US20130302821A1 - Method For Evaluating Atherosclerotic Lesion, And Kit - Google Patents
Method For Evaluating Atherosclerotic Lesion, And Kit Download PDFInfo
- Publication number
- US20130302821A1 US20130302821A1 US13/885,921 US201113885921A US2013302821A1 US 20130302821 A1 US20130302821 A1 US 20130302821A1 US 201113885921 A US201113885921 A US 201113885921A US 2013302821 A1 US2013302821 A1 US 2013302821A1
- Authority
- US
- United States
- Prior art keywords
- protein
- chain
- binding
- atherosclerotic lesion
- stage
- 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
- 230000003902 lesion Effects 0.000 title claims abstract description 190
- 230000003143 atherosclerotic effect Effects 0.000 title claims abstract description 147
- 238000000034 method Methods 0.000 title claims abstract description 86
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 274
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 273
- 238000001514 detection method Methods 0.000 claims abstract description 26
- 239000000126 substance Substances 0.000 claims description 90
- 206010003210 Arteriosclerosis Diseases 0.000 claims description 80
- 208000011775 arteriosclerosis disease Diseases 0.000 claims description 79
- 230000027455 binding Effects 0.000 claims description 73
- 239000003814 drug Substances 0.000 claims description 56
- 238000012360 testing method Methods 0.000 claims description 51
- 238000011156 evaluation Methods 0.000 claims description 41
- 229940124597 therapeutic agent Drugs 0.000 claims description 37
- 238000002560 therapeutic procedure Methods 0.000 claims description 35
- 239000003795 chemical substances by application Substances 0.000 claims description 31
- 239000002872 contrast media Substances 0.000 claims description 28
- 108060003951 Immunoglobulin Proteins 0.000 claims description 25
- 241001465754 Metazoa Species 0.000 claims description 25
- 102000018358 immunoglobulin Human genes 0.000 claims description 25
- 102100035182 Plastin-2 Human genes 0.000 claims description 22
- 101710081231 Plastin-2 Proteins 0.000 claims description 22
- 102100029219 Thrombospondin-4 Human genes 0.000 claims description 20
- 108010060815 thrombospondin 4 Proteins 0.000 claims description 20
- 102100026862 CD5 antigen-like Human genes 0.000 claims description 19
- 101710122347 CD5 antigen-like Proteins 0.000 claims description 19
- 102100038567 Properdin Human genes 0.000 claims description 19
- 108010005642 Properdin Proteins 0.000 claims description 19
- 102100027473 Cartilage oligomeric matrix protein Human genes 0.000 claims description 18
- 101710176668 Cartilage oligomeric matrix protein Proteins 0.000 claims description 18
- 108010013996 Fibromodulin Proteins 0.000 claims description 18
- 102000017177 Fibromodulin Human genes 0.000 claims description 18
- 102100037362 Fibronectin Human genes 0.000 claims description 18
- 108010067306 Fibronectins Proteins 0.000 claims description 18
- 239000012528 membrane Substances 0.000 claims description 18
- 102000007000 Tenascin Human genes 0.000 claims description 17
- 108010008125 Tenascin Proteins 0.000 claims description 17
- 101710170765 Fibrinogen beta chain Proteins 0.000 claims description 16
- 102100024783 Fibrinogen gamma chain Human genes 0.000 claims description 16
- 108010046722 Thrombospondin 1 Proteins 0.000 claims description 16
- 102100036034 Thrombospondin-1 Human genes 0.000 claims description 16
- 108010031318 Vitronectin Proteins 0.000 claims description 16
- 102100035140 Vitronectin Human genes 0.000 claims description 16
- 108010048325 fibrinopeptides gamma Proteins 0.000 claims description 16
- 102000014914 Carrier Proteins Human genes 0.000 claims description 15
- 108010016165 Matrix Metalloproteinase 2 Proteins 0.000 claims description 15
- 102000000424 Matrix Metalloproteinase 2 Human genes 0.000 claims description 15
- 108091008324 binding proteins Proteins 0.000 claims description 15
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 14
- 102100022980 ADAMTS-like protein 4 Human genes 0.000 claims description 14
- 101710188407 ADAMTS-like protein 4 Proteins 0.000 claims description 14
- 102100033312 Alpha-2-macroglobulin Human genes 0.000 claims description 14
- 108010053085 Complement Factor H Proteins 0.000 claims description 14
- 102100035432 Complement factor H Human genes 0.000 claims description 14
- 102100038938 Myosin-9 Human genes 0.000 claims description 14
- 101710204108 Myosin-9 Proteins 0.000 claims description 14
- 108010015078 Pregnancy-Associated alpha 2-Macroglobulins Proteins 0.000 claims description 14
- 102000003847 Carboxypeptidase B2 Human genes 0.000 claims description 13
- 108090000201 Carboxypeptidase B2 Proteins 0.000 claims description 13
- 102000004225 Cathepsin B Human genes 0.000 claims description 13
- 108090000712 Cathepsin B Proteins 0.000 claims description 13
- 102000003780 Clusterin Human genes 0.000 claims description 13
- 108090000197 Clusterin Proteins 0.000 claims description 13
- 102100024203 Collagen alpha-1(XIV) chain Human genes 0.000 claims description 13
- 101710106877 Collagen alpha-1(XIV) chain Proteins 0.000 claims description 13
- 102000003712 Complement factor B Human genes 0.000 claims description 13
- 108090000056 Complement factor B Proteins 0.000 claims description 13
- 102000030914 Fatty Acid-Binding Human genes 0.000 claims description 13
- 101710132152 Immunoglobulin J chain Proteins 0.000 claims description 13
- 102100029571 Immunoglobulin J chain Human genes 0.000 claims description 13
- 102100023490 Inter-alpha-trypsin inhibitor heavy chain H1 Human genes 0.000 claims description 13
- 101710083916 Inter-alpha-trypsin inhibitor heavy chain H1 Proteins 0.000 claims description 13
- 102000019211 Metalloproteinase inhibitor 3 Human genes 0.000 claims description 13
- 108050006600 Metalloproteinase inhibitor 3 Proteins 0.000 claims description 13
- 102100027378 Prothrombin Human genes 0.000 claims description 13
- 108010094028 Prothrombin Proteins 0.000 claims description 13
- 102000005937 Tropomyosin Human genes 0.000 claims description 13
- 108010030743 Tropomyosin Proteins 0.000 claims description 13
- 229930003448 Vitamin K Natural products 0.000 claims description 13
- 150000001875 compounds Chemical class 0.000 claims description 13
- 230000001419 dependent effect Effects 0.000 claims description 13
- 108091022862 fatty acid binding Proteins 0.000 claims description 13
- SHUZOJHMOBOZST-UHFFFAOYSA-N phylloquinone Natural products CC(C)CCCCC(C)CCC(C)CCCC(=CCC1=C(C)C(=O)c2ccccc2C1=O)C SHUZOJHMOBOZST-UHFFFAOYSA-N 0.000 claims description 13
- 229940039716 prothrombin Drugs 0.000 claims description 13
- 239000000758 substrate Substances 0.000 claims description 13
- 230000008685 targeting Effects 0.000 claims description 13
- 235000019168 vitamin K Nutrition 0.000 claims description 13
- 239000011712 vitamin K Substances 0.000 claims description 13
- 150000003721 vitamin K derivatives Chemical class 0.000 claims description 13
- 229940046010 vitamin k Drugs 0.000 claims description 13
- 101001056015 Homo sapiens Mannan-binding lectin serine protease 2 Proteins 0.000 claims description 12
- 108010090227 Immunoglobulin kappa-Chains Proteins 0.000 claims description 12
- 102100026046 Mannan-binding lectin serine protease 2 Human genes 0.000 claims description 12
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 claims description 12
- 101710138747 Receptor-type tyrosine-protein phosphatase C Proteins 0.000 claims description 12
- 102100023543 Vascular cell adhesion protein 1 Human genes 0.000 claims description 12
- 101710160666 Vascular cell adhesion protein 1 Proteins 0.000 claims description 12
- FEPMHVLSLDOMQC-UHFFFAOYSA-N virginiamycin-S1 Natural products CC1OC(=O)C(C=2C=CC=CC=2)NC(=O)C2CC(=O)CCN2C(=O)C(CC=2C=CC=CC=2)N(C)C(=O)C2CCCN2C(=O)C(CC)NC(=O)C1NC(=O)C1=NC=CC=C1O FEPMHVLSLDOMQC-UHFFFAOYSA-N 0.000 claims description 12
- 238000002591 computed tomography Methods 0.000 claims description 9
- 239000003446 ligand Substances 0.000 claims description 9
- 241000700605 Viruses Species 0.000 claims description 8
- 210000004027 cell Anatomy 0.000 claims description 8
- 239000002105 nanoparticle Substances 0.000 claims description 8
- 108091008104 nucleic acid aptamers Proteins 0.000 claims description 8
- 239000002245 particle Substances 0.000 claims description 8
- 150000002632 lipids Chemical class 0.000 claims description 7
- 238000010171 animal model Methods 0.000 claims description 6
- 239000000839 emulsion Substances 0.000 claims description 6
- 238000012632 fluorescent imaging Methods 0.000 claims description 6
- 238000004393 prognosis Methods 0.000 claims description 6
- 238000002603 single-photon emission computed tomography Methods 0.000 claims description 6
- 238000000376 autoradiography Methods 0.000 claims description 5
- 238000002595 magnetic resonance imaging Methods 0.000 claims description 5
- 238000002600 positron emission tomography Methods 0.000 claims description 5
- 239000000941 radioactive substance Substances 0.000 claims description 5
- 230000001225 therapeutic effect Effects 0.000 claims description 5
- 238000002525 ultrasonication Methods 0.000 claims description 5
- 229910052688 Gadolinium Inorganic materials 0.000 claims description 4
- UIWYJDYFSGRHKR-UHFFFAOYSA-N gadolinium atom Chemical compound [Gd] UIWYJDYFSGRHKR-UHFFFAOYSA-N 0.000 claims description 4
- WTFXARWRTYJXII-UHFFFAOYSA-N iron(2+);iron(3+);oxygen(2-) Chemical compound [O-2].[O-2].[O-2].[O-2].[Fe+2].[Fe+3].[Fe+3] WTFXARWRTYJXII-UHFFFAOYSA-N 0.000 claims description 4
- 239000002502 liposome Substances 0.000 claims description 4
- 239000000693 micelle Substances 0.000 claims description 4
- 239000011859 microparticle Substances 0.000 claims description 4
- 230000009149 molecular binding Effects 0.000 claims description 4
- 229920000642 polymer Polymers 0.000 claims description 4
- TXEYQDLBPFQVAA-UHFFFAOYSA-N tetrafluoromethane Chemical compound FC(F)(F)F TXEYQDLBPFQVAA-UHFFFAOYSA-N 0.000 claims description 4
- 200000000007 Arterial disease Diseases 0.000 claims description 3
- 239000003527 fibrinolytic agent Substances 0.000 claims description 3
- 238000002331 protein detection Methods 0.000 claims description 3
- 229960000103 thrombolytic agent Drugs 0.000 claims description 3
- 102100028313 Fibrinogen beta chain Human genes 0.000 claims 5
- 102000012960 Immunoglobulin kappa-Chains Human genes 0.000 claims 5
- 239000003550 marker Substances 0.000 abstract description 132
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 44
- 201000010099 disease Diseases 0.000 abstract description 38
- 230000001747 exhibiting effect Effects 0.000 abstract description 2
- 235000018102 proteins Nutrition 0.000 description 245
- 241000699670 Mus sp. Species 0.000 description 59
- 102000013918 Apolipoproteins E Human genes 0.000 description 31
- 108010025628 Apolipoproteins E Proteins 0.000 description 31
- 230000002950 deficient Effects 0.000 description 31
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 24
- 210000001519 tissue Anatomy 0.000 description 20
- 241000699666 Mus <mouse, genus> Species 0.000 description 16
- 102000004190 Enzymes Human genes 0.000 description 13
- 108090000790 Enzymes Proteins 0.000 description 13
- 229940088598 enzyme Drugs 0.000 description 13
- 238000010790 dilution Methods 0.000 description 12
- 239000012895 dilution Substances 0.000 description 12
- 238000011532 immunohistochemical staining Methods 0.000 description 12
- 238000005259 measurement Methods 0.000 description 12
- 102400001064 Fibrinogen beta chain Human genes 0.000 description 11
- 238000003018 immunoassay Methods 0.000 description 10
- 238000002372 labelling Methods 0.000 description 10
- 239000003153 chemical reaction reagent Substances 0.000 description 9
- 239000007790 solid phase Substances 0.000 description 9
- -1 152Eu Chemical class 0.000 description 8
- 102100029572 Immunoglobulin kappa constant Human genes 0.000 description 8
- 241000283973 Oryctolagus cuniculus Species 0.000 description 8
- 238000004949 mass spectrometry Methods 0.000 description 7
- 210000002381 plasma Anatomy 0.000 description 7
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 6
- 206010002383 Angina Pectoris Diseases 0.000 description 6
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 6
- 206010008118 cerebral infarction Diseases 0.000 description 6
- 238000003745 diagnosis Methods 0.000 description 6
- 208000035475 disorder Diseases 0.000 description 6
- 238000003384 imaging method Methods 0.000 description 6
- 230000002265 prevention Effects 0.000 description 6
- 238000012800 visualization Methods 0.000 description 6
- 201000008247 brain infarction Diseases 0.000 description 5
- 239000000872 buffer Substances 0.000 description 5
- 238000013500 data storage Methods 0.000 description 5
- 229940079593 drug Drugs 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 108020004999 messenger RNA Proteins 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 229940002612 prodrug Drugs 0.000 description 5
- 239000000651 prodrug Substances 0.000 description 5
- 108020003175 receptors Proteins 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 108060003393 Granulin Proteins 0.000 description 4
- 241000282412 Homo Species 0.000 description 4
- 102000004142 Trypsin Human genes 0.000 description 4
- 108090000631 Trypsin Proteins 0.000 description 4
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 4
- 230000000996 additive effect Effects 0.000 description 4
- 210000001367 artery Anatomy 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 4
- 230000004069 differentiation Effects 0.000 description 4
- 239000003480 eluent Substances 0.000 description 4
- 230000006372 lipid accumulation Effects 0.000 description 4
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 4
- 208000010125 myocardial infarction Diseases 0.000 description 4
- 108020004707 nucleic acids Proteins 0.000 description 4
- 150000007523 nucleic acids Chemical class 0.000 description 4
- 102000039446 nucleic acids Human genes 0.000 description 4
- 108090000765 processed proteins & peptides Proteins 0.000 description 4
- 239000012588 trypsin Substances 0.000 description 4
- 208000037260 Atherosclerotic Plaque Diseases 0.000 description 3
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 3
- 241000700159 Rattus Species 0.000 description 3
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 3
- 208000007718 Stable Angina Diseases 0.000 description 3
- 206010000891 acute myocardial infarction Diseases 0.000 description 3
- 239000000654 additive Substances 0.000 description 3
- 238000002583 angiography Methods 0.000 description 3
- 229940039231 contrast media Drugs 0.000 description 3
- 238000007796 conventional method Methods 0.000 description 3
- 230000007423 decrease Effects 0.000 description 3
- 238000011033 desalting Methods 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 239000002552 dosage form Substances 0.000 description 3
- 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 3
- 239000007850 fluorescent dye Substances 0.000 description 3
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 3
- 230000006872 improvement Effects 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 150000002500 ions Chemical class 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000011159 matrix material Substances 0.000 description 3
- 239000012188 paraffin wax Substances 0.000 description 3
- 235000004252 protein component Nutrition 0.000 description 3
- 238000011002 quantification Methods 0.000 description 3
- 230000005855 radiation Effects 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 208000024891 symptom Diseases 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- OBYNJKLOYWCXEP-UHFFFAOYSA-N 2-[3-(dimethylamino)-6-dimethylazaniumylidenexanthen-9-yl]-4-isothiocyanatobenzoate Chemical compound C=12C=CC(=[N+](C)C)C=C2OC2=CC(N(C)C)=CC=C2C=1C1=CC(N=C=S)=CC=C1C([O-])=O OBYNJKLOYWCXEP-UHFFFAOYSA-N 0.000 description 2
- 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 2
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 2
- 208000004476 Acute Coronary Syndrome Diseases 0.000 description 2
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 2
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 2
- 201000001320 Atherosclerosis Diseases 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 2
- 238000011740 C57BL/6 mouse Methods 0.000 description 2
- 241000282472 Canis lupus familiaris Species 0.000 description 2
- 241000282693 Cercopithecidae Species 0.000 description 2
- 206010008088 Cerebral artery embolism Diseases 0.000 description 2
- 206010008132 Cerebral thrombosis Diseases 0.000 description 2
- 101000573945 Coccidioides posadasii (strain C735) Neutral protease 2 homolog MEP2 Proteins 0.000 description 2
- 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 2
- 238000002965 ELISA Methods 0.000 description 2
- 241000282326 Felis catus Species 0.000 description 2
- 108010049003 Fibrinogen Proteins 0.000 description 2
- 102000008946 Fibrinogen Human genes 0.000 description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 2
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 2
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 2
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 2
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 2
- 102000017727 Immunoglobulin Variable Region Human genes 0.000 description 2
- 108010067060 Immunoglobulin Variable Region Proteins 0.000 description 2
- 201000001429 Intracranial Thrombosis Diseases 0.000 description 2
- 239000007836 KH2PO4 Substances 0.000 description 2
- 102000003992 Peroxidases Human genes 0.000 description 2
- 241000282887 Suidae Species 0.000 description 2
- 239000007983 Tris buffer Substances 0.000 description 2
- SXEHKFHPFVVDIR-UHFFFAOYSA-N [4-(4-hydrazinylphenyl)phenyl]hydrazine Chemical compound C1=CC(NN)=CC=C1C1=CC=C(NN)C=C1 SXEHKFHPFVVDIR-UHFFFAOYSA-N 0.000 description 2
- 230000001154 acute effect Effects 0.000 description 2
- 239000003708 ampul Substances 0.000 description 2
- 210000002376 aorta thoracic Anatomy 0.000 description 2
- 208000007474 aortic aneurysm Diseases 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 229960002685 biotin Drugs 0.000 description 2
- 239000011616 biotin Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 239000004202 carbamide Substances 0.000 description 2
- 208000026106 cerebrovascular disease Diseases 0.000 description 2
- 235000012000 cholesterol Nutrition 0.000 description 2
- 238000012790 confirmation Methods 0.000 description 2
- 238000007405 data analysis Methods 0.000 description 2
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 2
- 229940012952 fibrinogen Drugs 0.000 description 2
- 210000000497 foam cell Anatomy 0.000 description 2
- 235000019253 formic acid Nutrition 0.000 description 2
- 238000005194 fractionation Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 239000005090 green fluorescent protein Substances 0.000 description 2
- 235000009200 high fat diet Nutrition 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 238000011503 in vivo imaging Methods 0.000 description 2
- 238000011065 in-situ storage Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 201000010849 intracranial embolism Diseases 0.000 description 2
- 238000001990 intravenous administration Methods 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- 230000004807 localization Effects 0.000 description 2
- 238000004020 luminiscence type Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 230000000414 obstructive effect Effects 0.000 description 2
- GLDOVTGHNKAZLK-UHFFFAOYSA-N octadecan-1-ol Chemical compound CCCCCCCCCCCCCCCCCCO GLDOVTGHNKAZLK-UHFFFAOYSA-N 0.000 description 2
- 239000002674 ointment Substances 0.000 description 2
- 150000002894 organic compounds Chemical class 0.000 description 2
- 238000007911 parenteral administration Methods 0.000 description 2
- 108040007629 peroxidase activity proteins Proteins 0.000 description 2
- 230000007505 plaque formation Effects 0.000 description 2
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000002285 radioactive effect Effects 0.000 description 2
- 238000003127 radioimmunoassay Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 238000005063 solubilization Methods 0.000 description 2
- 239000007921 spray Substances 0.000 description 2
- 230000006641 stabilisation Effects 0.000 description 2
- 238000011105 stabilization Methods 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- STCOOQWBFONSKY-UHFFFAOYSA-N tributyl phosphate Chemical compound CCCCOP(=O)(OCCCC)OCCCC STCOOQWBFONSKY-UHFFFAOYSA-N 0.000 description 2
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- 238000000539 two dimensional gel electrophoresis Methods 0.000 description 2
- 238000001291 vacuum drying Methods 0.000 description 2
- DQJCDTNMLBYVAY-ZXXIYAEKSA-N (2S,5R,10R,13R)-16-{[(2R,3S,4R,5R)-3-{[(2S,3R,4R,5S,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}-5-(ethylamino)-6-hydroxy-2-(hydroxymethyl)oxan-4-yl]oxy}-5-(4-aminobutyl)-10-carbamoyl-2,13-dimethyl-4,7,12,15-tetraoxo-3,6,11,14-tetraazaheptadecan-1-oic acid Chemical compound NCCCC[C@H](C(=O)N[C@@H](C)C(O)=O)NC(=O)CC[C@H](C(N)=O)NC(=O)[C@@H](C)NC(=O)C(C)O[C@@H]1[C@@H](NCC)C(O)O[C@H](CO)[C@H]1O[C@H]1[C@H](NC(C)=O)[C@@H](O)[C@H](O)[C@@H](CO)O1 DQJCDTNMLBYVAY-ZXXIYAEKSA-N 0.000 description 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 description 1
- UAIUNKRWKOVEES-UHFFFAOYSA-N 3,3',5,5'-tetramethylbenzidine Chemical compound CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 UAIUNKRWKOVEES-UHFFFAOYSA-N 0.000 description 1
- UMCMPZBLKLEWAF-BCTGSCMUSA-N 3-[(3-cholamidopropyl)dimethylammonio]propane-1-sulfonate Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCCC[N+](C)(C)CCCS([O-])(=O)=O)C)[C@@]2(C)[C@@H](O)C1 UMCMPZBLKLEWAF-BCTGSCMUSA-N 0.000 description 1
- BTJIUGUIPKRLHP-UHFFFAOYSA-N 4-nitrophenol Chemical compound OC1=CC=C([N+]([O-])=O)C=C1 BTJIUGUIPKRLHP-UHFFFAOYSA-N 0.000 description 1
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 101710199744 Anionic trypsin-2 Proteins 0.000 description 1
- 102000004411 Antithrombin III Human genes 0.000 description 1
- 108090000935 Antithrombin III Proteins 0.000 description 1
- 238000013258 ApoE Receptor knockout mouse model Methods 0.000 description 1
- 108091023037 Aptamer Proteins 0.000 description 1
- 102100031680 Beta-catenin-interacting protein 1 Human genes 0.000 description 1
- 102100026189 Beta-galactosidase Human genes 0.000 description 1
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 1
- 206010008111 Cerebral haemorrhage Diseases 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 102000055157 Complement C1 Inhibitor Human genes 0.000 description 1
- 102100037085 Complement C1q subcomponent subunit B Human genes 0.000 description 1
- 101710112691 Complement C1q subcomponent subunit B Proteins 0.000 description 1
- 102000006912 Complement C4b-Binding Protein Human genes 0.000 description 1
- 108010047548 Complement C4b-Binding Protein Proteins 0.000 description 1
- 108050000891 Complement component C9 Proteins 0.000 description 1
- 102000008929 Complement component C9 Human genes 0.000 description 1
- 102100035436 Complement factor D Human genes 0.000 description 1
- 108090000059 Complement factor D Proteins 0.000 description 1
- 241000938605 Crocodylia Species 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
- IGXWBGJHJZYPQS-SSDOTTSWSA-N D-Luciferin Chemical compound OC(=O)[C@H]1CSC(C=2SC3=CC=C(O)C=C3N=2)=N1 IGXWBGJHJZYPQS-SSDOTTSWSA-N 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-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
- CYCGRDQQIOGCKX-UHFFFAOYSA-N Dehydro-luciferin Natural products OC(=O)C1=CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 CYCGRDQQIOGCKX-UHFFFAOYSA-N 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 206010061818 Disease progression Diseases 0.000 description 1
- 102000009024 Epidermal Growth Factor Human genes 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 102100031758 Extracellular matrix protein 1 Human genes 0.000 description 1
- 101710127949 Extracellular matrix protein 1 Proteins 0.000 description 1
- 108010086121 Fetuin-B Proteins 0.000 description 1
- 102000007437 Fetuin-B Human genes 0.000 description 1
- BJGNCJDXODQBOB-UHFFFAOYSA-N Fivefly Luciferin Natural products OC(=O)C1CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 BJGNCJDXODQBOB-UHFFFAOYSA-N 0.000 description 1
- 150000000921 Gadolinium Chemical class 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 108010015899 Glycopeptides Proteins 0.000 description 1
- 102000002068 Glycopeptides Human genes 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 229920000084 Gum arabic Polymers 0.000 description 1
- 108090000481 Heparin Cofactor II Proteins 0.000 description 1
- 102100030500 Heparin cofactor 2 Human genes 0.000 description 1
- 101000993469 Homo sapiens Beta-catenin-interacting protein 1 Proteins 0.000 description 1
- 101000840257 Homo sapiens Immunoglobulin kappa constant Proteins 0.000 description 1
- 101001002508 Homo sapiens Immunoglobulin-binding protein 1 Proteins 0.000 description 1
- 208000035150 Hypercholesterolemia Diseases 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- 101710138663 Ig gamma-2B chain C region Proteins 0.000 description 1
- 102100039352 Immunoglobulin heavy constant mu Human genes 0.000 description 1
- 102100021042 Immunoglobulin-binding protein 1 Human genes 0.000 description 1
- 102100039440 Inter-alpha-trypsin inhibitor heavy chain H2 Human genes 0.000 description 1
- 101710083919 Inter-alpha-trypsin inhibitor heavy chain H2 Proteins 0.000 description 1
- 238000008214 LDL Cholesterol Methods 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 108060001084 Luciferase Proteins 0.000 description 1
- 239000005089 Luciferase Substances 0.000 description 1
- DDWFXDSYGUXRAY-UHFFFAOYSA-N Luciferin Natural products CCc1c(C)c(CC2NC(=O)C(=C2C=C)C)[nH]c1Cc3[nH]c4C(=C5/NC(CC(=O)O)C(C)C5CC(=O)O)CC(=O)c4c3C DDWFXDSYGUXRAY-UHFFFAOYSA-N 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 102000009112 Mannose-Binding Lectin Human genes 0.000 description 1
- 108010087870 Mannose-Binding Lectin Proteins 0.000 description 1
- MQUQNUAYKLCRME-INIZCTEOSA-N N-tosyl-L-phenylalanyl chloromethyl ketone Chemical compound C1=CC(C)=CC=C1S(=O)(=O)N[C@H](C(=O)CCl)CC1=CC=CC=C1 MQUQNUAYKLCRME-INIZCTEOSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 108010038807 Oligopeptides Proteins 0.000 description 1
- 102000015636 Oligopeptides Human genes 0.000 description 1
- 239000002033 PVDF binder Substances 0.000 description 1
- 241000282579 Pan Species 0.000 description 1
- 208000031481 Pathologic Constriction Diseases 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 108010033276 Peptide Fragments Proteins 0.000 description 1
- 102000007079 Peptide Fragments Human genes 0.000 description 1
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 1
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 1
- 108050007539 Plasma protease C1 inhibitor Proteins 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 239000004372 Polyvinyl alcohol Substances 0.000 description 1
- 241000288906 Primates Species 0.000 description 1
- 101710098940 Pro-epidermal growth factor Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 1
- 229940096437 Protein S Drugs 0.000 description 1
- 108010066124 Protein S Proteins 0.000 description 1
- 102000029301 Protein S Human genes 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 102000016611 Proteoglycans Human genes 0.000 description 1
- 108010067787 Proteoglycans Proteins 0.000 description 1
- 108091030071 RNAI Proteins 0.000 description 1
- 241000978776 Senegalia senegal Species 0.000 description 1
- 101710151381 Serine protease 2 Proteins 0.000 description 1
- 229920002125 Sokalan® Polymers 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 235000021355 Stearic acid Nutrition 0.000 description 1
- 108010023197 Streptokinase Proteins 0.000 description 1
- 238000000692 Student's t-test Methods 0.000 description 1
- 208000007536 Thrombosis Diseases 0.000 description 1
- 108090000373 Tissue Plasminogen Activator Proteins 0.000 description 1
- 102000003978 Tissue Plasminogen Activator Human genes 0.000 description 1
- 102100034392 Trypsin-2 Human genes 0.000 description 1
- 108090000435 Urokinase-type plasminogen activator Proteins 0.000 description 1
- 102000003990 Urokinase-type plasminogen activator Human genes 0.000 description 1
- 206010072810 Vascular wall hypertrophy Diseases 0.000 description 1
- 102100029477 Vitamin K-dependent protein C Human genes 0.000 description 1
- 101710193900 Vitamin K-dependent protein C Proteins 0.000 description 1
- 102100021208 Vitamin K-dependent protein Z Human genes 0.000 description 1
- 101710194028 Vitamin K-dependent protein Z Proteins 0.000 description 1
- 239000003655 absorption accelerator Substances 0.000 description 1
- 235000010489 acacia gum Nutrition 0.000 description 1
- 239000000205 acacia gum Substances 0.000 description 1
- 230000035508 accumulation Effects 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- 230000001070 adhesive effect Effects 0.000 description 1
- 239000000443 aerosol Substances 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 235000010419 agar Nutrition 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 239000000556 agonist Substances 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 239000005557 antagonist Substances 0.000 description 1
- 230000000692 anti-sense effect Effects 0.000 description 1
- 230000002421 anti-septic effect Effects 0.000 description 1
- 229960005348 antithrombin iii Drugs 0.000 description 1
- 108010005774 beta-Galactosidase Proteins 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 230000036765 blood level Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 239000006172 buffering agent Substances 0.000 description 1
- 230000002308 calcification Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 210000000692 cap cell Anatomy 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 235000011089 carbon dioxide Nutrition 0.000 description 1
- 210000000845 cartilage Anatomy 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
- 235000021240 caseins Nutrition 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 230000021164 cell adhesion Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000001311 chemical methods and process Methods 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000005515 coenzyme Substances 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 238000010835 comparative analysis Methods 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 230000008602 contraction Effects 0.000 description 1
- 239000006071 cream Substances 0.000 description 1
- 239000003431 cross linking reagent Substances 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 239000007933 dermal patch Substances 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000001687 destabilization Effects 0.000 description 1
- 238000012631 diagnostic technique Methods 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 230000005750 disease progression Effects 0.000 description 1
- 239000002270 dispersing agent Substances 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 230000000857 drug effect Effects 0.000 description 1
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000003889 eye drop Substances 0.000 description 1
- 229940012356 eye drops Drugs 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 230000003176 fibrotic effect Effects 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 230000009368 gene silencing by RNA Effects 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 239000007902 hard capsule Substances 0.000 description 1
- 208000019622 heart disease Diseases 0.000 description 1
- 231100000086 high toxicity Toxicity 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 238000010324 immunological assay Methods 0.000 description 1
- 238000012744 immunostaining Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 210000004969 inflammatory cell Anatomy 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 150000002484 inorganic compounds Chemical class 0.000 description 1
- 108010093564 inter-alpha-inhibitor Proteins 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 239000011630 iodine Substances 0.000 description 1
- 229910052740 iodine Inorganic materials 0.000 description 1
- 125000002346 iodo group Chemical group I* 0.000 description 1
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 1
- 230000000155 isotopic effect Effects 0.000 description 1
- 238000001948 isotopic labelling Methods 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 229910052746 lanthanum Inorganic materials 0.000 description 1
- FZLIPJUXYLNCLC-UHFFFAOYSA-N lanthanum atom Chemical compound [La] FZLIPJUXYLNCLC-UHFFFAOYSA-N 0.000 description 1
- 239000000865 liniment Substances 0.000 description 1
- 230000037356 lipid metabolism Effects 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- HWYHZTIRURJOHG-UHFFFAOYSA-N luminol Chemical compound O=C1NNC(=O)C2=C1C(N)=CC=C2 HWYHZTIRURJOHG-UHFFFAOYSA-N 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 150000002739 metals Chemical class 0.000 description 1
- 238000002493 microarray Methods 0.000 description 1
- 239000003094 microcapsule Substances 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 102000035118 modified proteins Human genes 0.000 description 1
- 108091005573 modified proteins Proteins 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- GOQYKNQRPGWPLP-UHFFFAOYSA-N n-heptadecyl alcohol Natural products CCCCCCCCCCCCCCCCCO GOQYKNQRPGWPLP-UHFFFAOYSA-N 0.000 description 1
- 239000007923 nasal drop Substances 0.000 description 1
- 229940100662 nasal drops Drugs 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 235000021590 normal diet Nutrition 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
- 230000003287 optical effect Effects 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 239000003002 pH adjusting agent Substances 0.000 description 1
- 238000010979 pH adjustment Methods 0.000 description 1
- 230000005298 paramagnetic effect Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 229920001277 pectin Polymers 0.000 description 1
- 239000001814 pectin Substances 0.000 description 1
- 235000010987 pectin Nutrition 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 238000002823 phage display Methods 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 239000003186 pharmaceutical solution Substances 0.000 description 1
- 239000006187 pill Substances 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 229920002451 polyvinyl alcohol Polymers 0.000 description 1
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000001294 propane Substances 0.000 description 1
- 235000013772 propylene glycol Nutrition 0.000 description 1
- 238000003498 protein array Methods 0.000 description 1
- 239000010453 quartz Substances 0.000 description 1
- 210000003296 saliva Anatomy 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 235000010413 sodium alginate Nutrition 0.000 description 1
- 239000000661 sodium alginate Substances 0.000 description 1
- 229940005550 sodium alginate Drugs 0.000 description 1
- 239000007901 soft capsule Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 230000007928 solubilization Effects 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 235000010356 sorbitol Nutrition 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 238000010972 statistical evaluation Methods 0.000 description 1
- 239000008117 stearic acid Substances 0.000 description 1
- 230000036262 stenosis Effects 0.000 description 1
- 208000037804 stenosis Diseases 0.000 description 1
- 150000003431 steroids Chemical class 0.000 description 1
- 229960005202 streptokinase Drugs 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000000829 suppository Substances 0.000 description 1
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 229910052713 technetium Inorganic materials 0.000 description 1
- GKLVYJBZJHMRIY-UHFFFAOYSA-N technetium atom Chemical compound [Tc] GKLVYJBZJHMRIY-UHFFFAOYSA-N 0.000 description 1
- 230000008719 thickening Effects 0.000 description 1
- 210000000115 thoracic cavity Anatomy 0.000 description 1
- 229960000187 tissue plasminogen activator Drugs 0.000 description 1
- 239000012929 tonicity agent Substances 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 229960005356 urokinase Drugs 0.000 description 1
- 210000005167 vascular cell Anatomy 0.000 description 1
- 229940099259 vaseline Drugs 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 108010047303 von Willebrand Factor Proteins 0.000 description 1
- 102100036537 von Willebrand factor Human genes 0.000 description 1
- 229960001134 von willebrand factor Drugs 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
- 239000000230 xanthan gum Substances 0.000 description 1
- 235000010493 xanthan gum Nutrition 0.000 description 1
- 229920001285 xanthan gum Polymers 0.000 description 1
- 229940082509 xanthan gum Drugs 0.000 description 1
Images
Classifications
-
- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B5/00—Measuring for diagnostic purposes; Identification of persons
- A61B5/02—Detecting, measuring or recording pulse, heart rate, blood pressure or blood flow; Combined pulse/heart-rate/blood pressure determination; Evaluating a cardiovascular condition not otherwise provided for, e.g. using combinations of techniques provided for in this group with electrocardiography or electroauscultation; Heart catheters for measuring blood pressure
- A61B5/02007—Evaluating blood vessel condition, e.g. elasticity, compliance
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B5/00—Measuring for diagnostic purposes; Identification of persons
- A61B5/05—Detecting, measuring or recording for diagnosis by means of electric currents or magnetic fields; Measuring using microwaves or radio waves
- A61B5/055—Detecting, measuring or recording for diagnosis by means of electric currents or magnetic fields; Measuring using microwaves or radio waves involving electronic [EMR] or nuclear [NMR] magnetic resonance, e.g. magnetic resonance imaging
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B6/00—Apparatus or devices for radiation diagnosis; Apparatus or devices for radiation diagnosis combined with radiation therapy equipment
- A61B6/50—Apparatus or devices for radiation diagnosis; Apparatus or devices for radiation diagnosis combined with radiation therapy equipment specially adapted for specific body parts; specially adapted for specific clinical applications
- A61B6/508—Apparatus or devices for radiation diagnosis; Apparatus or devices for radiation diagnosis combined with radiation therapy equipment specially adapted for specific body parts; specially adapted for specific clinical applications for non-human patients
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B5/00—Measuring for diagnostic purposes; Identification of persons
- A61B5/0059—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence
- A61B5/0071—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence by measuring fluorescence emission
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B6/00—Apparatus or devices for radiation diagnosis; Apparatus or devices for radiation diagnosis combined with radiation therapy equipment
- A61B6/50—Apparatus or devices for radiation diagnosis; Apparatus or devices for radiation diagnosis combined with radiation therapy equipment specially adapted for specific body parts; specially adapted for specific clinical applications
- A61B6/504—Apparatus or devices for radiation diagnosis; Apparatus or devices for radiation diagnosis combined with radiation therapy equipment specially adapted for specific body parts; specially adapted for specific clinical applications for diagnosis of blood vessels, e.g. by angiography
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B8/00—Diagnosis using ultrasonic, sonic or infrasonic waves
- A61B8/08—Detecting organic movements or changes, e.g. tumours, cysts, swellings
- A61B8/0891—Detecting organic movements or changes, e.g. tumours, cysts, swellings for diagnosis of blood vessels
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/32—Cardiovascular disorders
- G01N2800/323—Arteriosclerosis, Stenosis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
Definitions
- the present invention relates to a method, a kit, and an apparatus for evaluating atherosclerotic lesions. More specifically, the present invention relates to a method, a kit, and an apparatus for evaluating atherosclerotic lesions based on novel marker protein(s). Furthermore, the present invention relates to agent(s) for visualizing atherosclerotic lesions and agent(s) for targeting atherosclerotic lesions using the marker protein(s), and a method for evaluating the effectiveness of the therapeutic agent(s) or therapeutic method(s) for atherosclerotic lesions.
- Arteriosclerosis proceeds over a long period of time without subjective symptoms, and induces cardiac diseases such as severe myocardial infarction or cerebrovascular diseases such as cerebral infarction or cerebral hemorrhage. Arteriosclerosis progresses to result in the formation of atherosclerotic lesions (atheroma or plaque) within arterial blood vessels. Plaques include stable plaques rich in calcified fibrous tissue and unstable plaques rich in lipids or inflammatory cells, in which fibrous caps are thinned. Most acute atherosclerotic diseases such as myocardial infarction and brain infarction are induced by rupture of unstable plaques and thrombus formation.
- Arteriosclerosis is currently detected mainly by angiography, which involves evaluation of the degree of stenosis, but it is problematic because information concerning intravascular plaque formation or the properties thereof can barely be obtained. Moreover, angiography is highly invasive. Hence, a low-invasive method for detecting atherosclerotic lesions has been required.
- Disease proteomics is a technique for exhaustively searching increases or decreases in protein levels that vary depending on specific diseases when compared with a healthy state, using a body fluid such as blood, saliva, or urine, or a tissue sample, as a material.
- Disease proteomics is a technique characterized by enabling simultaneous extraction and/or detection of a plurality of factors, the association of which with diseases is conventionally unknown.
- Application of multiple specimens to and increased sensitivity for a 2D electrophoresis method and measuring apparatuses such as protein microarrays and mass spectrometry have been realized.
- data analysis technology has been in place and is applied in searches for markers for various diseases including cancer, immunity disorders, infections, and the like (Non-patent document 1).
- an object of the present invention is to provide diagnosis, prevention, or evaluation for treatment of atherosclerotic lesions by identifying protein groups (maker proteins, the expression levels of which vary with the progression of atherosclerotic lesions) with a proteomic technique, recording the variations in an expression profile database, comparing the data with the variations in the expression levels of marker proteins in samples obtained from subjects, and thereby identifying the stage of progression of atherosclerotic lesion(s).
- Another object of the present invention is to provide a method for evaluating atherosclerotic lesions, comprising:
- administering e.g., via intravenous administration
- a substance having specific affinity for a marker protein group the expression of which varies with the progression of atherosclerotic lesions, wherein the substance is labeled in advance;
- the present inventors have obtained arterial tissue from ApoE-deficient mice, in which arteriosclerosis commonly occurs and progresses at an accelerated rate with administration of a high-fat diet and aging (age in weeks), and wild-type mice. They then exhaustively compared and analyzed the expression levels of proteins contained in the arterial tissue with a mass spectroscope.
- An expression profile database was constructed using the ratios of the measured expression levels of proteins contained in arterial tissue samples obtained at each age in weeks (12-week-old, 18-week-old, 25-week-old, and 35-week-old) and differing in the extent of the progression of arteriosclerosis.
- the onset or the progression (worsening) of arteriosclerosis correlates with the variation tendency for each protein expression (level) ratio, and the tendencies can be classified into a plurality of groups. Furthermore, the present inventors have succeeded in identifying proteins that exhibit expression tendencies characteristic of specific stages of arteriosclerotic lesions, and have found that the proteins can be used as markers for evaluation of the likelihood of the onset of arteriosclerosis, high or low risk of contraction of the disease, and possibility of progression, thereby completing the present invention.
- the present invention provides the following [1] to [39].
- a kit for evaluating an atherosclerotic lesion comprising at least one type of substance specifically binding to at least one type of protein selected from at least group (a) from among proteins in the following groups (a) to (d): (a) cartilage oligomeric matrix protein, fibromodulin, fibronectin, and thrombospondin-4; (b) plastin-2, tenascin, CD5 antigen-like protein, properdin, Cb4-binding protein, fibrinogen beta chain, and fibrinogen gamma chain; (c) vitronectin, matrix metalloproteinase-2, and thrombospondin-1; (d) membrane-bound form immunoglobulin mu chain C region, receptor-type tyrosine protein phosphatase C, immunoglobulin gamma-2B chain-C region, immunoglobulin J chain, immunoglobulin kappa chain C region, vascular cell adhesion protein 1, alpha-2-macroglobulin, ADAMTS
- kits according to [1] comprising at least two types of substance specifically binding to a protein selected from group (a) and a protein selected from at least one of groups (b) to (d).
- the kit according to [1] comprising at least three types of substance specifically binding to a protein selected from group (a) and proteins selected from at least two of groups (b) to (d).
- the kit according to [1] comprising at least four types of substance specifically binding to a protein selected from group (a) and proteins selected from each of groups (b) to (d).
- a kit for evaluating an atherosclerotic lesion at the intermediate stage or later comprising at least one type of substance specifically binding to at least one type of protein selected from the group consisting of cartilage oligomeric matrix protein, fibromodulin, fibronectin, and thrombospondin-4.
- a kit for evaluating an atherosclerotic lesion at the initial or the intermediate stage comprising at least one type of substance specifically binding to at least one type of protein selected from the group consisting of plastin-2, tenascin, CD5 antigen-like protein, properdin, Cb4-binding protein, fibrinogen beta chain, and fibrinogen gamma chain.
- kits for evaluating an atherosclerotic lesion at the late stage comprising at least one type of substance specifically binding to at least one type of protein selected from the group consisting of vitronectin, matrix metalloproteinase-2, and thrombospondin-1.
- the evaluation of an atherosclerotic lesion is selected from the group consisting of the determination of the presence of an atherosclerotic lesion in a subject, the determination of the stage of an atherosclerotic lesion in a subject, the determination of the location of an atherosclerotic lesion in a subject, the evaluation of the therapeutic effect of arteriosclerosis existing in a subject, and the prediction of prognosis of arteriosclerosis existing in a subject.
- kits according to any one of [1] to [8], wherein a substance specifically binding to a protein is selected from the group consisting of an antibody, an antibody-like molecule, a nucleic acid aptamer, a ligand, and a substrate.
- a substance specifically binding to a protein is bound to a contrast medium.
- the contrast medium is selected from the group consisting of a fluorescent substance, a luminescent substance, a radioactive substance, a liposome, a micelle, a cell, a virus particle, a virus, a microparticle, a nanoparticle, a microdevice with a space, an emulsion, a lipid disc, a polymer, a gadolinium-conjugated molecule, a superparamagnetic iron oxide particle, a perfluorocarbon nanoparticle, and an ultrafine bubble.
- PET positron emission tomography
- SPECT single photon emission computed tomography
- ⁇ scintigraphy autoradiography
- fluorescent imaging e.g., single photon emission computed tomography
- MRI magnetic resonance imaging
- ultrasonication e.g., ultrasonication
- CT computed tomography
- An apparatus for evaluating an atherosclerotic lesion comprising: (i) a means of detecting at least one type of protein selected from at least group (a) from among proteins shown in the following groups (a) to (d), in a subject: (a) cartilage oligomeric matrix protein, fibromodulin, fibronectin, and thrombospondin-4; (b) plastin-2, tenascin, CD5 antigen-like protein, properdin, Cb4-binding protein, fibrinogen beta chain, and fibrinogen gamma chain; (c) vitronectin, matrix metalloproteinase-2, and thrombospondin-1; and (d) membrane-bound form immunoglobulin mu chain C region, receptor-type tyrosine protein phosphatase C, immunoglobulin gamma-2B chain-C region, immunoglobulin J chain, immunoglobulin kappa chain C region, vascular cell adhesion protein 1, alpha-2-macroglob
- An agent for visualizing an atherosclerotic lesion comprising at least one type of protein selected from at least group (a) from among proteins in the following groups (a) to (d), and a contrast medium binding thereto: (a) cartilage oligomeric matrix protein, fibromodulin, fibronectin, and thrombospondin-4; (b) plastin-2, tenascin, CD5 antigen-like protein, properdin, Cb4-binding protein, fibrinogen beta chain, and fibrinogen gamma chain; (c) vitronectin, matrix metalloproteinase-2, and thrombospondin-1; and (d) membrane-bound form immunoglobulin mu chain C region, receptor-type tyrosine protein phosphatase C, immunoglobulin mu chain C region, receptor-type tyrosine protein phosphatase C, immunoglobulin mu chain C region, receptor-type tyrosine protein phosphatase C, immunoglobulin mu chain C region, receptor
- An agent for visualizing an atherosclerotic lesion at the initial or the intermediate stage comprising:
- An agent for visualizing an atherosclerotic lesion at the late stage comprising:
- a targeting agent for directing a compound or a molecule to an atherosclerotic lesion comprising a substance specifically binding to at least one type of protein selected from at least group (a) from among the following groups (a) to (d), and the compound or the molecule binding thereto:
- a method for evaluating the effectiveness of a therapeutic agent or a therapeutic method for an atherosclerotic lesion comprising the steps of: (i) detecting at least one type of protein selected from at least group (a) from among proteins shown in the following groups (a) to (d) in an animal with an atherosclerotic lesion treated by a test therapeutic agent or a test therapeutic method: (a) cartilage oligomeric matrix protein, fibromodulin, fibronectin, and thrombospondin-4; (b) plastin-2, tenascin, CD5 antigen-like protein, properdin, Cb4-binding protein, fibrinogen beta chain, and fibrinogen gamma chain; (c) vitronectin, matrix metalloproteinase-2, and thrombospondin-1; and (d) membrane-bound form immunoglobulin mu chain C region, receptor-type
- a method for evaluating an atherosclerotic lesion comprising the steps of: (i) detecting at least one type of protein in a subject selected from at least group (a) from among proteins shown in the following groups (a) to (d): (a) cartilage oligomeric matrix protein, fibromodulin, fibronectin, and thrombospondin-4; (b) plastin-2, tenascin, CD5 antigen-like protein, properdin, Cb4-binding protein, fibrinogen beta chain, and fibrinogen gamma chain; (c) vitronectin, matrix metalloproteinase-2, and thrombospondin-1; and (d) membrane-bound form immunoglobulin mu chain C region, receptor-type tyrosine protein phosphatase C, immunoglobulin gamma-2B chain-C region, immunoglobulin J chain, immunoglobulin kappa chain C region, vascular cell adhesion protein 1, alpha-2-macroglobul
- a method for evaluating an atherosclerotic lesion comprising the steps of: (i) detecting at least one type of protein selected from the group consisting of cartilage oligomeric matrix protein, fibromodulin, fibronectin, and thrombospondin-4 in a subject; and (ii) evaluating, based on the results of (i), whether or not the subject has an atherosclerotic lesion at the intermediate stage or later.
- a method for evaluating an atherosclerotic lesion comprising the steps of: (i) detecting at least one type of protein selected from the group consisting of plastin-2, tenascin, CD5 antigen-like protein, properdin, Cb4-binding protein, fibrinogen beta chain, and fibrinogen gamma chain, in a subject; and (ii) evaluating whether or not the subject has an atherosclerotic lesion at the initial or intermediate stage based on the results of (i).
- a method for evaluating an atherosclerotic lesion comprising the steps of: (i) detecting at least one type of protein selected from the group consisting of vitronectin, matrix metalloproteinase-2, and thrombospondin-1 in a subject; and (ii) evaluating whether or not the subject has an atherosclerotic lesion at the late stage based on the results of (i).
- step (i) is performed by administering a substance specifically binding to a protein to a subject, and then detecting the substance binding to the protein existing in the subject.
- step (i) is performed by preparing a subject-derived sample, bringing the sample into contact with a substance specifically binding to a protein, and then detecting binding between the protein and the substance.
- evaluation of an atherosclerotic lesion is selected from the group consisting of determination of the presence of an atherosclerotic lesion in a subject, determination of the stage of an atherosclerotic lesion existing in a subject, determination of the location of an atherosclerotic lesion existing in a subject, evaluation of the therapeutic effect on arteriosclerosis existing in a subject, and prediction of prognosis of arteriosclerosis existing in a subject.
- the contrast medium is selected from the group consisting of a fluorescent substance, a luminescent substance, a radioactive substance, a liposome, a micelle, a cell, a virus particle, a virus, a microparticle, a nanoparticle, a microdevice with a space, an emulsion, a lipid disc, a polymer, a gadolinium-conjugated molecule, a superparamagnetic iron oxide particle, a perfluorocarbon nanoparticle, and an ultrafine bubble.
- the present invention enables detection of arteriosclerotic lesions (destabilization) that have been missed by conventional methods in primary care or medical examination and enables assaying the possibility of the occurrence and progression of the resulting disorder.
- the ratio of the expression level of a protein group existing in a sample from a subject to the expression level of the same in a standard sample and variations in the ratio over time are measured, and then the results are compared with an expression profile database compiled in advance. This makes it possible to evaluate the possibility of the occurrence of arteriosclerosis and the resulting disorder and the possibility of the progression of the symptoms.
- the present invention can be used for precisely and conveniently testing or analyzing the possibility of the occurrence of arteriosclerotic lesions and the resulting disorder and the possibility of the progression thereof.
- the present invention can also be used for developing various reagents or remedies, and related apparatuses.
- a marker protein group selected in this study is visualized by a known means of imaging with a substance specifically binding to the protein (e.g., an antibody, an antibody-like substance, a ligand, and a substrate) and a contrast medium (e.g., a fluorescent substance and a radioactive substance).
- a substance specifically binding to the protein e.g., an antibody, an antibody-like substance, a ligand, and a substrate
- a contrast medium e.g., a fluorescent substance and a radioactive substance.
- the present invention can be used for precisely and conveniently assessing or analyzing the possibility of the occurrence of arteriosclerosis and the resulting disorder, and the possibility of the progression thereof.
- the present invention can also be used for developing various reagents or remedies, and related apparatuses.
- FIG. 1 is a graph showing the ratio of the expression level of a protein group in ApoE-deficient mice divided by that in wild-type mice at each disease stage, by which variation pattern 1 was exhibited.
- FIG. 2 is a graph showing the ratio of the expression level of a protein group in ApoE-deficient mice divided by that in wild-type mice at each disease stage, by which variation pattern 2 was exhibited.
- FIG. 3 is a graph showing the ratio of the expression level of a protein group in ApoE-deficient mice divided by that in wild-type mice at each disease stage, by which variation pattern 3 was exhibited.
- FIG. 4 is a graph showing the ratio of the expression level of a protein group in ApoE-deficient mice divided by that in wild-type mice at each disease stage, by which variation pattern 4 was exhibited.
- FIG. 5 shows the outline of the apparatus of the present invention for evaluating atherosclerotic lesions.
- FIG. 6 shows images showing the expression of markers (CD5 antigen-like protein and properdin) in atherosclerotic lesions.
- markers CD5 antigen-like protein and properdin
- “*” indicates the intravascular lumen side.
- FIG. 7 shows images showing the expression of markers (cartilage oligomeric matrix protein, fibromodulin, fibronectin, and thrombospondin-4) in atherosclerotic lesions.
- FIG. 8 shows images showing the expression of markers (plastin-2 and tenascin) in atherosclerotic lesions.
- the present invention provides novel markers and marker groups for evaluation and visualization of atherosclerotic lesions.
- the markers and marker groups provided by the present invention are proteins, the expression levels of which characteristically vary with the progression of atherosclerotic lesions. They are useful for prediction of the onset of diseases associated with arteriosclerosis such as stable angina pectoris and acute myocardial infarction, determination of the stages of atherosclerotic lesions, prediction of prognosis of atherosclerotic lesions, visualization of atherosclerotic lesions, and evaluation of the effectiveness of therapeutic agents or therapeutic methods for diseases associated with arteriosclerosis, for example.
- the method for evaluating atherosclerotic lesions comprises the steps of: detecting a marker protein exhibiting the expression pattern (variation in expression) characteristic to a specific disease stage of an atherosclerotic lesion in a subject; and evaluating the atherosclerotic lesion in the subject based on the detection result.
- Subjects to be evaluated according to the present invention are humans and non-human mammals, such as primates (e.g., monkeys and chimpanzees), livestock animals (cattle, horses, pigs, and sheep), pet animals (e.g., dogs and cats), and experimental animals (e.g., mice, rats, and rabbits). Such subjects may also be reptiles and birds.
- primates e.g., monkeys and chimpanzees
- livestock animals e.g., horses, pigs, and sheep
- pet animals e.g., dogs and cats
- experimental animals e.g., mice, rats, and rabbits
- the term “marker” as used herein refers to a protein to be detected for evaluation of arteriosclerosis according to the present invention.
- the term “marker group” refers to a combination comprising two or more markers.
- Markers to be used in the present invention are as summarized in the following Table 1 to Table 4.
- Table 1 to Table 4 the “Entry name” column and the “Accession No.” column are located under the “Mouse” column and the “Human” column, and they show the names and the accession numbers, respectively, of marker proteins of the “Mouse” and the “Human,” as in UniProtKB/Swiss-Prot (http://www.uniprot.org/).
- Table 1 to Table 4 also show the ratios of the expression levels of marker proteins at different disease stages as detected in examples.
- “12-week-old,” “18-week-old,” “25-week-old,” or “35-week-old” used in tables and the Description refer to a specific disease stage corresponding to the relevant age in weeks.
- An association between the age in weeks of an ApoE-deficient mouse and a disease stage has been reported in Nakashima, Y. et al., Arterioscler Thromb. 14:133-140, 1994; Zhao, Y. et al., J. Nucl. Med. 49:1707-1714, 2008, and the like.
- thickening of the intima of arteries and lipid accumulation are observed (stage 1).
- stage 2 foam cells are formed because of lipid accumulation.
- stage 3 many unstable atheromatous plaques are observed extensively in the arteries (stage 3), corresponding to the most dangerous stage in atherosclerotic arteriosclerosis.
- stage 4 many stable lesions with advanced fibrillization and/or calcification are observed.
- “12-week-old” (ApoE-deficient mouse) corresponds to the initial stage (lesion) of arteriosclerosis
- “18-week-old” corresponds to the early intermediate stage (lesion) of arteriosclerosis
- “25-week-old” corresponds to the intermediate stage (lesion) of arteriosclerosis
- “35-week-old” corresponds to the late stage (lesion) of arteriosclerosis.
- Proteins shown in Table 1 showed no significant difference in amounts until the mice reached the age of 18 weeks, but were characterized in that (i) the ratio of the amounts thereof in ApoE-deficient mice divided by the amounts thereof in wild-type mice (hereinafter, referred to as “the expression level ratio”) at the age of 25 weeks is at least 3, and (ii) the expression level ratio thereof at the age of 25 weeks is at least twice as large as the expression level ratio thereof at the age of 18 weeks. These proteins are also markers showing the risk of plaque rupture at the intermediate stage.
- Proteins shown in Table 2 are characterized in that: (i) the ratio of the amounts thereof in ApoE-deficient mice divided by the amounts thereof in wild-type mice at the age of 18 weeks is at least 2; (ii) the expression level ratio thereof at the age of 18 weeks is at least 1.3 times as large as the expression level ratio thereof at the age of 12 weeks; (iii) the expression level ratio at the age of 25 weeks is at least 4; and (iv) the expression level ratio at the age of 25 weeks is at least 1.3 times as large as the expression level ratio at the age of 18 weeks.
- These proteins are marker proteins showing the progression of plaques from the initial stage to the intermediate stage.
- a lesion at the age of 12 weeks (the initial stage) or a lesion at the age of 18 weeks (the early intermediate stage) or later is present can be evaluated based on the expression level of the marker.
- These proteins were marker proteins indicating the stabilization of plaques at the late stage. With the use of at least one type of these proteins as a marker protein, the presence or the absence of a lesion at the age of 35 weeks (the late stage) or later can be evaluated based on the expression level of the marker.
- Proteins shown in Table 4 are marker proteins, the expression levels of which in ApoE-deficient mice are varied depending on ages in weeks compared with those in wild-type mice. Of these proteins, at least one type thereof can be used as a marker, or this can be used in combination with markers shown in Table 1 to Table 3. Thus, the presence or the absence of a lesion at each age in week (at the initial stage, the early intermediate stage, the intermediate stage, or the late stage) can be evaluated based on the expression level(s) of the marker(s).
- Atherosclerotic lesions can be evaluated by detecting at least one type of markers shown in at least Table 1. Specifically, the presence or the absence of an atherosclerotic lesion with a risk of plaque rupture can be evaluated using each marker in Table 1. Furthermore, according to the present invention, at least two markers are used in combination, making it possible to more precise and highly accurate evaluation of atherosclerotic lesions. For example, at least one type of protein shown in Table 1 (cartilage oligomeric matrix protein, fibromodulin, fibronectin, or thrombospondin-4) and, among each group shown in Table 2 to Table 4, at least two types of protein selected from at least one, at least two, or at least three groups are combined.
- marker(s) shown in Table 1 and marker(s) shown in Table 2 are used in combination.
- differentiation becomes possible, such as whether the lesion is at the initial stage (12-week-old), the early intermediate stage (18-week-old), the intermediate stage (25-week-old), or later, based on the expression level(s) of the marker(s).
- a marker(s) shown in Table 1 and a marker(s) shown in Table 3 are used in combination.
- differentiation becomes possible such that if the lesion is at the intermediate stage (25-week-old), the late stage or later (35-week-old), based on the expression levels of these markers.
- a marker(s) shown in Table 1 and a marker(s) shown in Table 4 are used in combination.
- three or more markers shown in Table 1 to Table 4 can be used in combination.
- at least one marker each was selected from each one of Table 1 to Table 4 and then the markers are used in combination.
- the markers are used in combination.
- three or more markers, and more preferably, three to seven markers are combined.
- Such a combination can be adequately selected in accordance with subject type, sex, age, purpose of evaluation of arteriosclerosis, disease stage to be subjected to evaluation, and the like.
- the above markers or marker groups are expressed in patterns characteristic to specific disease stages of atherosclerotic lesions. Therefore, in the method for evaluating an atherosclerotic lesion according to the present invention, the above markers or marker groups in a subject are detected. When two or more markers are detected, the steps of detecting each marker can be performed simultaneously or at different times.
- the term “detection of marker (group)” may refer to the detection of a marker protein, a substance derived therefrom, or a derivative thereof, or a gene (mRNA) encoding the protein.
- the term “substance derived therefrom” and “a derivative thereof” refer to a substance derived or originated from a marker protein.
- fibrinogen shown in Table 2 contains ⁇ chain and ⁇ chain, and an intact molecule of fibrinogen may be detected, or any one of ⁇ chain or ⁇ chain that is a substance derived therefrom may be detected in the present invention.
- Detection of a marker protein preferably relates to semiquantitative or quantitative measurement of the amount or the concentration of the marker protein in a subject.
- the amount may be the absolute quantity or the relative quantity.
- Detection can be performed directly or indirectly.
- Direct detection relates to measurement of the amount or the concentration of a marker protein or mRNA existing in a subject based on a signal directly correlating with the number of molecules of the marker protein or mRNA. Such a signal is based on specific physical or chemical properties of a protein or mRNA, for example.
- Indirect measurement is measurement of signals obtained from secondary components (that is, components other than marker proteins or mRNA) such as ligands (e.g., antibodies), labels, or enzymatic reaction products.
- the step of detecting a marker protein can be performed by administering to a subject a substance specifically binding to a marker protein, and then detecting the substance bound to the marker protein existing in the subject, for example.
- the step of detecting a marker protein can be performed by preparing a subject-derived sample, bringing the sample into contact with a substance specifically binding to a marker protein, and then detecting the binding of the marker protein with the substance.
- substance specifically binding to a marker protein refers to a substance that binds to a marker protein, a substance derived therefrom, or a derivative thereof with high affinity, but binds (with only low affinity) or never bind to molecules unrelated to a marker protein, a substance derived therefrom, or a derivative thereof.
- a substance specifically binding to a marker protein is an antibody or an antibody fragment.
- an antibody against each marker protein may be either a monoclonal antibody or a polyclonal antibody, or may be particularly Fab, Fab′, F(ab′) 2 , scFv, Fv fragment, or the like that can bind to an epitope of each marker protein.
- An antibody and an antibody-like molecule can be prepared by methods known in the art or can be obtained as a commercially available product.
- An antibody-like molecule is a protein fragment containing a peptide equivalent to an antibody variable region capable of binding to a marker protein, which is prepared by a non-immunological technique such as a phage display method or a combinatorial chemistry method. An example thereof is an affibody.
- a nucleic acid aptamer is known in the art and is a nucleic acid molecule capable of specifically binding to a specific protein (specifically, a marker protein). Such a nucleic acid aptamer can be chemically synthesized in vitro and is advantageous in that it has low immunogenicity.
- a nucleic acid aptamer can be designed by a SELEX method or the like. Persons skilled in the art can design and synthesize an appropriate nucleic acid aptamer.
- the substrate or the substrate derivative thereof can be used as a substance capable of specifically binding to the enzyme.
- a protease inhibitor may be the one derived from nature or a synthetic molecule
- a marker protein is a receptor
- a ligand thereof an agonist, an antagonist, and a derivative thereof
- the receptor is advantageous in that a ligand to be administered to a subject can easily bind from outside the cell to the receptor.
- a marker protein When a marker protein is detected by administration of a substance specifically binding to the marker protein to a subject, the substance specifically binding to the marker protein binds to the marker protein in vivo in the subject when administered to the subject. Therefore, with the use of such a substance, localization of a marker protein in a subject can be examined and the location and disease stage of an atherosclerotic lesion(s) can be evaluated, for example. Moreover, such a substance is bound to a contrast medium in advance, making it possible to visualize the atherosclerotic lesions in the subject.
- a marker protein can be conveniently and noninvasively detected using such an agent for visualization. Therefore, the present invention also provides an agent for visualizing an atherosclerotic lesion.
- the agent for visualization contains a substance specifically binding to at least one type of marker protein and a contrast medium.
- a contrast medium an arbitrary contrast medium known in the in vivo imaging field can be used.
- contrast medium examples include: fluorescent substances such as fluorescein, FITC, and fluorescence emitting metals (e.g., 152 Eu, and lanthanum series); chemical or biological luminescent substances such as luminol, imidazole, luciferin, luciferase, and green fluorescent protein (GFP); radioactive substances such as 99m Tc, 123 I, 131 I, 97 Ru, 67 Cu, 11 C, and 13 N; paramagnetic isotopes such as 153 Gd, 157 Gd, 55 Mn, 162 Dy, 52 Cr, 56 Fe, and superparamagnetic iron oxide particles; and other contrast media such as gadolinium, gadolinium complexes, iodo contrast media, liposomes, micelles, cells, virus particles, viruses, microparticles, nanoparticles, microdevices with spaces, emulsions, lipid discs, polymers, perfluorocarbon nanoparticles, and ultrafine bubbles.
- fluorescent substances such
- a medicine to be used in the present invention containing the above agent for visualizing an atherosclerotic lesion may contain, in addition to a substance specifically binding to a marker protein, a pharmaceutically acceptable carrier or additive.
- a pharmaceutically acceptable carrier or additive include water, a pharmaceutically acceptable organic solvent, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, xanthan gum, gum Arabic, casein, gelatin, agar, glycerin, propylene glycol, polyethylene glycol, Vaseline, paraffin, stearyl alcohol, stearic acid, mannitol, sorbitol, and lactose.
- An additive to be used herein can be adequately selected or selected in combination depending on dosage forms.
- the method for administration of the medicine of the present invention is not particularly limited.
- the medicine can be administered via peroral administration or parenteral administration such as subcutaneous administration, intradermal administration, intramuscular administration, intravenous administration, transdermal administration, rectal administration, and intranasal administration.
- the medicine of the present invention When the medicine of the present invention is administered via peroral administration, it may be in the form of tablets, capsules (e.g., hard capsules, soft capsules, and microcapsules), granules, powders, pills, troches, pharmaceutical solution as internal medicine, liquid agents, suspensions, emulsions, syrups, or the like.
- the medicine may also be a dry product that is dissolved again when it is used.
- examples of dosage forms that can be selected herein include injection preparations (e.g., solutions, emulsions, and suspensions) for intravenous injection (including drip infusion), intramuscular injection, intraperitoneal injection, and subcutaneous injection, and external preparations such as ointments (particularly, an ointment for eyes), cream pharmaceuticals, suppositories, adhesive skin patches, eye drops, nasal drops, inhalers, liniments, and aerosol.
- injection preparations e.g., solutions, emulsions, and suspensions
- intravenous injection including drip infusion
- intramuscular injection intraperitoneal injection
- subcutaneous injection and external preparations
- ointments particularly, an ointment for eyes
- cream pharmaceuticals suppositories
- adhesive skin patches e.g., eye drops, nasal drops, inhalers, liniments, and aerosol.
- injection preparations they are provided in unit dosage ampules or vessels for high dose administration.
- compositions can be produced by conventional methods by appropriately selecting an excipient, an extending agent, a binder, a wetting agent, a disintegrator, a lubricant, a surfactant, a dispersing agent, a buffering agent, a pH adjuster, a preservative, a solubilizing agent, an antiseptic, a taste and flavor corrigent, an absorption accelerator, a soothing agent, a stabilizer, a tonicity agent, and the like that are generally used in medicaments.
- the amount of a substance specifically binding to a marker protein that is to be incorporated into the medicine of the present invention differs depending on the type of substance, the type of contrast medium to be bound with the substance, the purpose of use thereof, dosage form, the route of administration, and the like.
- the amount ranges from 1% to 99% of the total weight, and preferably ranges from 5% to 90% of the total weight.
- the dose and the dosing interval of the medicine of the present invention differ depending on the type of a substance specifically binding to a marker protein, the type of a contrast medium to be bound with the substance, a subject of administration, the age and the body weight of a subject, the route of administration, and the frequency of administration.
- the dose and the dosing interval thereof can be varied widely.
- an agent for visualizing an atherosclerotic lesion after administration of the medicine, the presence or the location of a substance specifically binding to a marker protein in a subject is visualized using a contrast medium as an indicator.
- a contrast medium As an indicator.
- the presence or the location of a substance specifically binding to a marker protein is visualized by a known imaging technique.
- a known imaging technique differs depending on a label to be used herein, the type of a subject, a site for imaging, and the like.
- Positron emission tomography (PET), single photon emission computed tomography (SPECT), ⁇ scintigraphy, autoradiography, fluorescent imaging, magnetic resonance imaging (MRI), ultrasonication, computed tomography (CT), and other in vivo imaging systems can be used.
- determination of the disease stage of an atherosclerotic lesion, prediction of the prognosis of an atherosclerotic lesion, and evaluation of the therapeutic effect against arteriosclerosis in a subject, and the like can also be evaluated based on the type and the amount of the marker protein.
- Samples to be used herein are not particularly limited, as long as they are samples derived from subjects to be evaluated for atherosclerotic lesions. Samples are adequately selected in accordance with methods or types of means of detecting markers. Examples thereof include tissue or cell samples (e.g., arterial tissues and cells). Also, a tissue or cell sample is preferably frozen and cryopreserved immediately after collection by a method known or generally used in the art using liquid nitrogen, dry ice, or the like.
- a marker protein can be detected by a means of measuring the amount of a protein in a sample. Such means are known in the art, such as immunoassay methods and reagents. Also, a marker protein can be detected by a means of measuring physical or chemical properties peculiar to the marker protein, such as a means of measuring accurate molecular weights, rNMR spectra, or the like. Examples of a means of detecting a marker protein include analyzers such as a biosensor, a protein chip, an optical unit combined with immunoassay, a mass spectrometer, an NMR analyzer, a 2D electrophoresis apparatus, and a chromatography apparatus.
- analyzers such as a biosensor, a protein chip, an optical unit combined with immunoassay, a mass spectrometer, an NMR analyzer, a 2D electrophoresis apparatus, and a chromatography apparatus.
- a marker protein can be detected using mass spectrometry (MS).
- MS mass spectrometry
- analysis made by a mass spectrometer combined with liquid chromatography (LC/MS) is sensitive and thus is advantageous.
- isotope labeling reagents known in the art can be used. Appropriate labeling reagents can be commercially obtained.
- fractionation can be performed by a method known in the art. For example, fractionation can be performed using a commercially available strong cationic column or the like.
- a marker protein in a sample can be detected by immunoassay (immunological assay). Specifically, the protein in a sample is detected based on a reaction between the marker protein in the sample and an antibody specifically binding to the protein. Immunoassay may be performed with either a liquid phase system or a solid phase system, as long as the method is generally employed in the art. In view of ease of detection, a solid phase system is preferably used.
- the forms of immunoassay are not limited and may be, in addition to a direct solid phase method, a sandwich method, a competition assay, a Western blotting method, an ELISA (enzyme linked immunosorbent assay) method, or the like.
- An antibody (to be used in the method of the present invention) against each marker protein may be either a monoclonal antibody or a polyclonal antibody, or may be Fab, Fab′, F(ab′) 2 , scFv, Fv fragment, or the like capable of binding to the epitope of each marker protein.
- a primary antibody and a secondary antibody both antibodies may be monoclonal antibodies.
- either a primary antibody or a secondary antibody may be a polyclonal antibody.
- Such an antibody can be prepared by a method known in the art or can be commercially obtained.
- Binding of a marker protein with an antibody can be measured according to a known method. Persons skilled in the art can determine an effective and optimum measurement method for each assay according to the type and form of immunoassay to be employed, or the type of a label to be used herein, for example. For example, for easy detection of the binding of a marker protein in a sample with an antibody, the binding is directly detected by labeling the antibody, or the binding is indirectly detected using a labeled secondary antibody, a biotin-avidin conjugate, or the like.
- an antibody or a protein component in a sample is immobilized onto a solid phase (e.g., a plate, a membrane, or beads), and then immunological binding of a marker protein with a solid is tested on the solid phase.
- a solid phase e.g., a plate, a membrane, or beads
- Such a solid phase is not particularly limited, as long as it is conventionally used in the art.
- a commercially available nitrocellulose membrane or PVDF membrane can be used.
- An antibody or a protein component in a sample is immobilized on a solid phase, so that unbound components in the sample or a reagent can be easily removed.
- the presence of many types of marker protein can be analyzed within a short time using a small amount of a subject-derived sample (e.g., plasma).
- a subject-derived sample e.g., plasma
- Such immunoassay can also be performed with test strips, the operation with which can be conveniently performed.
- liquid-phase immunoassay for example, a labeled antibody is brought into contact with a sample so as to bind the labeled antibody to a marker protein, the conjugate is separated, and thus the signal of the label is detected.
- an antibody (primary antibody) against a marker protein is brought into contact with a sample so as to bind the primary antibody to the marker protein, a labeled antibody (secondary antibody) is bound to the conjugate, and thus the signal of the label is detected from the conjugate of these three substances.
- an unlabeled secondary antibody is first bound to an antibody+marker protein conjugate, so as to bind a labeling substance to the secondary antibody.
- binding of a labeling substance to a secondary antibody can be performed by biotinylating the secondary antibody and avidinylating the labeling substance in advance, for example.
- a marker protein can also be detected in situ, as in the case of using an immunohistochemical staining method (e.g., an immunostaining method) or immunoelectron microscopy.
- In situ detection can be performed by excising a histological sample from a subject (e.g., a biopsy sample and a tissue paraffin embedded section), and then bringing a labeled antibody or antibody fragment into contact therewith.
- enzymes for labeling antibodies to be used in immunoassay, enzymes, radio isotopes, fluorescent dyes, or avidin-biotin systems can be used.
- enzymes enzymes that are used for general enzyme immunoassay (EIA) can be used, such as peroxidase, ⁇ -galactosidase, and alkaline phosphatase.
- EIA enzyme immunoassay
- an enzyme inhibition substance, a coenzyme, and the like can be used. Binding of these enzymes with antibodies can be performed by known methods using cross-linking agents such as a maleimide compound.
- radio isotopes 125 I, 3 H, and the like, which are used for general radio immunoassay (RIA) can be used.
- fluorescent dyes fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), and the like, which are used for general fluorescent antibody techniques, can be used.
- the signal of a label can also be detected according to a method known in the art.
- a substrate that is degraded by enzymatic action to develop color is added, the amount of the substrate degraded is optically measured to find enzyme activity, the resulting value is converted to the amount of the antibody bound, the result is compared with a standard value, and thus the amount of the antibody is calculated.
- a substrate differs depending on the type of enzyme to be used herein. For example, when peroxidase is used as an enzyme, 3,3′,5,5′-tetramethylbenzidine can be used as a substrate, and when alkaline phosphatase is used as an enzyme, para-nitrophenol or the like can be used as a substrate.
- a radioactive label the radiation dose from the radioactive label is measured by a scintillation counter or the like.
- a fluorescent label can be detected or quantitatively determined using a fluorescent microscope, a plate reader, a fluorescent imaging apparatus, or the like.
- a marker protein in a subject-derived sample is detected, and then arteriosclerotic lesion(s) in the subject can be evaluated based on the results.
- evaluation of atherosclerotic lesions refers to determination of the presence of atherosclerotic lesions in a subject, determination of the stage of atherosclerotic lesions existing in a subject, determination of the location of atherosclerotic lesions existing in a subject, evaluation of therapeutic effects on arteriosclerosis existing in a subject, and prediction of the prognosis of arteriosclerosis existing in a subject. Also, the term “evaluation” in the present invention is meant to include continuous monitoring of arteriosclerosis that has already been evaluated or diagnosed and confirmation of the past evaluation or diagnosis of arteriosclerosis.
- arteriosclerosis e.g., angina pectoris (e.g., stable angina pectoris), acute myocardial infarction, brain infarction, encephalorrhagy, cerebral thrombosis, cerebral embolism, aortic aneurysm, obstructive arteriosclerosis, and acute coronary syndromes
- angina pectoris e.g., stable angina pectoris
- acute myocardial infarction e.g., stable angina pectoris
- brain infarction e.g., encephalorrhagy
- cerebral thrombosis e.g., cerebral embolism
- aortic aneurysm e.g., obstructive arteriosclerosis
- acute coronary syndromes e.g., acute coronary syndromes
- the “evaluation” is intended to allow evaluation of subjects accounting for a statistically significant proportion of all subjects. Such a statistically significant proportion can be determined using various known statistical evaluation tools such as determination of a confidence interval, determination of p value, student's t-test, Mann-Whitney test, and the like.
- a preferable confidence interval is at least 90%.
- the p value is preferably 0.1, 0.01, 0.005, or 0.0001. More preferably, at least 60%, at least 80%, or at least 90% of subjects can be appropriately evaluated by the present invention.
- a standard level is: the measured level of a marker protein in a healthy-subject-derived sample, the measured level of a marker protein in a sample from a subject diagnosed as having arteriosclerosis at a specific stage, the measured level of a marker protein in a sample from a subject before treatment, or the like.
- a standard level for an individual subject may be varied depending on various physiological parameters such as subject type, age, and sex.
- individual subjects with arteriosclerosis are classified according to the degree of the progression of the disease (stage), and the expression levels of marker proteins are obtained from individual subjects belonging to each stage and individual subjects in a normal state. Then, correlations between variations in the expression of a marker protein and progression stages are recorded into an expression profile database. The ratio of the expression level of a marker protein in a subject-derived sample divided by the expression level thereof in a standard sample (prepared in advance) is measured. The expression profile database can be referred for the thus obtained measured value.
- Such an expression profile database is useful as “standard levels” or “standard range” that can be indicators of the presence or the absence of arteriosclerosis in a subject, the progression stage, and the onset or the degree of progression of arteriosclerosis.
- Such a standard level may be the only one cutoff value (e.g., average value or median value), or standard levels may be within a specific range (e.g., quartile).
- R(i) the ratio of the expression level of a protein in a subject-derived sample to the same in a standard sample is measured.
- Each of these measured values is compared with the value at each progression stage (Rj(i), wherein “j” denotes stage No. ranging from 0-4) and then the results are recorded in the database, so that the progression stage of arteriosclerosis in the subject can be determined.
- a standard level is also preferably the measured expression level of a marker protein in a human.
- a standard level obtained with the use of experimental animals such as mice can also be extrapolated.
- the level of a marker protein shown in Table 1 in a subject indicates the presence of atherosclerotic lesions at the intermediate stage or later in the subject.
- the level of a marker protein shown in Table 2 in a subject indicates the presence of atherosclerotic lesions at the initial or early intermediate stage or later in the subject.
- the level of a marker protein shown in Table 3 in a subject which is higher than a standard level, indicates the presence of atherosclerotic lesions at the late stage or later in a subject.
- Marker proteins shown in Table 4 in a subject are combined with other marker proteins, and then variations in the expression levels thereof are examined, indicating the presence of atherosclerotic lesions in the subject.
- the method for evaluating atherosclerotic lesions according to the present invention may be performed in combination with other known diagnosis methods for arteriosclerosis.
- known diagnosis methods for arteriosclerosis include measurement of physiological and biochemical markers of arteriosclerosis (e.g., high blood pressure, cholesterol level, and triglyceride level), measurement with electrocardiogram, measurement of arterial pulse wave velocity (PWV), and angiography.
- PWV arterial pulse wave velocity
- the method for evaluating atherosclerotic lesions of the present invention can also be combined with other arteriosclerosis markers.
- the present inventors have revealed that: an increase in the expression level of von Willebrand factor and/or a decrease in the expression level of complement factor D in blood plasma can be an indicator for arteriosclerosis at the early intermediate stage; and an increase in the expression level of complement component C8 and/or a an increase in the expression level of vitamin K-dependent protein Z in blood plasma can be an indicator for arteriosclerosis at the intermediate stage (WO 2011/136080).
- a CD5 antigen-like protein and an immunoglobulin mu chain C region exhibited maximum expression levels at the early intermediate stage
- a blood plasma protease C1 inhibitor, thrombospondin-4, fetuin-B, and complement factor H exhibited minimum expression levels at the early intermediate stage or the intermediate stage.
- Heparin cofactor 2 alpha-2-macroglobulin, complement component C9, vitamin K-dependent protein C, antithrombin-III, EGF-containing fibrin-like extracellular matrix protein 1, inter alpha trypsin inhibitor heavy chain H1, inter alpha trypsin inhibitor heavy chain H2, and properdin exhibited decreased expression levels with the progression of the disease stage.
- Complement C1q subcomponent subunit B exhibited the expression level that increased with the progression of the disease stage. Therefore, the above blood plasma arteriosclerosis markers are used in combination with the evaluation of atherosclerotic lesions of the present invention, so that evaluation can be performed with higher accuracy.
- the presence of atherosclerotic (unstable) lesions can be determined at an early stage by the method for evaluating atherosclerotic lesions according to the present invention.
- the present invention enables determination of the presence or the absence of unstable atherosclerotic lesions, which cannot be recognized by currently available diagnostic techniques or criteria.
- the disease stage and the location of each lesion can be specified in detail. Accordingly, the subject can timely receive treatment (for diseases associated with arteriosclerosis such as angina pectoris, myocardial infarction, and brain infarction) suitable for the disease stage thereof precisely.
- the method of the present invention is also advantageous in that arteriosclerosis can be reliably and rapidly evaluated by techniques with low invasiveness.
- the method for evaluating atherosclerotic lesions according to the present invention can be easily and conveniently performed using a kit and/or an apparatus provided with a means of detecting the above markers.
- the kit for evaluating atherosclerotic lesions according to the present invention contains at least one type of substance specifically binding to at least one type of protein selected from at least group (a) from among proteins shown in the following groups (a) to (d):
- kits of the present invention is composed of antibodies specifically binding to marker proteins, contrast media or labels, buffers for dilution and washing, and if necessary labeled secondary antibodies, substrate reagents for causing color development, luminescence and fluorescence generation, manuals describing procedures and evaluation methods, and the like.
- the kit may further contain a means of administration, such as vials, ampules, and syringes.
- an antibody immobilized on a solid-phase support e.g., a membrane or beads
- a solid-phase support e.g., a membrane or beads
- the kit of the present invention may contain instructions in which procedures and protocols for implementing the method of the present invention are described and tables showing standard levels or reference ranges to be employed for evaluation of arteriosclerotic lesions, for example.
- kit of the present invention may be individually provided or provided within a single container.
- the kit of the present invention contains all the components required for implementation of the method of the present invention, as components with adjusted concentrations, for example, so that they can be used immediately.
- the apparatus for evaluating atherosclerotic lesions comprises the following means of:
- the apparatus of present invention may further comprise the following means of:
- the apparatus of the present invention is a system, wherein the means (i) and (ii) are combined with further preferably means (iii) and (iv) so that the means can be implemented in a mutual manner, so as to be able to perform the method of the present invention.
- FIG. 5 shows the outline of the apparatus of the present invention.
- the means (i) can be a protein detection part 101 provided with a quantification part 104 for detecting and quantifying signals following the operation of a protein specific signal detection part 102 (e.g., mass spectroscopy part), or a quantification part 104 or a imaging part 103 for detecting, quantifying or imaging signals from radioactivity, color development, luminescence, or fluorescence, for example.
- the means (ii) can be a part 301 for evaluation of atherosclerotic lesions, which is provided with an image.numeric data storage part 302 for storing the detection results (images ⁇ numeric data) obtained from the detection part 101 , for example, a software 307 for processing the detection results, and a computer part 308 .
- a preferable apparatus is an apparatus that can be used without the knowledge of an experienced clinician.
- a specific apparatus comprises the detection part 101 (provided with a protein-specific signal detection part 102 such as PET, SPECT, ⁇ scintigraphy, autoradiography, fluorescent imaging, MRI, ultrasonication, CT, a biosensor, a solid-phase support to which an antibody against a marker has been immobilized, mass spectroscopy, a surface plasmon resonance sensor, and an NMR analyzer), and an evaluation part 301 (e.g., an image.numeric data storage part 302 , standard level data storage part 303 , a data comparison part 304 and a data analysis part 305 for analysis of data, data output.display part 306 , a software 307 and a computer unit for activating a computer part 308 ).
- a protein-specific signal detection part 102 such as PET, SPECT, ⁇ scintigraphy, autoradiography, fluorescent imaging, MRI, ultrasonication, CT,
- the means (iii) can be a protein standard level database 201 , which is provided with expression data 202 containing pathological (arteriosclerosis) stage-specific expression data and may also be provided as necessary with at least one type of data selected from among arterial tissue data 203 , blood plasma data 204 , and data of differences in males and females 205 .
- Necessary data are loaded from the standard level database 201 to the standard level data storage part 303 of the means (ii), and then the standard level data and the data of image.numeric data storage part 302 are compared and analyzed.
- a compound or a molecule such as a drug or a prodrug is bound to a substance specifically binding to a marker protein, instead of a contrast medium, and thus the compound or the molecule can be delivered to a site of a subject where the marker protein is present; that is, an atherosclerotic lesion.
- a drug or prodrug include known thrombolytic agents (e.g., urokinase, streptokinase, and a tissue plasminogen activator).
- thrombolytic agents e.g., urokinase, streptokinase, and a tissue plasminogen activator.
- Such a targeting agent for delivery of such a drug or a prodrug to an atherosclerotic lesion is also encompassed within the present invention.
- Such a targeting agent has the effects of: binding to a marker protein existing in an atherosclerotic lesion so as to be able to deliver a compound or a molecule (e.g., a drug or a prodrug) to an atherosclerotic lesion, remaining for a long time within the atherosclerotic lesion, and thus allowing the drug effects to be continuously exhibited for a long time. Therefore, the targeting agent of the present invention can be used as an agent for prevention or treatment of arteriosclerosis. Specifically, the effective dose of the targeting agent of the present invention is administered to a subject, so that arteriosclerosis in the subject can be prevented or treated.
- a compound or a molecule e.g., a drug or a prodrug
- a subject to which the targeting agent of the present invention is administered is not particularly limited. Examples thereof include mammals such as humans, domestic animals (e.g., cattle and pigs), pet animals (e.g., dogs and cats), and experimental animals (e.g., mice, rats, and monkeys).
- the target agent is preferably used for a subject suspected of having arteriosclerosis and a subject having arteriosclerosis.
- arteriosclerosis to be treated or prevented in the present invention include, but are not limited to, arteriosclerosis and diseases associated with arteriosclerosis such as angina pectoris (e.g., stable angina pectoris), acute myocardial infarction, brain infarction, encephalorrhagy, cerebral thrombosis, cerebral embolism, aortic aneurysm, obstructive arteriosclerosis, and acute coronary syndromes.
- angina pectoris e.g., stable angina pectoris
- acute myocardial infarction e.g., brain infarction
- encephalorrhagy e.g., cerebral thrombosis
- cerebral embolism e.g., cerebral embolism
- aortic aneurysm e.g., obstructive arteriosclerosis
- acute coronary syndromes e.g., acute coronary syndromes.
- the targeting agent of the present invention can be prepared and administered in a manner as described above in the case of an agent for visualization.
- Persons skilled in the art can adequately select methods in view of a compound or a molecule (e.g., a drug or a prodrug) to be contained in the targeting agent.
- Such a method of the present invention comprises the steps of:
- a marker protein is detected in an animal with arteriosclerosis, that is, an animal developing arteriosclerosis or an animal at a risk of arteriosclerosis.
- a marker protein is detected in an animal with arteriosclerosis, and then the disease stage of the atherosclerotic lesion is confirmed before treatment.
- a marker protein is detected in the animal at an appropriate time.
- detection is performed immediately after treatment, 30 minutes after, 1 hour after, 3 hours after, 5 hours after, 10 hours after, 15 hours after, 20 hours after, 24 hours (1 day) after, 2 to 10 days after, 10 to 20 days after, 20 to 30 days after, or 1 to 6 months after treatment.
- Detection of a marker protein(s) and evaluation of atherosclerotic lesions can be performed in a manner similar to the above.
- Subject animals in the present invention may be humans with arteriosclerotic lesions, arterial disease model animals, or preferably arteriosclerosis model experimental animals (e.g., mice, rats, and rabbits), for example.
- arteriosclerosis model experimental animals e.g., mice, rats, and rabbits
- effectiveness can be evaluated in humans by clinical trials, for example.
- test therapeutic agent or test therapeutic method for evaluation or screening are not particularly limited.
- test therapeutic agents or test therapeutic methods include arbitrary material factors, specifically, naturally occurring molecules such as amino acid, peptide, oligo peptide, polypeptide, protein, nucleic acid, lipid, carbohydrate (e.g., sugar), steroid, glycopeptide, glycoprotein, and proteoglycan; synthetic analogs or derivatives of naturally occurring molecules such as peptide mimic or nucleic acid molecule (e.g., aptamer, antisense nucleic acid, and double-stranded RNA (RNAi)); molecules that do not occur naturally such as low-molecular-weight organic compound (e.g., inorganic and organic compound libraries, or combinatorial library); and mixtures thereof.
- naturally occurring molecules such as amino acid, peptide, oligo peptide, polypeptide, protein, nucleic acid, lipid, carbohydrate (e.g., sugar), steroid, glycopeptide, glycoprotein, and proteogly
- test therapeutic agent or a test therapeutic method may be a single substance, a complex composed of a plurality of substances, a food, a feed, or the like.
- test therapeutic agents or test therapeutic methods may be, in addition to the above material factors, radiation, ultraviolet radiation, and the like.
- Treatment of an animal using a test therapeutic agent or a test therapeutic method differs depending on the type of therapeutic agent or therapeutic method, and can be easily determined by persons skilled in the art. For example, persons skilled in the art can appropriately determine administration conditions such as the dosage, the dosage period, and the route of administration of a test therapeutic agent.
- test therapeutic agent or a test therapeutic method can be examined under several conditions.
- conditions include the time or the period of treatment with the test therapeutic agent or by the test therapeutic method, the amount thereof (high or low), and frequency.
- a dilution series of a test therapeutic agent is prepared, so that a plurality of doses can be determined.
- the therapeutic agents or therapeutic methods may be used in combination.
- the amount and/or the location of a marker protein(s) in an animal after treatment with a test therapeutic agent or by a test therapeutic method is compared with the amount and/or the location of the same before treatment, so as to allow evaluation of whether or not the test therapeutic agent or the test therapeutic method is effective for improvement of atherosclerotic lesions (instability thereof) (e.g., improvement from the intermediate stage to the early intermediate stage or the initial stage), or for stopping or slowing down the progression of atherosclerotic lesions (instability thereof) (e.g., the disease progression from the intermediate stage to the late stage is stopped, unlike a case in which no treatment has been performed).
- a therapeutic agent or a therapeutic method for treating or preventing arteriosclerosis can be found, and then the effectiveness of the therapeutic agent or the therapeutic method can be confirmed.
- atherosclerotic lesions (instablility thereof) at the early stage in particular the initial stage and the early intermediate stage
- no sign indicating the disease can be detected.
- the method of the present invention is particularly effective for identifying a therapeutic agent or a therapeutic method effective for treatment of arteriosclerosis at the early stage.
- the effects of a substance or a factor on arteriosclerosis can also be determined, and thus a substance or a factor that causes the progression of atherosclerotic lesions (instability thereof) can be identified.
- ApoE-deficient mice (ApoED; B6.KOR/StmSlc-Apoeshl), which were deficient in the lipid metabolism-related ApoE protein, were used as atherosclerosis models (Matsushima, Y. et al., J. Atheroscler. Thromb. 8: 71-79, 2001; Japan SLC, Inc.).
- Administration of a high-fat diet accelerates lesion progression in ApoED mice.
- lesion progression is accelerated depending on age in weeks.
- lesion progression is observed to such an extent that arterial intimal thickening and lipid accumulation are observed (stage 1).
- lipid accumulation results in foam cell formation (stage 2).
- stage 3 In 25-week-old mice, many forms of unstable atherosclerotic plaque are widely observed in arteries (stage 3), and this corresponds to the most dangerous stage in the atherosclerosis process. In 35-week-old mice, many stable lesions that are highly fibrotic and calcified are observed (stage 4).
- arterial tissue was collected from 12, 18, 25, and 35-week-old wild-type mice (WT; C57BL/6) and ApoED mice (male and female mice: 5 animals each).
- WT and ApoED mouse arterial tissue samples were crushed in liquid nitrogen and then solubilized with a solubilization solution (9.5 M urea, 2% sulfuric acid-3-[(3-cholamidopropyl)dimethylammonio]-1-propane (CHAPS), 65 mM dithiothreitol (DTT), 40 mM Tris HCl, pH 8.3).
- solubilization solution 9.5 M urea, 2% sulfuric acid-3-[(3-cholamidopropyl)dimethylammonio]-1-propane (CHAPS), 65 mM dithiothreitol (DTT), 40 mM Tris HCl, pH 8.3
- the protein fractions (1 mg each) extracted from arterial tissues were adjusted in a manner such that each sample contained 6M urea, 0.05% SDS, 50 mM Tris (pH 8.5), 5 mM ethylenediamine tetraacetic acid (EDTA), and 10 mM tri-n-butyl phosphate (TBP) (final concentrations) in a total volume of 800 ⁇ l, followed by degeneration treatment at 37° C. for 30 minutes.
- Each SCX fraction was analyzed using a mass spectrometry apparatus and an accompanying LC system device (NanoFrontier LD; Hitachi High-Technologies Corporation). Each obtained sample was dissolved in a buffer A (water: 98%; ACN: 2%; formic acid: 0.1%) (4 ⁇ l to 10 ⁇ l). One microliter of each obtained solution was applied to the apparatus.
- a MonoCap for Fast-flow 50 ⁇ m ⁇ 150 mm; C18; GL Sciences
- IBA information based acquisition
- IBA is a technique involving storing target information (m/z, charge number, retention time) obtained by the first analysis in a database within an apparatus, and analyzing ions that do not correspond to the target information in the second analysis. It was expected that weak ions would be analyzed with the use of such technique so as to increase the number of identified proteins.
- the measurement data were processed using software that had been developed for ICAT comparative quantification. Thus, comparative analysis data for two groups (the WT mouse group and the ApoED mouse group) were obtained.
- proteins belonging to the pattern 3 From among proteins belonging to the pattern 3, four proteins characterized in that: (i) the ratio of the amounts thereof in ApoE-deficient mice divided by the amounts thereof in wild-type mice (herein after referred to as “the expression level ratio”) at the age of 25 weeks was at least 3; and (ii) the expression level ratio at the age of 25 weeks was at least twice as large as the expression level ratio at the age of 18 weeks, were selected as markers indicating the risk of plaque rupture at the intermediate stage (Table 1).
- proteins belonging to patterns 1 and 2 seven proteins were selected as markers indicating the progression of plaques from the initial to the intermediate stages (Table 2). These proteins were characterized in that: (i) the expression level ratio at the age of 18 weeks was at least 2; (ii) the expression level ratio at the age of 18 weeks was at least 1.3 times as large as the expression level ratio at the age of 12 weeks; (iii) the expression level ratio at the age of 25 weeks was at least 4; and (iv) the expression level ratio at the age of 25 weeks was at least 1.3 times as large as the expression level ratio at the age of 18 weeks.
- the lipid concentrations in blood of a subject were: total cholesterol of 539 mg/dl; triglyceride of 49 mg/dl; LDL cholesterol of 404 mg/dl; and HDL cholesterol of 143 mg/dl.
- the progression of arteriosclerosis was suspected.
- the ratios of the expression levels of proteins contained in tissue samples were exhaustively analyzed by the method of Example 1 (that had been partially modified) using an arterial tissue obtained from a wild-type mouse (WT; C57BL/6) at the same age in weeks as a standard level.
- the partial modification in the method of Example 1 is that the number of SCX fractions was 7 instead of 25 for simplifying experimental procedures.
- Example 1 Ten typical proteins were selected from the candidate marker protein group that exhibited variations in Example 1 (characteristic in ApoE-deficient mice) in the amounts thereof in arterial tissues. The expression of the proteins in atherosclerotic lesion sites was confirmed by immunohistochemical staining of lesion sites (plaque formation sites) in arterial tissues of ApoE-deficient mice at different ages in weeks.
- aortic sample subjected to 10% formalin fixation was coronally excised at the aortic arch, and then a paraffin embedded block was prepared. Subsequently, it was sliced to a thickness of 3 ⁇ m, and then the slices were subjected to immunohistochemical staining according to a conventional method using hematoxylin.eosin (H&E) staining and antibodies.
- Primary antibodies used for immunohistochemical staining are as follows.
- Anti-properdin antibody 50-fold dilution, sc68367; Santa Cruz
- Anti-CD5 antigen-like protein antibody 100-fold dilution, ab45408; Abcam
- Anti-fibronectin antibody 50-fold dilution, sc59826; Santa Cruz
- Anti-fibromodulin antibody 50-fold dilution, sc33772; Santa Cruz
- Anti-thrombospondin-4 antibody 50-fold dilution, sc68367; Santa Cruz
- Anti-plastin-2 antibody 50-fold dilution, 13025-1-AP; Proteintech
- Anti-ADAMTS-like protein 4 antibody 50-fold dilution, 15304-1-AP; Proteintech
- Anti-matrix metalloproteinase-2 antibody 50-fold dilution, BS1236; Bioworld Technology
- Anti-tenascin antibody 50-fold dilution, 3118-1; Epitomics
- Anti-cartilage oligomeric matrix protein antibody 50-
- DAB diaminobenzidine
- FIG. 6 shows the results of immunohistochemical staining using the anti-CD5 antigen-like protein antibody and the anti-properdin antibody.
- FIG. 7 shows the results of immunohistochemical staining using the anti-cartilage oligomeric matrix protein antibody, the anti-fibromodulin antibody, the anti-fibronectin antibody, and the anti-thrombospondin-4 antibody.
- FIG. 8 shows the results of immunohistochemical staining using the anti-plastin-2 antibody and the anti-tenascin antibody.
- Example 1 Since 35-week-old mouse arterial tissues contain intermediate stage lesions in addition to late stage lesions, the result that the cartilage oligomeric matrix protein exhibited strong expression in the intermediate stage lesions is consistent with the result of mass spectroscopy in Example 1. Moreover, as a result of immunohistochemical staining using the anti-matrix metalloproteinase-2antibody, high signals were detected for lesion sites at the late stage in particular, the expression of the target protein to be recognized by the antibody in the lesions was confirmed, and thus the results of Example 1 were supported. As a result of immunohistochemical staining using the anti-ADAMTS-like protein 4 antibody, an increase in signal intensity was observed with the progression of stages. The expression of target proteins to be recognized by these antibodies in lesions was confirmed, and thus the results of Example 1 were supported.
- WHHL arteriosclerosis model rabbits formed atherosclerotic lesions, the progression degree of which differs depending on age in months.
- 7-week-old and 13-week-old WHHL rabbits male, Kitayama Labes
- a 99m technetium-labeled anti-properdin antibody and a 125 iodine-labeled anti-CD5L antibody were mixed, the mixture was administered (under anesthesia) into the marginal vein of the ear of each rabbit, and then the ways in which signals from both antibodies were accumulated in lesions were examined. Signal accumulation 6 hours after administration was visualized using an animal SPECT device (R1; Hitach, Ltd.).
- the present invention technology for evaluation of the presence or the absence of the onset of arteriosclerosis and the progression thereof is provided.
- the presence or the absence, the state or the progression degree, and the location of arteriosclerosis can be conveniently determined with high accuracy according to the present invention.
- a method for screening for a medicine useful for prevention and/or treatment of arteriosclerosis is provided.
- the present invention is useful in the field of diagnosis, prevention, and/or treatment of arteriosclerosis.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Pathology (AREA)
- Medical Informatics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Surgery (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Biophysics (AREA)
- Heart & Thoracic Surgery (AREA)
- Veterinary Medicine (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Radiology & Medical Imaging (AREA)
- High Energy & Nuclear Physics (AREA)
- Biochemistry (AREA)
- Food Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- General Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Physiology (AREA)
- Dentistry (AREA)
- Vascular Medicine (AREA)
- Optics & Photonics (AREA)
- Cardiology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Apparatus For Radiation Diagnosis (AREA)
- Magnetic Resonance Imaging Apparatus (AREA)
Abstract
The present invention provides a method and a means for evaluating atherosclerotic lesions by identifying a protein (marker protein) group, the expression level of which varies according to the progression of the atherosclerotic lesion, and using the proteins. Specifically, the present invention relates to a method for evaluating atherosclerotic lesions, comprising the steps of detecting a marker protein exhibiting an expression pattern (expression variation) characteristic at a specific disease stage of atherosclerotic lesions in a subject, and evaluating the atherosclerotic lesions in the subject based on the detection result.
Description
- The present invention relates to a method, a kit, and an apparatus for evaluating atherosclerotic lesions. More specifically, the present invention relates to a method, a kit, and an apparatus for evaluating atherosclerotic lesions based on novel marker protein(s). Furthermore, the present invention relates to agent(s) for visualizing atherosclerotic lesions and agent(s) for targeting atherosclerotic lesions using the marker protein(s), and a method for evaluating the effectiveness of the therapeutic agent(s) or therapeutic method(s) for atherosclerotic lesions.
- Arteriosclerosis proceeds over a long period of time without subjective symptoms, and induces cardiac diseases such as severe myocardial infarction or cerebrovascular diseases such as cerebral infarction or cerebral hemorrhage. Arteriosclerosis progresses to result in the formation of atherosclerotic lesions (atheroma or plaque) within arterial blood vessels. Plaques include stable plaques rich in calcified fibrous tissue and unstable plaques rich in lipids or inflammatory cells, in which fibrous caps are thinned. Most acute atherosclerotic diseases such as myocardial infarction and brain infarction are induced by rupture of unstable plaques and thrombus formation. Therefore, it is important to appropriately evaluate and diagnose the formation and growth conditions of unstable plaques and the rupture risk thereof in acute atherosclerotic diseases. Arteriosclerosis is currently detected mainly by angiography, which involves evaluation of the degree of stenosis, but it is problematic because information concerning intravascular plaque formation or the properties thereof can barely be obtained. Moreover, angiography is highly invasive. Hence, a low-invasive method for detecting atherosclerotic lesions has been required.
- Disease proteomics is a technique for exhaustively searching increases or decreases in protein levels that vary depending on specific diseases when compared with a healthy state, using a body fluid such as blood, saliva, or urine, or a tissue sample, as a material. Disease proteomics is a technique characterized by enabling simultaneous extraction and/or detection of a plurality of factors, the association of which with diseases is conventionally unknown. Application of multiple specimens to and increased sensitivity for a 2D electrophoresis method and measuring apparatuses such as protein microarrays and mass spectrometry have been realized. Thus, data analysis technology has been in place and is applied in searches for markers for various diseases including cancer, immunity disorders, infections, and the like (Non-patent document 1).
- For example, methods for diagnosing arteriosclerosis or diseases associated therewith by using biochemical markers have been reported, wherein the blood levels of the biochemical markers vary as atherosclerotic lesions develop, (
patent documents 1 and 2). However, further development of marker groups is desired in order to precisely diagnose atherosclerotic lesions. -
- Patent Document 1: JP Patent Publication (Kokai) No. 2007-278907 A
- Patent document 2: JP Patent Publication (Kokai) No. 2010-85242 A
-
- Non-patent document 1: Hanash S., Nature, vol. 422, pp. 226-232, 2003
- Formation of atherosclerotic lesions, and unstable lesions in particular, induces severe diseases such as myocardial infarction and brain infarction. Hence, precise diagnosis thereof is an important issue. If precise evaluation of unstable arteriosclerotic lesions becomes possible, this would enable prevention of a disease through improvement of lifestyle or the like, prevention of progression and/or worsening of the disease, and treatment of the disease.
- Therefore, an object of the present invention is to provide diagnosis, prevention, or evaluation for treatment of atherosclerotic lesions by identifying protein groups (maker proteins, the expression levels of which vary with the progression of atherosclerotic lesions) with a proteomic technique, recording the variations in an expression profile database, comparing the data with the variations in the expression levels of marker proteins in samples obtained from subjects, and thereby identifying the stage of progression of atherosclerotic lesion(s).
- Another object of the present invention is to provide a method for evaluating atherosclerotic lesions, comprising:
- administering (e.g., via intravenous administration) to a subject a substance having specific affinity for a marker protein group, the expression of which varies with the progression of atherosclerotic lesions, wherein the substance is labeled in advance;
- detecting the affinity substance binding to the marker protein in vivo in the subject; and measuring the level of the affinity substance binding to the marker protein.
- The present inventors have obtained arterial tissue from ApoE-deficient mice, in which arteriosclerosis commonly occurs and progresses at an accelerated rate with administration of a high-fat diet and aging (age in weeks), and wild-type mice. They then exhaustively compared and analyzed the expression levels of proteins contained in the arterial tissue with a mass spectroscope. An expression profile database was constructed using the ratios of the measured expression levels of proteins contained in arterial tissue samples obtained at each age in weeks (12-week-old, 18-week-old, 25-week-old, and 35-week-old) and differing in the extent of the progression of arteriosclerosis. It was discovered that the onset or the progression (worsening) of arteriosclerosis correlates with the variation tendency for each protein expression (level) ratio, and the tendencies can be classified into a plurality of groups. Furthermore, the present inventors have succeeded in identifying proteins that exhibit expression tendencies characteristic of specific stages of arteriosclerotic lesions, and have found that the proteins can be used as markers for evaluation of the likelihood of the onset of arteriosclerosis, high or low risk of contraction of the disease, and possibility of progression, thereby completing the present invention.
- The present invention provides the following [1] to [39].
- [1] A kit for evaluating an atherosclerotic lesion, comprising at least one type of substance specifically binding to at least one type of protein selected from at least group (a) from among proteins in the following groups (a) to (d):
(a) cartilage oligomeric matrix protein, fibromodulin, fibronectin, and thrombospondin-4;
(b) plastin-2, tenascin, CD5 antigen-like protein, properdin, Cb4-binding protein, fibrinogen beta chain, and fibrinogen gamma chain;
(c) vitronectin, matrix metalloproteinase-2, and thrombospondin-1;
(d) membrane-bound form immunoglobulin mu chain C region, receptor-type tyrosine protein phosphatase C, immunoglobulin gamma-2B chain-C region, immunoglobulin J chain, immunoglobulin kappa chain C region, vascularcell adhesion protein 1, alpha-2-macroglobulin, ADAMTS-like protein 4, cathepsin B, carboxy peptidase-B2, complement factor-H, clusterin, collagen alpha-1 (XIV) chain, epidermal fatty acid-binding protein, mannose-binding lectin-associated serine protease-2, prothrombin, complement factor B, inter-alpha-trypsin inhibitor-heavy chain-H1, myosin-9, vitamin K-dependent factor S,metalloproteinase inhibitor 3, andtropomyosin alpha 4.
[2] The kit according to [1], comprising at least two types of substance specifically binding to a protein selected from group (a) and a protein selected from at least one of groups (b) to (d).
[3] The kit according to [1], comprising at least three types of substance specifically binding to a protein selected from group (a) and proteins selected from at least two of groups (b) to (d).
[4] The kit according to [1], comprising at least four types of substance specifically binding to a protein selected from group (a) and proteins selected from each of groups (b) to (d).
[5] A kit for evaluating an atherosclerotic lesion at the intermediate stage or later, comprising at least one type of substance specifically binding to at least one type of protein selected from the group consisting of cartilage oligomeric matrix protein, fibromodulin, fibronectin, and thrombospondin-4.
[6] A kit for evaluating an atherosclerotic lesion at the initial or the intermediate stage, comprising at least one type of substance specifically binding to at least one type of protein selected from the group consisting of plastin-2, tenascin, CD5 antigen-like protein, properdin, Cb4-binding protein, fibrinogen beta chain, and fibrinogen gamma chain. - [7] A kit for evaluating an atherosclerotic lesion at the late stage, comprising at least one type of substance specifically binding to at least one type of protein selected from the group consisting of vitronectin, matrix metalloproteinase-2, and thrombospondin-1.
- [8] The kit according to any one of [1] to [7], the evaluation of an atherosclerotic lesion is selected from the group consisting of the determination of the presence of an atherosclerotic lesion in a subject, the determination of the stage of an atherosclerotic lesion in a subject, the determination of the location of an atherosclerotic lesion in a subject, the evaluation of the therapeutic effect of arteriosclerosis existing in a subject, and the prediction of prognosis of arteriosclerosis existing in a subject.
- [9] The kit according to any one of [1] to [8], wherein a substance specifically binding to a protein is selected from the group consisting of an antibody, an antibody-like molecule, a nucleic acid aptamer, a ligand, and a substrate.
[10] The kit according to any one of [1] to [9], wherein the substance specifically binding to a protein is bound to a contrast medium.
[11] The kit according to [10], wherein the contrast medium is selected from the group consisting of a fluorescent substance, a luminescent substance, a radioactive substance, a liposome, a micelle, a cell, a virus particle, a virus, a microparticle, a nanoparticle, a microdevice with a space, an emulsion, a lipid disc, a polymer, a gadolinium-conjugated molecule, a superparamagnetic iron oxide particle, a perfluorocarbon nanoparticle, and an ultrafine bubble.
[12] The kit according to [10] or [11], wherein the atherosclerotic lesion is visualized by a means selected from the group consisting of positron emission tomography (PET), single photon emission computed tomography (SPECT), γ scintigraphy, autoradiography, fluorescent imaging, magnetic resonance imaging (MRI), ultrasonication, and computed tomography (CT).
[13] An apparatus for evaluating an atherosclerotic lesion, comprising:
(i) a means of detecting at least one type of protein selected from at least group (a) from among proteins shown in the following groups (a) to (d), in a subject:
(a) cartilage oligomeric matrix protein, fibromodulin, fibronectin, and thrombospondin-4;
(b) plastin-2, tenascin, CD5 antigen-like protein, properdin, Cb4-binding protein, fibrinogen beta chain, and fibrinogen gamma chain;
(c) vitronectin, matrix metalloproteinase-2, and thrombospondin-1; and
(d) membrane-bound form immunoglobulin mu chain C region, receptor-type tyrosine protein phosphatase C, immunoglobulin gamma-2B chain-C region, immunoglobulin J chain, immunoglobulin kappa chain C region, vascularcell adhesion protein 1, alpha-2-macroglobulin, ADAMTS-like protein 4, cathepsin B, carboxy peptidase-B2, complement factor-H, clusterin, collagen alpha-1 (XIV) chain, epidermal fatty acid-binding protein, mannose-binding lectin-associated serine protease-2, prothrombin, complement factor B, inter-alpha-trypsin inhibitor-heavy chain-H1, myosin-9, vitamin K-dependent factor S,metalloproteinase inhibitor 3, andtropomyosin alpha 4; and
(ii) a means of evaluating the atherosclerotic lesion in the subject based on the detection results obtained by means (i).
[14] The apparatus according to [13], further comprising:
(iii) a means of storing the standard levels of the above proteins; and
(iv) a means of comparing the protein detection results in the subject with the standard levels.
[15] An agent for visualizing an atherosclerotic lesion, comprising at least one type of protein selected from at least group (a) from among proteins in the following groups (a) to (d), and a contrast medium binding thereto:
(a) cartilage oligomeric matrix protein, fibromodulin, fibronectin, and thrombospondin-4;
(b) plastin-2, tenascin, CD5 antigen-like protein, properdin, Cb4-binding protein, fibrinogen beta chain, and fibrinogen gamma chain;
(c) vitronectin, matrix metalloproteinase-2, and thrombospondin-1; and
(d) membrane-bound form immunoglobulin mu chain C region, receptor-type tyrosine protein phosphatase C, immunoglobulin gamma-2B chain-C region, immunoglobulin J chain, immunoglobulin kappa chain C region, vascularcell adhesion protein 1, alpha-2-macroglobulin, ADAMTS-like protein 4, cathepsin B, carboxy peptidase-B2, complement factor-H, clusterin, collagen alpha-1 (XIV) chain, epidermal fatty acid-binding protein, mannose-binding lectin-associated serine protease-2, prothrombin, complement factor B, inter-alpha-trypsin inhibitor-heavy chain-H1, myosin-9, vitamin K-dependent factor S,metalloproteinase inhibitor 3, andtropomyosin alpha 4.
[16] An agent for visualizing an atherosclerotic lesion at the intermediate stage or later, comprising: - at least one type of substance specifically binding to at least one type of protein selected from the group consisting of cartilage oligomeric matrix protein, fibromodulin, fibronectin, and thrombospondin-4; and
- a contrast medium binding thereto.
- [17] An agent for visualizing an atherosclerotic lesion at the initial or the intermediate stage, comprising:
- at least one type of substance specifically binding to at least one type of protein selected from the group consisting of plastin-2, tenascin, CD5 antigen-like protein, properdin, Cb4-binding protein, fibrinogen beta chain, and fibrinogen gamma chain; and
- a contrast medium binding thereto.
- [18] An agent for visualizing an atherosclerotic lesion at the late stage, comprising:
- at least one type of substance specifically binding to at least one type of protein selected from the group consisting of vitronectin, matrix metalloproteinase-2, and thrombospondin-1; and
- a contrast medium binding thereto.
- [19] A targeting agent for directing a compound or a molecule to an atherosclerotic lesion, comprising a substance specifically binding to at least one type of protein selected from at least group (a) from among the following groups (a) to (d), and the compound or the molecule binding thereto:
- (a) cartilage oligomeric matrix protein, fibromodulin, fibronectin, and thrombospondin-4;
(b) plastin-2, tenascin, CD5 antigen-like protein, properdin, Cb4-binding protein, fibrinogen beta chain, and fibrinogen gamma chain;
(c) vitronectin, matrix metalloproteinase-2, and thrombospondin-1; and
(d) membrane-bound form immunoglobulin mu chain C region, receptor-type tyrosine protein phosphatase C, immunoglobulin gamma-2B chain-C region, immunoglobulin J chain, immunoglobulin kappa chain C region, vascularcell adhesion protein 1, alpha-2-macroglobulin, ADAMTS-like protein 4, cathepsin B, carboxy peptidase-B2, complement factor-H, clusterin, collagen alpha-1 (XIV) chain, epidermal fatty acid-binding protein, mannose-binding lectin-associated serine protease-2, prothrombin, complement factor B, inter-alpha-trypsin inhibitor-heavy chain-H1, myosin-9, vitamin K-dependent factor S,metalloproteinase inhibitor 3, andtropomyosin alpha 4.
[20] The targeting agent according to [19], wherein the compound or the molecule is a thrombolytic agent.
[21] A method for evaluating the effectiveness of a therapeutic agent or a therapeutic method for an atherosclerotic lesion, comprising the steps of:
(i) detecting at least one type of protein selected from at least group (a) from among proteins shown in the following groups (a) to (d) in an animal with an atherosclerotic lesion treated by a test therapeutic agent or a test therapeutic method:
(a) cartilage oligomeric matrix protein, fibromodulin, fibronectin, and thrombospondin-4;
(b) plastin-2, tenascin, CD5 antigen-like protein, properdin, Cb4-binding protein, fibrinogen beta chain, and fibrinogen gamma chain;
(c) vitronectin, matrix metalloproteinase-2, and thrombospondin-1; and
(d) membrane-bound form immunoglobulin mu chain C region, receptor-type tyrosine protein phosphatase C, immunoglobulin gamma-2B chain-C region, immunoglobulin J chain, immunoglobulin kappa chain C region, vascularcell adhesion protein 1, alpha-2-macroglobulin, ADAMTS-like protein 4, cathepsin B, carboxy peptidase-B2, complement factor-H, clusterin, collagen alpha-1 (XIV) chain, epidermal fatty acid-binding protein, mannose-binding lectin-associated serine protease-2, prothrombin, complement factor B, inter-alpha-trypsin inhibitor-heavy chain-H1, myosin-9, vitamin K-dependent factor S,metalloproteinase inhibitor 3, andtropomyosin alpha 4; and
(ii) evaluating the effectiveness of the test therapeutic agent or the test therapeutic method on the atherosclerotic lesion based on the results of (i).
[22] The method according to [21], further comprising a step of detecting the protein in an animal with an atherosclerotic lesion before treatment using the test therapeutic agent or the test therapeutic method.
[23] The method according to [21] or [22], wherein an animal having an atherosclerotic lesion is an arterial disease animal model.
[24] A method for evaluating an atherosclerotic lesion, comprising the steps of:
(i) detecting at least one type of protein in a subject selected from at least group (a) from among proteins shown in the following groups (a) to (d):
(a) cartilage oligomeric matrix protein, fibromodulin, fibronectin, and thrombospondin-4;
(b) plastin-2, tenascin, CD5 antigen-like protein, properdin, Cb4-binding protein, fibrinogen beta chain, and fibrinogen gamma chain;
(c) vitronectin, matrix metalloproteinase-2, and thrombospondin-1; and
(d) membrane-bound form immunoglobulin mu chain C region, receptor-type tyrosine protein phosphatase C, immunoglobulin gamma-2B chain-C region, immunoglobulin J chain, immunoglobulin kappa chain C region, vascularcell adhesion protein 1, alpha-2-macroglobulin, ADAMTS-like protein 4, cathepsin B, carboxy peptidase-B2, complement factor-H, clusterin, collagen alpha-1 (XIV) chain, epidermal fatty acid-binding protein, mannose-binding lectin-associated serine protease-2, prothrombin, complement factor B, inter-alpha-trypsin inhibitor-heavy chain-H1, myosin-9, vitamin K-dependent factor S,metalloproteinase inhibitor 3, andtropomyosin alpha 4; and
(ii) evaluating the atherosclerotic lesion in the subject based on the results of (i).
[25] The method according to [24], wherein a protein selected from group (a), and at least two types of protein selected from at least one of groups (b) to (d), are detected.
[26] The method according to [24], wherein a protein selected from group (a), and at least three types of protein selected from at least two of groups (b) to (d), are detected.
[27] The method according to [24], wherein a protein selected from group (a), and at least four types of protein selected from each of groups (b) to (d), are detected.
[28] A method for evaluating an atherosclerotic lesion, comprising the steps of:
(i) detecting at least one type of protein selected from the group consisting of cartilage oligomeric matrix protein, fibromodulin, fibronectin, and thrombospondin-4 in a subject; and
(ii) evaluating, based on the results of (i), whether or not the subject has an atherosclerotic lesion at the intermediate stage or later.
[29] A method for evaluating an atherosclerotic lesion, comprising the steps of:
(i) detecting at least one type of protein selected from the group consisting of plastin-2, tenascin, CD5 antigen-like protein, properdin, Cb4-binding protein, fibrinogen beta chain, and fibrinogen gamma chain, in a subject; and
(ii) evaluating whether or not the subject has an atherosclerotic lesion at the initial or intermediate stage based on the results of (i).
[30] A method for evaluating an atherosclerotic lesion, comprising the steps of:
(i) detecting at least one type of protein selected from the group consisting of vitronectin, matrix metalloproteinase-2, and thrombospondin-1 in a subject; and
(ii) evaluating whether or not the subject has an atherosclerotic lesion at the late stage based on the results of (i).
[31] The method according to any one of [24] to [30], wherein step (i) is performed by administering a substance specifically binding to a protein to a subject, and then detecting the substance binding to the protein existing in the subject.
[32] The method according to any one of [24] to [30], wherein the step (i) is performed by preparing a subject-derived sample, bringing the sample into contact with a substance specifically binding to a protein, and then detecting binding between the protein and the substance.
[33] The method according to any one of [24] to [32], wherein evaluation of an atherosclerotic lesion is selected from the group consisting of determination of the presence of an atherosclerotic lesion in a subject, determination of the stage of an atherosclerotic lesion existing in a subject, determination of the location of an atherosclerotic lesion existing in a subject, evaluation of the therapeutic effect on arteriosclerosis existing in a subject, and prediction of prognosis of arteriosclerosis existing in a subject.
[34] The method according to [31] or [32], wherein a substance specifically binding to a protein is selected from the group consisting of an antibody, an antibody-like molecule, a nucleic acid aptamer, a ligand, and a substrate.
[35] The method according to any one of [31], [32], and [34], wherein a substance specifically binding to a protein is bound to a contrast medium.
[36] The method according to [35], wherein the contrast medium is selected from the group consisting of a fluorescent substance, a luminescent substance, a radioactive substance, a liposome, a micelle, a cell, a virus particle, a virus, a microparticle, a nanoparticle, a microdevice with a space, an emulsion, a lipid disc, a polymer, a gadolinium-conjugated molecule, a superparamagnetic iron oxide particle, a perfluorocarbon nanoparticle, and an ultrafine bubble.
[37] The method according to [35] or [36], wherein an atherosclerotic lesion is visualized by a means selected from the group consisting of positron emission tomography (PET), single photon emission computed tomography (SPECT), γ scintigraphy, autoradiography, fluorescent imaging, magnetic resonance imaging (MRI), ultrasonication, and computed tomography (CT).
[38] The method according to any one of [24] to [37], wherein the evaluation step (ii) comprises comparing a measured protein level in a healthy subject with a standard level selected from measured protein levels in subjects with arteriosclerosis at known stages.
[39] The method according to [38], wherein the evaluation step (ii) comprises evaluating whether or not the measured protein level in the subject is higher than the standard level. - The present invention enables detection of arteriosclerotic lesions (destabilization) that have been missed by conventional methods in primary care or medical examination and enables assaying the possibility of the occurrence and progression of the resulting disorder. With the use of the present invention, the ratio of the expression level of a protein group existing in a sample from a subject to the expression level of the same in a standard sample and variations in the ratio over time are measured, and then the results are compared with an expression profile database compiled in advance. This makes it possible to evaluate the possibility of the occurrence of arteriosclerosis and the resulting disorder and the possibility of the progression of the symptoms. Furthermore, the present invention can be used for precisely and conveniently testing or analyzing the possibility of the occurrence of arteriosclerotic lesions and the resulting disorder and the possibility of the progression thereof. The present invention can also be used for developing various reagents or remedies, and related apparatuses.
- Furthermore, variations in the expression of a marker protein group selected in this study are visualized by a known means of imaging with a substance specifically binding to the protein (e.g., an antibody, an antibody-like substance, a ligand, and a substrate) and a contrast medium (e.g., a fluorescent substance and a radioactive substance). The progression stage can then be evaluated based on the aspect of the expression of the protein group in the relevant lesions (amount, localization, density, or relative distribution). Thus, it becomes possible to use the present invention as an indicator of the possibility of the formation of atherosclerotic lesions, the occurrence of the resulting disorders, and the progression of symptoms. Furthermore, the present invention can be used for precisely and conveniently assessing or analyzing the possibility of the occurrence of arteriosclerosis and the resulting disorder, and the possibility of the progression thereof. The present invention can also be used for developing various reagents or remedies, and related apparatuses.
-
FIG. 1 is a graph showing the ratio of the expression level of a protein group in ApoE-deficient mice divided by that in wild-type mice at each disease stage, by whichvariation pattern 1 was exhibited. -
FIG. 2 is a graph showing the ratio of the expression level of a protein group in ApoE-deficient mice divided by that in wild-type mice at each disease stage, by whichvariation pattern 2 was exhibited. -
FIG. 3 is a graph showing the ratio of the expression level of a protein group in ApoE-deficient mice divided by that in wild-type mice at each disease stage, by whichvariation pattern 3 was exhibited. -
FIG. 4 is a graph showing the ratio of the expression level of a protein group in ApoE-deficient mice divided by that in wild-type mice at each disease stage, by whichvariation pattern 4 was exhibited. -
FIG. 5 shows the outline of the apparatus of the present invention for evaluating atherosclerotic lesions. -
FIG. 6 shows images showing the expression of markers (CD5 antigen-like protein and properdin) in atherosclerotic lesions. InFIG. 6 , “*” indicates the intravascular lumen side. -
FIG. 7 shows images showing the expression of markers (cartilage oligomeric matrix protein, fibromodulin, fibronectin, and thrombospondin-4) in atherosclerotic lesions. -
FIG. 8 shows images showing the expression of markers (plastin-2 and tenascin) in atherosclerotic lesions. - The present invention is explained in detail below. This application claims a priority of Japanese patent application No. 2010-257065 filed on Nov. 17, 2010 and the content described in the description and/or drawings of this patent application is incorporated herein by reference.
- The present invention provides novel markers and marker groups for evaluation and visualization of atherosclerotic lesions. The markers and marker groups provided by the present invention are proteins, the expression levels of which characteristically vary with the progression of atherosclerotic lesions. They are useful for prediction of the onset of diseases associated with arteriosclerosis such as stable angina pectoris and acute myocardial infarction, determination of the stages of atherosclerotic lesions, prediction of prognosis of atherosclerotic lesions, visualization of atherosclerotic lesions, and evaluation of the effectiveness of therapeutic agents or therapeutic methods for diseases associated with arteriosclerosis, for example. Therefore, the method for evaluating atherosclerotic lesions according to the present invention comprises the steps of: detecting a marker protein exhibiting the expression pattern (variation in expression) characteristic to a specific disease stage of an atherosclerotic lesion in a subject; and evaluating the atherosclerotic lesion in the subject based on the detection result.
- Subjects to be evaluated according to the present invention are humans and non-human mammals, such as primates (e.g., monkeys and chimpanzees), livestock animals (cattle, horses, pigs, and sheep), pet animals (e.g., dogs and cats), and experimental animals (e.g., mice, rats, and rabbits). Such subjects may also be reptiles and birds.
- In addition, the term “marker” as used herein refers to a protein to be detected for evaluation of arteriosclerosis according to the present invention. Also, the term “marker group” refers to a combination comprising two or more markers.
- Markers to be used in the present invention are as summarized in the following Table 1 to Table 4. In Table 1 to Table 4, the “Entry name” column and the “Accession No.” column are located under the “Mouse” column and the “Human” column, and they show the names and the accession numbers, respectively, of marker proteins of the “Mouse” and the “Human,” as in UniProtKB/Swiss-Prot (http://www.uniprot.org/). Table 1 to Table 4 also show the ratios of the expression levels of marker proteins at different disease stages as detected in examples.
-
TABLE 1 Expression level ratio (ApoE-deficient/wild-type) Mouse Human 12-week-old 18-week-old 25-week-old 35-week-old Accession Accession Initial Early intermediate Intermediate Late Protein name Entry name No. Entry name No. stage stage stage stage Cartilage oligomeric COMP_MOUSE Q9R0G6 COMP_HUMAN P49747 ND 0.855 4.918 13.744 matrix protein Fibromodulin FMOD_MOUSE P50608 FMOD_HUMAN Q06828 ND 1.838 4.668 7.632 Fibronectin FINC_MOUSE P11276 FINC_HUMAN P02751 1.474 1.634 4.358 4.468 Thrombospondin-4 TSP4_MOUSE Q9Z1T2 TSP4_HUMAN P35443 ND 1.174 3.911 3.353 -
TABLE 2 Expression level ratio (ApoE-deficient/wild-type) 12- 35- Mouse Human week-old 18-week-old 25-week-old week-old Accession Accession Initial Early intermediate Intermediate Late Protein name Entry name No. Entry name No. stage stage stage stage Plastin-2 PLSL_MOUSE Q61233 PLSL_HUMAN P13796 ND 3.675 7.454 7.828 Tenascin TENA_MOUSE Q80YX1 TENA_HUMAN P24821 ND 3.754 5.065 7.436 CD5 antigen-like protein CD5L_MOUSE Q9QWK4 CD5L_HUMAN O43866 ND 4.512 7.573 8.895 Properdin PROP_MOUSE P11680 PROP_HUMAN P27918 2.060 4.118 9.759 6.621 C4b-binding protein C4BPA_MOUSE P08607 C4BPA_HUMAN P04003 2.025 3.430 6.884 6.482 Fibrinogen beta chain FIBB_MOUSE Q8K0E8 FIBB_HUMAN P02675 0.678 2.383 6.418 6.834 Fibrinogen gamma chain FIBG_MOUSE Q8VCM7 FIBG_HUMAN P02679 1.116 2.496 8.092 6.745 -
TABLE 3 Expression level ratio (ApoE-deficient/wild-type) Mouse Human 12-week-old 18-week-old 25-week-old 35-week-old Accession Accession Initial Early intermediate Intermediate Late Protein name Entry name No. Entry name No. stage stage stage stage Vitronectin VTNC_MOUSE P29788 VTNC_HUMAN P04004 1.785 1.699 1.789 5.031 Matrix MMP2_MOUSE P33434 MMP2_HUMAN P08253 1.028 2.979 3.633 12.156 metalloproteinase-2 Thrombospondin-1 TSP1_MOUSE P35441 TSP1_MOUSE P35441 0.853 2.684 4.527 11.370 -
TABLE 4 Ratio of expression level (ApoE-deficient/wild-type) 12-week- 18-week-old 25-week- 35-week- Mouse Human old Early old old Accession Accession Initial intermediate Intermediate Late Protein name Entry name No. Entry name No. stage stage stage stage membrane-bound form Ig mu MUCM_MOUSE P01873 IGHM_HUMAN P01871 2.505 3.721 4.857 6.460 chain C region Receptor-type tyrosine-protein PTPRC_MOUSE P06800 PTPRC_HUMAN P08575 0.676 4.633 4.738 4.202 phosphatase C Ig gamma-2B chain C region IGG2B_MOUSE P01867 ND 2.945 4.673 6.901 Immunoglobulin J chain IGJ_MOUSE P01592 IGJ_HUMAN P01591 ND 2.519 3.860 6.182 Ig kappa chain C region IGKC_MOUSE P01837 IGKC_HUMAN P01834 1.570 2.803 3.247 5.915 Vascular cell adhesion VCAM1_MOUSE P29533 VCAM1_HUMAN P19320 1.980 2.968 3.327 4.947 protein 1 Alpha-2-macroglobulin A2M_MOUSE Q61838 A2MG_HUMAN P01023 1.031 1.704 3.150 3.632 ADAMTS-like protein 4 ATL4_MOUSE Q80T21 ATL4_HUMAN Q6UY14 1.430 1.275 2.633 2.853 Cathepsin B CATB_MOUSE P10605 CATB_HUMAN P07858 1.134 1.680 2.365 2.847 Carboxypeptidase B2 CBPB2_MOUSE Q9JHH6 CBPB2_HUMAN Q96IY4 0.755 1.405 2.097 3.163 Complement factor H CFAH_MOUSE P06909 CFAH_HUMAN P08603 1.228 1.800 2.709 4.957 Clusterin CLUS_MOUSE Q06890 CLUS_HUMAN P10909 ND 1.497 2.883 3.743 Collagen alpha-1(XIV) chain COEA1_MOUSE Q80X19 COEA1_HUMAN Q05707 1.110 1.470 2.504 3.057 Epidermal fatty acid-binding FABP5_MOUSE Q05816 FABP5_HUMAN Q01469 1.282 1.837 3.547 3.471 protein Mannan-binding lectin MASP2_MOUSE Q91WP0 MASP2_HUMAN O00187 0.861 1.998 2.846 4.525 associated serine protease 2 Prothrombin THRB_MOUSE P19221 THRB_HUMAN P00734 1.018 1.572 2.852 5.903 Complement factor B CFAB_MOUSE P04186 CFAB_HUMAN P00751 1.057 1.570 1.611 2.479 Inter-alpha-trypsin inhibitor ITIH1_MOUSE Q61702 ITIH1_HUMAN P19827 ND 1.409 1.864 3.347 heavy chain H1 Myosin-9 MYH9_MOUSE Q8VDD5 MYH9_HUMAN P35579 0.876 1.438 1.569 2.374 Vitamin K-dependent PROS_MOUSE Q08761 PROS_HUMAN P07225 ND 1.415 1.940 2.376 protein S Metalloproteinase inhibitor 3 TIMP3_MOUSE P39876 TIMP3_HUMAN P35625 ND 0.941 1.034 2.745 Tropomyosin alpha-4 TPM4_MOUSE Q6IRU2 TPM4_HUMAN P67936 ND 1.329 1.408 2.785 - In addition, “12-week-old,” “18-week-old,” “25-week-old,” or “35-week-old” (ApoE-deficient mouse) used in tables and the Description refer to a specific disease stage corresponding to the relevant age in weeks. An association between the age in weeks of an ApoE-deficient mouse and a disease stage has been reported in Nakashima, Y. et al., Arterioscler Thromb. 14:133-140, 1994; Zhao, Y. et al., J. Nucl. Med. 49:1707-1714, 2008, and the like. Specifically, at the age of 12 weeks, thickening of the intima of arteries and lipid accumulation are observed (stage 1). At the age of 18 weeks, foam cells are formed because of lipid accumulation (stage 2). At the age of 25 weeks, many unstable atheromatous plaques are observed extensively in the arteries (stage 3), corresponding to the most dangerous stage in atherosclerotic arteriosclerosis. At the age of 35 weeks, many stable lesions with advanced fibrillization and/or calcification are observed (stage 4). Therefore, in the Description, “12-week-old” (ApoE-deficient mouse) corresponds to the initial stage (lesion) of arteriosclerosis, “18-week-old” corresponds to the early intermediate stage (lesion) of arteriosclerosis, “25-week-old” corresponds to the intermediate stage (lesion) of arteriosclerosis, and “35-week-old” corresponds to the late stage (lesion) of arteriosclerosis.
- Proteins shown in Table 1 showed no significant difference in amounts until the mice reached the age of 18 weeks, but were characterized in that (i) the ratio of the amounts thereof in ApoE-deficient mice divided by the amounts thereof in wild-type mice (hereinafter, referred to as “the expression level ratio”) at the age of 25 weeks is at least 3, and (ii) the expression level ratio thereof at the age of 25 weeks is at least twice as large as the expression level ratio thereof at the age of 18 weeks. These proteins are also markers showing the risk of plaque rupture at the intermediate stage. With the use of at least one type of these proteins as a marker, whether or not lesions at the age of 18 weeks (early intermediate stage) or later are present, and particularly whether or not there is a risk of plaque rupture can be evaluated based on the expression level of the marker.
- Proteins shown in Table 2 are characterized in that: (i) the ratio of the amounts thereof in ApoE-deficient mice divided by the amounts thereof in wild-type mice at the age of 18 weeks is at least 2; (ii) the expression level ratio thereof at the age of 18 weeks is at least 1.3 times as large as the expression level ratio thereof at the age of 12 weeks; (iii) the expression level ratio at the age of 25 weeks is at least 4; and (iv) the expression level ratio at the age of 25 weeks is at least 1.3 times as large as the expression level ratio at the age of 18 weeks. These proteins are marker proteins showing the progression of plaques from the initial stage to the intermediate stage. With the use of at least one type thereof as a marker, whether or not a lesion at the age of 12 weeks (the initial stage) or a lesion at the age of 18 weeks (the early intermediate stage) or later is present can be evaluated based on the expression level of the marker.
- Proteins shown in Table 3, and particularly: proteins, for which no significant difference was observed in amount (expression level) until the mice reached the age of 12 weeks, but the amounts thereof in ApoE-deficient mice increase to twofold or more than the amounts thereof in wild-type mice at the age of 18 weeks and older; and proteins, for which no significant difference is observed in amount (expression level) until the mice reached the age of 25 weeks, but the amounts thereof in ApoE-deficient mice increase to two fold or more than the amounts thereof in wild-type mice at the age of 35 weeks is at least, are characterized in that: (i) the expression level ratio at the age of 35 weeks is at least 4; and (ii) the expression level ratio at the age of 35 weeks is at least twice as large as the expression level ratio at the age of 25 weeks. These proteins were marker proteins indicating the stabilization of plaques at the late stage. With the use of at least one type of these proteins as a marker protein, the presence or the absence of a lesion at the age of 35 weeks (the late stage) or later can be evaluated based on the expression level of the marker.
- Proteins shown in Table 4 are marker proteins, the expression levels of which in ApoE-deficient mice are varied depending on ages in weeks compared with those in wild-type mice. Of these proteins, at least one type thereof can be used as a marker, or this can be used in combination with markers shown in Table 1 to Table 3. Thus, the presence or the absence of a lesion at each age in week (at the initial stage, the early intermediate stage, the intermediate stage, or the late stage) can be evaluated based on the expression level(s) of the marker(s).
- In the present invention, atherosclerotic lesions can be evaluated by detecting at least one type of markers shown in at least Table 1. Specifically, the presence or the absence of an atherosclerotic lesion with a risk of plaque rupture can be evaluated using each marker in Table 1. Furthermore, according to the present invention, at least two markers are used in combination, making it possible to more precise and highly accurate evaluation of atherosclerotic lesions. For example, at least one type of protein shown in Table 1 (cartilage oligomeric matrix protein, fibromodulin, fibronectin, or thrombospondin-4) and, among each group shown in Table 2 to Table 4, at least two types of protein selected from at least one, at least two, or at least three groups are combined.
- For example, marker(s) shown in Table 1 and marker(s) shown in Table 2 are used in combination. Thus, differentiation becomes possible, such as whether the lesion is at the initial stage (12-week-old), the early intermediate stage (18-week-old), the intermediate stage (25-week-old), or later, based on the expression level(s) of the marker(s). Furthermore, a marker(s) shown in Table 1 and a marker(s) shown in Table 3 are used in combination. Thus, differentiation becomes possible such that if the lesion is at the intermediate stage (25-week-old), the late stage or later (35-week-old), based on the expression levels of these markers. Furthermore, a marker(s) shown in Table 1 and a marker(s) shown in Table 4 are used in combination. Thus, differentiation becomes possible such that if the lesion is at the initial stage (12-week-old), the early intermediate stage (18-week-old), the intermediate stage (25-week-old) or the late stage (35-week-old), based on the expression levels of these markers.
- Alternatively, three or more markers shown in Table 1 to Table 4 can be used in combination. For example, at least one marker each was selected from each one of Table 1 to Table 4 and then the markers are used in combination. Thus, detailed and precise differentiation becomes possible, such that if a lesion is at the initial stage (12-week-old), the early intermediate stage (18-week-old), the intermediate stage (25-week-old), or the late stage (35-week-old) based on the expression levels of these markers. Preferably, three or more markers, and more preferably, three to seven markers are combined. Such a combination can be adequately selected in accordance with subject type, sex, age, purpose of evaluation of arteriosclerosis, disease stage to be subjected to evaluation, and the like.
- As described above, the above markers or marker groups are expressed in patterns characteristic to specific disease stages of atherosclerotic lesions. Therefore, in the method for evaluating an atherosclerotic lesion according to the present invention, the above markers or marker groups in a subject are detected. When two or more markers are detected, the steps of detecting each marker can be performed simultaneously or at different times. Also, in the present invention, the term “detection of marker (group)” may refer to the detection of a marker protein, a substance derived therefrom, or a derivative thereof, or a gene (mRNA) encoding the protein. The term “substance derived therefrom” and “a derivative thereof” refer to a substance derived or originated from a marker protein. Examples thereof include, but are not limited to, a protein containing a signal peptide, a specific subunit molecule of a protein, a modified protein, and a protein fragment. For example, fibrinogen shown in Table 2 contains β chain and γ chain, and an intact molecule of fibrinogen may be detected, or any one of β chain or γ chain that is a substance derived therefrom may be detected in the present invention.
- Detection of a marker protein preferably relates to semiquantitative or quantitative measurement of the amount or the concentration of the marker protein in a subject. The amount may be the absolute quantity or the relative quantity. Detection can be performed directly or indirectly. Direct detection relates to measurement of the amount or the concentration of a marker protein or mRNA existing in a subject based on a signal directly correlating with the number of molecules of the marker protein or mRNA. Such a signal is based on specific physical or chemical properties of a protein or mRNA, for example. Indirect measurement is measurement of signals obtained from secondary components (that is, components other than marker proteins or mRNA) such as ligands (e.g., antibodies), labels, or enzymatic reaction products.
- The step of detecting a marker protein can be performed by administering to a subject a substance specifically binding to a marker protein, and then detecting the substance bound to the marker protein existing in the subject, for example. Alternatively, the step of detecting a marker protein can be performed by preparing a subject-derived sample, bringing the sample into contact with a substance specifically binding to a marker protein, and then detecting the binding of the marker protein with the substance.
- The term “substance specifically binding to a marker protein” refers to a substance that binds to a marker protein, a substance derived therefrom, or a derivative thereof with high affinity, but binds (with only low affinity) or never bind to molecules unrelated to a marker protein, a substance derived therefrom, or a derivative thereof. Examples of such a substance include antibodies, antibody-like molecules, nucleic acid aptamers, substrates and other ligands. Preferably, a substance specifically binding to a marker protein is an antibody or an antibody fragment. For example, an antibody against each marker protein may be either a monoclonal antibody or a polyclonal antibody, or may be particularly Fab, Fab′, F(ab′)2, scFv, Fv fragment, or the like that can bind to an epitope of each marker protein. An antibody and an antibody-like molecule can be prepared by methods known in the art or can be obtained as a commercially available product. An antibody-like molecule is a protein fragment containing a peptide equivalent to an antibody variable region capable of binding to a marker protein, which is prepared by a non-immunological technique such as a phage display method or a combinatorial chemistry method. An example thereof is an affibody. An antibody-like molecule is effective in a case where preparation of an antibody with a general immunization method is difficult because of high toxicity. A nucleic acid aptamer is known in the art and is a nucleic acid molecule capable of specifically binding to a specific protein (specifically, a marker protein). Such a nucleic acid aptamer can be chemically synthesized in vitro and is advantageous in that it has low immunogenicity. A nucleic acid aptamer can be designed by a SELEX method or the like. Persons skilled in the art can design and synthesize an appropriate nucleic acid aptamer. When a marker protein has enzyme activity, the substrate or the substrate derivative thereof can be used as a substance capable of specifically binding to the enzyme. For example, when a marker protein is protease, a protease inhibitor (may be the one derived from nature or a synthetic molecule) can be used. When a marker protein is a receptor, a ligand thereof (an agonist, an antagonist, and a derivative thereof) can be used as a substance specifically binding to the receptor. In particular, in the case of a receptor localized in cell membrane, a ligand-binding site is located extracellularly, and thus the receptor is advantageous in that a ligand to be administered to a subject can easily bind from outside the cell to the receptor.
- When a marker protein is detected by administration of a substance specifically binding to the marker protein to a subject, the substance specifically binding to the marker protein binds to the marker protein in vivo in the subject when administered to the subject. Therefore, with the use of such a substance, localization of a marker protein in a subject can be examined and the location and disease stage of an atherosclerotic lesion(s) can be evaluated, for example. Moreover, such a substance is bound to a contrast medium in advance, making it possible to visualize the atherosclerotic lesions in the subject.
- A marker protein can be conveniently and noninvasively detected using such an agent for visualization. Therefore, the present invention also provides an agent for visualizing an atherosclerotic lesion. The agent for visualization contains a substance specifically binding to at least one type of marker protein and a contrast medium. As a contrast medium, an arbitrary contrast medium known in the in vivo imaging field can be used. Examples of such a contrast medium include: fluorescent substances such as fluorescein, FITC, and fluorescence emitting metals (e.g., 152Eu, and lanthanum series); chemical or biological luminescent substances such as luminol, imidazole, luciferin, luciferase, and green fluorescent protein (GFP); radioactive substances such as 99mTc, 123I, 131I, 97Ru, 67Cu, 11C, and 13N; paramagnetic isotopes such as 153Gd, 157Gd, 55Mn, 162Dy, 52Cr, 56Fe, and superparamagnetic iron oxide particles; and other contrast media such as gadolinium, gadolinium complexes, iodo contrast media, liposomes, micelles, cells, virus particles, viruses, microparticles, nanoparticles, microdevices with spaces, emulsions, lipid discs, polymers, perfluorocarbon nanoparticles, and ultrafine bubbles. A substance specifically binding to a marker protein can be bound to a contrast medium by a method known in the art. For example, such a substance and a medium can be directly chemically bound or indirectly bound via an appropriate linker.
- A medicine to be used in the present invention containing the above agent for visualizing an atherosclerotic lesion may contain, in addition to a substance specifically binding to a marker protein, a pharmaceutically acceptable carrier or additive. Examples of such a carrier or an additive include water, a pharmaceutically acceptable organic solvent, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, xanthan gum, gum Arabic, casein, gelatin, agar, glycerin, propylene glycol, polyethylene glycol, Vaseline, paraffin, stearyl alcohol, stearic acid, mannitol, sorbitol, and lactose. An additive to be used herein can be adequately selected or selected in combination depending on dosage forms.
- The method for administration of the medicine of the present invention is not particularly limited. The medicine can be administered via peroral administration or parenteral administration such as subcutaneous administration, intradermal administration, intramuscular administration, intravenous administration, transdermal administration, rectal administration, and intranasal administration.
- When the medicine of the present invention is administered via peroral administration, it may be in the form of tablets, capsules (e.g., hard capsules, soft capsules, and microcapsules), granules, powders, pills, troches, pharmaceutical solution as internal medicine, liquid agents, suspensions, emulsions, syrups, or the like. The medicine may also be a dry product that is dissolved again when it is used. Also, when the medicine of the present invention is administered via parenteral administration, examples of dosage forms that can be selected herein include injection preparations (e.g., solutions, emulsions, and suspensions) for intravenous injection (including drip infusion), intramuscular injection, intraperitoneal injection, and subcutaneous injection, and external preparations such as ointments (particularly, an ointment for eyes), cream pharmaceuticals, suppositories, adhesive skin patches, eye drops, nasal drops, inhalers, liniments, and aerosol. In the case of injection preparations, they are provided in unit dosage ampules or vessels for high dose administration.
- These various preparations can be produced by conventional methods by appropriately selecting an excipient, an extending agent, a binder, a wetting agent, a disintegrator, a lubricant, a surfactant, a dispersing agent, a buffering agent, a pH adjuster, a preservative, a solubilizing agent, an antiseptic, a taste and flavor corrigent, an absorption accelerator, a soothing agent, a stabilizer, a tonicity agent, and the like that are generally used in medicaments.
- The amount of a substance specifically binding to a marker protein that is to be incorporated into the medicine of the present invention, differs depending on the type of substance, the type of contrast medium to be bound with the substance, the purpose of use thereof, dosage form, the route of administration, and the like. For example, the amount ranges from 1% to 99% of the total weight, and preferably ranges from 5% to 90% of the total weight.
- Also, the dose and the dosing interval of the medicine of the present invention differ depending on the type of a substance specifically binding to a marker protein, the type of a contrast medium to be bound with the substance, a subject of administration, the age and the body weight of a subject, the route of administration, and the frequency of administration. The dose and the dosing interval thereof can be varied widely.
- In the case of an agent for visualizing an atherosclerotic lesion, after administration of the medicine, the presence or the location of a substance specifically binding to a marker protein in a subject is visualized using a contrast medium as an indicator. Preferably, the presence or the location of a substance specifically binding to a marker protein is visualized by a known imaging technique. Such an imaging technique differs depending on a label to be used herein, the type of a subject, a site for imaging, and the like. Positron emission tomography (PET), single photon emission computed tomography (SPECT), γ scintigraphy, autoradiography, fluorescent imaging, magnetic resonance imaging (MRI), ultrasonication, computed tomography (CT), and other in vivo imaging systems can be used. Thus, based on such a contrast medium to which a substance specifically binding to a marker protein has been bound, the presence or the location of an atherosclerotic lesion in the subject can be visualized and determined.
- In addition to the above-visualized presence or the location of a marker protein, determination of the disease stage of an atherosclerotic lesion, prediction of the prognosis of an atherosclerotic lesion, and evaluation of the therapeutic effect against arteriosclerosis in a subject, and the like can also be evaluated based on the type and the amount of the marker protein.
- Moreover, visualization of an atherosclerotic lesion as described above makes it possible to conveniently and precisely specify a lesion site upon surgery.
- Another embodiment that involves preparing a subject-derived sample and detecting a marker protein is explained as follows. Samples to be used herein are not particularly limited, as long as they are samples derived from subjects to be evaluated for atherosclerotic lesions. Samples are adequately selected in accordance with methods or types of means of detecting markers. Examples thereof include tissue or cell samples (e.g., arterial tissues and cells). Also, a tissue or cell sample is preferably frozen and cryopreserved immediately after collection by a method known or generally used in the art using liquid nitrogen, dry ice, or the like.
- In an embodiment of the present invention, a marker protein can be detected by a means of measuring the amount of a protein in a sample. Such means are known in the art, such as immunoassay methods and reagents. Also, a marker protein can be detected by a means of measuring physical or chemical properties peculiar to the marker protein, such as a means of measuring accurate molecular weights, rNMR spectra, or the like. Examples of a means of detecting a marker protein include analyzers such as a biosensor, a protein chip, an optical unit combined with immunoassay, a mass spectrometer, an NMR analyzer, a 2D electrophoresis apparatus, and a chromatography apparatus.
- In a preferred embodiment, a marker protein can be detected using mass spectrometry (MS). In particular, analysis made by a mass spectrometer combined with liquid chromatography (LC/MS) is sensitive and thus is advantageous. As labels, isotope labeling reagents known in the art can be used. Appropriate labeling reagents can be commercially obtained. Also, fractionation can be performed by a method known in the art. For example, fractionation can be performed using a commercially available strong cationic column or the like.
- In another embodiment, a marker protein in a sample can be detected by immunoassay (immunological assay). Specifically, the protein in a sample is detected based on a reaction between the marker protein in the sample and an antibody specifically binding to the protein. Immunoassay may be performed with either a liquid phase system or a solid phase system, as long as the method is generally employed in the art. In view of ease of detection, a solid phase system is preferably used. The forms of immunoassay are not limited and may be, in addition to a direct solid phase method, a sandwich method, a competition assay, a Western blotting method, an ELISA (enzyme linked immunosorbent assay) method, or the like.
- An antibody (to be used in the method of the present invention) against each marker protein may be either a monoclonal antibody or a polyclonal antibody, or may be Fab, Fab′, F(ab′)2, scFv, Fv fragment, or the like capable of binding to the epitope of each marker protein. When a primary antibody and a secondary antibody are used, both antibodies may be monoclonal antibodies. Alternatively, either a primary antibody or a secondary antibody may be a polyclonal antibody. Such an antibody can be prepared by a method known in the art or can be commercially obtained.
- Binding of a marker protein with an antibody can be measured according to a known method. Persons skilled in the art can determine an effective and optimum measurement method for each assay according to the type and form of immunoassay to be employed, or the type of a label to be used herein, for example. For example, for easy detection of the binding of a marker protein in a sample with an antibody, the binding is directly detected by labeling the antibody, or the binding is indirectly detected using a labeled secondary antibody, a biotin-avidin conjugate, or the like.
- With a solid phase system, an antibody or a protein component in a sample is immobilized onto a solid phase (e.g., a plate, a membrane, or beads), and then immunological binding of a marker protein with a solid is tested on the solid phase. Such a solid phase is not particularly limited, as long as it is conventionally used in the art. For example, a commercially available nitrocellulose membrane or PVDF membrane can be used. An antibody or a protein component in a sample is immobilized on a solid phase, so that unbound components in the sample or a reagent can be easily removed. Also, in particular, in the case of a protein array method using a membrane with dozens of types of antibody immobilized thereto, the presence of many types of marker protein can be analyzed within a short time using a small amount of a subject-derived sample (e.g., plasma). Such immunoassay can also be performed with test strips, the operation with which can be conveniently performed.
- When liquid-phase immunoassay is selected, for example, a labeled antibody is brought into contact with a sample so as to bind the labeled antibody to a marker protein, the conjugate is separated, and thus the signal of the label is detected. Alternatively, an antibody (primary antibody) against a marker protein is brought into contact with a sample so as to bind the primary antibody to the marker protein, a labeled antibody (secondary antibody) is bound to the conjugate, and thus the signal of the label is detected from the conjugate of these three substances. Alternatively, for further enhancement of signals, an unlabeled secondary antibody is first bound to an antibody+marker protein conjugate, so as to bind a labeling substance to the secondary antibody. Such binding of a labeling substance to a secondary antibody can be performed by biotinylating the secondary antibody and avidinylating the labeling substance in advance, for example.
- A marker protein can also be detected in situ, as in the case of using an immunohistochemical staining method (e.g., an immunostaining method) or immunoelectron microscopy. In situ detection can be performed by excising a histological sample from a subject (e.g., a biopsy sample and a tissue paraffin embedded section), and then bringing a labeled antibody or antibody fragment into contact therewith.
- As labels for labeling antibodies to be used in immunoassay, enzymes, radio isotopes, fluorescent dyes, or avidin-biotin systems can be used. As enzymes, enzymes that are used for general enzyme immunoassay (EIA) can be used, such as peroxidase, β-galactosidase, and alkaline phosphatase. Also, an enzyme inhibition substance, a coenzyme, and the like can be used. Binding of these enzymes with antibodies can be performed by known methods using cross-linking agents such as a maleimide compound. As radio isotopes, 125I, 3H, and the like, which are used for general radio immunoassay (RIA), can be used. As fluorescent dyes, fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), and the like, which are used for general fluorescent antibody techniques, can be used.
- The signal of a label can also be detected according to a method known in the art. For example, when an enzyme label is used, a substrate that is degraded by enzymatic action to develop color is added, the amount of the substrate degraded is optically measured to find enzyme activity, the resulting value is converted to the amount of the antibody bound, the result is compared with a standard value, and thus the amount of the antibody is calculated. A substrate differs depending on the type of enzyme to be used herein. For example, when peroxidase is used as an enzyme, 3,3′,5,5′-tetramethylbenzidine can be used as a substrate, and when alkaline phosphatase is used as an enzyme, para-nitrophenol or the like can be used as a substrate. When a radioactive label is used, the radiation dose from the radioactive label is measured by a scintillation counter or the like. A fluorescent label can be detected or quantitatively determined using a fluorescent microscope, a plate reader, a fluorescent imaging apparatus, or the like.
- As described above, a marker protein in a subject-derived sample is detected, and then arteriosclerotic lesion(s) in the subject can be evaluated based on the results.
- The term “evaluation of atherosclerotic lesions” as used herein refers to determination of the presence of atherosclerotic lesions in a subject, determination of the stage of atherosclerotic lesions existing in a subject, determination of the location of atherosclerotic lesions existing in a subject, evaluation of therapeutic effects on arteriosclerosis existing in a subject, and prediction of the prognosis of arteriosclerosis existing in a subject. Also, the term “evaluation” in the present invention is meant to include continuous monitoring of arteriosclerosis that has already been evaluated or diagnosed and confirmation of the past evaluation or diagnosis of arteriosclerosis.
- Moreover, through evaluation of atherosclerotic lesions, the risk of developing diseases associated with arteriosclerosis, such as angina pectoris (e.g., stable angina pectoris), acute myocardial infarction, brain infarction, encephalorrhagy, cerebral thrombosis, cerebral embolism, aortic aneurysm, obstructive arteriosclerosis, and acute coronary syndromes can also be evaluated.
- Persons skilled in the art understand that accurate results cannot be always obtained for all (that is, 100% of) subjects to be evaluated by the present invention (evaluation is an object of the present invention). In the present invention, the “evaluation” is intended to allow evaluation of subjects accounting for a statistically significant proportion of all subjects. Such a statistically significant proportion can be determined using various known statistical evaluation tools such as determination of a confidence interval, determination of p value, student's t-test, Mann-Whitney test, and the like. A preferable confidence interval is at least 90%. The p value is preferably 0.1, 0.01, 0.005, or 0.0001. More preferably, at least 60%, at least 80%, or at least 90% of subjects can be appropriately evaluated by the present invention.
- When evaluation is performed, the detection result of a marker protein in a subject is compared with a standard level. Such a standard level is: the measured level of a marker protein in a healthy-subject-derived sample, the measured level of a marker protein in a sample from a subject diagnosed as having arteriosclerosis at a specific stage, the measured level of a marker protein in a sample from a subject before treatment, or the like. A standard level for an individual subject may be varied depending on various physiological parameters such as subject type, age, and sex.
- Preferably, individual subjects with arteriosclerosis are classified according to the degree of the progression of the disease (stage), and the expression levels of marker proteins are obtained from individual subjects belonging to each stage and individual subjects in a normal state. Then, correlations between variations in the expression of a marker protein and progression stages are recorded into an expression profile database. The ratio of the expression level of a marker protein in a subject-derived sample divided by the expression level thereof in a standard sample (prepared in advance) is measured. The expression profile database can be referred for the thus obtained measured value. Such an expression profile database is useful as “standard levels” or “standard range” that can be indicators of the presence or the absence of arteriosclerosis in a subject, the progression stage, and the onset or the degree of progression of arteriosclerosis. Such a standard level may be the only one cutoff value (e.g., average value or median value), or standard levels may be within a specific range (e.g., quartile). When evaluation is performed using a database, for example, the ratio of the expression level R(i) of a protein in a subject-derived sample to the same in a standard sample is measured. One, two, or more marker proteins (i=1−N) thereof are subjected to measurement. Each of these measured values is compared with the value at each progression stage (Rj(i), wherein “j” denotes stage No. ranging from 0-4) and then the results are recorded in the database, so that the progression stage of arteriosclerosis in the subject can be determined. Comparison can be performed by finding stage “j” at which the result of Σ{R(i)−Rj(i)}2(i=1−N) is minimum, for example.
- In addition, when evaluation is performed using a human as a subject, a standard level is also preferably the measured expression level of a marker protein in a human. A standard level obtained with the use of experimental animals such as mice can also be extrapolated.
- Specific examples of evaluation of arteriosclerosis are as follows. The level of a marker protein shown in Table 1 in a subject, which is higher than a standard level, indicates the presence of atherosclerotic lesions at the intermediate stage or later in the subject. Also, the level of a marker protein shown in Table 2 in a subject, which is higher than a standard level, indicates the presence of atherosclerotic lesions at the initial or early intermediate stage or later in the subject. The level of a marker protein shown in Table 3 in a subject, which is higher than a standard level, indicates the presence of atherosclerotic lesions at the late stage or later in a subject. Marker proteins shown in Table 4 in a subject are combined with other marker proteins, and then variations in the expression levels thereof are examined, indicating the presence of atherosclerotic lesions in the subject.
- Furthermore, the method for evaluating atherosclerotic lesions according to the present invention may be performed in combination with other known diagnosis methods for arteriosclerosis. Examples of such known diagnosis methods for arteriosclerosis include measurement of physiological and biochemical markers of arteriosclerosis (e.g., high blood pressure, cholesterol level, and triglyceride level), measurement with electrocardiogram, measurement of arterial pulse wave velocity (PWV), and angiography.
- Furthermore, the method for evaluating atherosclerotic lesions of the present invention can also be combined with other arteriosclerosis markers. For example, the present inventors have revealed that: an increase in the expression level of von Willebrand factor and/or a decrease in the expression level of complement factor D in blood plasma can be an indicator for arteriosclerosis at the early intermediate stage; and an increase in the expression level of complement component C8 and/or a an increase in the expression level of vitamin K-dependent protein Z in blood plasma can be an indicator for arteriosclerosis at the intermediate stage (WO 2011/136080). Moreover, as arteriosclerosis markers in blood plasma, a CD5 antigen-like protein and an immunoglobulin mu chain C region exhibited maximum expression levels at the early intermediate stage, and a blood plasma protease C1 inhibitor, thrombospondin-4, fetuin-B, and complement factor H exhibited minimum expression levels at the early intermediate stage or the intermediate stage.
Heparin cofactor 2, alpha-2-macroglobulin, complement component C9, vitamin K-dependent protein C, antithrombin-III, EGF-containing fibrin-likeextracellular matrix protein 1, inter alpha trypsin inhibitor heavy chain H1, inter alpha trypsin inhibitor heavy chain H2, and properdin exhibited decreased expression levels with the progression of the disease stage. Complement C1q subcomponent subunit B exhibited the expression level that increased with the progression of the disease stage. Therefore, the above blood plasma arteriosclerosis markers are used in combination with the evaluation of atherosclerotic lesions of the present invention, so that evaluation can be performed with higher accuracy. - The presence of atherosclerotic (unstable) lesions can be determined at an early stage by the method for evaluating atherosclerotic lesions according to the present invention. Specifically, the present invention enables determination of the presence or the absence of unstable atherosclerotic lesions, which cannot be recognized by currently available diagnostic techniques or criteria. Also, when atherosclerotic lesions are present, the disease stage and the location of each lesion can be specified in detail. Accordingly, the subject can timely receive treatment (for diseases associated with arteriosclerosis such as angina pectoris, myocardial infarction, and brain infarction) suitable for the disease stage thereof precisely. The method of the present invention is also advantageous in that arteriosclerosis can be reliably and rapidly evaluated by techniques with low invasiveness.
- The method for evaluating atherosclerotic lesions according to the present invention can be easily and conveniently performed using a kit and/or an apparatus provided with a means of detecting the above markers.
- The kit for evaluating atherosclerotic lesions according to the present invention contains at least one type of substance specifically binding to at least one type of protein selected from at least group (a) from among proteins shown in the following groups (a) to (d):
- (a) cartilage oligomeric matrix protein, fibromodulin, fibronectin, and thrombospondin-4;
(b) plastin-2, tenascin, CD5 antigen-like protein, properdin, Cb4-binding protein, fibrinogen beta chain, and fibrinogen gamma chain;
(c) vitronectin, matrix metalloproteinase-2, and thrombospondin-1; and
(d) membrane-bound form immunoglobulin mu chain C region, receptor-type tyrosine protein phosphatase C, immunoglobulin gamma-2B chain-C region, immunoglobulin J chain, immunoglobulin kappa chain C region, vascularcell adhesion protein 1, alpha-2-macroglobulin, ADAMTS-like protein 4, cathepsin B, carboxy peptidase-B2, complement factor-H, clusterin, collagen alpha-1 (XIV) chain, epidermal fatty acid-binding protein, mannose-binding lectin-associated serine protease-2, prothrombin, complement factor B, inter-alpha-trypsin inhibitor-heavy chain-H1, myosin-9, vitamin K-dependent factor S,metalloproteinase inhibitor 3, andtropomyosin alpha 4. - An example of the kit of the present invention is composed of antibodies specifically binding to marker proteins, contrast media or labels, buffers for dilution and washing, and if necessary labeled secondary antibodies, substrate reagents for causing color development, luminescence and fluorescence generation, manuals describing procedures and evaluation methods, and the like. When a marker protein in a subject is detected in vivo, the kit may further contain a means of administration, such as vials, ampules, and syringes. When a marker protein in a subject-derived sample is detected, an antibody immobilized on a solid-phase support (e.g., a membrane or beads) may be used.
- The kit of the present invention may contain instructions in which procedures and protocols for implementing the method of the present invention are described and tables showing standard levels or reference ranges to be employed for evaluation of arteriosclerotic lesions, for example.
- Components contained in the kit of the present invention may be individually provided or provided within a single container. Preferably, the kit of the present invention contains all the components required for implementation of the method of the present invention, as components with adjusted concentrations, for example, so that they can be used immediately.
- The apparatus for evaluating atherosclerotic lesions according to the present invention comprises the following means of:
- (i) detecting at least one type of protein selected from at least group (a) from among proteins shown in the above groups (a) to (d), in a subject; and
(ii) evaluating an atherosclerotic lesion in the subject based on the detection result obtained by means (i). - The apparatus of present invention may further comprise the following means of:
- (iii) storing the standard levels of the above proteins; and
(iv) comparing the detection results of proteins in a subject with the standard levels. - The apparatus of the present invention is a system, wherein the means (i) and (ii) are combined with further preferably means (iii) and (iv) so that the means can be implemented in a mutual manner, so as to be able to perform the method of the present invention.
FIG. 5 shows the outline of the apparatus of the present invention. - The means (i) can be a
protein detection part 101 provided with aquantification part 104 for detecting and quantifying signals following the operation of a protein specific signal detection part 102 (e.g., mass spectroscopy part), or aquantification part 104 or aimaging part 103 for detecting, quantifying or imaging signals from radioactivity, color development, luminescence, or fluorescence, for example. Also, the means (ii) can be apart 301 for evaluation of atherosclerotic lesions, which is provided with an image.numericdata storage part 302 for storing the detection results (images·numeric data) obtained from thedetection part 101, for example, asoftware 307 for processing the detection results, and acomputer part 308. A preferable apparatus is an apparatus that can be used without the knowledge of an experienced clinician. A specific apparatus comprises the detection part 101 (provided with a protein-specificsignal detection part 102 such as PET, SPECT, γ scintigraphy, autoradiography, fluorescent imaging, MRI, ultrasonication, CT, a biosensor, a solid-phase support to which an antibody against a marker has been immobilized, mass spectroscopy, a surface plasmon resonance sensor, and an NMR analyzer), and an evaluation part 301 (e.g., an image.numericdata storage part 302, standard leveldata storage part 303, adata comparison part 304 and adata analysis part 305 for analysis of data, dataoutput.display part 306, asoftware 307 and a computer unit for activating a computer part 308). - Furthermore, the means (iii) can be a protein
standard level database 201, which is provided withexpression data 202 containing pathological (arteriosclerosis) stage-specific expression data and may also be provided as necessary with at least one type of data selected from amongarterial tissue data 203,blood plasma data 204, and data of differences in males andfemales 205. Necessary data are loaded from thestandard level database 201 to the standard leveldata storage part 303 of the means (ii), and then the standard level data and the data of image.numericdata storage part 302 are compared and analyzed. - Further in the present invention, a compound or a molecule such as a drug or a prodrug is bound to a substance specifically binding to a marker protein, instead of a contrast medium, and thus the compound or the molecule can be delivered to a site of a subject where the marker protein is present; that is, an atherosclerotic lesion. Examples of such a drug or prodrug include known thrombolytic agents (e.g., urokinase, streptokinase, and a tissue plasminogen activator). Such a targeting agent for delivery of such a drug or a prodrug to an atherosclerotic lesion is also encompassed within the present invention. Such a targeting agent has the effects of: binding to a marker protein existing in an atherosclerotic lesion so as to be able to deliver a compound or a molecule (e.g., a drug or a prodrug) to an atherosclerotic lesion, remaining for a long time within the atherosclerotic lesion, and thus allowing the drug effects to be continuously exhibited for a long time. Therefore, the targeting agent of the present invention can be used as an agent for prevention or treatment of arteriosclerosis. Specifically, the effective dose of the targeting agent of the present invention is administered to a subject, so that arteriosclerosis in the subject can be prevented or treated.
- A subject to which the targeting agent of the present invention is administered is not particularly limited. Examples thereof include mammals such as humans, domestic animals (e.g., cattle and pigs), pet animals (e.g., dogs and cats), and experimental animals (e.g., mice, rats, and monkeys). In particular, the target agent is preferably used for a subject suspected of having arteriosclerosis and a subject having arteriosclerosis.
- Examples of arteriosclerosis to be treated or prevented in the present invention include, but are not limited to, arteriosclerosis and diseases associated with arteriosclerosis such as angina pectoris (e.g., stable angina pectoris), acute myocardial infarction, brain infarction, encephalorrhagy, cerebral thrombosis, cerebral embolism, aortic aneurysm, obstructive arteriosclerosis, and acute coronary syndromes.
- The targeting agent of the present invention can be prepared and administered in a manner as described above in the case of an agent for visualization. Persons skilled in the art can adequately select methods in view of a compound or a molecule (e.g., a drug or a prodrug) to be contained in the targeting agent.
- Furthermore, the effectiveness of therapeutic agents or therapeutic methods for arteriosclerosis can be evaluated and test therapeutic agents for arteriosclerosis can be screened for using the above markers or marker groups. Specifically, such a method of the present invention comprises the steps of:
- (i) detecting a marker protein in an animal having arteriosclerosis, which has been treated with a test therapeutic agent or by a test therapeutic method; and
(ii) evaluating the effectiveness of the test therapeutic agent or therapeutic method against arteriosclerosis based on the result of (i). - According to the method of the present invention, a marker protein is detected in an animal with arteriosclerosis, that is, an animal developing arteriosclerosis or an animal at a risk of arteriosclerosis. Preferably, before treatment with a test therapeutic agent or by a test therapeutic method, a marker protein is detected in an animal with arteriosclerosis, and then the disease stage of the atherosclerotic lesion is confirmed before treatment. After treatment of an animal with arteriosclerosis using a test therapeutic agent or by a test therapeutic method, a marker protein is detected in the animal at an appropriate time. For example, detection is performed immediately after treatment, 30 minutes after, 1 hour after, 3 hours after, 5 hours after, 10 hours after, 15 hours after, 20 hours after, 24 hours (1 day) after, 2 to 10 days after, 10 to 20 days after, 20 to 30 days after, or 1 to 6 months after treatment. Detection of a marker protein(s) and evaluation of atherosclerotic lesions can be performed in a manner similar to the above.
- Subject animals in the present invention may be humans with arteriosclerotic lesions, arterial disease model animals, or preferably arteriosclerosis model experimental animals (e.g., mice, rats, and rabbits), for example. In general, after confirmation of the effectiveness of a test therapeutic agent or a test therapeutic method in a model animal, effectiveness can be evaluated in humans by clinical trials, for example.
- Types of test therapeutic agent or test therapeutic method for evaluation or screening are not particularly limited. Examples of test therapeutic agents or test therapeutic methods include arbitrary material factors, specifically, naturally occurring molecules such as amino acid, peptide, oligo peptide, polypeptide, protein, nucleic acid, lipid, carbohydrate (e.g., sugar), steroid, glycopeptide, glycoprotein, and proteoglycan; synthetic analogs or derivatives of naturally occurring molecules such as peptide mimic or nucleic acid molecule (e.g., aptamer, antisense nucleic acid, and double-stranded RNA (RNAi)); molecules that do not occur naturally such as low-molecular-weight organic compound (e.g., inorganic and organic compound libraries, or combinatorial library); and mixtures thereof. Furthermore, a test therapeutic agent or a test therapeutic method may be a single substance, a complex composed of a plurality of substances, a food, a feed, or the like. Moreover, test therapeutic agents or test therapeutic methods may be, in addition to the above material factors, radiation, ultraviolet radiation, and the like.
- Treatment of an animal using a test therapeutic agent or a test therapeutic method differs depending on the type of therapeutic agent or therapeutic method, and can be easily determined by persons skilled in the art. For example, persons skilled in the art can appropriately determine administration conditions such as the dosage, the dosage period, and the route of administration of a test therapeutic agent.
- Furthermore, the effectiveness of a test therapeutic agent or a test therapeutic method can be examined under several conditions. Examples of such conditions include the time or the period of treatment with the test therapeutic agent or by the test therapeutic method, the amount thereof (high or low), and frequency. For example, a dilution series of a test therapeutic agent is prepared, so that a plurality of doses can be determined.
- Furthermore, when the additive effects, synergistic effects, and the like of a plurality of test therapeutic agents or test therapeutic methods are examined, the therapeutic agents or therapeutic methods may be used in combination.
- The amount and/or the location of a marker protein(s) in an animal after treatment with a test therapeutic agent or by a test therapeutic method is compared with the amount and/or the location of the same before treatment, so as to allow evaluation of whether or not the test therapeutic agent or the test therapeutic method is effective for improvement of atherosclerotic lesions (instability thereof) (e.g., improvement from the intermediate stage to the early intermediate stage or the initial stage), or for stopping or slowing down the progression of atherosclerotic lesions (instability thereof) (e.g., the disease progression from the intermediate stage to the late stage is stopped, unlike a case in which no treatment has been performed).
- As described above, according to the method for evaluating the effectiveness of a therapeutic agent or a therapeutic method for atherosclerotic lesions (instability thereof) according to the present invention, a therapeutic agent or a therapeutic method for treating or preventing arteriosclerosis can be found, and then the effectiveness of the therapeutic agent or the therapeutic method can be confirmed. In the case of atherosclerotic lesions (instablility thereof) at the early stage in particular (the initial stage and the early intermediate stage), no sign indicating the disease can be detected. If a subject is evaluated as having arteriosclerosis at the initial stage or the early intermediate stage and some treatment is performed for the subject, it is difficult to evaluate the effectiveness of the treatment. Therefore, the method of the present invention is particularly effective for identifying a therapeutic agent or a therapeutic method effective for treatment of arteriosclerosis at the early stage.
- In a manner similar to the above method, the effects of a substance or a factor on arteriosclerosis can also be determined, and thus a substance or a factor that causes the progression of atherosclerotic lesions (instability thereof) can be identified.
- The present invention is hereinafter illustrated in greater detail with reference to the following examples. The examples are provided merely for the explanation of the present invention and are not intended to limit or restrict the scope of the present invention disclosed in the present application. It is apparently understood that various changes and modifications to the present invention can be made based on the concept of the present invention described herein.
- ApoE-deficient mice (ApoED; B6.KOR/StmSlc-Apoeshl), which were deficient in the lipid metabolism-related ApoE protein, were used as atherosclerosis models (Matsushima, Y. et al., J. Atheroscler. Thromb. 8: 71-79, 2001; Japan SLC, Inc.). Administration of a high-fat diet accelerates lesion progression in ApoED mice. In addition, lesion progression is accelerated depending on age in weeks. In 12-week-old mice, lesion progression is observed to such an extent that arterial intimal thickening and lipid accumulation are observed (stage 1). In 18-week-old mice, lipid accumulation results in foam cell formation (stage 2). In 25-week-old mice, many forms of unstable atherosclerotic plaque are widely observed in arteries (stage 3), and this corresponds to the most dangerous stage in the atherosclerosis process. In 35-week-old mice, many stable lesions that are highly fibrotic and calcified are observed (stage 4).
- Therefore, for this example, arterial tissue was collected from 12, 18, 25, and 35-week-old wild-type mice (WT; C57BL/6) and ApoED mice (male and female mice: 5 animals each). WT and ApoED mouse arterial tissue samples were crushed in liquid nitrogen and then solubilized with a solubilization solution (9.5 M urea, 2% sulfuric acid-3-[(3-cholamidopropyl)dimethylammonio]-1-propane (CHAPS), 65 mM dithiothreitol (DTT), 40 mM Tris HCl, pH 8.3). After 20 minutes of centrifugation at 12,000 rpm and 4° C., the supernatants were recovered, acetone precipitation was performed with the addition of ice-cold acetone, and thus the protein components were concentrated. Precipitates subjected to vacuum drying were solubilized again in a re-solubilization solution (0.2% sodium dodecyl sulfate (SDS), 50 mM Tris-HCl, pH 8.5). Protein concentrations were measured and then the resultants were subjected to isotopic labeling.
- The protein fractions (1 mg each) extracted from arterial tissues were adjusted in a manner such that each sample contained 6M urea, 0.05% SDS, 50 mM Tris (pH 8.5), 5 mM ethylenediamine tetraacetic acid (EDTA), and 10 mM tri-n-butyl phosphate (TBP) (final concentrations) in a total volume of 800 μl, followed by degeneration treatment at 37° C. for 30 minutes. A “Light labeling reagent” and a “Heavy labeling reagent,” each of which had been dissolved with acetonitrile (200 μl), were added to a WT mouse sample and an ApoED mouse sample, respectively, followed by a labeling reaction at 37° C. for 2 hours. 800 μl of 10 mM Tris buffer (pH 8.0) was added to each sample for pH adjustment. A trypsin solution (Trypsin, TPCK Treated; Cat. No. 4352157; Applied Biosystems) (160 μl) adjusted to 125 μg/ml was added thereto. Then, both types of samples were mixed in equivalent volumes, followed by a trypsin digestion reaction at 37° C. for 16 hours. Further, peptide fragments obtained by trypsin treatment were introduced into an SCX column (poly Sulfoethyl A; 4.6×100 mm; PolyLC Inc.), followed by separation of the eluate into 25 fractions. Separation was carried out with the use of an eluent A [10 mM KH2PO4 (pH 2.8), 25% acetonitrile (ACN)] and an eluent B [10 mM KH2PO4 (pH 2.8), 25% ACN, 0.5 M KCl] with a linear gradient (% B: 10 minutes-0%, 70 minutes-20%, 85 minutes-50%, 90 minutes-60%, 95 minutes-60%, and 100 minutes-100%). Each fraction was subjected to vacuum concentration so as to result in a volume that was approximately one-fourth (¼) the initial volume. Then, desalting with a desalting column (CAPCELL C18 MG; 2.0×10 mm; Shiseido) and vacuum drying were performed. An eluent A (2% ACN, 0.05% trifluoroacetic acid (TFA)) and an eluent B (80% ACN, 0.05% TFA) were used for desalting.
- Each SCX fraction was analyzed using a mass spectrometry apparatus and an accompanying LC system device (NanoFrontier LD; Hitachi High-Technologies Corporation). Each obtained sample was dissolved in a buffer A (water: 98%; ACN: 2%; formic acid: 0.1%) (4 μl to 10 μl). One microliter of each obtained solution was applied to the apparatus. A MonoCap for Fast-flow (50 μmφ×150 mm; C18; GL Sciences) was used as a sample separation column in the LC system. Analysis was carried out with a linear gradient of buffer A and buffer B (water: 2%; ACN: 98%; formic acid: 0.1%) at a flow rate of 200 mL/min, provided that the buffer B concentration reached 2% to 30% in 120 minutes. A Monolith Trap (50 μmφ×150 mm; Cat. No. C18-50-150; Hitachi High-Technologies Corporation) was used as a trap column in the apparatus. A quartz spray chip (Picotip; outer diameter: 360 μm; inner diameter: 50 μm; tip inner diameter: 10 μm; New Objective) was used as a column tip. Electrospray ionization mass spectrometry was performed in the positive ion mode. Samples obtained from 25 fractions were subjected twice to IBA (information based acquisition) analysis. IBA is a technique involving storing target information (m/z, charge number, retention time) obtained by the first analysis in a database within an apparatus, and analyzing ions that do not correspond to the target information in the second analysis. It was expected that weak ions would be analyzed with the use of such technique so as to increase the number of identified proteins. The following are additional apparatus conditions: Curtain Gas Flow: 0.7 L/min; Spray potential: 1700 V; Detector potential: 2200 V; Isolation Time: 5 ms; Isolation Width: 10 Da; and CID Time: 10 ms. The measurement data were processed using software that had been developed for ICAT comparative quantification. Thus, comparative analysis data for two groups (the WT mouse group and the ApoED mouse group) were obtained.
- As a result, at least 568 proteins were confirmed to express in all 4 types (age in weeks). Proteins, the expression levels of which tended to increase or decrease stepwise in arterial tissue at the age of 12 weeks or later, were classified into the following four types of pattern. The expression level ratio for each protein was plotted against lesion progression stages (
FIG. 1 toFIG. 4 ). Three proteins were found for which amounts thereof in 12-week-old or older ApoE-deficient mice increased to twice or more the amounts in 12-week-old or older WT (wild-type) mice (pattern 1,FIG. 1 ). Twelve proteins were found for which amounts thereof exhibited no significant difference until the age of 12 weeks, while the amounts thereof in 18-week-old or older ApoE-deficient mice increased to twice or more the amounts in 18-week-old or older wild-type mice (pattern 2,FIG. 2 ). Fourteen proteins were found for which amounts exhibited no significant difference until the age of 18 weeks, while the amounts thereof in 25-week-old or older ApoE-deficient mice increased to twice or more the amounts in 25-week-old or older wild type mice (pattern 3,FIG. 3 ). Seven proteins were found for which amounts exhibited no significant difference until the age of 25 weeks, while the amounts thereof in 35 week-old ApoE-deficient mice increased to twice or more the amounts in 35 week-old wild-type mice (pattern 4,FIG. 4 ). From these protein groups, marker proteins related to the progression of atherosclerotic lesions were extracted according to the following criteria. - From among proteins belonging to the
pattern 3, four proteins characterized in that: (i) the ratio of the amounts thereof in ApoE-deficient mice divided by the amounts thereof in wild-type mice (herein after referred to as “the expression level ratio”) at the age of 25 weeks was at least 3; and (ii) the expression level ratio at the age of 25 weeks was at least twice as large as the expression level ratio at the age of 18 weeks, were selected as markers indicating the risk of plaque rupture at the intermediate stage (Table 1). - From among proteins belonging to
patterns - From among proteins belonging to the
patterns - In addition, from among proteins belonging to the
patterns 1 to 4, 22 proteins not selected in Table 1 to Table 3 are categorized into another marker group relating to the progression of atherosclerotic lesions, as listed in Table 4. - Arterial tissues were excised from five 12-week-old ApoE knockout mice (female) from different lines (ApoEKO; B6.129P2-ApoetmlUnc/J) fed with a normal diet, and then protein fractions (pool) were prepared in a manner similar to that in Example 1. The ApoEKO line differing from the ApoED line described in Example 1 (Piedrahita, J A. et al., Proc. Natl. Acad. Sci. U.S.A., 89: 4471-4475, 1992; Charles River Laboratories, Japan Inc.) were confirmed in advance in a preliminary study to have no lesion formed in arteries (data not shown). The lipid concentrations in blood of a subject were: total cholesterol of 539 mg/dl; triglyceride of 49 mg/dl; LDL cholesterol of 404 mg/dl; and HDL cholesterol of 143 mg/dl. Thus, the progression of arteriosclerosis was suspected. Hence, the ratios of the expression levels of proteins contained in tissue samples were exhaustively analyzed by the method of Example 1 (that had been partially modified) using an arterial tissue obtained from a wild-type mouse (WT; C57BL/6) at the same age in weeks as a standard level. The partial modification in the method of Example 1 is that the number of SCX fractions was 7 instead of 25 for simplifying experimental procedures.
- As a result, 247 proteins were identified, which were the same as those included in the proteins identified in Example 1. Of these proteins, a protein group, the ratio of the amounts of which in ApoE-deficient mice divided by those in wild-type mice was at least 2 in Example 1, was examined for behavior. Three proteins (C4BPA_MOUSE, MUCM_MOUSE, PROP_MOUSE), the amounts of which in 12-week-old or older ApoE-deficient mice increased to twice or more the amounts in 12-week-old or older wild-type mice in Example 1, were not identified in this measurement. Of the proteins (total of 32 proteins), the amounts of which in 18-week-old or older ApoE-deficient mice increased to twice or more the amounts in 18-week-old or older wild-type mice in Example 1, eleven proteins could be identified in this measurement. However, the ratio of the amount of each of these eleven proteins in ApoE-deficient mice divided by the same in wild-type mice was always 1.35 or less. Therefore, the mice subjected to this measurement were most likely at the progression stage of arteriosclerosis corresponding to the age of 12 weeks (initial lesion). Thus, the effectiveness of the present invention was demonstrated.
- Ten typical proteins were selected from the candidate marker protein group that exhibited variations in Example 1 (characteristic in ApoE-deficient mice) in the amounts thereof in arterial tissues. The expression of the proteins in atherosclerotic lesion sites was confirmed by immunohistochemical staining of lesion sites (plaque formation sites) in arterial tissues of ApoE-deficient mice at different ages in weeks.
- Each aortic sample subjected to 10% formalin fixation was coronally excised at the aortic arch, and then a paraffin embedded block was prepared. Subsequently, it was sliced to a thickness of 3 μm, and then the slices were subjected to immunohistochemical staining according to a conventional method using hematoxylin.eosin (H&E) staining and antibodies. Primary antibodies used for immunohistochemical staining are as follows.
- Anti-properdin antibody (50-fold dilution, sc68367; Santa Cruz),
Anti-CD5 antigen-like protein antibody (100-fold dilution, ab45408; Abcam),
Anti-fibronectin antibody (50-fold dilution, sc59826; Santa Cruz),
Anti-fibromodulin antibody (50-fold dilution, sc33772; Santa Cruz),
Anti-thrombospondin-4 antibody (50-fold dilution, sc68367; Santa Cruz),
Anti-plastin-2 antibody (50-fold dilution, 13025-1-AP; Proteintech),
Anti-ADAMTS-like protein 4 antibody (50-fold dilution, 15304-1-AP; Proteintech),
Anti-matrix metalloproteinase-2 antibody (50-fold dilution, BS1236; Bioworld Technology),
Anti-tenascin antibody (50-fold dilution, 3118-1; Epitomics),
Anti-cartilage oligomeric matrix protein antibody (50-fold dilution, PC-140; Kamiya Biomedical Company). - After reaction with a biotin-labeled secondary antibody, signals were visualized with the addition of diaminobenzidine (DAB). Stained specimens were observed under a microscope (Biozero, BZ-8000; KEYENCE CORPORATION) and then the thus obtained images were stored as digital data.
-
FIG. 6 shows the results of immunohistochemical staining using the anti-CD5 antigen-like protein antibody and the anti-properdin antibody. Moreover,FIG. 7 shows the results of immunohistochemical staining using the anti-cartilage oligomeric matrix protein antibody, the anti-fibromodulin antibody, the anti-fibronectin antibody, and the anti-thrombospondin-4 antibody. Furthermore,FIG. 8 shows the results of immunohistochemical staining using the anti-plastin-2 antibody and the anti-tenascin antibody. - As shown in these figures, as a result of immunohistochemical staining using the anti-properdin antibody, the anti-CD5 antigen-like protein antibody, the anti-plastin-2antibody, and the anti-tenascin antibody, high signals were detected for the lesion sites at particularly the intermediate stage, the expression of the target protein group to be recognized by each antibody in lesions was confirmed, and thus the results obtained by mass spectroscopy in Example 1 were supported (
FIG. 6 andFIG. 8 ). Also, as a result of immunohistochemical staining using the anti-fibromodulin antibody, the anti-fibronectin antibody, and the anti-thrombospondin-4 antibody, high signals were detected for lesion sites at the intermediate stage in particular and the late stage, the expression of the target protein group to be recognized by each antibody in the lesions was confirmed, and thus the results of Example 1 were supported (FIG. 7 ). Also, as a result of immunohistochemical staining using the anti-cartilage oligomeric matrix protein antibody, higher signals were detected for lesion sites at the intermediate stage than the same at the late stage, and the expression of the target protein in lesions was confirmed (FIG. 7 ). Since 35-week-old mouse arterial tissues contain intermediate stage lesions in addition to late stage lesions, the result that the cartilage oligomeric matrix protein exhibited strong expression in the intermediate stage lesions is consistent with the result of mass spectroscopy in Example 1. Moreover, as a result of immunohistochemical staining using the anti-matrix metalloproteinase-2antibody, high signals were detected for lesion sites at the late stage in particular, the expression of the target protein to be recognized by the antibody in the lesions was confirmed, and thus the results of Example 1 were supported. As a result of immunohistochemical staining using the anti-ADAMTS-like protein 4 antibody, an increase in signal intensity was observed with the progression of stages. The expression of target proteins to be recognized by these antibodies in lesions was confirmed, and thus the results of Example 1 were supported. - As described above, it was confirmed that the ratio of the amounts of the proteins in ApoE-deficient mice to those in wild-type mice had a high correlation with the amounts of the proteins in atherosclerotic lesions (
FIG. 6 toFIG. 8 ). - WHHL arteriosclerosis model rabbits formed atherosclerotic lesions, the progression degree of which differs depending on age in months. In this example, 7-week-old and 13-week-old WHHL rabbits (male, Kitayama Labes) were used. A 99mtechnetium-labeled anti-properdin antibody and a 125iodine-labeled anti-CD5L antibody were mixed, the mixture was administered (under anesthesia) into the marginal vein of the ear of each rabbit, and then the ways in which signals from both antibodies were accumulated in lesions were examined.
Signal accumulation 6 hours after administration was visualized using an animal SPECT device (R1; Hitach, Ltd.). - Only the anti-properdin antibody-derived signals were detected in 7-week-old WHHL rabbits; however, in the case of 13-week-old WHHL rabbits, anti-properdin antibody-derived signals and anti-CD5L antibody-derived signals were detected mainly in the thoracic aortic arch. On the other hand, no signals from either antibody were ever detected in the case of 13-week-old wild-type rabbits (Japanese white; Kitayama Labes).
- All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety
- According to the present invention, technology for evaluation of the presence or the absence of the onset of arteriosclerosis and the progression thereof is provided. The presence or the absence, the state or the progression degree, and the location of arteriosclerosis can be conveniently determined with high accuracy according to the present invention. Furthermore, according to the present invention, a method for screening for a medicine useful for prevention and/or treatment of arteriosclerosis is provided. The present invention is useful in the field of diagnosis, prevention, and/or treatment of arteriosclerosis.
Claims (23)
1.-23. (canceled)
24. A kit for evaluating an atherosclerotic lesion at the initial or the intermediate stage, comprising at least one type of substance specifically binding to plastin-2.
25. A kit for evaluating the stage of an atherosclerotic lesion, comprising
at least one type of substance specifically binding to plastin-2, and
at least one type of substance specifically binding to at least one type of protein selected from at least group (a), (b), or (d) from among proteins in the following groups (a) to (d):
(a) thrombospondin-4, fibromodulin, fibronectin, and cartilage oligomeric matrix protein;
(b) tenascin, CD5 antigen-like protein, properdin, Cb4-binding protein, fibrinogen beta chain, and fibrinogen gamma chain;
(c) vitronectin, matrix metalloproteinase-2, and thrombospondin-1;
(d) membrane-bound form immunoglobulin mu chain C region, receptor-type tyrosine protein phosphatase C, immunoglobulin gamma-2B chain-C region, immunoglobulin J chain, immunoglobulin kappa chain C region, vascular cell adhesion protein 1, alpha-2-macroglobulin, ADAMTS-like protein 4, cathepsin B, carboxy peptidase-B2, complement factor-H, clusterin, collagen alpha-1 (XIV) chain, epidermal fatty acid-binding protein, mannose-binding lectin-associated serine protease-2, prothrombin, complement factor B, inter-alpha-trypsin inhibitor-heavy chain-H1, myosin-9, vitamin K-dependent factor S, metalloproteinase inhibitor 3, and tropomyosin alpha 4.
26. The kit according to claim 25 , wherein the protein selected from group (a) is thrombospondin-4.
27. The kit according to claim 25 , wherein the protein selected from group (b) is a protein selected from properdin and CD5 antigen-like protein.
28. The kit according to claim 25 , wherein the protein selected from group (d) is a protein selected from epidermal fatty acid-binding protein, myosin-9 and ADAMTS-like protein 4.
29. The kit according to claim 25 , the evaluation of the stage of an atherosclerotic lesion is selected from the group consisting of the determination of the presence of an atherosclerotic lesion at a certain stage in a subject, the determination of the stage of an atherosclerotic lesion in a subject, the determination of the location of an atherosclerotic lesion at a certain stage in a subject, the evaluation of the therapeutic effect on arteriosclerosis at a certain stage existing in a subject, and the prediction of prognosis of arteriosclerosis existing in a subject.
30. The kit according to claim 24 , wherein a substance specifically binding to a protein is selected from the group consisting of an antibody, an antibody-like molecule, a nucleic acid aptamer, a ligand, and a substrate.
31. The kit according to claim 24 , wherein the substance specifically binding to a protein is bound to a contrast medium.
32. The kit according to claim 31 , wherein the contrast medium is selected from the group consisting of a fluorescent substance, a luminescent substance, a radioactive substance, a liposome, a micelle, a cell, a virus particle, a virus, a microparticle, a nanoparticle, a microdevice with a space, an emulsion, a lipid disc, a polymer, a gadolinium-conjugated molecule, a superparamagnetic iron oxide particle, a perfluorocarbon nanoparticle, and an ultrafine bubble.
33. The kit according to claim 31 , wherein the atherosclerotic lesion is visualized by a means selected from the group consisting of positron emission tomography (PET), single photon emission computed tomography (SPECT), γ scintigraphy, autoradiography, fluorescent imaging, magnetic resonance imaging (MRI), ultrasonication, and computed tomography (CT).
34. An apparatus for evaluating an atherosclerotic lesion at the initial or the intermediate stage, comprising:
(i) a means of detecting plastin-2 in a subject:
(ii) a means of evaluating the atherosclerotic lesion at the initial or the intermediate stage in the subject based on the detection results obtained by means (i).
35. An apparatus for evaluating the stage of an atherosclerotic lesion, comprising:
(i) a means of detecting plastin-2 in a subject and a means of detecting at least one type of protein selected from at least group (a), (b), or (d) from among proteins shown in the following groups (a) to (d) in a subject:
(a) thrombospondin-4, fibromodulin, fibronectin, and cartilage oligomeric matrix protein;
(b) tenascin, CD5 antigen-like protein, properdin, Cb4-binding protein, fibrinogen beta chain, and fibrinogen gamma chain;
(c) vitronectin, matrix metalloproteinase-2, and thrombospondin-1; and
(d) membrane-bound form immunoglobulin mu chain C region, receptor-type tyrosine protein phosphatase C, immunoglobulin gamma-2B chain-C region, immunoglobulin J chain, immunoglobulin kappa chain C region, vascular cell adhesion protein 1, alpha-2-macroglobulin, ADAMTS-like protein 4, cathepsin B, carboxy peptidase-B2, complement factor-H, clusterin, collagen alpha-1 (XIV) chain, epidermal fatty acid-binding protein, mannose-binding lectin-associated serine protease-2, prothrombin, complement factor B, inter-alpha-trypsin inhibitor-heavy chain-H1, myosin-9, vitamin K-dependent factor S, metalloproteinase inhibitor 3, and tropomyosin alpha 4; and
(ii) a means of evaluating the stage of the atherosclerotic lesion in the subject based on the detection results obtained by means (i).
36. The apparatus according to claim 34 , further comprising:
(iii) a means of storing the standard levels of the above proteins; and
(iv) a means of comparing the protein detection results in the subject with the standard levels.
37. An agent for visualizing an atherosclerotic lesion at the initial or the intermediate stage, comprising at least one type of substance specifically binding to plastin-2, and a contrast medium binding thereto.
38. An agent for visualizing an atherosclerotic lesion at a certain stage, comprising
at least one type of substance specifically binding to plastin-2 and a contrast medium binding thereto, and
at least one type of protein selected from at least group (a), (b), or (d) from among proteins in the following groups (a) to (d) and a contrast medium binding thereto:
(a) thrombospondin-4, fibromodulin, fibronectin, and cartilage oligomeric matrix protein;
(b) tenascin, CD5 antigen-like protein, properdin, Cb4-binding protein, fibrinogen beta chain, and fibrinogen gamma chain;
(c) vitronectin, matrix metalloproteinase-2, and thrombospondin-1; and
(d) membrane-bound form immunoglobulin mu chain C region, receptor-type tyrosine protein phosphatase C, immunoglobulin gamma-2B chain-C region, immunoglobulin J chain, immunoglobulin kappa chain C region, vascular cell adhesion protein 1, alpha-2-macroglobulin, ADAMTS-like protein 4, cathepsin B, carboxy peptidase-B2, complement factor-H, clusterin, collagen alpha-1 (XIV) chain, epidermal fatty acid-binding protein, mannose-binding lectin-associated serine protease-2, prothrombin, complement factor B, inter-alpha-trypsin inhibitor-heavy chain-H1, myosin-9, vitamin K-dependent factor S, metalloproteinase inhibitor 3, and tropomyosin alpha 4.
39. A targeting agent for directing a compound or a molecule to an atherosclerotic lesion at the initial or the intermediate stage, comprising a substance specifically binding to plastin-2 and the compound or the molecule binding thereto.
40. A targeting agent for directing a compound or a molecule to an atherosclerotic lesion at a certain stage, comprising
a substance specifically binding to plastin-2 and the compound or the molecule binding thereto, and
a substance specifically binding to at least one type of protein selected from at least group (a),
(b), or (d) from among the following groups (a) to (d), and the compound or the molecule binding thereto:
(a) thrombospondin-4, fibromodulin, fibronectin, and cartilage oligomeric matrix protein;
(b) tenascin, CD5 antigen-like protein, properdin, Cb4-binding protein, fibrinogen beta chain, and fibrinogen gamma chain;
(c) vitronectin, matrix metalloproteinase-2, and thrombospondin-1; and
(d) membrane-bound form immunoglobulin mu chain C region, receptor-type tyrosine protein phosphatase C, immunoglobulin gamma-2B chain-C region, immunoglobulin J chain, immunoglobulin kappa chain C region, vascular cell adhesion protein 1, alpha-2-macroglobulin, ADAMTS-like protein 4, cathepsin B, carboxy peptidase-B2, complement factor-H, clusterin, collagen alpha-1 (XIV) chain, epidermal fatty acid-binding protein, mannose-binding lectin-associated serine protease-2, prothrombin, complement factor B, inter-alpha-trypsin inhibitor-heavy chain-H1, myosin-9, vitamin K-dependent factor S, metalloproteinase inhibitor 3, and tropomyosin alpha 4.
41. The targeting agent according to claim 39 , wherein the compound or the molecule is a thrombolytic agent.
42. A method for evaluating the effectiveness of a therapeutic agent or a therapeutic method for an atherosclerotic lesion at the initial or the intermediate stage, comprising the steps of:
(i) detecting plastin-2 in an animal with an atherosclerotic lesion at the initial or the intermediate stage treated by a test therapeutic agent or a test therapeutic method:
(ii) evaluating the effectiveness of the test therapeutic agent or the test therapeutic method on the atherosclerotic lesion at the initial or the intermediate stage based on the results of (i).
43. A method for evaluating the effectiveness of a therapeutic agent or a therapeutic method for an atherosclerotic lesion at a certain stage, comprising the steps of:
(i) detecting plastin-2 and at least one type of protein selected from at least group (a), (b), or (d) from among proteins shown in the following groups (a) to (d) in an animal with an atherosclerotic lesion treated by a test therapeutic agent or a test therapeutic method:
(a) thrombospondin-4, fibromodulin, fibronectin, and cartilage oligomeric matrix protein;
(b) tenascin, CD5 antigen-like protein, properdin, Cb4-binding protein, fibrinogen beta chain, and fibrinogen gamma chain;
(c) vitronectin, matrix metalloproteinase-2, and thrombospondin-1; and
(d) membrane-bound form immunoglobulin mu chain C region, receptor-type tyrosine protein phosphatase C, immunoglobulin gamma-2B chain-C region, immunoglobulin J chain, immunoglobulin kappa chain C region, vascular cell adhesion protein 1, alpha-2-macroglobulin, ADAMTS-like protein 4, cathepsin B, carboxy peptidase-B2, complement factor-H, clusterin, collagen alpha-1 (XIV) chain, epidermal fatty acid-binding protein, mannose-binding lectin-associated serine protease-2, prothrombin, complement factor B, inter-alpha-trypsin inhibitor-heavy chain-H1, myosin-9, vitamin K-dependent factor S, metalloproteinase inhibitor 3, and tropomyosin alpha 4; and
(ii) evaluating the effectiveness of the test therapeutic agent or the test therapeutic method on the atherosclerotic lesion at a certain stage based on the results of (i).
44. The method according to claim 42 , further comprising a step of detecting the protein in an animal with an atherosclerotic lesion before treatment using the test therapeutic agent or the test therapeutic method.
45. The method according to claim 42 , wherein an animal having an atherosclerotic lesion is an arterial disease animal model.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2010257065A JP5066599B2 (en) | 2010-11-17 | 2010-11-17 | Method and kit for evaluating arteriosclerotic lesions |
JP2010-257065 | 2010-11-17 | ||
PCT/JP2011/076499 WO2012067180A1 (en) | 2010-11-17 | 2011-11-17 | Method for evaluating atherosclerotic lesion, and kit |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2011/076499 A-371-Of-International WO2012067180A1 (en) | 2010-11-17 | 2011-11-17 | Method for evaluating atherosclerotic lesion, and kit |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US14/935,994 Division US9575074B2 (en) | 2010-11-17 | 2015-11-09 | Method for evaluating atherosclerotic lesion, and kit |
Publications (1)
Publication Number | Publication Date |
---|---|
US20130302821A1 true US20130302821A1 (en) | 2013-11-14 |
Family
ID=46084101
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US13/885,921 Abandoned US20130302821A1 (en) | 2010-11-17 | 2011-11-17 | Method For Evaluating Atherosclerotic Lesion, And Kit |
US14/935,994 Active US9575074B2 (en) | 2010-11-17 | 2015-11-09 | Method for evaluating atherosclerotic lesion, and kit |
Family Applications After (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US14/935,994 Active US9575074B2 (en) | 2010-11-17 | 2015-11-09 | Method for evaluating atherosclerotic lesion, and kit |
Country Status (3)
Country | Link |
---|---|
US (2) | US20130302821A1 (en) |
JP (1) | JP5066599B2 (en) |
WO (1) | WO2012067180A1 (en) |
Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN107110867A (en) * | 2014-12-12 | 2017-08-29 | 首尔大学校产学协力团 | Biomarker for diagnosing cancer of liver and application thereof |
US9952213B2 (en) | 2013-10-15 | 2018-04-24 | Hitachi, Ltd. | Method for evaluating ischemic heart disease by assaying complement factor H or D |
CN111458512A (en) * | 2019-01-21 | 2020-07-28 | 中国科学院分子细胞科学卓越创新中心 | Atherosclerosis biomarker and application thereof |
CN111690727A (en) * | 2019-03-12 | 2020-09-22 | 南方医科大学南方医院 | FABP5 as a novel biomarker for diagnosing atherosclerosis |
CN112904020A (en) * | 2021-01-25 | 2021-06-04 | 上海市第六人民医院 | Application of FAM172A in screening and treating diabetic macroangiopathy |
CN113287019A (en) * | 2018-10-31 | 2021-08-20 | 卡洛斯三世国家心血管研究中心 | Subclinical atherosclerosis biomarkers |
US11337931B2 (en) * | 2018-05-31 | 2022-05-24 | University Of South Florida | Nanoparticle delivery system for diseases associated with major basement membrane components of blood vessels accessible from blood stream |
US11604196B2 (en) | 2014-04-04 | 2023-03-14 | Mayo Foundation For Medical Education And Research | Isotyping immunoglobulins using accurate molecular mass |
US11946937B2 (en) | 2017-09-13 | 2024-04-02 | Mayo Foundation For Medical Education And Research | Identification and monitoring of apoptosis inhibitor of macrophage |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP5066599B2 (en) | 2010-11-17 | 2012-11-07 | 株式会社日立製作所 | Method and kit for evaluating arteriosclerotic lesions |
WO2020101251A1 (en) * | 2018-11-14 | 2020-05-22 | 이화여자대학교 산학협력단 | Marker for diagnosing atherosclerosis severity, and diagnostic method using same |
US20210080466A1 (en) * | 2019-06-26 | 2021-03-18 | Berg Llc | Markers for the diagnosis of prostate cancer |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4444879A (en) * | 1981-01-29 | 1984-04-24 | Science Research Center, Inc. | Immunoassay with article having support film and immunological counterpart of analyte |
US8603829B2 (en) * | 2009-09-09 | 2013-12-10 | Hitachi Ltd. | Atherosclerosis marker and use thereof |
Family Cites Families (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2007278907A (en) | 2006-04-07 | 2007-10-25 | Takeda Chem Ind Ltd | Arteriosclerosis marker protein and use thereof |
JP5453667B2 (en) | 2008-09-30 | 2014-03-26 | 株式会社バイオス医科学研究所 | Arteriosclerosis detection method and arteriosclerosis marker |
JP5337096B2 (en) | 2010-04-28 | 2013-11-06 | 株式会社日立製作所 | Evaluation of arteriosclerosis |
JP5066599B2 (en) | 2010-11-17 | 2012-11-07 | 株式会社日立製作所 | Method and kit for evaluating arteriosclerotic lesions |
JP5417464B2 (en) | 2012-02-13 | 2014-02-12 | 株式会社日立製作所 | Method, kit, device and medicament for evaluating arteriosclerotic lesions |
-
2010
- 2010-11-17 JP JP2010257065A patent/JP5066599B2/en active Active
-
2011
- 2011-11-17 WO PCT/JP2011/076499 patent/WO2012067180A1/en active Application Filing
- 2011-11-17 US US13/885,921 patent/US20130302821A1/en not_active Abandoned
-
2015
- 2015-11-09 US US14/935,994 patent/US9575074B2/en active Active
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4444879A (en) * | 1981-01-29 | 1984-04-24 | Science Research Center, Inc. | Immunoassay with article having support film and immunological counterpart of analyte |
US8603829B2 (en) * | 2009-09-09 | 2013-12-10 | Hitachi Ltd. | Atherosclerosis marker and use thereof |
Non-Patent Citations (4)
Title |
---|
Fach et al. (Molecular & Cellular Proteomics Vol.3, pages 1200-1210, 2004) * |
Frolova et al., (Circ Res. 2010 Nov 26;107(11):1313-25. doi: 10.1161/CIRCRESAHA.110.232371. Epub 2010 Sep 30) * |
Kang et al. (Journal of Proteome Research, 2010, Vol.9, pages 1157-1164). * |
Kang et al. Molecular proteomics imaging of tumor interfaces by mass spectrometry. Journal of Proteome Research, Vol. 9, pages 1157-1164, 2010, published online 10/12/2009. * |
Cited By (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9952213B2 (en) | 2013-10-15 | 2018-04-24 | Hitachi, Ltd. | Method for evaluating ischemic heart disease by assaying complement factor H or D |
US11604196B2 (en) | 2014-04-04 | 2023-03-14 | Mayo Foundation For Medical Education And Research | Isotyping immunoglobulins using accurate molecular mass |
CN107110867A (en) * | 2014-12-12 | 2017-08-29 | 首尔大学校产学协力团 | Biomarker for diagnosing cancer of liver and application thereof |
CN107110867B (en) * | 2014-12-12 | 2020-08-11 | 首尔大学校产学协力团 | Biomarker for liver cancer diagnosis and application thereof |
US11946937B2 (en) | 2017-09-13 | 2024-04-02 | Mayo Foundation For Medical Education And Research | Identification and monitoring of apoptosis inhibitor of macrophage |
US11337931B2 (en) * | 2018-05-31 | 2022-05-24 | University Of South Florida | Nanoparticle delivery system for diseases associated with major basement membrane components of blood vessels accessible from blood stream |
CN113287019A (en) * | 2018-10-31 | 2021-08-20 | 卡洛斯三世国家心血管研究中心 | Subclinical atherosclerosis biomarkers |
CN111458512A (en) * | 2019-01-21 | 2020-07-28 | 中国科学院分子细胞科学卓越创新中心 | Atherosclerosis biomarker and application thereof |
CN111690727A (en) * | 2019-03-12 | 2020-09-22 | 南方医科大学南方医院 | FABP5 as a novel biomarker for diagnosing atherosclerosis |
CN112904020A (en) * | 2021-01-25 | 2021-06-04 | 上海市第六人民医院 | Application of FAM172A in screening and treating diabetic macroangiopathy |
Also Published As
Publication number | Publication date |
---|---|
US20160061846A1 (en) | 2016-03-03 |
WO2012067180A1 (en) | 2012-05-24 |
JP2012107989A (en) | 2012-06-07 |
US9575074B2 (en) | 2017-02-21 |
JP5066599B2 (en) | 2012-11-07 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US9575074B2 (en) | Method for evaluating atherosclerotic lesion, and kit | |
Bar et al. | Degradation of glycocalyx and multiple manifestations of endothelial dysfunction coincide in the early phase of endothelial dysfunction before atherosclerotic plaque development in apolipoprotein E/low‐density lipoprotein receptor‐deficient mice | |
US11071770B2 (en) | Methods for detecting cardiac damage | |
US10551372B2 (en) | Trimethylamine compounds as risk predictors of cardiovascular disease | |
US9175346B2 (en) | Evaluation method for arteriosclerosis | |
US8759095B2 (en) | Diagnostic and therapeutic tools for diseases altering vascular function | |
CN106461681B (en) | Biomarker and application thereof for diagnosing vascular diseases | |
US20190094205A1 (en) | Method for disease diagnosis based on metabolite in urine | |
US10989720B2 (en) | Method for clinically and pathologically monitoring Alzheimer's disease through concentration of amyloidbeta in plasma | |
Ta et al. | A dual magnetic resonance imaging/fluorescent contrast agent for Cathepsin‐D detection | |
JP2020064055A (en) | Detection or risk estimation method of fatty liver disease, diagnostic reagent kit to detect fatty liver disease and biomarker, method for determining progress of target liver fibrosis, as well as biomarker for determining progress of target liver fibrosis | |
JP5417464B2 (en) | Method, kit, device and medicament for evaluating arteriosclerotic lesions | |
Bankstahl et al. | A novel PET imaging protocol identifies seizure-induced regional overactivity of P-glycoprotein at the blood-brain barrier | |
EP3997244A1 (en) | Targeting ligands for tau pathology | |
Pedretti et al. | Structure-based probe reveals the presence of large transthyretin aggregates in plasma of ATTR amyloidosis patients | |
US20140154711A1 (en) | Detection of defensins to diagnose cancer | |
JP2021181890A (en) | Biomarkers for determining adult Still's disease (ASD) | |
CN116973427A (en) | Biomarkers for atherosclerosis diagnosis and uses thereof |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: NATIONAL UNIVERSITY CORPORATION HOKKAIDO UNIVERSIT Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:SAKAMOTO, TAKESHI;HANZAWA, HIROKO;MANRI, NAOMI;AND OTHERS;SIGNING DATES FROM 20130701 TO 20130705;REEL/FRAME:030921/0706 Owner name: HITACHI, LTD., JAPAN Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:SAKAMOTO, TAKESHI;HANZAWA, HIROKO;MANRI, NAOMI;AND OTHERS;SIGNING DATES FROM 20130701 TO 20130705;REEL/FRAME:030921/0706 |
|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION |