from the right arm using standard sphygmomanometers with the woman in the seated position after 3-5 minutes of rest. For each patient the proper cuff size was selected based on right midarm circumference.
Measurements of blood pressure that coincided with the timing of the first (systolic) and fifth (diastolic) Korotkoff sounds were recorded. All subjects for the current study had no history of preexisting hypertension or diabetes mellitus, initiated and completed their prenatal care and pregnancy within the MOMS network, delivered a live infant, and had no evidence of hypertension 6 weeks after delivery.
Preeclampsia was defined as systolic blood pressure elevation of at least 140 mm Hg or diastolic blood pressure of at least 90 mm Hg after 20 wk gestation, in association with proteinuria, either 2+ or greater by dipstick or at least 300mg/24 h in the absence of urinary tract infection (ACOG Committee on Practice Bulletins--Obstetrics.
ACOG
practice bulletin. diagnosis and management of preeclampsia and eclampsia.
Number 33, January 2002. Obstet Gynecol. 99:159-67). Preeclamptics were analyzed as term preeclampsia (? 37 weeks) and preterm preeclampsia (<37 weeks).
Statistical analysis Demographic and clinical characteristics were compared using Chi Square tests or Student's t test, as appropriate. Log transformation was needed for the primary exposures given their skewed distributions (Levine et al., (2004), supra, Levine et al., N.
Engl. J. Med. 355:992-1005 (2006)). Primary exposures were examined as continuous variables, and with cut points and tertile analyses based on the natural distributions of the controls and simplified for clinical interpretation. Multiple regression analysis was performed using logistic regression techniques. All P values were two-tailed, and a P
value < 0.05 was considered statistically significant.
Secondary Analysis of angiogenic markers in the CPEP study We also performed a secondary analysis of angiogenic factor changes during early pregnancy from the recently published nested case controlled study within the CPEP cohort, described above. We analyzed samples from normotensive women, women who developed preeclampsia prior to 37 weeks, and women who developed preeclampsia after 37 weeks at three different time intervals - 10-12 weeks,.13-16 weeks, and 17-20 weeks.
Results Demographic and Clinical Characteristics Baseline=and delivery characteristics of the MOMS study population are displayed in Table 9. There were no significant differences in age and body mass index between the two groups. Women who subsequently developed preeclampsia had higher systolic and diastolic blood pressures at the first prenatal visit. At the time of presentation of preeclampsia, systolic and diastolic blood pressures were higher in preeclamptic group as expected.
Table 9. Demographics Variable Normal (n=147) Preeclampsia P value (n=39) Age ( yrs) 31.4 5.4 32.8 t 5.4 0.06 BIvII(kg/m ) 28.5 6.3 29.8 9.1 0.40 Smoker % Never) 47% 60% 0.21 Parity 0.7 0.9 0.8 1.0 0.47 Baseline characteristics DBP(mmHg) 70 7 75 8 <0.01 SBP(mmHg) 113 7 120 13 <0.01 UTP(mg/dl) NA 614 547 Gestational age at 39.3 1.9 37.2 2.3 <0.001 delive Birth weight 3444 532 3300 809 0.35 Characteristics at presentation Maximum DBP 78 5 93 7 <0,01 (mmHg) Maximum SBP 122 t 7 147 ~ 10 <0.01 (mmHg) Maximum SPOT 0.6 0.3 1.4 0.9 <0.01 1'Jg) BMI = body mass index; DBP = diastolic blood pressure; SBP = systolic blood pressure UTP = urine total protein in mg/L; SPOT = urine protein/creatinine ratio Values are mean S.D.
First and Second Trimester Levels of sFltl and sEng in Normal and Preeclamptic Pregnancy The mean serum levels of sFltl were higher in women with preeclampsia compared to women with normal pregnancies, 3.49 0.35 ng/ml versus 3.03 0.13 ng/ml respectively, in first trimester (P=NS). In the second trimester the mean serum levels of sFltl were significantly higher in preeclamptic group, mean value of 4.12+-0.5ng/ml compared to normal group 3.10 0.15 ng/ml (P<0.0 1) (Table 10).
The mean serum levels of sEng thought not significantly different in the first trimester were significantly altered in the second trimester among women with preeclampsia, as compared to normal women, 6.9:h 0.32 ng/ml versus 6.57:L 0.
17 ng/ml (P=NS) in first trimester and 6.37j= 0.38 ng/mi versus 5.23:~= 0.12 ng/ml in second trimester, (P=0.004), respectively (Table 10).
Table 10. Serum levels of sFlt and sEng.
Angiogenic Factor Normal Pregnancy Preeclampsia P value N mean SE N Mean SE
sFltl (ng/ml) 147 3.03 0.13 39 3.496 0.35 0.14 (first trimester) sFltl (ng/ml) 144 3.10 0.15 35 4.12 0.5 0.01*
(second trimester) sEng (ng/ml) 147 6.57 0.17 39 6.9 0.32 0.37 (first trimester) sEng (ng/ml) 144 5.23 0.12 35 6.37 0.38 0.004*
(second trimester) Sequential changes in angiogenic factors Figure 37 graphically displays the delta or d (difference between first and second trimester values of sFltl and sEng) in normal, all preeclamptic women and in women with preeclampsia less than 37 weeks. In normal pregnancy, there is very little change in sFltl between first and second trimester (dsFltl=0.05 0.15 ng/ml). dsFltl is relatively higher in women who develop pre-eclampsia 0.713 =b 0.47 ng/ml versus 0.0497 ZE
0.15 ng/ml in normal women (P=0.08). Similarly in women with pre-eclampsia less than 37 weeks, dsFltl was higher at 0.634 t 0.91 ng/ml.
There is a fall in the level of sEng between the first and second trimester in normal pregnancy (-1.322 =~ 0.18 ng/ml). This fall is blunted in patients with pre-eclampsia, with dsEng of -0.441t0.42 ng/ml (P=0.04). In women with pre-eclampsia less than 37 weeks, the fall in the level of soluble endoglin is blunted and appeard to trend in the opposite direction (0.732+0.77 ng/ml, P <0.01 compared with controls).
Predictive Algorithnzs To see if these alterations can be used as a predictive test for severe premature pre-eclampsia, we looked at the product (value of sFItl x sEng) as product-I
(in first trimester) and product-2 (in second trimester) in normotensive women and women who developed pre-eclampsia and specifically in preterm pre-eclampsia. Both product-1 and product-2 were significantly elevated in patients with pre-eclampsia and importantly the delta of the product (dproduct) were strikingly positive in contrast with a negative number in normal controls (Figure 38). Furthermore, the dproduct were greatly amplified in patients with preterm pre-eclampsia as compared to normal controls (P=0.004).
To assess the relationship between altered levels of angiogenic factors and risk of preterm pre-eclampsia, we computed adjusted odds ratios (aOR) and 95 percent confidence intervals (95% CI) for preterm pre-eclampsia in the highest category of the distribution of dproduct concentrations with respect to the lower two categories after adjustment for race/ethnicity, body-mass index, and gestational age at specimen collection (Figure 39). Substantial increases in risk of preterm were observed in the , group whose delta product levels were greater than +1 [aOR 5.5, 95% CI 1.4 -22.4], compared to women whose delta product was less than -1.
Secondary anal,ysis of tlie CPEP nested case control study In the subset of women in the CPEP trial, among the cohort of women who remained normotensive, the mean values of sFltl increased from 3.68 ng/ml to 4.92 ng/ml between 10-12wks and 13-16 weeks and decreased to 4.29 ng/ml at 17-20 weeks, while in women with preterm pre-eclampsia, the mean values of sFltl increased from 3.44 ng/ml to 4.22 ng/ml and further increased to 5.39 ng/ml at 10-12, 13-16 and 17-20 weeks of gestation. This pattern was not obvious in women with term pre-eclampsia (Table 11).
Table 11. Serum levels of sFlt and sEng in CPEP trial.
sFltl (ng/ml) sEng (ng/ml) wks wks wks wks wks wks Normal 3.68 4.99 4.29 6.8 7.07 5.78 PE <37 wks 3.44 4.22 5.39 7.15 7.9 10.19 N 13 28 32 ' 13 28 32 PE >37 wks 4.06 4.48 4.25 7.41 7.8 8.34 Similar results were observed in the levels of sEng. In normotensive women the levels of sEng increased from 6.8 ng/ml to 7.07 ng/ml at 10-12 wks and 13-16 wks and then decreased to 5.78 ng/ml at 17-20 wks while in women with preterm preeclampsia the levels of endoglin increased from 7.15 ng/ml to 7.95 ng/ml and further increased to 10.19 ng/ml between 10-12 wks, 13-16 wks and 17-20 weeks. The same trend was noted in women with term preeclampsia where the mean levels of sEng increased from 7.41 ng/ml to 7.80 ng/ml to 8.34 ng/ml at 10-12 weeks, 13-16 weeks and 17-20 weeks (see Table 11).
Summary Both sFltl and sEng are elevated during second trimester in patients destined to develop pre-eclampsia. Normal pregnancy is characterized by a fall in sEng from first to second trimester without significant change in sFltl. However, in patients who develop pre-eclampsia, particularly preterm pre-eclampsia, both sFltl and sEng continue to rise from first to second trimester. The changes in sFltl and sEng during first and second trimesters are useful for screening patients at high risk for subsequent development of preterm pre-eclampsia.
These findings have important implications for the prediction of preterm preeciampsia. The pursuit of a safe, reliable screening test for preeclampsia has been a goal of researchers for many years. Previous efforts have focused on detecting early manifestations of disease such as microalbuminuria, weight gain and plasma volume changes. In a large metaanalysis, Conde-Agudelo A et al analyzed eighty-seven of 7,191 (211,369 women) potentially relevant articles to assess the usefulness of clinical, biophysical, and biochemical tests in the prediction of pre-eclampsia. They concluded that as of 2004, there was no clinically useful screening test for predicting the development of pre-eclampsia (12). In the present study we have shown that the levels of sFltl and sEng are elevated in women who are destined to become pre-eclamptic, in their first and second trimester (as measured as a delta in individual patients), weeks to months before the clinical onset of disease. These changes are more substantial in women who develop preterm pre-eclampsia.
An imbalance in angiogenic factors is thought to play an intimate role in the pathogenesis of pre-eclampsia. Pre-eclamptic placentas are characterized by shallow implantation and abnormal vascular remodeling including impaired pseudo-vasculogenesis (Fisher et al., Semin Cell Biol. 4:183-8 (1993)). It is believed that these placental changes occur between 12-18 weeks of pregnancy and is important in the pathogenesis of the vast majority of severe early onset preeclampsia. It is thought that these placentation abnormalities lead to the elaboration of systemic factors that induce the maternal syndrome of preeclarnpsia. As described herein and in PCT application publication numbers WO 2004/008946, WO 2005/077007, and WO 2006/034507, both sFltl and sEng, two anti-angiogenic proteins have been found to be elevated in preeclampsia not only during clinical disease but also several weeks before onset of symptoms (Levine et al., (2006), supra). Importantly, both factors have been implicated in inducing a preeclampsia-like syndrome in rats (Maynard et al., supra, Venkatesha et al., supra). However, since alterations in concentrations of angiogenic factors in the matemal circulation occur relatively late in pregnancy, increased production of these anti-angiogenic factors may be a secondary phenomenon that occurs in response to abnormal placentation. In vitro data using placental villous explants and primary cytotrophoblast culture studies suggest that in addition to its role in inducing maternal endothelial dysfunction, anti-angiogenic factors may be involved in cytotrophoblast migration and differentiation. Our findings that levels of anti-angiogenic factors decrease from the first to the second trimester in normal pregnancies, but not in pregnancies in which preterm preeclampsia later develops, suggests that abnormalities of circulating angiogenic factors are occurring at the same time as abnormalities in placental differentiation.
The etiology of the increased concentrations of circulating sFlt 1 and sEng in preeclam.ptic patients is unknown. Hypoxia, genes, or immunological factors are believed to play a role. It is worth noting that expression of both sFltl and sEng are elevated in response to hypoxia in vivo and in vitro models of placental hypoxia where increased expression is mediated by HIF-1 (Nevo et al., Am JPhysiol Regul Integr Comp Physiol. 291:R1085-93 (2006)). Furthermore, it is believed that during normal pregnancy the placenta is hypoxic early in pregnancy and this hypoxia disappears with increased 20' blood flow to the placenta during second trimesters. Although hypoxia has never been formally documented in pre-eclamptic pregnancies, it is believed that hypoxia is central to most pre-eclamptic pregnancies based on surrogate evidence of increased hypoxia induced transcription factor expression and impaired Doppler blood flow to the placentas.
Our findings that both sFltl and sEng remain elevated in patients with severe pre-eclampsia in contrast to normal pregnancies where there is a fall between first and second trimesters suggests that placental ischemia may in fact play a role in the increased production of these anti-angiogenic proteins in pre-eclamptic patients.
In summary, sequential changes of sFltl and sEng appear to identify women destined to develop preeclampsia, especially women who subsequently develop preterm pre-eclampsia. Our findings are reproduced in cross-sectional studies with much larger sample size (Table 11).
Example 12. Endoglin is necessary for TGF-P1-induced eNOS Thr495 dephosphorylation.
Murine endothelial cells were derived from Ene and Eng"" mouse embryos (E8.5) and grown as described in Balconi et al. (Arterioscler. Thromb. Vasc.
Biol.
20:1443-51 (2000)). Confluent monolayers were serum starved for 2 hours and stimulated with or without TGF-P 1 (125 and 250 pM) for 15 minutes. Cell extracts were immediately prepared in 10mM Tris-HCI containing 1% Triton X-100 and supplemented with protease and phosphatase inhibitors. Protein concentrations were quantified and samples were analyzed by western blot using phospho-specific pAbs to Thr495 of eNOS
(Cell Signaling) and a mAb for total eNOS (BD Biosciences).
The representative western blots shown in Figure 40A and associated graph shown in Figure 40B (mean of n=3 experiments) demonstrate that TGF-(31-induced (**p<0.01 versus baseline) eNOS Thr495 dephosphorylation in mouse Eng+"+
endothelial cells but not in Eng'l"cells. This result suggests a critical role for endoglin in linking TGF-01 signaling to eNOS Thr495 dephosphorylation and activation. Moreover, given that this process is endoglin-dependent, soluble endoglin can be used to inhibit the process, presumably by binding to and inhibiting TGF-p I.
Other Embodiments The description of the specific embodiments of the invention is presented for the purposes of illustration. It is not intended to be exhaustive or to limit the scope of the invention to the specific fornls described herein. Although the invention has been described with reference to several embodiments, it will be understood by one of ordinary skill in the art that various modifications can be made without departing from the spirit and the scope of the invention, as set forth in the claims.
All patents, patent applications, and publications referenced herein, including PCT Application Publication Numbers WO 2004/008946, WO 2005/077007, and WO
2006/034507; U.S Patent Application Publication Numbers 20060067937 and 20070104707; and U.S. Provisional Patent Application Number 60/852,761 are hereby incorporated by reference.
Other embodiments are in the claims.
What is claimed is:
Soluble Endoglin (ng/ml) 24.5 d: 3.1 24.2 ~ 3.3 0.8 Gestational age (weeks) 17.6- 27.9 17.7 - 28.0 Range n=19 n=19 13.2 t 1.3 Interval before clinical manifestation (weeks) 16-25 weeks before clinical manifestation 3.44 ~ 1.07 3.69 =L 1.18 0.3 Soluble Endoglin (ng/ml) 17.6 3.5 16.5 :b 4.5 0.2 Gestational age (weeks) 9.1- 23.4 8.0 -22.7 Range n=42 n=42 Interval before clinical manifestation 20.6 :h 3.6 (weeks) Value expressed as mean sd 2 pre-eclamptic patients had no blood samples available at clinical manifestation To examine the diagnostic potential of plasma soluble endoglin concentrations to identify those destined to develop pre-eclampsia, patients were stratified into early onset pre-eclampsia (PE<34 weeks) and late onset pre-eclampsia (PE>34 weeks). For patients with early-onset pre-eclampsia, the mean plasma soluble endoglin levels was significantly higher in pre-eclampsia (before clinical diagnosis) than in normal pregnancy starting around 16-24 weeks of gestation (Table 6) with very dramatic differences in 24-28 week and 28-32 week gestational windows. In contrast, for patients with late-onset pre-eclampsia, plasma soluble endoglin concentrations in pre-clinical pre-eclampsia was significantly higher than in nonnal pregnancy only at 28-32 weeks with very dramatic differences at 32-36 week of gestation (Table 7).
Table 6. Plasma soluble endoglin concentrations in normal pregnant women and patients who developed clinical Pre-eclam sia at 34 weeks of gestation or less.
Normal p Pre-olinical samples p Clinical samples pregnancy Pre-eclampsia pre-colampsia l" blood samplin (7.1-16 weeks) =
Soluble Endoglin (ng/ml) 3.89 t.928 0.7 3.81 * 1.11 Gestational age (weeks) 12.3 :L 2.2 0.4 11.6 f 2.6 Range 8.4-15.9 8.0 -15.1 n=37 n=8 2ad blood sampling (16.1-24 weeks) Soluble Endoglin (ng/ml) 3.36 f 1.11 0.02* 4.60 d:1.72 Gestational age (weeks) 19.4 f 1.7 0.7 19.8 f 2.9 Range 16.3 -23.4 17.3 -23.9 n=44 n=7 3'd blood sampling (24.1-28 weeks) Soluble Endoglin (ng/ml) 3.189 f.729 <01001 10.22 :E 6.17 Gestational age (weeks) 25.9 f 1.3 0.03* 26.8 t 0.6 Range 24. l -28.0 26.0 -27.3 n=38 n=6 4N blood sampling (28.1-32 weeks) = Soluble Endoglin (ng/ml) 3.70 t 1.10 0.01* 17.66 8.9 0.008* 96.10 t 25.76 0.05 Gestational age (weeks) 29.9 :k 1.1 1.0 29.7 t 1.1 1.0 30.4 :1: 1.4 1.0 Range 28.3-32.0 28.7-31.3 29.4-31.4 n=42 n=6 n=2 5ei blood sampling (32.1-36.9 weeks) Soluble Endoglin (ng/ml) 5.79 t 2.42 53.38 zk 32.09 0.001 *
Gestational age (weeks) 34.7 :~ 1.3 33.5 f 0.5 <0.001 *
Range 32.4 -36.6 32.6 -34.0 n=37 n=6 p :compared between samples at clinical manifestation of pre-eclampsia and normal pregnancy Value expressed as mean sd 6:2 pre-eclamptic patients had no blood samples available at clinical manifestation Table 7. Plasma soluble endoglin concentrations in normal pregnant women and pre-eclamptics (34 weeks of gestation) Normal p Pre-clinical samples p Clinical pp, pregnancy Pre-eclampsia samples Pre-eclampsia )A1 blood sam hnp,(7.l-16 weeks) Soluble Endoglin (ng/ml) 3.89 ,928 0.9 4.01f 1.35 Gestational age (weeks) 12.3 t 2.2 0.2 1 S.6 f 2.4 Range 8.4-15.9 7.7 - 15.1 n=37 n=26 2"d blood samplina (16.1-24 weeks) Soluble Endoglin (ng/ml) 3.36 f 1.11 0.4 3.59 t 1.23 Gestational age (weeks) 19.4 t 1.7 0.04* 20.3 t 1.9 Range 16.3 -23.4 16.7 -24.0 n=44 n=29 3'd blood samulinp (24.1-28 weeks) Soluble Endoglin (ng/ml) 3.18 t.729 0.1 3.98 ~z 2.13 Gestational age (weeks) 25.9 11.3 0.4 ' 26.3 d: 1.1 Range 24.1 -28.0 24.6 -28.0 n=38 n=23 4'h blood sampling (28.1-32 weeks) Soluble Endoglin (ng/ml) 3.70 t 1.10 0.001* 8.57 f 9.45 Gestational age (weeks) 29.9 1.1 0.2 30.3 t 1.0 Range 28.3-32.0 28.7 -32.0 n=42 n=27 5'h blood sampling (32.1-36.9 weeks) Soluble Endoglin (ng/ml) 5.79 :h 2.42 <0.001 * 10.51 f 6.59 <0,001 34,36 f 16.30 <0.001 *
Gestational age (weeks) 34.7 f 1.3 1.0 34.8 t 1.5 0.9 35.4 f 0.9 0.7 Range 32.4 -36.6 32.6 -36.7 34.3 -36.6 n=37 n=20 n=7 6 i blood samplinp (>=37weeks) Soluble Endoglin (ng/mi) 8.98 t 45.12 -- 15.23 10.61 0.006*
Gestational age (weeks) 39.411.0 38.8 1.1 0.05 Range 37.0 - 40.7 37.6 - 41.4 n=27 n=27 p:compared between samples at clinical manifestation of pre-eclampsia and normal pregnancy Value expressed as mean. sd ~
Summary The results of these experiments demonstrate that women with clinical pre-eclarnpsia have very high levels of circulating soluble endoglin when compared to gestational age matched controls. The results also demonstrate that women destined to develop pre-eclampsia (pre-clinical pre-eclampsia) have higher plasma soluble endoglin levels than those who are predicted to have a normal pregnancy. The increase in soluble endoglin levels is detectable at least 6-10 weeks prior to onset of clinical symptoms.
Finally, these results demonstrate that both early onset and late onset pre-eclampsia have elevated circulating soluble endoglin concentrations, but the alterations are more dramatic in the early onset pre-eclampsia.
Example 6. Soluble endoglin protein levels as a diagnostic indicator of pre-eclampsia and eclampsia in women (CPEP Study).
As described above, we have discovered that soluble endoglin, a cell surface receptor for the pro-angiogenic protein TGF-(3 and expressed on endothelium and syncytiotrophoblast, is upregulated in pre-eclarnptic placentas. In the experiments described above, we have shown that in pre-eclampsia excess soluble endoglin is released from the placenta into the circulation through shedding of the extracellular domain;
soluble endoglin may then synergize with sFltl, an anti-angiogenic factor which binds placental growth factor (PIGF) and VEGF, to cause endothelial dysfunction. To test this hypothesis, we compared serum concentrations of soluble endoglin, sFltl, and free PIGF
throughout pregnancy in women who developed pre-eclampsia and in those women with other pregnancy complication such as gestational hypertension (GH) and pregnancies complicated by small-for-gestational (SGA) infants to those of women with nonnotensive control pregnancies. This study was done in collaboration with the Dr.
Richard Levine at the NIH.
There were two principal objectives of this study. The first objective was to determine whether, in comparison with normotensive controls, elevated serum concentrations of soluble endoglin, sFltl, and reduced levels of P1GF can be detected before the onset of pre-eclampsia and other gestational disorders such as gestational hypertension or pregnancies complicated by small-for-gestational (SGA) infants. The second objective was to describe the time course of maternal serum concentrations of soluble endoglin, sFlt-1, and free PIGF with respect to gestational age in women with pre-eclampsia, gestational hypertension, or SGA with separate examination of specimens obtained before and after onset of clinical symptoms, and in normotensive controls.
Methods Clinical information This study was a case control study of pregnancy complications (premature pre-eclampsia, term pre-eclampsia, gestational hypertension, pregnancies with SGA
infants, normotensive control pregnancies) nested within the cohort of 4,589 healthy nulliparous women who participated in the Calcium for Pre-eclampsia Prevention trial (CPEP). 120 random cases were selected from each of the study groups. The study methods were identical to the nested case control study recently performed for pre-eclampsia (Levine et al, N. Eng. J. Med. 2004, 350:672-83). From each woman blood specimens were obtained before study enrollment (13-21 wks), at 26-29 weeks, at 36 weeks, and on suspicion of hypertension or proteinuria. All serum specimens collected at any time during pregnancy before onset of labor and delivery were eligible for the study. Cases included 120 women who developed term pre-eclampsia, gestational hypertension, or SGA and who delivered a liveborn or stillborn male baby without known major structural or chromosomal abnormalities, and from whom a baseline serum specimen was obtained.
For premature pre-eclampsia, defined as (PE<37 weeks) all 72 patients from the CPEP
cohort were studied. The clinical criterion for the diagnosis of pre-eclampsia is described in Levine et al., (2004), surpa. All cases of gestational hypertension were required to have a normal urine protein measurement within the interval from 1 day prior to onset of gestational hypertension through 7 days following. SGA was defined as <10th and <5th (severe SGA) percentile, using Zhang & Bowes' tables of birthweight for gestational age, specific for race, nulliparity, and infant gender. Controls were randomly selected from women without pre-eclampsia or gestational hypertension or SGA who delivered a liveborn or stillborn baby without known major structural malformations or chromosomal anomalies and matched, one control to one case, by the clinical center, gestational age at collection of the first serum specimen I wk), by freezer storage time (zL 1 year), and by number of freeze-thaws. A total of 1674 serum speciinens were studied.
Matching by gestational age was done to control for gestational age-related differences in levels of sFlt-1, VEGF, and P1GF. Matching for freezer storage time was done to minimize differences due to possible degradation during freezer storage. Matching by clinical center was done to control for the fact that pre-eclampsia rates differed significantly between centers, perhaps due to differences in the pathophysiology of the disease. In addition, the centers may have used slightly different procedures for collecting, preparing, and storing specimens. Matching by number of thaws was also performed to ensure that cases and controls will have been subjected equally to freeze thaw degradation.
ELISA measurements ELISA for the various angiogenic markers were performed at the Karumanchi laboratory by a single research assistant that was blinded to the clinical outcomes.
Commercially available ELISA kits for soluble endoglin (DNDGOO), sFItl (DVR 100), PIGF (DPGOO) were obtained from R&D systems, (Minneapolis, MN).
Statistical analysis T-test was used for the comparison of the various measurements after logarithm tic transformation to determine significance. P<0.05 was considered as statistically significant.
Results The mean soluble endoglin (FIGURE 6), sFltl (FIGURE 7) and P1GF (FIGURE
8) concentrations for the five different study groups of pregnant women throughout pregnancy during the various gestational age group windows= as described in the methods are shown in FIGURES 6-8. For the pre-eclampsia groups and gestational hypertensive groups, specimens taken after onset of clinical symptoms are not shown here.
Compared with gestational age-matched control specimens, soluble endoglin and sFitl increased and free P1GF decreased beginning 9-11 weeks before preterm pre-eclampsia, reaching levels 5-fold (46.4 vs 9.8 ng/ml, P<.0001) and 3-fold higher (6356 vs 2316 pg/ml, P<.0001) and 4-fold lower (144 vs 546 pg/ml, P<.0001), respectively, after pre-eclampsia onset. For term pre-eclampsia, soluble endoglin increased beginning 12-14 weeks, free PIGF decreased beginning 9-11 weeks, and sFltl increased <5 weeks'before pre-eclampsia onset. Serum concentrations of sFlt1 and free PIGF did not differ significantly between pregnancies with SGA or average for gestation age/large for gestation age (AGA/LGA) infants from 10-42 weeks of gestation. Serum soluble endoglin was modestly increased in SGA pregnancies beginning at 17-20 weeks (7.2 vs 5.8 ng/ml, P=.03), attaining concentrations of 15.7 and 43.7 ng/ml at 37-42 weeks for mild and severe SGA, respectively, as compared with 12.9 ng/ml in AGA/LGA pregnancies (severe SGA vs AGA/LGA, P=.002). In the gestational hypertension study, compared with GA-matched control specimens, modest increases in soluble endoglin were apparent <1-5 weeks before gestational hypertension, reaching levels 2-fold higher for soluble endoglin (29.7 vs 12.5 ng/ml, P=.002) after onset of gestational hypertension.
The adjusted odds ratio for subsequent preterm PE for specimens obtained at 21-32 weeks which were in the highest quartile of control soluble endoglin concentrations (>7.2 ng/ml), as compared to all other quartiles, was 9.8 (95% CI 4.5-21.5).
The soluble endoglin anti-angiogenic index for pre-eclampsia was defined as (sFltl + 0.25 soluble endoglin)/P1GF. The index was calculated throughout the various gestational age groups for the five different study groups. The soluble endoglin anti-angiogenic index for pre-eclampsia anti-angiogenesis for samples taken prior to clinical symptoms=is shown in FIGURE 9. Elevated values for the soluble endoglin anti-angiogenic index were noted as early as 17-20 weeks of pregnancies and seemed to get more dramatic with advancing gestation in severe pre-mature pre-eclampsia. In term pre-eclampsia, SGA and GH, there was a modest elevation during the end of pregnancy (33-36 weeks) when compared to control women.
FIGURES 10 and 11 depict the mean concentrations of soluble endoglin (FIGURE 10) and soluble endoglin anti-angiogenic index (FIGURE 11) according to the number of weeks before clinical premature pre-eclampsia (PE <37 weeks). Even as early 9-11 weeks prior to the onset of premature pre-eclampsia, there was a 2-3 fold elevation in soluble endoglin and soluble endoglin anti-angiogenic index in women destined to develop pre-eclampsia with dramatic elevations (>5 fold) in 1-5 weeks preceding clinical symptoms.
FIGURES 12 and 13 show the alteration in soluble endoglin (FIGURE 12) and the soluble endoglin anti-angiogenic index (FIGURE 13) throughout pregnancy for term pre-eclampsia (PE>37 weeks) before and after symptoms. Elevation in soluble endoglin and the soluble endoglin anti-angiogenic index are noted starting at 33-36 weeks of pregnancy reaching on average 2-fold higher levels at the time of clinical pre-eclampsia.
FIGURES 14 and 15 show a modest elevation in soluble endoglin (FIGURE 14) and the soluble endoglin anti-angiogenic index (FIGURE 15) detected in women during gestational hypertension, and 1-5 weeks preceding gestational hypertension (during 33-36 week of pregnancy) when compared to normotensive controls.
FIGURES 16 and 17 show a modest elevations in soluble endoglin (FIGURE 16) and the soluble endoglin anti-angiogenic index (FIGURE 17) detected during the week gestational windows in women with severe SGA and not in all women with SGA
when compared to control pregnancies.
Summary The results of this study show that the soluble endoglin levels and soluble endoglin anti-angiogenic index levels, when measured prior to 33 weeks of pregnancy, was dramatically elevated in women destined to develop premature pre-eclampsia and in women with clinical premature pre-eclampsia (PE <37 weeks) when compared to normal control pregnancy. Therefore, soluble endoglin levels and soluble endoglin anti-angiogenic index levels (prior to 33 weeks) can not only be used for the diagnosis of premature pre-eclampsia, but also for the prediction of pre-eclampsia. It appears that elevations in soluble endoglin levels and soluble endoglin anti-angiogenic index levels start as early as 10-12 weeks prior to symptoms of pre-eclampsia.
The soluble endoglin levels and soluble endoglin anti-angiogenic index levels were also significantly elevated in term pre-eclampsia (PE >37 weeks) and modestly elevated in gestational hypertension and severe SGA when measured late in pregnancy (33-36 week gestational windows). Therefore, soluble endoglin levels and soluble endoglin anti-angiogenic index levels can also be used to identify other pregnancy complications such as SGA and gestation hypertension when measured after 33 weeks of pregnancy.
Example 7. Involvement of soluble endoglin in the pathogenesis of pre-eclampsia.
We have shown that endoglin, a cell surface receptor for the pro-angiogenic protein TGF-0 and expressed on endothelium and syncytiotrophoblast, is upregulated in pre-eclamptic placentas. We have also shown that in pre-eclampsia, excess soluble endoglin is released from the placenta into the circulation through shedding of the extracellular domain. The experiments described below were designed to test the hypothesis that soluble endoglin may synergize with sFltl, an anti-angiogenic factor which binds placental growth factor (PIGF) and VEGF, to cause endothelial dysfunction.
Materials and Methods Reagents Recombinant Human endoglin, human sFltl, mouse endoglin, mouse sFltl, human TGF-pl, human TGF-P3, mouse VEGF were obtained from R&D systems (Minneapolis, MN). Mouse monoclonal antibody (catalog # sc 20072) and polyclonal antibody (sc 20632) against the N-terminal region of human endoglin was obtained from Santa Cruz Biotechnology, Inc. ELISA kits for human sFlt1, mouse sFltl and human soluble endoglin were obtained from R&D systems, MN.
Generation ofadenoviruses Adenoviruses against sFltl and control adenovirus (CMV) have been previously described (Maynard et al, J. Clin. Invest. 111: 649:658 (2003)) and were generated at the Harvard Medical Core facility in collaboration with Dr. Richard Mulligan. To create the soluble endoglin adenovirus, we used the Adeasy Kit (Stratagene). Briefly, human soluble endoglin (encoding the entire extracellular region of the endoglin protein) was PCR amplified using human cDNA full length endoglin clone (Invitrogen, CA) as the template and the following oligonucleotides as primers: forward 5'-ACG AAG CTT
GAA ACA GTC CAT TGT GAC CTT-3' (SEQ ID NO: 3) and reverse 5'TTA GAT
ATC TGG CCT TTG CTT GTG CAA CC-3' (SEQ ID NO: 4). Amplified PCR
fragments were initially subcloned into pSecTag2-B (Invitrogen, CA) and the DNA
sequence was confirmed. A mammalian expression construct encoding His-tagged human soluble endoglin was PCR amplified using pSecTag2 B-soluble endoglin as the template and subcloned into pShuttle-CMV vector (Stratagene; Kpnl and Scal sites), an adenovirus transfer vector, for adenovirus generation. Adenovirus expressing soluble endoglin (sE) was -then. generated using the standard protocol per manufacturer instructions and confirmed for expression by western blotting. The confirmed clone was then amplified on 293 cells and purified on a CsC12 density gradient as previously described (Kuo et al, Proc. Natl. Acad. Sci. USA 98:4605-4610 (2001)). The final products were titered by an optical absorbance method (Sweeney et al, Virology, 2002, 295:284-288). The titer is expressed as plaque forming units (pfu)/mL based on a formula derived from previous virus preps that were titered using the standard plaque dilution based titration assay kit (BD Biosciences Clontech, Palo Alto, CA, Cat. No.
K1653-1) and the optical absorbance method.
Western blots Western blots were used for checking the expression of adenoviral-infected transgenes in the rat plasma as described elsewhere (Maynard et al, supra).
Imrrzunoprecipitation (IP) experiments IP followed by western blots were used to identify and characterize soluble endoglin in the placental tissue and serum specimens from patients with pre-eclampsia.
Human placental tissue was washed with cold PBS and lysed in homogenization buffer [10 mM Tris-HCI, pH 7.4; 15 mM NaCI; 60 mM KCI; 1 mM EDTA; 0.1 mM EGTA;
0.5% Nonidet P-40; 5% sucrose; protease mixture from Roche (Indianapolis, IN)]
for 10 minutes. Placental lysates were then subjected to immunoprecipitation with an anti-human monoclonal mouse endoglin antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Immunoaffinity columns were prepared by the directional coupling of mg of the purified antibody to 2ml protein A-Sepharose using an immunopure IgG
orientation kit (Pierce Chemical Co., Rockford, Illinois, USA) according to the manufacturer's instructions. Columns were then washed extensively with RIPA
buffer containing protease mixture, and bound proteins were eluted with 0.1 mol/L
glycine-HC1 buffer, pH 2.8. The eluent was collected in 0.5-mi fractions containing 1 mol/L Tris-HCl buffer. Protein-containing fractions were pooled and concentrated 9- to 10-fold with CENTRICON Centrifugal Concentrator (Millipore Corp., Bedford, Massachusetts, USA).
The immunoprecipitated samples were separated on a 4-12% gradient gel (Invitrogen) and proteins were transferred to polyvinylidene difluoride (PVDF) membranes.
Endoglin protein was detected by western blots using rabbit polyclonal antibody to human endoglin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).
Endothelial tube assay Growth factor reduced matrigel (7mg/mL, Collaborative Biomedical Products, Bedford, MA) was placed in wells (1001/well) of a pre-chilled 48-well cell culture plate and incubated at 37 C for 30 minutes to allow polymerization. HUVEC cells (30,000 +
in 300 l of endothelial basal medium with no serum, Clonetics, Walkersville, MD) were treated with various combinations of recombinant protein (soluble endoglin, sFltl, or both) and plated onto the Matrigel coated wells, and incubated at 37 C for 12-16 hours.
Tube formation was then assessed through an inverted phase contrast microscope at 4X
(Nikon Corporation, Tokyo, Japan) and quantitatively analyzed (tube area and total length) using the Simple PCI imaging analysis software.
Microvascular permeability experiments Balb-C mice were injected through the retro-orbital venous plexus with lx 108 pfu of adenovirus expressing GFP or soluble endoglin or sFitl or combinations and microvascular permeability assay was performed 48 hours later. Mice were anesthetized by IP injection of 0.5 ml Avertin. 100 ml of 1% Evans blue dye (in PBS) was injected into the tail vein. 40 minutes later, mice were perfused via heart puncture with PBS
containing 2 mM EDTA for 20 minutes. Organs (brain, lung, liver, kidney) were harvested and incubated in fornnamide for 3 days to elute Evans blue dye. OD
of formamide solution was measured using 620 nm wave length.
Renal microvascular reactivity experiments Microvascular reactivity experiments were done as described previously (Maynard et al., supra) using rat renal microvessels (70-170 }tm internal diameter). In all experimental groups, the relaxation responses of kidney microvessels were examined after pre-contraction of the microvessels with U46619 (thromboxane agonist) to 40-60%
of their baseline diameter at a distending pressure of 40 mmHg. Once the steady-state tone was reached, the responses to various reagents such as TGF-(31 or TGF-03 or VEGF
were examined in a standardized order. All drugs were applied extraluminally.
Animal models Both pregnant and non-pregnant Sprague-Dawley rats were injected with 2 x 109 pfu of adenoviruses (Ad CMV or Ad sFItl or Ad sE or Ad sFltl +Ad sE) by tail vein injections. Pregnant rats were injected at day 8-9 of pregnancy (early second trimester) and blood pressure measured at day 16-17 of pregnancy (early third trimester).
Blood pressures were measured in the rats after anesthesia with pentobarbital sodium (60 mg/kg, i.p.). The carotid artery was isolated and cannulated with a 3-Fr high-fidelity microtip catheter connected to a pressure transducer (Millar Instruments, Houston, TX).
Blood pressure was recorded and averaged over a 10-minute period. Blood, tissue and urine samples were then obtained before euthanasia. Plasma levels were measured on the day of blood pressure measurement (day 8 after injection of the adenoviruses), recognizing that 7-10 days after adenoviral injection corresponds to the peak level of expression of these proteins. Circulating sFlt-I and soluble endoglin levels were confirmed initially by western blotting and then quantified using commercially available murine ELISA kits (R & D Systems, Minneapolis, MN). Urinary albumin was measured both by both standard dipstick and quantified by competitive enzyme-linked immunoassay using a commercially available rat albumin ELISA kit (Nephrat kit, Exocell Inc, Philadelphia, PA). Urinary creatinine was measured by a picric acid colorimetric procedure kit (Metra creatinine assay kit, Quidel Corp, San Diego, CA).
AST and LDH were measured using the commercially available kits (Thermo Electron, Louisville, CO). Platelet counts from rat blood were measured using an automated hemocytometer (Hemavet 850, Drew Scientific Inc, Oxford, CT). A peripheral smear of the blood with Wright's stain was performed for t.he detection of schistocytes in circulating blood. After the measurement of blood pressure and collection of specimens, the rats were sacrificed and organs harvested for histology. The litter was counted and individual placentas and fetuses weighed. Harvested kidneys were placed in Bouin's solution, paraffin embedded, sectioned and stained with H&E, PAS or Masson's trichrome stain.
Statistical comparisons Results are presented as mean standard error of mean (SEM) and comparisons between multiple groups were made by analysis of variance using ANOVA.
Significant differences are reported when p< 0.05.
Results Soluble endoglin is an anti-angiogenic molecule and induces vascular dysfunction We used an in vitro model of angiogenesis,to understand the function of the.
soluble endoglin. Soluble endoglin modestly inhibits endothelial tube fonnation, that is further enhanced by the presence of sFltl (FIGURE 22 and FIGURE 31). In pre-eclampsia, it has been reported that in addition to endothelial dysfunction, there is also enhanced microvascular permeability as evidenced by edema and enhanced leakage of Evan's blue bound albumin extracellularly. In order to see if soluble endoglin induces microvascular leak, we used mice treated for 48 hours with soluble endoglin and sFlt adenoviruses. A combination of soluble endoglin and sFltl induced a dramatic increase in albumin leakage in the lungs, liver and the kidney and a modest leakage in the brain as demonstrated using Evan's blue assay (FIGURE 23). Soluble endoglin alone induced a modest leakage in the liver. Importantly, the combination of soluble endoglin and sFlt-1 showed an additive effect in the liver, indicating that these soluble receptors may act in concert to disrupt endothelial integrity and induce significant vascular damage and leak.
These data suggest that soluble endoglin and sFltl combination are potent anti-angiogenic molecules and can induce significant vascular leakage.
To assess the hemodynamic effects of soluble endoglin, a series of microvascular reactivity experiments in rat renal microvessels were performed. We studied first the effects of TGF-0 1 and TGF-(33 -two known ligands of endoglin. Both TGF-(31 and TGF-(33 induced a dose-dependent increase in vascular diameter. Both TGF-(31 and (33 induced a dose-dependent increase in arterial diameter, whereas, TGF-(32, which is not a ligand for endoglin, failed to produce any significant vasodilation. (<2% at 0.1 and 1 g/ml). Importantly in the presence of excess soluble endoglin, the effect of both the TGF-(is were significantly attenuated (FIGURE 24). This acute effect of TGF-01 and TGF-03 isoforms on vascular tone was also seen in mesenteric vessels (FIGURE
32).
Finally, the combination of VEGF and TGF-01 induced vasodilation which was blocked by excess soluble endoglin and sFltl (FIGURE 25). This suggests that the sFltl and soluble endoglin may oppose the physiological vasodilation induced by angiogenic growth factors such as VEGF and TGF-(31 and induce hypertension.
In vivo effects of soluble endoglin and sFltl In order to assess the vascular effects of soluble endoglin and sFltl, we resorted to adenoviral expression system in pregnant rats. Adenovirus encoding a control gene (CMV) or soluble endoglin or sFltl or sFltl + soluble endoglin were injected by tail vein on day 8 of pregnancy in Sprague Dawley rats. On day 17, animals were examined for pre-eclampsia phenotype. Table 8 includes the hemodynamic and biochemical data.
Table 8. Hemodynamic and biochemical data for adenovirus treated rat animal models.
Groups N MAP in Urine Platelet LDH AST Fetal mm Hg Alb/creat count x U/L U/L weight (g) /m 1000/pl Control 6 83 5 186 94 1,098 +_ 75 156 + 54 + 4 2.1 +_ 0.5 (CMV) 32 sFltl 6 117 + 7* 2,295 867* 1,131 + 91 172 + 94 +_ 4* 1.75 + 0.4 SEng 6 104+6* 432+249 1,195+78 188--- 110+13* 1.6+0.4 sFltl + 6 121 + 9* 9,029 4043* 615 + 67* 1,952 + 210 + 92* 0.75 + 0.3*
sEng - 784* -Data are presented as mean s.e.m. MAP- mean arterial pressure (diastolic pressure +
1/3 pulse pressure); Fetal weight is the average weight of the litter for each group in grams; Alb/Creat - Albumin/creatinine ratios; LDH- Lactate dehyrogenase; AST-Aspartate Aminotransferase.
*P<0.05 when compared to control group.
Expression of sFltl and sEng were first confirmed in rat plasma by.Western blots (FIGURE 36) and circulating concentration quantified using commercially available ELISA kits.
The mean plasma concentrations of sFltl in the control, sEng, sFltl and sFltl+
sEng groups were 0.64 ng/ml, 0.66 ng/ml, 249 ng/ml, and 204 ng/ml respectively. The concentrations of sEng in these four groups were 0.39 ng/ml, 129 ng/ml, 0.37 ng/ml, and 123 ng/ml, respectively.
Soluble endoglin alone induced a mild hypertension. sFltl induced both hypertension and proteinuria, as previously reported. Fetal growth restriction was observed in litters born to the sFltl+sEng group, probably related to the placental vascular ischemia and damage. Importantly, the combination of sFlt'l and soluble endoglin induced severe hypertension, nephrotic range proteinuria, growth restriction of the fetuses and biochemical evidence of the development of the HELLP syndrome (elevated LDH, elevated AST and decreasing platelet counts) (Table 8).
Evidence of hemolysis in the soluble endoglin+sFltl group was confirmed by peripheral smear which revealed schistocytes and reticulocytosis (FIGURES 26A-B). Finally, renal histology also revealed focal endotheliosis in the soluble endoglin group and a severe glomerular endotheliosis in the soluble endoglin+sFltl group (FIGURES 27A-27D, and 33).
Note that in FIGURE 33, the control group is within normal limits. Note open capillary loops with fenestrated endothelium. The soluble endoglin panel shows endothelial swelling with loss of fenestrae and partial luminal occlusion. Note a red blood cell squeezing through the compromised lumen. While light microscopy of the kidneys of soluble endoglin treated rats was not striking for significant endotheliosis, electron microscopy revealed focal endotheliosis. Importantly, the animals that received both soluble endoglin and sFlt-1 had severe glomerular endotheliosis. The combination therapy group (lower panel) shows massive endocapillary occlusion with swollen endothelial cells.
Note the relative preservation of podocyte foot processes (shown as arrows) despite severe proteinuria. Extensive vascular damage of the placenta including infarction at the maternal-fetal junction was observed in the sFltl+sEng group, but not in control rats or in those treated with either agent alone (FIGURES 34A-H). Diffuse inflammation in the giant cell layer (corresponding to human invasive trophoblasts) was noted in the sFitl and sEng groups, and was higher in the combined group. Liver histology revealed signs of ischemia and areas of necrosis in the sFitl+sEng group, similar to those seen in patients with the HELLP syndrome (FIGURES 34A-H). Signs of severe maternal vascular damage were also seen when sFltl+sEng were injected to non-pregnant rats, suggesting that the observed phenotype in pregnant rats was due to a direct effect on the maternal vessels and did not require the placenta.
Summary These results demonstrate that soluble endoglin is up-regulated in pre-eclamptic placentas and is present at extremely high levels in patients with pre-eclampsia. The highest levels of soluble endoglin were present in patients with HELLP
syndrome, one of the most severe forms of pre-eclampsia. These results also demonstrate that soluble endoglin levels correlated with the elevated sFltl in pregnant patients and was higher in those patients in whom there is a higher circulating sFltl levels. In addition, the results indicate that soluble endoglin is an anti-angiogenic molecule and disrupts endothelial function in multiple endothelial assays such as angiogenesis assays, microvascular permeability assays, and microvascular reactivity experiments. Importantly, soluble endoglin can amplify the toxic consequence of sFltl in these in vitro endothelial assays.
Further, in in vivo assays, adenoviral expression of soluble endoglin induces mild hypertension without any significant proteinuria. However, in the presence of sFltl, soluble endoglin induces significant vascular damage as evidenced by the presence of severe hypertension, proteinuria, glomerular endotheliosis, development of the HELLP
syndrome and fetal growth restriction.
The mechanism of soluble endoglin release is likely proteolytic cleavage of the extracellular region of the endoglin molecule. Specific proteases that are up-regulated in the pre-eclamptic tissue may serve as candidate molecules. One example would be the membrane type matrix metalloproteinase-1 (MT1-MMP) that has been shown to cleave betaglycan, a molecule that shares similarity to endoglin (Velasco-Loyden G et al, J.
Biol. Chem. 279:7721-33 (2004)). Therefore, inhibitors of such proteases can serve as valuable targets for the treatment of pre-eclampsia.
Example S. Soluble endoglin inhibits TGF-(31 and TGF-P3 mediated NOS-dependent vasodilation.
eNOS is a Ca2+/calmodulin-regulated nitric oxide (NO) synthase that can be activated by fluid shear stress and neurohumoral stimuli. Endothelium-derived NO is a very potent vasorelaxant contributing to systemic blood pressure regulation, vascular permeability, and angiogenesis. In fact, the effects of VEGF ori angiogenesis and vascular tone are partly mediated by activation of eNOS, through increased eNOS/Hsp90 association and Akt-dependent eNOS phosphorylation at Ser1177. Our recent demonstration that increased placenta-derived sFltl in sera of preeclamptic patients is anti-angiogenic and induces hypertension may in fact reflect impaired VEGF-dependent eNOS activation (Maynard et al., supra). More recently, dephosphorylation of eNOS
Thr495 has been shown to precede Ser1177 phosphorylation and these coordinated events determine eNOS activity in endothelial cells (Fleming et al., Cir. Res.
88:E68-75 (2001)). Given the known effect of VEGF on reducing vascular reactivity via eNOS
activation and the recent demonstration that endoglin modulates eNOS-dependent vasomotor activity (Toporsian et al., Circ. Res. 96:684-692 (2005)), we assessed the hemodynamic effects of TGF-(3 isoforms and soluble endoglin in isolated rat renal microvessels. As described in Example 7 and in FIGURES 24 and 25, both TGF-(3]
and -03 induced a dose-dependent increase in arterial diameter which was significantly attenuated by soluble endoglin. This acute effect of TGF-f31 and -P3 isoforms on vascular tone has not been previously recognized and was also seen in mesenteric vessels (FIGURE 32). VEGF and TGF-01 had additive effects on vasodilation, which were blocked by sEng+sFltl at concentrations noted in patients with preeclampsia (FIGURE
and 35A). L-NAME blocked the vasodilation mediated by TGF-(31 and VEGF
25 indicating a NOS dependent response (FIGURE 35A). These data suggest that circulating sFItl and sEng may oppose the physiological NO-dependent vasodilatation elicited by these angiogenic growth factors, contributirng to the development of hypertension seen in preeclampsia.
Example 9. Soluble endoglin inhibits TGF-P1 binding and signaling in endothelial cells.
Given *that endoglin is a co-receptor for TGF-0 1 and -03 isoforms, we hypothesized that soluble endoglin acts by interfering with cell surface receptor binding.
Pre-incubating radio-labeled TGF-01 with recombinant soluble endoglin significantly reduced its binding to TGF-0 receptor type II (T(3RII) at both 50 and 100 pM
(FIGURE
35B). Thus soluble endoglin competes for TGF-0 1 binding to its receptors on endothelial cells. To test whether this leads to impaired signaling, the activity of a CAGA-Luc reporter construct was assessed in human endothelial cells. TGF-P 1 induced the activation of the Smad 2/3-dependent CAGA-Luc reporter and this response was abolished by treatment with soluble endoglin (FIGURE 35C).
Example 10. Soluble endoglin blocks TGF-P1 mediated eNOS activation.
Given our findings that TGF-(31 induces a NOS-dependent vasorelaxation in both renal and mesenteric resistance vessels, we explored its immediate effects on eNOS
activation. While TGF-0 I had no effect on eNOS Ser1177 phosphorylation, it induced a significant dephosphorylation at Thr495 (FIGURE 35D) suggesting that TGF-0 regulates the phosphorylation status of a key residue in eNOS activation. This effect was significantly attenuated by soluble endoglin (FIGURE 35D).
Taken together, the results in Examples 8-10 demonstrate that soluble endoglin interferes with TGF-0 receptor binding and downstream signaling in endothelial cells and attenuates eNOS activation. Soluble endoglin and sFlt-1 may be working in concert to inhibit endothelial dependent NO activation and vasomotor effects by both the VEGF and the TGF-(3 signaling pathways.
Example 11. Sequential changes in angiogenic factos can identify women at risk for pre-eclampsia or eclampsia.
In the examples described above, we have shown that both sFltl and soluble endoglin (sEng) are intimately related to the pathogenesis of preeclampsia. In the example described below we measured the concentrations of sFlt 1, and sEng in paired serum specimens collected in first and second trimesters from women followed prospectively during pregnancy and whose pregnancy outcomes were characterized in detail in order to determine if sequential changes of these markers between first and second trimesters are associated with the development of pre-eclampsia.
Materials and Methods Study Population We performed a prospective, nested case-control study of women who enrolled in the Massachusetts General Hospital Obstetrical Maternal Study (MOMS) whose methods have been described previously (Thadhani et al., Obstet. Gynecol. 97:515-20 (2001) and Wolf et al., Obstet. Gynecol. 98:757-62 (2001)). In brief, the MOMS cohort was established in 1998 for the prospective study of early gestational risk factors for adverse outcomes that occur later in pregnancy. Women who received prenatal care at Massachusetts General Hospital and affiliated health centers were eligible for inclusion in the cohort. For the current study, consecutive women with singleton gestations between June 1, 2001, and May 1, 2003, who enrolled in the MOMS cohort at or before 12 weeks of gestation and who delivered after 20 weeks were eligible for inclusion.
Cases (n=39) were defined as those with blood collections in the first and second trimester who subsequently developed pre-eclampsia, and controls (n=147) were consecutive contemporaneous women enrolled in the same cohort who delivered at term (>37 weeks) and remained normotensive, normoglycemic, and without evidence of proteinuria throughout pregnancy. Cases and controls were matched by age (f 2 years) and body mass index (+ I kg/m2) given potential for confounding by these exposures (Thadhani et al., Obstet. Gynecol. 94:543-50 (1999)). All subjects provided written informed consent, and this study was approved by the Institutional Review Board of the Massachusetts General Hospital.
Primary Exposures Blood samples were collected at the first prenatal visit (11-13 wks) and again in the second trimester (17-20 wks) in all women. Following collection, samples were stored at -80C for future analysis. The primary exposures were serum sFltl and sEng that were measured using commercial ELISA kits (R&D systems, Minneapolis, MN) (Maynard et al., supra and Venkatesha et al., Nat Med. 12:642-9 (2006)). The intraassay precision coefficients of variation for sFltl and sEng were 3.5 and 3.2%
respectively.
The interassay precision coefficients of variation for sFltl and endoglin were 8.1 and 9.5% respectively. All samples were run in duplicate, and if more than 10%
variation existed between duplicates, the assay was repeated, and averages were reported. All assays were performed by someone who was blinded to case status. Samples were randomly ordered for analysis.
Covariates and Confounders The electronic medical record (EMR), which is the medical record used at the Massachusetts General Hospital, provides clinical and demographic data that prospectively details the events of pregnancy through the early postpartum period.
Specific information obtained from the EMR collected at baseline (first prenatal visit) and at all subsequent prenatal visits included age, race, height, weight, smoking status, gestational age estimated from the last menstrual period and verified by ultrasound, blood pressure, and the results of urine analysis and fetal gestational age estimation. All pregnancy outcome information is also entered in the EMR including results of glucose tolerance tests and other routinely measured laboratory values, and delivery characteristics such as birth weight, route of delivery, and diagnosis of preeclampsia.
Primary Outcomes All pregnancy outcomes were verified by detailed examination of medical records, including prenatal flow sheets and laboratory measurements. At each prenatal visit blood pressure was obtained from the right arm using standard sphygmomanometers with the woman in the seated position after 3-5 minutes of rest. For each patient the proper cuff size was selected based on right midarm circumference.
Measurements of blood pressure that coincided with the timing of the first (systolic) and fifth (diastolic) Korotkoff sounds were recorded. All subjects for the current study had no history of preexisting hypertension or diabetes mellitus, initiated and completed their prenatal care and pregnancy within the MOMS network, delivered a live infant, and had no evidence of hypertension 6 weeks after delivery.
Preeclampsia was defined as systolic blood pressure elevation of at least 140 mm Hg or diastolic blood pressure of at least 90 mm Hg after 20 wk gestation, in association with proteinuria, either 2+ or greater by dipstick or at least 300mg/24 h in the absence of urinary tract infection (ACOG Committee on Practice Bulletins--Obstetrics.
ACOG
practice bulletin. diagnosis and management of preeclampsia and eclampsia.
Number 33, January 2002. Obstet Gynecol. 99:159-67). Preeclamptics were analyzed as term preeclampsia (? 37 weeks) and preterm preeclampsia (<37 weeks).
Statistical analysis Demographic and clinical characteristics were compared using Chi Square tests or Student's t test, as appropriate. Log transformation was needed for the primary exposures given their skewed distributions (Levine et al., (2004), supra, Levine et al., N.
Engl. J. Med. 355:992-1005 (2006)). Primary exposures were examined as continuous variables, and with cut points and tertile analyses based on the natural distributions of the controls and simplified for clinical interpretation. Multiple regression analysis was performed using logistic regression techniques. All P values were two-tailed, and a P
value < 0.05 was considered statistically significant.
Secondary Analysis of angiogenic markers in the CPEP study We also performed a secondary analysis of angiogenic factor changes during early pregnancy from the recently published nested case controlled study within the CPEP cohort, described above. We analyzed samples from normotensive women, women who developed preeclampsia prior to 37 weeks, and women who developed preeclampsia after 37 weeks at three different time intervals - 10-12 weeks,.13-16 weeks, and 17-20 weeks.
Results Demographic and Clinical Characteristics Baseline=and delivery characteristics of the MOMS study population are displayed in Table 9. There were no significant differences in age and body mass index between the two groups. Women who subsequently developed preeclampsia had higher systolic and diastolic blood pressures at the first prenatal visit. At the time of presentation of preeclampsia, systolic and diastolic blood pressures were higher in preeclamptic group as expected.
Table 9. Demographics Variable Normal (n=147) Preeclampsia P value (n=39) Age ( yrs) 31.4 5.4 32.8 t 5.4 0.06 BIvII(kg/m ) 28.5 6.3 29.8 9.1 0.40 Smoker % Never) 47% 60% 0.21 Parity 0.7 0.9 0.8 1.0 0.47 Baseline characteristics DBP(mmHg) 70 7 75 8 <0.01 SBP(mmHg) 113 7 120 13 <0.01 UTP(mg/dl) NA 614 547 Gestational age at 39.3 1.9 37.2 2.3 <0.001 delive Birth weight 3444 532 3300 809 0.35 Characteristics at presentation Maximum DBP 78 5 93 7 <0,01 (mmHg) Maximum SBP 122 t 7 147 ~ 10 <0.01 (mmHg) Maximum SPOT 0.6 0.3 1.4 0.9 <0.01 1'Jg) BMI = body mass index; DBP = diastolic blood pressure; SBP = systolic blood pressure UTP = urine total protein in mg/L; SPOT = urine protein/creatinine ratio Values are mean S.D.
First and Second Trimester Levels of sFltl and sEng in Normal and Preeclamptic Pregnancy The mean serum levels of sFltl were higher in women with preeclampsia compared to women with normal pregnancies, 3.49 0.35 ng/ml versus 3.03 0.13 ng/ml respectively, in first trimester (P=NS). In the second trimester the mean serum levels of sFltl were significantly higher in preeclamptic group, mean value of 4.12+-0.5ng/ml compared to normal group 3.10 0.15 ng/ml (P<0.0 1) (Table 10).
The mean serum levels of sEng thought not significantly different in the first trimester were significantly altered in the second trimester among women with preeclampsia, as compared to normal women, 6.9:h 0.32 ng/ml versus 6.57:L 0.
17 ng/ml (P=NS) in first trimester and 6.37j= 0.38 ng/mi versus 5.23:~= 0.12 ng/ml in second trimester, (P=0.004), respectively (Table 10).
Table 10. Serum levels of sFlt and sEng.
Angiogenic Factor Normal Pregnancy Preeclampsia P value N mean SE N Mean SE
sFltl (ng/ml) 147 3.03 0.13 39 3.496 0.35 0.14 (first trimester) sFltl (ng/ml) 144 3.10 0.15 35 4.12 0.5 0.01*
(second trimester) sEng (ng/ml) 147 6.57 0.17 39 6.9 0.32 0.37 (first trimester) sEng (ng/ml) 144 5.23 0.12 35 6.37 0.38 0.004*
(second trimester) Sequential changes in angiogenic factors Figure 37 graphically displays the delta or d (difference between first and second trimester values of sFltl and sEng) in normal, all preeclamptic women and in women with preeclampsia less than 37 weeks. In normal pregnancy, there is very little change in sFltl between first and second trimester (dsFltl=0.05 0.15 ng/ml). dsFltl is relatively higher in women who develop pre-eclampsia 0.713 =b 0.47 ng/ml versus 0.0497 ZE
0.15 ng/ml in normal women (P=0.08). Similarly in women with pre-eclampsia less than 37 weeks, dsFltl was higher at 0.634 t 0.91 ng/ml.
There is a fall in the level of sEng between the first and second trimester in normal pregnancy (-1.322 =~ 0.18 ng/ml). This fall is blunted in patients with pre-eclampsia, with dsEng of -0.441t0.42 ng/ml (P=0.04). In women with pre-eclampsia less than 37 weeks, the fall in the level of soluble endoglin is blunted and appeard to trend in the opposite direction (0.732+0.77 ng/ml, P <0.01 compared with controls).
Predictive Algorithnzs To see if these alterations can be used as a predictive test for severe premature pre-eclampsia, we looked at the product (value of sFItl x sEng) as product-I
(in first trimester) and product-2 (in second trimester) in normotensive women and women who developed pre-eclampsia and specifically in preterm pre-eclampsia. Both product-1 and product-2 were significantly elevated in patients with pre-eclampsia and importantly the delta of the product (dproduct) were strikingly positive in contrast with a negative number in normal controls (Figure 38). Furthermore, the dproduct were greatly amplified in patients with preterm pre-eclampsia as compared to normal controls (P=0.004).
To assess the relationship between altered levels of angiogenic factors and risk of preterm pre-eclampsia, we computed adjusted odds ratios (aOR) and 95 percent confidence intervals (95% CI) for preterm pre-eclampsia in the highest category of the distribution of dproduct concentrations with respect to the lower two categories after adjustment for race/ethnicity, body-mass index, and gestational age at specimen collection (Figure 39). Substantial increases in risk of preterm were observed in the , group whose delta product levels were greater than +1 [aOR 5.5, 95% CI 1.4 -22.4], compared to women whose delta product was less than -1.
Secondary anal,ysis of tlie CPEP nested case control study In the subset of women in the CPEP trial, among the cohort of women who remained normotensive, the mean values of sFltl increased from 3.68 ng/ml to 4.92 ng/ml between 10-12wks and 13-16 weeks and decreased to 4.29 ng/ml at 17-20 weeks, while in women with preterm pre-eclampsia, the mean values of sFltl increased from 3.44 ng/ml to 4.22 ng/ml and further increased to 5.39 ng/ml at 10-12, 13-16 and 17-20 weeks of gestation. This pattern was not obvious in women with term pre-eclampsia (Table 11).
Table 11. Serum levels of sFlt and sEng in CPEP trial.
sFltl (ng/ml) sEng (ng/ml) wks wks wks wks wks wks Normal 3.68 4.99 4.29 6.8 7.07 5.78 PE <37 wks 3.44 4.22 5.39 7.15 7.9 10.19 N 13 28 32 ' 13 28 32 PE >37 wks 4.06 4.48 4.25 7.41 7.8 8.34 Similar results were observed in the levels of sEng. In normotensive women the levels of sEng increased from 6.8 ng/ml to 7.07 ng/ml at 10-12 wks and 13-16 wks and then decreased to 5.78 ng/ml at 17-20 wks while in women with preterm preeclampsia the levels of endoglin increased from 7.15 ng/ml to 7.95 ng/ml and further increased to 10.19 ng/ml between 10-12 wks, 13-16 wks and 17-20 weeks. The same trend was noted in women with term preeclampsia where the mean levels of sEng increased from 7.41 ng/ml to 7.80 ng/ml to 8.34 ng/ml at 10-12 weeks, 13-16 weeks and 17-20 weeks (see Table 11).
Summary Both sFltl and sEng are elevated during second trimester in patients destined to develop pre-eclampsia. Normal pregnancy is characterized by a fall in sEng from first to second trimester without significant change in sFltl. However, in patients who develop pre-eclampsia, particularly preterm pre-eclampsia, both sFltl and sEng continue to rise from first to second trimester. The changes in sFltl and sEng during first and second trimesters are useful for screening patients at high risk for subsequent development of preterm pre-eclampsia.
These findings have important implications for the prediction of preterm preeciampsia. The pursuit of a safe, reliable screening test for preeclampsia has been a goal of researchers for many years. Previous efforts have focused on detecting early manifestations of disease such as microalbuminuria, weight gain and plasma volume changes. In a large metaanalysis, Conde-Agudelo A et al analyzed eighty-seven of 7,191 (211,369 women) potentially relevant articles to assess the usefulness of clinical, biophysical, and biochemical tests in the prediction of pre-eclampsia. They concluded that as of 2004, there was no clinically useful screening test for predicting the development of pre-eclampsia (12). In the present study we have shown that the levels of sFltl and sEng are elevated in women who are destined to become pre-eclamptic, in their first and second trimester (as measured as a delta in individual patients), weeks to months before the clinical onset of disease. These changes are more substantial in women who develop preterm pre-eclampsia.
An imbalance in angiogenic factors is thought to play an intimate role in the pathogenesis of pre-eclampsia. Pre-eclamptic placentas are characterized by shallow implantation and abnormal vascular remodeling including impaired pseudo-vasculogenesis (Fisher et al., Semin Cell Biol. 4:183-8 (1993)). It is believed that these placental changes occur between 12-18 weeks of pregnancy and is important in the pathogenesis of the vast majority of severe early onset preeclampsia. It is thought that these placentation abnormalities lead to the elaboration of systemic factors that induce the maternal syndrome of preeclarnpsia. As described herein and in PCT application publication numbers WO 2004/008946, WO 2005/077007, and WO 2006/034507, both sFltl and sEng, two anti-angiogenic proteins have been found to be elevated in preeclampsia not only during clinical disease but also several weeks before onset of symptoms (Levine et al., (2006), supra). Importantly, both factors have been implicated in inducing a preeclampsia-like syndrome in rats (Maynard et al., supra, Venkatesha et al., supra). However, since alterations in concentrations of angiogenic factors in the matemal circulation occur relatively late in pregnancy, increased production of these anti-angiogenic factors may be a secondary phenomenon that occurs in response to abnormal placentation. In vitro data using placental villous explants and primary cytotrophoblast culture studies suggest that in addition to its role in inducing maternal endothelial dysfunction, anti-angiogenic factors may be involved in cytotrophoblast migration and differentiation. Our findings that levels of anti-angiogenic factors decrease from the first to the second trimester in normal pregnancies, but not in pregnancies in which preterm preeclampsia later develops, suggests that abnormalities of circulating angiogenic factors are occurring at the same time as abnormalities in placental differentiation.
The etiology of the increased concentrations of circulating sFlt 1 and sEng in preeclam.ptic patients is unknown. Hypoxia, genes, or immunological factors are believed to play a role. It is worth noting that expression of both sFltl and sEng are elevated in response to hypoxia in vivo and in vitro models of placental hypoxia where increased expression is mediated by HIF-1 (Nevo et al., Am JPhysiol Regul Integr Comp Physiol. 291:R1085-93 (2006)). Furthermore, it is believed that during normal pregnancy the placenta is hypoxic early in pregnancy and this hypoxia disappears with increased 20' blood flow to the placenta during second trimesters. Although hypoxia has never been formally documented in pre-eclamptic pregnancies, it is believed that hypoxia is central to most pre-eclamptic pregnancies based on surrogate evidence of increased hypoxia induced transcription factor expression and impaired Doppler blood flow to the placentas.
Our findings that both sFltl and sEng remain elevated in patients with severe pre-eclampsia in contrast to normal pregnancies where there is a fall between first and second trimesters suggests that placental ischemia may in fact play a role in the increased production of these anti-angiogenic proteins in pre-eclamptic patients.
In summary, sequential changes of sFltl and sEng appear to identify women destined to develop preeclampsia, especially women who subsequently develop preterm pre-eclampsia. Our findings are reproduced in cross-sectional studies with much larger sample size (Table 11).
Example 12. Endoglin is necessary for TGF-P1-induced eNOS Thr495 dephosphorylation.
Murine endothelial cells were derived from Ene and Eng"" mouse embryos (E8.5) and grown as described in Balconi et al. (Arterioscler. Thromb. Vasc.
Biol.
20:1443-51 (2000)). Confluent monolayers were serum starved for 2 hours and stimulated with or without TGF-P 1 (125 and 250 pM) for 15 minutes. Cell extracts were immediately prepared in 10mM Tris-HCI containing 1% Triton X-100 and supplemented with protease and phosphatase inhibitors. Protein concentrations were quantified and samples were analyzed by western blot using phospho-specific pAbs to Thr495 of eNOS
(Cell Signaling) and a mAb for total eNOS (BD Biosciences).
The representative western blots shown in Figure 40A and associated graph shown in Figure 40B (mean of n=3 experiments) demonstrate that TGF-(31-induced (**p<0.01 versus baseline) eNOS Thr495 dephosphorylation in mouse Eng+"+
endothelial cells but not in Eng'l"cells. This result suggests a critical role for endoglin in linking TGF-01 signaling to eNOS Thr495 dephosphorylation and activation. Moreover, given that this process is endoglin-dependent, soluble endoglin can be used to inhibit the process, presumably by binding to and inhibiting TGF-p I.
Other Embodiments The description of the specific embodiments of the invention is presented for the purposes of illustration. It is not intended to be exhaustive or to limit the scope of the invention to the specific fornls described herein. Although the invention has been described with reference to several embodiments, it will be understood by one of ordinary skill in the art that various modifications can be made without departing from the spirit and the scope of the invention, as set forth in the claims.
All patents, patent applications, and publications referenced herein, including PCT Application Publication Numbers WO 2004/008946, WO 2005/077007, and WO
2006/034507; U.S Patent Application Publication Numbers 20060067937 and 20070104707; and U.S. Provisional Patent Application Number 60/852,761 are hereby incorporated by reference.
Other embodiments are in the claims.
What is claimed is:
Claims (57)
1. A method of treating or preventing a pregnancy related hypertensive disorder in a subject, said method comprising the step of administering to said subject a compound capable of increasing the expression level or biological activity of NOS, wherein said administering is for a time and in an amount sufficient to treat or prevent said pregnancy related hypertensive disorder in said subject.
2. The method of claim 1, wherein said NOS is eNOS.
3. The method of claim 2, wherein said compound increases the phosphorylation of Ser 1177 of eNOS.
4. The method of claim 1, wherein said compound is VEGF or biologically active fragments thereof.
5. The method of claim 4, wherein said VEGF is VEGF121, VEGF165, or a modified form of VEGF, or biologically active fragments thereof.
6. The method of claim 25, wherein said compound is P1GF or biologically active fragments thereof.
7. The method of claim 2, wherein said compound increases the dephosphorylation of Thr495 of eNOS.
8. The method of claim 7, wherein said compound is selected from the group consisting of TGF-.beta.1, TGF-.beta.3, activin A, BMP-2, BMP-7, and biologically active fragments thereof.
9. The method of claim 1, further comprising administering to said subject a compound capable of reducing soluble endoglin expression or soluble endoglin biological activity, wherein said administering is sufficient to treat or prevent said pregnancy related hypertensive disorder in said subject.
10. The method of claim 9, wherein said compound is a purified antibody that specifically binds soluble endoglin or a soluble endoglin antigen-binding fragment.
11. The method of claim 10, wherein said antibody binds to a soluble endoglin polypeptide or fragment thereof comprising an amino acid sequence selected from the group consisting of amino acids 26 to 437, 40 to 406, or 26 to 587 of the human endoglin sequence shown in Figure 30B.
12. The method of claim 10, wherein said antibody binds to an epitope on soluble endoglin comprising amino acids 40 to 86, 144 to199, 206 to 222, 289 to 304, or 375 to 381 of the human endoglin sequence shown in Figure 30B.
13. The method of claim 9, wherein said compound is a compound that inhibits a proteolytic enzyme selected from the group consisting of a matrix metalloproteinase (MMP), cathepsin, and elastase.
14. The method of claim 1, wherein said pregnancy related hypertensive disorder is characterized by increased levels of sFlt-1 as compared to a normal reference.
15. The method of claim 1, further comprising administering to said subject a compound capable of reducing sFlt-1 expression levels or sFlt-1 biological activity, wherein said administering is sufficient to treat or prevent said pregnancy related hypertensive disorder in said subject.
16. The method of claim 15, wherein said compound is a purified growth factor or antibody or antigen binding fragment that binds sFlt-1.
17. The method of claim 1, wherein said subject is a pregnant human, a post-partum human, or a pregnant non-human.
18. The method of claim 1, wherein said method further comprises monitoring said pregnancy related hypertensive disorder in said subject, wherein said monitoring comprises measuring the level of soluble endoglin polypeptide in a serum or plasma sample from said subject, wherein a level of soluble endoglin polypeptide less than 25 ng/ml indicates an improvement in said pregnancy related hypertensive disorder.
19. The method of claim 1, wherein said method further comprises monitoring said pregnancy related hypertensive disorder in said subject, wherein said monitoring comprises measuring the level of soluble endoglin polypeptide in a sample from said subject, wherein said measuring of levels is done on two or more occasions and a decrease in said soluble endoglin levels between measurements is an indicator of an improvement in said pregnancy related hypertensive disorder.
20. The method of claim 1, wherein said method further comprises monitoring said pregnancy related hypertensive disorder in said subject, wherein said monitoring comprises measuring the level of soluble endoglin polypeptide in a sample from said subject and comparing said level to a positive reference sample, wherein a decrease in the level of soluble endoglin relative to said positive reference sample indicates an improvement in said pregnancy related hypertensive disorder in said subject.
21. The method of any one of claims 18-20, wherein said monitoring is used to determine the therapeutic dosage of the compound.
22. The method of any one of claims 18-20, wherein said measuring comprises the use of an immunological assay.
23. The method of any one of claims 18-20, wherein the level of soluble endoglin is the level of free, bound, or total soluble endoglin or the level of an endoglin polypeptide resulting from degradation or enzymatic cleavage.
24. The method of any one of claims 18-20, wherein said monitoring further comprises measuring the level of at least one of sFlt-1, VEGF, or P1GF
polypeptide in a sample from said subject.
polypeptide in a sample from said subject.
25. The method of claim 24, further comprising calculating the relationship between said levels of soluble endoglin, sFlt-1, VEGF, or P1GF using a metric, wherein an alteration in the relationship between said levels in the subject sample relative to a reference sample, indicates an improvement in said pregnancy related hypertensive disorder in said subject.
26. The method of claim 25, wherein said metric is selected from the group consisting of: [(sFlt-1 + 0.25 soluble endoglin)/P1GF], [(soluble endoglin +
sFlt-1)/P1GF], and [sFlt-1 x soluble endoglin].
sFlt-1)/P1GF], and [sFlt-1 x soluble endoglin].
27. The method of claim 25, wherein said metric is [dproduct =(sFlt1 x sEng) in the second trimester -(sFlt1 x sEng) in the first trimester], wherein a dproduct value greater than 0 indicates a pregnancy related hypertensive disorder in said subject and a decrease in said dproduct value as compared to a positive reference sample indicates an improvement in said pregnancy related hypertensive disorder in said subject.
28. An antibody or antigen-binding fragment thereof that specifically binds a soluble endoglin polypeptide, wherein said antibody binds to an epitope comprising amino acids 40 to 86, 144 to 199, 206 to 222, 289 to 304, or 375 to 381 of the human endoglin sequence shown in Figure 30B.
29. The antibody or antigen-binding fragment thereof of claim 28, wherein said antibody or antigen-binding fragment prevents binding of a growth factor to soluble endoglin.
30. The antibody or antigen-binding fragment thereof of claim 29, wherein said growth factor is selected from the group consisting of TGF-.beta.1, TGF-.beta.3, activin A, BMP-2, and BMP-7.
31. The antibody or antigen-binding fragment thereof of claim 28, wherein said antibody is a monoclonal antibody, chimeric antibody, humanized antibody, or human antibody.
32. The antibody or antigen-binding fragment thereof of claim 28, wherein said antibody lacks an Fc portion, is an F(ab')2, an Fab, or an Fv structure.
33. The antibody or antigen-binding fragment thereof of claim 28, wherein said antibody or antigen-binding fragment thereof is present in a pharmaceutically acceptable carrier.
34. A method of diagnosing a subject as having, or having a predisposition to, a pregnancy related hypertensive disorder, said method comprising measuring the level of a soluble endoglin polypeptide and at least one additional polypeptide selected from the group consisting of TGF-.beta.1, TGF-.beta.3, activin A, BMP2, BMP7, eNOS, and PGI2 in a sample from said subject, wherein an increase in the soluble endoglin level and a decrease in the level of said at least one additional polypeptide as compared to a normal reference sample, standard, or level is a diagnostic indicator of a pregnancy related hypertensive disorder or a propensity to develop a pregnancy related hypertensive disorder.
35. The method of claims 34, wherein said measuring comprises the use of an immunological assay.
36. The method of claim 35, wherein said immunological assay is an ELISA.
37. The method of claim 34, wherein said normal reference sample is a prior sample from said subject.
38. A method of diagnosing a subject as having, or having a predisposition to, a pregnancy related hypertensive disorder, said method comprising measuring the level of a soluble endoglin polypeptide and an sFlt-1 polypeptide from said subject and calculating the relationship between said levels of soluble endoglin and sFlt-1 using a [soluble endoglin x sFlt-1] metric, wherein an increase in the metric in the subject sample relative to a normal reference sample, is a diagnostic indicator of a pregnancy related hypertensive disorder in said subject.
39. The method of claim 38, wherein said metric further comprises the body mass index of the mother or the gestational age of the fetus.
40. A method of diagnosing a subject as having, or having a predisposition to, a pregnancy related hypertensive disorder, said method comprising measuring the level of a soluble endoglin polypeptide and an sFlt-1 polypeptide from said subject during the first trimester and the second trimester and calculating the relationship between said levels of soluble endoglin and sFlt-1 using the following metric: [dproduct =(sFlt1 x sEng) in the second trimester - (sFlt1 x sEng) in the first trimester], wherein a dproduct value greater than zero is a diagnostic indicator of a pregnancy related hypertensive disorder in said subject.
41. The method of claim 40, wherein a dproduct value greater than one is a diagnostic indicator of a pregnancy related hypertensive disorder in said subject.
42. The method of claim 41, wherein the dproduct value greater than one is a diagnostic indicator of pre-term pre-eclampsia.
43. The method of claim 34, 38, or 40, wherein said sample is a bodily fluid, cell, or a tissue of said subject in which said soluble endoglin is normally detectable.
44. The method of claim 43, wherein said bodily fluid is selected from the group consisting of urine, amniotic fluid, blood, serum, and plasma.
45. The method of claim 43, wherein said cell is selected from the group consisting of an endothelial cell, a leukocyte, a monocyte, and a cell derived from the placenta.
46. The method of claim 43, wherein said tissue is a placental tissue.
47. The method of claim 34, 38, or 40, wherein said subject is a non-pregnant human, a pregnant human, a post-partum human, or a non-human and said method diagnoses a propensity to develop a pregnancy related hypertensive disorder.
48. The method of claim 47, wherein said non-human is selected from the group consisting of a cow, a horse, a sheep, a pig, a goat, a dog, or a cat.
49. The method of claim 34, 38, or 40, wherein said method is used to diagnose a pregnancy related hypertensive disorder or a propensity to develop a pregnancy related hypertensive disorder at least four weeks prior to the onset of symptoms.
50. The method of claim 1, 34, 38, or 40, wherein said pregnancy related hypertensive disorder is pre-eclampsia, eclampsia, gestational hypertension, chronic hypertension, HELLP syndrome, and pregnancy with a SGA infant.
51. The method of claim 50, wherein said pregnancy related hypertensive disorder is pre-eclampsia or eclampsia.
52. A kit for the diagnosis of a pregnancy related hypertensive disorder in a subject comprising (i) a soluble endoglin binding agent and (ii) at least one additional binding agent that binds to a polypeptide selected from the group consisting of TGF-.beta.1, TGF-.beta.3, activin A, BMP2, BMP7, eNOS, and PG12 and (iii) instructions for the use of the binding agent of (i) and the at least one binding agent of (ii) for the diagnosis of a pregnancy related hypertensive disorder or a propensity to develop a pregnancy related hypertensive disorder.
53. The kit of claim 52, wherein said binding agent of (i) is an antibody, or antigen-binding fragment thereof, that specifically binds soluble endoglin and the at least one binding agent of (ii) is an antibody, or antigen binding fragment thereof, that specifically binds TGF-.beta.1, TGF-.beta.33, activin A, BMP2, BMP7, eNOS, or PGI2.
54. The kit of claim 52, wherein said kit further comprises a VEGF, sFlt-1, or P1GF binding molecule.
55. The kit of claim 52, wherein said pregnancy related hypertensive disorder is pre-eclampsia, eclampsia, chronic hypertension, HELLP syndrome, gestational hypertension, or pregnancy with an SGA infant.
56. A method of identifying a compound that ameliorates a pregnancy related hypertensive disorder, said method comprising (a) contacting a cell with a soluble endoglin compound;
(b) determining the phosphorylation state of Thr495 of eNOS in said cell of step (a);
(c) contacting said cell with a candidate compound;
(d) determining the phosphorylation state of Thr495 of eNOS in said cell of step (c); and (e) comparing the phosphorylation state determined in step (b) and step (d), wherein an increase in the dephosphorylation of Thr 495 of eNOS in step (d) as compared to step (b) identifies said candidate compound as a compound that ameliorates a pregnancy related hypertensive disorder.
(b) determining the phosphorylation state of Thr495 of eNOS in said cell of step (a);
(c) contacting said cell with a candidate compound;
(d) determining the phosphorylation state of Thr495 of eNOS in said cell of step (c); and (e) comparing the phosphorylation state determined in step (b) and step (d), wherein an increase in the dephosphorylation of Thr 495 of eNOS in step (d) as compared to step (b) identifies said candidate compound as a compound that ameliorates a pregnancy related hypertensive disorder.
57. A method of identifying a compound that ameliorates a pregnancy related hypertensive disorder, said method comprising (a) contacting a cell with a Smad2/3 dependent reporter construct and a soluble endoglin compound;
(b) determining the level of activation of the Smad2/3 reporter construct in the cell of step (a);
(c) contacting said cell of step (a) with a candidate compound;
(d) determining the level of activation of the Smad2/3 reporter construct in the cell of step (c); and (e) comparing the level of activation of the Smad2/3 reporter construct determined in step (b) and step (d), wherein an increase in the level of activation of the Smad2/3 reporter construct in step (d) as compared to step (b) identifies said candidate compound as a compound that ameliorates a pregnancy related hypertensive disorder.
(b) determining the level of activation of the Smad2/3 reporter construct in the cell of step (a);
(c) contacting said cell of step (a) with a candidate compound;
(d) determining the level of activation of the Smad2/3 reporter construct in the cell of step (c); and (e) comparing the level of activation of the Smad2/3 reporter construct determined in step (b) and step (d), wherein an increase in the level of activation of the Smad2/3 reporter construct in step (d) as compared to step (b) identifies said candidate compound as a compound that ameliorates a pregnancy related hypertensive disorder.
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US11/443,920 US7740849B2 (en) | 2004-09-24 | 2006-05-31 | Use of compounds that bind soluble endoglin and SFLT-1 for the treatment of pregnancy related hypertensive disorders |
US11/443,920 | 2006-05-31 | ||
US85276106P | 2006-10-19 | 2006-10-19 | |
US60/852,761 | 2006-10-19 | ||
PCT/US2007/012787 WO2008030283A1 (en) | 2006-05-31 | 2007-05-31 | Methods of diagnosing and treating complications of pregnancy |
Publications (1)
Publication Number | Publication Date |
---|---|
CA2654283A1 true CA2654283A1 (en) | 2008-03-13 |
Family
ID=39157545
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA002654283A Abandoned CA2654283A1 (en) | 2006-05-31 | 2007-05-31 | Methods of diagnosing and treating complications of pregnancy |
Country Status (9)
Country | Link |
---|---|
EP (1) | EP2029743A4 (en) |
JP (1) | JP2009538915A (en) |
KR (1) | KR20090040874A (en) |
CN (1) | CN101517078A (en) |
AU (1) | AU2007293475A1 (en) |
BR (1) | BRPI0713122A2 (en) |
CA (1) | CA2654283A1 (en) |
MX (1) | MX2008015197A (en) |
WO (1) | WO2008030283A1 (en) |
Families Citing this family (21)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7740849B2 (en) | 2004-09-24 | 2010-06-22 | Beth Israel Deaconess Medical Center | Use of compounds that bind soluble endoglin and SFLT-1 for the treatment of pregnancy related hypertensive disorders |
DK1804836T3 (en) | 2004-09-24 | 2011-01-24 | Beth Israel Hospital | Methods for diagnosing and treating pregnancy complications |
CN101299962A (en) | 2004-12-15 | 2008-11-05 | 貝丝以色列女执事医疗中心 | Nucleic acids and polypeptides useful for diagnosing and treating complications of pregnancy |
US20100016173A1 (en) * | 2008-01-30 | 2010-01-21 | Proteogenix, Inc. | Maternal serum biomarkers for detection of pre-eclampsia |
EP2300825A4 (en) * | 2008-06-18 | 2012-04-25 | Abbott Lab | P/gf-1 companion diagnostic methods and products |
CA2759534C (en) * | 2009-04-23 | 2018-03-27 | Wallac Oy | Method for determining maternal health risks |
AU2011231982B2 (en) * | 2010-03-24 | 2014-02-20 | Preelumina Diagnostics Ab | HbF and A1M as early stage markers for preeclampsia |
CA2793487A1 (en) * | 2010-04-13 | 2011-10-20 | Pronota N.V. | Biomarkers for hypertensive disorders of pregnancy |
US20110251094A1 (en) | 2010-04-13 | 2011-10-13 | Pronota N.V. | Biomarkers for hypertensive disorders of pregnancy |
EP2569253A4 (en) | 2010-05-14 | 2013-10-02 | Beth Israel Hospital | Extracorporeal devices and methods of treating complications of pregnancy |
CN102058435B (en) * | 2011-01-17 | 2012-05-09 | 北京工业大学 | Monitor for gestational hypertension risk on basis of physiology, biochemistry and blood dynamics information |
WO2013000992A1 (en) | 2011-06-28 | 2013-01-03 | Vitateq Biotechnology Gmbh | Method for diagnosing preeclampsia |
CN104011069A (en) * | 2011-08-01 | 2014-08-27 | 塔夫茨医学中心有限公司 | Treatment of heat failure and related conditions |
EP2890816B1 (en) * | 2012-08-30 | 2019-06-05 | Ansh Labs LLC | Papp-a2 as a marker for monitoring, predicting and diagnosing preeclampsia |
KR101417379B1 (en) * | 2012-10-30 | 2014-07-10 | 곽호석 | Diagnostic methods for Fetal Lung Maturity using Mass Spectrometer |
CN103235142B (en) * | 2013-04-28 | 2015-04-15 | 成都中医药大学 | First pregnancy screening kit for pregnant woman |
CN103207274B (en) * | 2013-04-28 | 2015-02-25 | 成都中医药大学 | Screening kit for seventh chromosome abnormality diseases of fetus |
MX2018011739A (en) * | 2016-03-31 | 2018-12-19 | Immutrix Therapeutics Inc | Method for extracorporeal treatment of preeclampsia and related disorders. |
CN113919453B (en) * | 2021-09-13 | 2024-06-11 | 深圳大学龙华生物产业创新研究院 | Blockchain integration system for recording biological characteristics |
CN113933129B (en) * | 2021-09-14 | 2024-01-12 | 深圳大学 | Preeclampsia diagnosis kit and application of fluorescent dye |
CN115791340B (en) * | 2023-01-17 | 2023-05-02 | 北京水木济衡生物技术有限公司 | Epilepsy composite quality control product and preparation method and application thereof |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20020031513A1 (en) * | 1997-11-24 | 2002-03-14 | Shamir Leibovitz | Method and pharmaceutical composition for inhibiting premature rapture of fetal membranes, ripening of uterine cervix and preterm labor in mammals |
HUP0200799A3 (en) * | 1999-04-16 | 2004-11-29 | Univ Yale New Haven | Enos mutations useful for gene therapy and therapeutic screening |
EP1407276B1 (en) * | 2001-04-24 | 2009-10-21 | Vlaams Interuniversitair Instituut voor Biotechnologie vzw. | Use of hypoxia inducible factor 2alpha for curing neonatal respiratory distress syndrome |
DK1804836T3 (en) * | 2004-09-24 | 2011-01-24 | Beth Israel Hospital | Methods for diagnosing and treating pregnancy complications |
-
2007
- 2007-05-31 CN CNA2007800268583A patent/CN101517078A/en active Pending
- 2007-05-31 BR BRPI0713122-4A patent/BRPI0713122A2/en not_active IP Right Cessation
- 2007-05-31 JP JP2009513263A patent/JP2009538915A/en active Pending
- 2007-05-31 CA CA002654283A patent/CA2654283A1/en not_active Abandoned
- 2007-05-31 MX MX2008015197A patent/MX2008015197A/en active IP Right Grant
- 2007-05-31 AU AU2007293475A patent/AU2007293475A1/en not_active Abandoned
- 2007-05-31 WO PCT/US2007/012787 patent/WO2008030283A1/en active Application Filing
- 2007-05-31 EP EP07795515A patent/EP2029743A4/en not_active Withdrawn
- 2007-05-31 KR KR1020087032270A patent/KR20090040874A/en not_active Application Discontinuation
Also Published As
Publication number | Publication date |
---|---|
EP2029743A1 (en) | 2009-03-04 |
AU2007293475A1 (en) | 2008-03-13 |
WO2008030283A1 (en) | 2008-03-13 |
MX2008015197A (en) | 2009-06-23 |
KR20090040874A (en) | 2009-04-27 |
JP2009538915A (en) | 2009-11-12 |
EP2029743A4 (en) | 2010-12-08 |
CN101517078A (en) | 2009-08-26 |
BRPI0713122A2 (en) | 2012-04-17 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20170242022A1 (en) | Methods of diagnosing and treating complications of pregnancy | |
CA2654283A1 (en) | Methods of diagnosing and treating complications of pregnancy | |
US10413591B2 (en) | Methods of diagnosing and treating complications of pregnancy | |
US7740849B2 (en) | Use of compounds that bind soluble endoglin and SFLT-1 for the treatment of pregnancy related hypertensive disorders | |
US7407659B2 (en) | Methods of diagnosing pre-eclampsia or eclampsia | |
EP2172220A1 (en) | Methods of diagnosing and treating pre-eclampsia or eclampsia | |
ZA200501478B (en) | Methods of diagnosing and treating pre-eclampsia or eclampsia |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
EEER | Examination request | ||
FZDE | Discontinued |
Effective date: 20150602 |