CN114606307A - Application of fibroblast activation protein-alpha as drug target - Google Patents
Application of fibroblast activation protein-alpha as drug target Download PDFInfo
- Publication number
- CN114606307A CN114606307A CN202011519396.2A CN202011519396A CN114606307A CN 114606307 A CN114606307 A CN 114606307A CN 202011519396 A CN202011519396 A CN 202011519396A CN 114606307 A CN114606307 A CN 114606307A
- Authority
- CN
- China
- Prior art keywords
- fap
- myocardial infarction
- alpha
- fibroblast activation
- ischemia
- 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.)
- Granted
Links
- 108010072257 fibroblast activation protein alpha Proteins 0.000 title claims abstract description 29
- 102100023832 Prolyl endopeptidase FAP Human genes 0.000 title claims abstract description 28
- 239000003596 drug target Substances 0.000 title claims abstract description 8
- 208000010125 myocardial infarction Diseases 0.000 claims abstract description 74
- 230000000694 effects Effects 0.000 claims abstract description 50
- 239000003814 drug Substances 0.000 claims abstract description 31
- 206010063837 Reperfusion injury Diseases 0.000 claims abstract description 28
- 208000012947 ischemia reperfusion injury Diseases 0.000 claims abstract description 27
- 230000014509 gene expression Effects 0.000 claims abstract description 24
- 229940079593 drug Drugs 0.000 claims abstract description 22
- 238000012216 screening Methods 0.000 claims abstract description 12
- 108091005804 Peptidases Proteins 0.000 claims abstract description 5
- 239000004365 Protease Substances 0.000 claims abstract description 5
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims abstract description 5
- 239000003112 inhibitor Substances 0.000 claims description 14
- 239000000090 biomarker Substances 0.000 claims description 4
- 238000011282 treatment Methods 0.000 abstract description 44
- 108090000623 proteins and genes Proteins 0.000 abstract description 20
- 230000002401 inhibitory effect Effects 0.000 abstract description 3
- 230000000144 pharmacologic effect Effects 0.000 abstract description 2
- 230000004952 protein activity Effects 0.000 abstract 1
- 241000699670 Mus sp. Species 0.000 description 49
- 101800000407 Brain natriuretic peptide 32 Proteins 0.000 description 32
- 102400000667 Brain natriuretic peptide 32 Human genes 0.000 description 32
- 101800002247 Brain natriuretic peptide 45 Proteins 0.000 description 32
- HPNRHPKXQZSDFX-OAQDCNSJSA-N nesiritide Chemical compound C([C@H]1C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@H](C(N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CSSC[C@@H](C(=O)N1)NC(=O)CNC(=O)[C@H](CO)NC(=O)CNC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](CCSC)NC(=O)[C@H](CCCCN)NC(=O)[C@H]1N(CCC1)C(=O)[C@@H](N)CO)C(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1N=CNC=1)C(O)=O)=O)[C@@H](C)CC)C1=CC=CC=C1 HPNRHPKXQZSDFX-OAQDCNSJSA-N 0.000 description 32
- 230000006870 function Effects 0.000 description 30
- 101500026751 Mus musculus Brain natriuretic peptide 45 Proteins 0.000 description 29
- 230000000747 cardiac effect Effects 0.000 description 29
- 102000004190 Enzymes Human genes 0.000 description 21
- 108090000790 Enzymes Proteins 0.000 description 21
- 241000699666 Mus <mouse, genus> Species 0.000 description 21
- 229940088598 enzyme Drugs 0.000 description 21
- 102000004169 proteins and genes Human genes 0.000 description 15
- 230000033115 angiogenesis Effects 0.000 description 14
- 230000015572 biosynthetic process Effects 0.000 description 14
- 230000002861 ventricular Effects 0.000 description 14
- 238000003209 gene knockout Methods 0.000 description 13
- 238000000034 method Methods 0.000 description 13
- 238000011813 knockout mouse model Methods 0.000 description 12
- 206010016654 Fibrosis Diseases 0.000 description 11
- 210000002950 fibroblast Anatomy 0.000 description 11
- 230000004761 fibrosis Effects 0.000 description 11
- 239000000758 substrate Substances 0.000 description 11
- 230000003205 diastolic effect Effects 0.000 description 10
- 230000005764 inhibitory process Effects 0.000 description 10
- 238000011160 research Methods 0.000 description 10
- 210000002966 serum Anatomy 0.000 description 10
- 102000012422 Collagen Type I Human genes 0.000 description 9
- 108010022452 Collagen Type I Proteins 0.000 description 9
- 238000001976 enzyme digestion Methods 0.000 description 9
- 230000001681 protective effect Effects 0.000 description 9
- 231100000241 scar Toxicity 0.000 description 9
- 208000024172 Cardiovascular disease Diseases 0.000 description 8
- 210000005003 heart tissue Anatomy 0.000 description 8
- 230000001225 therapeutic effect Effects 0.000 description 8
- 206010019280 Heart failures Diseases 0.000 description 7
- 230000033228 biological regulation Effects 0.000 description 7
- 230000004217 heart function Effects 0.000 description 7
- 208000017722 immune hydrops fetalis Diseases 0.000 description 7
- 230000002107 myocardial effect Effects 0.000 description 7
- 230000002829 reductive effect Effects 0.000 description 7
- 238000007634 remodeling Methods 0.000 description 7
- 241000282414 Homo sapiens Species 0.000 description 6
- 210000001054 cardiac fibroblast Anatomy 0.000 description 6
- 230000000302 ischemic effect Effects 0.000 description 6
- 230000005012 migration Effects 0.000 description 6
- 238000013508 migration Methods 0.000 description 6
- 230000008439 repair process Effects 0.000 description 6
- 230000006378 damage Effects 0.000 description 5
- 238000000338 in vitro Methods 0.000 description 5
- 238000011534 incubation Methods 0.000 description 5
- 239000003550 marker Substances 0.000 description 5
- 230000001575 pathological effect Effects 0.000 description 5
- 229920001184 polypeptide Polymers 0.000 description 5
- 108090000765 processed proteins & peptides Proteins 0.000 description 5
- 102000004196 processed proteins & peptides Human genes 0.000 description 5
- 230000001737 promoting effect Effects 0.000 description 5
- 238000010186 staining Methods 0.000 description 5
- 230000035882 stress Effects 0.000 description 5
- 206010061216 Infarction Diseases 0.000 description 4
- UUOZISWTWURDGU-SSDOTTSWSA-N [(2S)-1-(2-acetamidoacetyl)pyrrolidin-2-yl]boronic acid Chemical compound CC(=O)NCC(=O)N1CCC[C@@H]1B(O)O UUOZISWTWURDGU-SSDOTTSWSA-N 0.000 description 4
- 230000008828 contractile function Effects 0.000 description 4
- 230000034994 death Effects 0.000 description 4
- 230000003247 decreasing effect Effects 0.000 description 4
- 230000008021 deposition Effects 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 230000018109 developmental process Effects 0.000 description 4
- 230000012010 growth Effects 0.000 description 4
- 230000007574 infarction Effects 0.000 description 4
- 208000028867 ischemia Diseases 0.000 description 4
- 108020004999 messenger RNA Proteins 0.000 description 4
- 206010028980 Neoplasm Diseases 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 230000001154 acute effect Effects 0.000 description 3
- 230000004071 biological effect Effects 0.000 description 3
- 230000037396 body weight Effects 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 238000003776 cleavage reaction Methods 0.000 description 3
- 229940096422 collagen type i Drugs 0.000 description 3
- 230000008602 contraction Effects 0.000 description 3
- 206010020871 hypertrophic cardiomyopathy Diseases 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- 238000011369 optimal treatment Methods 0.000 description 3
- 210000000056 organ Anatomy 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 230000010410 reperfusion Effects 0.000 description 3
- 230000007017 scission Effects 0.000 description 3
- 150000003384 small molecules Chemical class 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 102000029816 Collagenase Human genes 0.000 description 2
- 108060005980 Collagenase Proteins 0.000 description 2
- 108090001081 Dipeptidases Proteins 0.000 description 2
- 102000004860 Dipeptidases Human genes 0.000 description 2
- 108010016626 Dipeptides Proteins 0.000 description 2
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 2
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 101710151321 Melanostatin Proteins 0.000 description 2
- 102400000064 Neuropeptide Y Human genes 0.000 description 2
- 208000006117 ST-elevation myocardial infarction Diseases 0.000 description 2
- 208000033774 Ventricular Remodeling Diseases 0.000 description 2
- 208000027418 Wounds and injury Diseases 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 206010000891 acute myocardial infarction Diseases 0.000 description 2
- 230000000996 additive effect Effects 0.000 description 2
- 150000001413 amino acids Chemical group 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004413 cardiac myocyte Anatomy 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 229960002424 collagenase Drugs 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 210000002889 endothelial cell Anatomy 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000003197 gene knockdown Methods 0.000 description 2
- 208000019622 heart disease Diseases 0.000 description 2
- 238000003125 immunofluorescent labeling Methods 0.000 description 2
- 208000014674 injury Diseases 0.000 description 2
- 208000031225 myocardial ischemia Diseases 0.000 description 2
- URPYMXQQVHTUDU-OFGSCBOVSA-N nucleopeptide y Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(N)=O)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CCCCN)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@@H](N)CC=1C=CC(O)=CC=1)C1=CC=C(O)C=C1 URPYMXQQVHTUDU-OFGSCBOVSA-N 0.000 description 2
- 230000001991 pathophysiological effect Effects 0.000 description 2
- 238000004393 prognosis Methods 0.000 description 2
- 230000000250 revascularization Effects 0.000 description 2
- -1 small molecule compounds Chemical class 0.000 description 2
- 238000002604 ultrasonography Methods 0.000 description 2
- 210000003606 umbilical vein Anatomy 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- ZOOGRGPOEVQQDX-UUOKFMHZSA-N 3',5'-cyclic GMP Chemical compound C([C@H]1O2)OP(O)(=O)O[C@H]1[C@@H](O)[C@@H]2N1C(N=C(NC2=O)N)=C2N=C1 ZOOGRGPOEVQQDX-UUOKFMHZSA-N 0.000 description 1
- 206010067484 Adverse reaction Diseases 0.000 description 1
- 206010065687 Bone loss Diseases 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- COXVTLYNGOIATD-HVMBLDELSA-N CC1=C(C=CC(=C1)C1=CC(C)=C(C=C1)\N=N\C1=C(O)C2=C(N)C(=CC(=C2C=C1)S(O)(=O)=O)S(O)(=O)=O)\N=N\C1=CC=C2C(=CC(=C(N)C2=C1O)S(O)(=O)=O)S(O)(=O)=O Chemical compound CC1=C(C=CC(=C1)C1=CC(C)=C(C=C1)\N=N\C1=C(O)C2=C(N)C(=CC(=C2C=C1)S(O)(=O)=O)S(O)(=O)=O)\N=N\C1=CC=C2C(=CC(=C(N)C2=C1O)S(O)(=O)=O)S(O)(=O)=O COXVTLYNGOIATD-HVMBLDELSA-N 0.000 description 1
- 108010077544 Chromatin Proteins 0.000 description 1
- 208000032544 Cicatrix Diseases 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 206010056370 Congestive cardiomyopathy Diseases 0.000 description 1
- 206010052337 Diastolic dysfunction Diseases 0.000 description 1
- 201000010046 Dilated cardiomyopathy Diseases 0.000 description 1
- 108010059378 Endopeptidases Proteins 0.000 description 1
- 102000005593 Endopeptidases Human genes 0.000 description 1
- 229940122586 Enkephalinase inhibitor Drugs 0.000 description 1
- 229940121772 Fibroblast activation protein inhibitor Drugs 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 206010018691 Granuloma Diseases 0.000 description 1
- 208000000435 Heart Rupture Diseases 0.000 description 1
- 208000013875 Heart injury Diseases 0.000 description 1
- 206010019668 Hepatic fibrosis Diseases 0.000 description 1
- 101000783712 Homo sapiens Alpha-2-antiplasmin Proteins 0.000 description 1
- 101500026735 Homo sapiens Brain natriuretic peptide 32 Proteins 0.000 description 1
- 238000012404 In vitro experiment Methods 0.000 description 1
- 101150018665 MAPK3 gene Proteins 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 206010028594 Myocardial fibrosis Diseases 0.000 description 1
- 206010028851 Necrosis Diseases 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- 108091008611 Protein Kinase B Proteins 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 108091006088 activator proteins Proteins 0.000 description 1
- 208000019269 advanced heart failure Diseases 0.000 description 1
- 230000006838 adverse reaction Effects 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 102000003801 alpha-2-Antiplasmin Human genes 0.000 description 1
- 108090000183 alpha-2-Antiplasmin Proteins 0.000 description 1
- 208000007502 anemia Diseases 0.000 description 1
- 230000000702 anti-platelet effect Effects 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 102100022422 cGMP-dependent protein kinase 1 Human genes 0.000 description 1
- 101710143429 cGMP-dependent protein kinase 1 Proteins 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000009787 cardiac fibrosis Effects 0.000 description 1
- 230000003293 cardioprotective effect Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 210000003483 chromatin Anatomy 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 239000000084 colloidal system Substances 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000002792 enkephalinase inhibitor Substances 0.000 description 1
- 229960003699 evans blue Drugs 0.000 description 1
- 230000017188 evasion or tolerance of host immune response Effects 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000035876 healing Effects 0.000 description 1
- 210000002064 heart cell Anatomy 0.000 description 1
- ZASXKEGREHRXDL-CAWNUZPDSA-H hexasodium;4-[[(2s,4r)-5-ethoxy-4-methyl-5-oxo-1-(4-phenylphenyl)pentan-2-yl]amino]-4-oxobutanoate;(2s)-3-methyl-2-[pentanoyl-[[4-[2-(1,2,3-triaza-4-azanidacyclopenta-2,5-dien-5-yl)phenyl]phenyl]methyl]amino]butanoate;pentahydrate Chemical compound O.O.O.O.O.[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].C1=CC(C[C@H](C[C@@H](C)C(=O)OCC)NC(=O)CCC([O-])=O)=CC=C1C1=CC=CC=C1.C1=CC(C[C@H](C[C@@H](C)C(=O)OCC)NC(=O)CCC([O-])=O)=CC=C1C1=CC=CC=C1.C1=CC(CN(C(=O)CCCC)[C@@H](C(C)C)C([O-])=O)=CC=C1C1=CC=CC=C1C1=NN=N[N-]1.C1=CC(CN(C(=O)CCCC)[C@@H](C(C)C)C([O-])=O)=CC=C1C1=CC=CC=C1C1=NN=N[N-]1 ZASXKEGREHRXDL-CAWNUZPDSA-H 0.000 description 1
- 230000001969 hypertrophic effect Effects 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 210000005240 left ventricle Anatomy 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 230000002101 lytic effect Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 238000010603 microCT Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 208000037891 myocardial injury Diseases 0.000 description 1
- 210000004165 myocardium Anatomy 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 230000009437 off-target effect Effects 0.000 description 1
- 201000008482 osteoarthritis Diseases 0.000 description 1
- 230000036542 oxidative stress Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 230000002085 persistent effect Effects 0.000 description 1
- 230000009038 pharmacological inhibition Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000004853 protein function Effects 0.000 description 1
- 230000006337 proteolytic cleavage Effects 0.000 description 1
- 208000005069 pulmonary fibrosis Diseases 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 206010039073 rheumatoid arthritis Diseases 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 230000037387 scars Effects 0.000 description 1
- 238000002603 single-photon emission computed tomography Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000007447 staining method Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- ADNPLDHMAVUMIW-CUZNLEPHSA-N substance P Chemical compound C([C@@H](C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(N)=O)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CCCCN)NC(=O)[C@H]1N(CCC1)C(=O)[C@@H](N)CCCN=C(N)N)C1=CC=CC=C1 ADNPLDHMAVUMIW-CUZNLEPHSA-N 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 210000002303 tibia Anatomy 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 230000004580 weight loss Effects 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
-
- 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
- G01N2800/324—Coronary artery diseases, e.g. angina pectoris, myocardial infarction
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Urology & Nephrology (AREA)
- Analytical Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Immunology (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Physics & Mathematics (AREA)
- Biomedical Technology (AREA)
- Animal Behavior & Ethology (AREA)
- Hematology (AREA)
- Pathology (AREA)
- Pharmacology & Pharmacy (AREA)
- Microbiology (AREA)
- Vascular Medicine (AREA)
- Food Science & Technology (AREA)
- Epidemiology (AREA)
- Cell Biology (AREA)
- General Engineering & Computer Science (AREA)
- Biophysics (AREA)
- General Physics & Mathematics (AREA)
- Cardiology (AREA)
- Heart & Thoracic Surgery (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention provides application of fibroblast activation protein-alpha as a target spot in screening medicines for treating myocardial infarction or ischemia-reperfusion injury. The drug is a drug that inhibits the expression level of fibroblast activation protein-alpha. The drug is a drug that inhibits the activity of fibroblast activation protein-alpha. The invention provides application of gene expression and protein activity of fibroblast activation protein-alpha as a drug target in screening drugs for treating myocardial infarction or ischemia-reperfusion injury. By systematically analyzing the expression condition of the Fap, the invention firstly intervenes the expression of the Fap from a gene level, finds that the Fap can be used as a potential target point for treating myocardial infarction and ischemia-reperfusion injury, and then finds that the protease activity for inhibiting the Fap has good treatment effect in treating myocardial infarction and ischemia-reperfusion injury through a series of pharmacological interventions.
Description
Technical Field
The invention belongs to the field of biological medicine, and relates to application of a drug target in screening and treating a medicine for myocardial infarction, in particular to application of fibroblast activation protein-alpha serving as a target in screening a medicine for treating myocardial infarction or ischemia-reperfusion injury.
Background
Cardiovascular diseases are the first lethal factor in the world at present, and the mortality rate of acute myocardial infarction in China is shown to be in a rapidly rising trend from 2005 according to the summary of Chinese cardiovascular disease report 2019. The treatment measures aiming at the myocardial infarction comprise drug intervention modes such as revascularization, antiplatelet, blood fat control and the like, but the death rate of cardiovascular diseases in China is not reduced by the current comprehensive treatment scheme, and the morbidity and the death rate of the myocardial infarction are still continuously increased year by year along with the aggravation of the aging of population.
Myocardial infarction is one of the most serious and most fatal diseases in cardiovascular diseases, and a large number of mature myocardial cells with contractile ability rapidly die after myocardial infarction, so that the structure and the function of the heart are seriously damaged. Because the proliferation capacity of the cardiac muscle cells is weak, the damaged heart cannot be quickly repaired, so that the fibroblasts play a great role in the injury repair after myocardial infarction. After myocardial infarction, the resident fibroblasts in the heart are stimulated by local necrosis factors, metabolites, inflammatory factors and the like, and are quickly activated and start to proliferate in large quantities, and the activated and proliferated cardiac fibroblasts can secrete a large quantity of extracellular matrix proteins, so that granuloma is formed at the damaged part, angiogenesis is promoted, and finally, fibrous scars are formed, so that the damaged heart is repaired, and the structure and the function of the heart are protected. Fibrosis and angiogenesis play important roles in the repair of myocardial infarction. Excessive fibrosis can lead to diastolic dysfunction, decreased ventricular compliance, leading to advanced heart failure; but insufficient fibrosis may also lead to heart rupture, scar enlargement. Therefore, it is very important to effectively regulate the function of the fibroblast after myocardial infarction.
Fibroblast activation protein-alpha (Fap) is a two-type transmembrane integration glycoprotein located on chromosome II of human, mouse, cattle and sheep, and 89% of the amino acid sequence is shared by human and mouse. Fap mainly exists on a fibroblast membrane, has structures of 6, 18 and 736 amino acids in cytoplasm, transmembrane and extracellular, has collagenase and dipeptide enzyme activity, and can degrade dipeptide, collagen I and the like. Previous studies show that Fap is mainly highly expressed in Cancer Associated Fibroblasts (CAFs), and is widely involved in tumor growth, invasion, metastasis, tumor extracellular matrix reconstruction, angiogenesis, immune escape process, and formation and regulation of tumor microenvironment. The expression of Fap is also closely related to damage repair and the like, and plays an important role in related diseases such as rheumatoid arthritis, osteoarthritis, pulmonary fibrosis, hepatic fibrosis and the like. In 2015, Tillmanns and the like firstly discover that Fap can be used as a marker for activating fibroblasts after myocardial infarction; in 2017, Bauersacs and the like firstly detect the change of the Fap concentration in the peripheral blood of patients with acute ST-elevation myocardial infarction (STEMI), and firstly suggest that the Fap change is related to myocardial injury and inflammatory response and can be used as a biomarker for diagnosing the myocardial infarction. In 2019, Aghajanian et al sequenced heart tissues of 238 normal adults and heart failure patients, found that Fap is a fibroblast marker protein with the highest expression level in heart failure patients with ischemic heart failure, hypertrophic cardiomyopathy, dilated cardiomyopathy and the like, and in 2020, Monika Litvi ň ukov a and the like draw the most comprehensive human heart cell map so far, and the result shows that Fap specifically marks a group of heart fibroblasts. In summary, Fap is mostly used as a surface marker protein of cardiac fibroblasts in previous researches, the action mechanism of the Fap protein in cardiovascular diseases is not clear, and after the Fap-positive cardiac fibroblasts are directionally killed by using a CAR-T method, myocardial fibrosis can be remarkably relieved, the contraction and relaxation functions of the heart are improved, and the important pathophysiological effect of the Fap-marked fibroblasts is suggested, but meanwhile, the CAR-T treatment method also causes serious adverse reactions such as anemia, weight loss, muscle and bone loss and the like, so that how to effectively regulate and control the functions of the Fap-positive fibroblasts is of great importance.
After myocardial infarction, promoting angiogenesis is an important treatment means for promoting myocardial repair after myocardial infarction, preventing excessive cardiac remodeling and correcting heart failure, and Fap is used as a protease and has the activity of dipeptidase and endopeptidase, and the action mechanism of Fap is probably to play a role due to the enzyme digestion of a substrate by Fap. It is therefore not known whether Fap has an important role after myocardial infarction, whether its role is due to its proteolytic cleavage or lytic function, leading to an increase or decrease in the biological protective effect of the protective polypeptide (or inhibitory polypeptide). The previous research shows that the Fap enzyme digestion substrate comprises classical collagen type I, gelatin and the like, and in addition, in the in vitro research, the Fap can also shear human alpha 2-antiplasmin, neuropeptide Y, P substance, polypeptide YY and Brain Natriuretic Peptide (BNP). BNP has strong regulation and control effects on pathological states after heart stress, can further influence pathways such as cGKI, Akt, Erk1/2 and the like by activating cGMP so as to influence angiogenesis, and can reduce the biological activity of BNP to a great extent by the enzyme digestion of Fap on human BNP. However, whether or not the BNP can be cleaved efficiently in vivo, whether or not BNP has similar biological activity after cleavage, and whether or not in vivo Fap inhibition has therapeutic potential are not clear at present. The discovery of the basic research provides a new thought for researching the function of the Fap protein and provides an important theoretical basis for effectively regulating and controlling the function of the Fap protein subsequently.
Disclosure of Invention
Aiming at the technical problems in the prior art, the invention provides application of fibroblast activation protein-alpha as a target spot in screening medicines for treating myocardial infarction or ischemia-reperfusion injury, and the application of the fibroblast activation protein-alpha as the target spot in screening the medicines for treating myocardial infarction or ischemia-reperfusion injury aims to solve the technical problems of poor effect and limited means for treating myocardial infarction or ischemia-reperfusion injury in the prior art.
The invention provides application of fibroblast activation protein-alpha as a target spot in screening medicines for treating myocardial infarction or ischemia-reperfusion injury.
Further, the drug is a drug that inhibits the gene and protein expression level of fibroblast activation protein-alpha.
Further, the drug is a drug that inhibits the protease activity of fibroblast activation protein- α.
The invention provides application of gene expression of fibroblast activation protein-alpha as a drug target in screening drugs for treating myocardial infarction or ischemia-reperfusion injury.
The invention provides application of the protease activity of fibroblast activation protein-alpha as a drug target in screening drugs for treating myocardial infarction or ischemia-reperfusion injury.
The invention provides an application of an inhibitor of targeted fibroblast activation protein-alpha in preparing a medicament for treating myocardial infarction or ischemia-reperfusion injury.
The invention provides an application of targeted fibroblast activation protein-alpha in preparing a biomarker for diagnosis and prognosis judgment of a myocardial infarction patient.
In view of the fact that the death rate cannot be effectively reduced by the existing comprehensive treatment means for cardiovascular diseases, the invention discovers a new treatment target point, is used for improving the treatment means for major cardiovascular diseases such as myocardial infarction, ischemia-reperfusion injury and the like, and improves the effective rate and success rate of treatment so as to reduce the death rate of the cardiovascular diseases.
The application method of the target inhibition fibroblast activation protein-alpha (Fap) in the treatment of myocardial infarction mainly comprises the following steps:
fap is highly expressed in situ in adult pathologic cardiac remodeling, the concentration of free Fap in serum is reduced, and the concentration of free Fap in serum has good correlation with indexes such as cardiac function, cardiac injury and the like, so that Fap can be used as a biomarker to diagnose and prognosticate pathologic cardiac remodeling, particularly myocardial infarction patients.
The invention intervenes the function of the Fap protein by various methods, specifically comprises gene-level knockout, knockdown and pharmacological-level inhibition, and researches show that the intervention of the expression and the function of the Fap can effectively play a good role in treating myocardial infarction and ischemia-reperfusion injury.
Fap inhibition protects against post-stress cardiac function primarily because its enzymatic activity is inhibited, resulting in protective polypeptides that function without being cleared. The BNP is a novel Fap endogenous substrate, a plurality of substrates discovered in the past, such as type I collagen, alpha 2-antiplasmin, neuropeptide Y, P substances and the like, can be expressed and changed after heart stress injury, and the intervention of the Fap function can influence the activity of the substrate to play a potential protective role, so the Fap is an important intervention target discovered in the invention.
Compared with the prior art, the invention has remarkable technical progress. The invention provides a brand-new intervention target for treating myocardial infarction and ischemia-reperfusion injury, wherein the intervention target is Fibroblast activation protein alpha (Fap), and the intervention mode is mainly to knock down the expression and inhibit the protein function of the Fibroblast activation protein alpha. A series of in vivo and in vitro experiments show that a new endogenous substrate of the Fap, namely BNP, is discovered, and the Fap inhibitor further defines the potential therapeutic action by comparing with the marketed positive drugs, has stronger transformation potential to a certain extent, can effectively protect cardiac remodeling and functional damage after myocardial infarction and ischemia-reperfusion injury, and promotes angiogenesis in the infarct area.
Drawings
FIG. 1 shows the expression of Fap in adults and mice in example 1 and its correlation with prognosis.
FIG. 2 shows the effect of the Fap gene knock-out in example 2 on the growth and development and cardiac function of mice under steady state conditions.
FIG. 3 is a graph showing the protective effect of the Fap gene knockout on cardiac function and cardiac structure after myocardial infarction in example 2.
FIG. 4 shows the therapeutic effect of the small molecule compounds in example 3 on the inhibition of Fap.
FIG. 5 shows the Fap inhibitor specificity assay in example 4.
FIG. 6 shows the therapeutic effect of Fap intervention on ischemia reperfusion injury in example 5.
FIG. 7 shows the effect of Fap intervention on type I collagen and angiogenesis in example 6.
FIG. 8 shows the in vitro validation of the cleavage of Fap with BNP in example 7.
FIG. 9 shows the effect of BNP on HUVECs tube formation in example 8.
FIG. 10 shows the effect of Fap on BNP to promote tube formation and migration of HUVECs in example 8.
Fig. 11 shows the therapeutic effect of FAPi on substrate BNP knockout mice in example 9 compared to the positive drug LCZ 696.
Detailed Description
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments, and is not intended to limit the scope of the present invention; in the description and claims of the present application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.
When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.
Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts. These techniques are well described in the literature.
Fibroblast activator protein Fap
Fibroblast activation protein inhibitor FAPi
BNP for brain natriuretic peptide abbreviation
HUVECs for human umbilical vein endothelial cell abbreviation
Acute myocardial infarction abbreviation AMI
Ischemic heart failure abbreviation IHF
HCM for hypertrophic cardiomyopathy
Ejection fraction abbreviation EF
Short axis shrinkage abbreviation FS
Left ventricular end diastolic diameter abbreviated LVID, D
Left ventricular end diastolic volume abbreviation LVEDV
End diastolic Chamber Interval thickness abbreviation IVS, D
LVPW for posterior wall thickness of the left ventricle at end diastole, D.
Example 1 confirmation of high expression of Fap in pathologically remodeled hearts in adults and mice
First, we collected heart tissues of healthy donors, patients with Ischemic heart disease and patients with hypertrophic cardiomyopathy, and analyzed the mRNA and protein levels of Fap in heart tissues, and the results showed that Fap was in Ischemic heart failure (Ischemic heart failure) compared to healthy donorsre, IHF), and Hypertrophic heart disease (HCM) patients showed significantly increased mRNA and protein expression in myocardial tissues (fig. 1A-1C), and adult myocardial tissues were stained with Masson and Fap immunofluorescence to show significantly increased cardiac fibrosis and significantly increased Fap expression in IHF and HCM patients (fig. 1D-1G). Subsequently, we examined the serum of normal subjects, AMI patients and IHF patients, and then determined the content of free Fap in the serum, and found that the serum free Fap content of the AMI patients and the serum free Fap content of the IHF patients are reduced compared with the normal subjects, and at the same time, the activity of the Fap enzyme in the serum of the AMI patients and the IHF patients is also significantly reduced (FIGS. 1H-1I). After linear regression analysis of the concentration of free Fap in serum and the EF value of the systolic function index of patients, we found that the concentration of free Fap in serum was positively correlated with the EF values of AMI and IHF patients (FIGS. 1J-1K). Subsequently, we divided AMI patients into high-concentration Fap and low-concentration Fap groups by median serum free Fap concentration, and after D-SPECT examination of all patients, it was found that patients in the low-concentration Fap group had higher ischemia scores (SRS score, SSS score) and greater Total reperfusion defects (TPD%) at rest and load conditions (fig. 1L-fig. 1O). To further study the function of Fap, we also observed the expression of Fap after myocardial infarction in mice, and the results showed that the infarct zone was localized after myocardial infarction in miceFapThe expression level of mRNA gradually increased at an early stage to reach a peak 7 days after myocardial infarction, and thenFapThe mRNA expression level gradually decreased and almost decreased to a normal level by 28 days (FIG. 1P). Since the previous research shows that Fap is the surface marker protein of activated cardiac fibroblasts, we found that Fap and alpha-SMA are both significantly highly expressed in the cardiac tissue 7 days after myocardial infarction of mice by co-staining with the previously recognized surface marker protein alpha-SMA of activated cardiac fibroblasts, and that the two are more co-localized (FIG. 1Q), suggesting that the expression level of Fap is increased after myocardial infarction, and a group of specific activated cardiac fibroblasts are marked.
Example 2 utilization ofFapFunctional verification of Fap by knockout mice
To further explore the specific function of Fap after myocardial infarction, we purchased using Jackson labFapKnockout mice (Fap -/- ) And performing subsequent function verification. First, we analyzeFapThe influence of gene knockout on the growth and development of mice under normal conditions is found inFapAfter the gene is knocked out, the weight, organ weight and tibia length of the mouse have no obvious difference (figure 2A-2F), and the suggestion is that Fap-/-The growth and development of the mice are not affected. Subsequently, we treated 8 weeksFap -/- The mice were Masson stained for cardiac sections and found to be in steady stateFapThe degree of interstitial fibrosis in myocardium was not significantly different in mice expressing normal expression compared to the two (FIGS. 2G-2H). At the same time, we are right toFap -/- As a result of WGA staining of cardiac sections of mice and control mice, no significant difference was observed in the sizes of cardiomyocytes between the mice and the control mice under the steady-state condition (FIGS. 2I to 2J). In terms of cardiac structure and function, we found by cardiac ultrasonography that both had similar diastolic ventricular septal thickness (IVS; D), diastolic left ventricular posterior wall thickness (LVPW; D), cardiac Ejection Fraction (EF), short-axis contraction rate (FS), and end-diastolic left ventricular volume (LVEDV) (FIGS. 2K-2O). Therefore, the fact that the knockout Fap gene has no obvious influence on the growth and development of mice, the degree of myocardial interstitial fibrosis, the size of myocardial cells, the cardiac structure and the function under the steady state is determined.
To further investigate whether Fap has biological function under stress conditions, we utilizedFap -/- Mice and permanently ligated anterior descending myocardial infarction model, pairFapThe function is explored, and the result shows that the myocardial infarction of the miceFapThe gene knockout can effectively slow down the ventricular remodeling and the cardiac hypofunction after the myocardial infarction of the mice, and the cardiac ultrasonic results show that,Fap -/- the mice had better left ventricular contraction function 1, 3, 7, 14 days after myocardial infarction, EF and FS were both significantly higher than the control group (FIGS. 3A-3C), and at the same time,Fap -/- the mice also had lower left ventricular end diastolic volume, smaller heart/body weightThe ratio, 7 days after myocardial infarction,Fap -/- mouse IVS; D, LVPW; D is higher, and these results all show thatFapThe heart reconstruction of the mouse is less after gene knockout,Fapknock-out is effective in protecting cardiac structure and function (fig. 3D-3F, 3K). In order to better observe the scar and fibrosis condition of mice after myocardial infarction, the heart tissues 7 days after myocardial infarction of two groups of mice are subjected to section staining, and the result shows thatFap -/- The overall scar area was smaller and the degree of interstitial fibrosis was higher in the mice (FIGS. 3G-3J). In order to fully evaluate the size of scar tissue after myocardial infarction of mice as much as possible, the hearts 7 days after myocardial infarction of two groups of mice are taken, fixed by 4% PFA, soaked in 25% lugol's solution for 24 hours and then subjected to Micro-CT scanning, and the results show that, from the whole,Fap -/- mice also had smaller overall scar volume (fig. 3L-3M), and therefore, we believe that the Fap gene knockout exerts a protective effect after myocardial infarction, resulting in better pathological remodeling of the heart and better scar repair in the acute phase, resulting in better cardiac contractile function of the mice after myocardial infarction.
Example 3 pharmacological inhibition of Fap enzyme Activity is effective in protecting cardiac architecture and function in mice after myocardial infarction
Fap has collagenase and dipeptidase activities, and to further investigate whether Fap's cardiac regulation function depends on enzyme activity, we intervene in Fap enzyme activity using Fap enzyme activity specific small molecule compound inhibitor (FAPi) Ac-Gly-Boropror (an acetylated glycine borated proline, purchased from MCE (MedChemexpress) Inc., Cat. No.: 886992-99-0), to verify whether Fap enzyme activity inhibitor has protection similar to Fap gene knock-out.
Firstly, we test the dose curve of FAPi, and according to the enzyme activity Ki value of Ac-Gly-Boropro, we select 0.15 mg/kg, 0.5 mg/kg and 1.5 mg/kg as 3 treatment dose groups, fix the treatment time for 7 days, administer the daily intraperitoneal injection mode to the mice after myocardial infarction, and analyze the mortality, cardiac structure and function, histology, organ weight and other multiple layers of the mice (the specific experimental flow is as shown in fig. 4A), and as a result, the 0.5 mg/kg FAPi treatment dose group can effectively reduce the mortality of the mice after myocardial infarction (fig. 4B), and by measuring the cardiac contractile function, we find that the EF value of the mice in the 0.5 mg/kg treatment group is higher (fig. 4C). Then we sliced and stained the mouse hearts 7 days after FAPi treatment, and found that at the level close to the ligation site, there was no significant difference in scar area between the vehicle-treated group and the 0.5 mg/kg FAPi-treated group, but the FAPi-treated group had more interstitial fibrosis, and the heart/body weight ratio after FAPi treatment was also significantly decreased compared to the control group (fig. 4D-4G), suggesting that the heart reconstruction was less after FAPi treatment, and the optimal treatment effect was achieved by 0.5 mg/kg FAPi treatment.
Subsequently, we further tested the time curve of FAPi treatment, and in the case of determining the optimal treatment dose, we determined the treatment dose to be 0.5 mg/kg, and the treatment time to be respectively set at 7 days, 14 days and 28 days of treatment, and collected the cardiac structure and function data, histological data, organ weight data, etc. of the mouse (the specific experimental process is shown in fig. 4H), and as a result, it was found that FAPi treatment for 7 days can effectively protect the systolic function, the proportion of mouse cardiac remodeling is greatly reduced with the increase of the treatment time, and the left ventricular end diastolic volume of the mouse is significantly reduced compared with vehicle after 14 days and 28 days of treatment (fig. 4J). We then further analyzed the extent of mouse cardiac interstitial fibrosis at various time points of FAPi treatment, and the results showed that the extent of mouse cardiac interstitial fibrosis gradually increased with treatment time (fig. 4K-4L).
Example 4 FAPi acts in a Fap enzyme activity-dependent manner and specifically inhibits Fap enzyme activity
To further exclude that the Fap inhibitor Ac-Gly-Boropro exerts cardioprotective effects after myocardial infarction in a manner independent of Fap enzyme activity, we further selected another small molecule compound SP-13786 (purchased from MCE (MedChemExpress) company, Cat. No.: 1448440-52-5) with completely different action mechanism from Ac-Gly-Boropro as a new Fap enzyme activity inhibitor and treated mice myocardial infarction model, and found that SP-13786 treatment for 7 days also effectively protected the systolic function of mice, and the EF and FS of SP-13786 treatment group mice were higher (FIGS. 5A-5C), and inhibited the pathological remodeling of the mouse heart after myocardial infarction, which was shown as smaller left ventricular end-diastolic volume of SP-13786 treatment group mice (FIG. 5D). The novel inhibitor SP-13786 can also play an effective protective role after myocardial infarction, and the inhibition of the activity of the Fap enzyme is suggested to have potential therapeutic value.
To further exclude that small molecule inhibitors may non-specifically target and inhibit the enzyme activity of other enzymes when inhibiting the Fap enzyme activity, we therefore further used small molecule compoundsFap -/- The specificity of a small molecular compound inhibitor is detected in a mouse, and after 7 days of FAPi treatment, the FAPi treatment group can effectively protect the cardiac contractile function of the mouse and improve the rational reconstruction of the heart disease after the wild mouse myocardial infarction, but the FAPi treatment is carried outFap -/- There was no additive effect in mice (fig. 5E-5H), suggesting that FAPi specifically acts on Fap enzymatic activity to exert a therapeutic effect, and there was no off-target effect of the inhibitor.
Example 5FapGene knockout and inhibition of Fap enzyme activity may also play a protective role in ischemia reperfusion injury
Ischemia reperfusion injury due to rapid revascularization after myocardial infarction is more common in clinical practice. Different from persistent ischemia caused by permanent ligation, more damages such as oxidative stress and the like can be caused by blood reperfusion in the ischemia reperfusion process, and in order to further simulate the actual conditions encountered in clinical treatment, we further explored whether Fap gene knockout and Fap enzyme activity inhibition can also play a protective role in a mouse myocardial ischemia reperfusion injury model. Firstly, by an Evans blue/TTC staining method, the influence of Fap gene knockout and FAPi treatment on an ischemia danger area and an infarct area 72 hours after ischemia-reperfusion injury is researched, and the result shows that the Fap gene knockout and the FAPi treatment can reduce the ischemia-reperfusion injury area of a mouse and show that compared with a control group,Fap -/- group and FAPi therapyThe group mice had smaller ratios of ischemic risk zone/left ventricular area, infarct area/ischemic risk zone area (fig. 6A-6D). At the same time, we also observe by cardiac ultrasoundFap -/- The change conditions of the heart function and the heart structure of the mice in the group and FAPi treatment group are shown by the resultsFap -/- Mice in the group and FAPi treated groups had better contractile function at the time points of 7 days and 28 days, both EF values and FS were significantly elevated, ventricular remodeling was less in both groups of mice, and left ventricular end diastolic volume was less (fig. 6E-6H). We then performed microtome Masson staining of heart tissue from three groups of mice, which showed that 7 days after ischemia reperfusion injury,Fap -/- both group and FAPi treated group showed more myocardial interstitial fibrosis, suggesting stronger cardiac scar repair (fig. 6I-6J). Finally, we analyzed the ratio of heart weight to body weight of mice, suggesting that after 7 and 28 days of ischemia-reperfusion injury,Fap -/- both cohort and FAPi treatment cohort had smaller heart weight/body weight ratio (fig. 6K).
Example 6 Fap Gene knockout and Fap enzyme Activity inhibition promote type I collagen deposition and angiogenesis
In the acute phase of myocardial infarction, ECM deposition and angiogenesis such as collagen play an important role in promoting cardiac function recovery and protecting cardiac structure and function, and because Fap can effectively degrade type I collagen, the method further researches whether Fap gene knockout and Fap enzyme activity inhibition can influence type I collagen deposition and angiogenesis at the edge of myocardial infarction of mice after myocardial infarction. After immunofluorescent staining was performed on heart sections of wild-type mice, Fap knockout mice and FAPi-treated mice, we found that both collagen type I deposition and angiogenesis in the myocardial infarction margin region of Fap knockout mice and FAPi-treated mice increased significantly, suggesting that Fap affected collagen type I and angiogenesis after myocardial infarction (fig. 7A-7D).
Example 7 determination that Fap can effectively cleave mouse BNP
BNP plays an important role in regulation after various pathophysiological stresses of heart, and previous researches show that Fap can perform regulation on human-derived BNPThe effective enzyme digestion is carried out, thereby reducing the biological activity of BNP and leading the BNP to lose the heart regulation and control function after stress. However, it is unknown whether mouse BNP can be effectively digested by Fap, and in order to further explore the digestion effect of Fap on mouse BNP, we firstly carried out,FapThe results of immunofluorescence staining of heart sections of knockout mice and FAPi treated mice show thatFapThe expression of BNP in the myocardial infarction marginal zone in a heart slice of a knockout mouse and a FAPi-treated mouse is obviously increased, which indicates that the BNP is possibly an endogenous substrate of the Fap, and then we further verify the expression amount of the BNP in the marginal zone heart tissue after the myocardial infarction of the three groups of mice through western blot, and the result shows that the BNP is expressedFapThe BNP expression level in heart tissues of knockout mice and FAPi treated mice was significantly increased (FIGS. 8A-8D). Therefore, to further verify the enzyme digestion of the Fap to the mouse BNP, we incubated two proteins and polypeptides in vitro, firstly, the dosage of the Fap is fixed, it is found that in a 10 ul enzyme digestion system, 10 ng of recombinant Fap protein can effectively enzyme-digest the mouse BNP after 48 hours, it can be found that the mouse BNP band fades and disappears after 48 hours of co-incubation through colloid blue staining (fig. 8E-8F), then we tested the enzyme digestion condition of the mouse BNP within 24 hours by different dosages of Fap, and as a result, the enzyme digestion of the mouse BNP gradually increases with the increase of the dosage of the Fap, and the Fap protein is pretreated by the Fap inhibitor and then incubated with the mouse BNP for 24 hours, and the enzyme digestion of the Fap to the mouse BNP disappears (fig. 8G-8H), which suggests that the Fap can effectively enzyme-digest the mouse BNP.
Example 8 in vitro angiogenesis promoting effects of mouse BNP can be blocked by Fap
To further explore the effect of cleavage of BNP by Fap on angiogenesis, we investigated the effect of mouse BNP on the tube forming ability of HUVECs using Human Umbilical Vein Endothelial Cells (HUVECs). First, we tested the effect of different concentrations of mouse BNP on the ability of HUVECs to promote tube formation, we set four dose groups of vehicle, 1 nM, 10 nM, and 100 nM, add mouse BNP to the culture medium of HUVECs, and analyze the effect of mouse BNP on the ability of HUVECs to promote tube formation after 16 hours, compared with vehicle1, the 1 nM and 10 nM dose groups can both significantly promote the ability of HUVECs to tube, the 1 nM and 10 nM dose groups can tube more HUVECs, the total cavity area is larger, the total branch length is longer, and the 10 nM dose group has better effect (fig. 9A-9B). Subsequently, we further tested the effect of 10 nM mouse BNP on HUVECs at different time points of tube formation, and found that mouse BNP significantly promoted tube formation of HUVECs at 8 hours, 16 hours and 24 hours, and that mouse BNP-treated groups had more junctions, more lacunae formation, larger total lacunae area and longer total branch length, compared to vehicle, with the 16-hour two groups having the most significant difference (fig. 9C-9G).
After the optimal dose and the optimal treatment time are determined, 10 nM mouse BNP is selected to treat HUVECs for 16 hours, vehicle, recombinant Fap protein, mouse BNP and the influence of co-incubation of mouse BNP and Fap on HUVECs tube formation are observed, and research results show that, compared with vehicle, Fap has no obvious influence on HUVECs tube formation, the mouse BNP can effectively promote HUVECs tube formation, the tube formation promoting effect disappears after the Fap and the mouse are co-incubated, and the co-incubation has no influence on the HUVECs tube formation (FIGS. 10A-10B).
In addition to the effect of the tube-forming ability of HUVECs, the migration ability also has an important effect on the formation of blood vessels, so we further explored the effect of mouse BNP on the tube-forming ability of HUVECs. Through a transwell experimental system, we found that the recombinant Fap protein and 10 nM mouse BNP can effectively promote the migration of HUVECs, but the ability of Fap to promote the migration of HUVECs is lost after co-incubation with mouse BNP (FIGS. 10C-10D). In order to further clarify the influence of Fap and mouse BNP on the migration capability of HUVECs, we further verified the results of transwell by using scratch experiments, and consistent with the results of transwell, Fap and mouse BNP alone can promote the healing of scratches, but the promotion capability of Fap and mouse BNP disappears after co-incubation (fig. 10E-10F), further verifying the regulation of the BNP on the migration capability of HUVECs, and suggesting that mouse BNP is an in vitro substrate of Fap.
Example 9 determination of FAPi treatment inefficiency in BNP knock-out mice
In order to further verify that BNP is an endogenous substrate of Fap, a substrate-BNP knockout mouse is subjected to myocardial infarction modeling, and FAP enzyme activity inhibitor treatment is given to determine the treatment effect of FAPi in the BNP knockout mouse (the specific experimental flow is shown in fig. 11A), the research result shows that FAPi can obviously improve the cardiac structure and the systolic function of a wild type mouse, and a single FAPi treatment group mouse has higher EF and FS, smaller left ventricular end diastolic inner diameter and left ventricular end diastolic volume, but has no obvious treatment effect after being treated with FAPi for 7 days in the BNP knockout mouse (fig. 11B-11F). Subsequently, the therapeutic effect of the marketed enkephalinase inhibitor LCZ696 capable of cleaving BNP is used as a positive drug and is compared with that of FAPi in parallel (FIG. 11G), and the result shows that FAPi and LCZ696 can both protect the cardiac function and cardiac structure of mice after myocardial infarction, and the combined treatment of FAPi and LCZ696 has no additive effect (FIGS. 11H-11L), further suggesting that the two acts on a common target point, namely BNP, and providing more powerful evidence for BNP to be an endogenous substrate of Fap.
Claims (7)
1. Application of fibroblast activation protein-alpha as a target spot in screening of drugs for treating myocardial infarction or ischemia-reperfusion injury.
2. The use according to claim 1, wherein the medicament is a medicament that inhibits the expression level of fibroblast activation protein-alpha.
3. The use according to claim 1, wherein the medicament is a medicament that inhibits the activity of fibroblast activation protein-alpha.
4. The application of the gene expression of the fibroblast activation protein-alpha as a drug target in screening drugs for treating myocardial infarction or ischemia-reperfusion injury.
5. The application of the protease activity of the fibroblast activation protein-alpha as a drug target in screening drugs for treating myocardial infarction or ischemia-reperfusion injury.
6. Application of an inhibitor of targeted fibroblast activation protein-alpha in preparing a medicament for treating myocardial infarction or ischemia-reperfusion injury.
7. Application of fibroblast activation protein-alpha in preparing a biomarker for diagnosing or prognostically judging a patient with myocardial infarction.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011426974 | 2020-12-09 | ||
| CN2020114269748 | 2020-12-09 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114606307A true CN114606307A (en) | 2022-06-10 |
| CN114606307B CN114606307B (en) | 2023-11-21 |
Family
ID=81856846
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011519396.2A Active CN114606307B (en) | 2020-12-09 | 2020-12-21 | Application of fibroblast activation protein-alpha as drug target |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114606307B (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090010919A1 (en) * | 2004-07-29 | 2009-01-08 | Bayer Healthcare Ag | Diagnostics and Therapeutics for Diseases Associated With Fibroblast Activation Protein (Fap) |
-
2020
- 2020-12-21 CN CN202011519396.2A patent/CN114606307B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090010919A1 (en) * | 2004-07-29 | 2009-01-08 | Bayer Healthcare Ag | Diagnostics and Therapeutics for Diseases Associated With Fibroblast Activation Protein (Fap) |
Non-Patent Citations (4)
| Title |
|---|
| JOCHEN TILLMANNS等: "Changes in concentrations of circulating fibroblast activation protein alpha are associated with myocardial damage in patients with acute ST-elevation MI", INT J CARDIOL, vol. 232, pages 155 - 159, XP029920889, DOI: 10.1016/j.ijcard.2017.01.037 * |
| JOCHEN TILLMANNS等: "Fibroblast activation protein alpha expression identifies activated fibroblasts after myocardial infarction", JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY, vol. 87 * |
| 廉虹等: "成纤维细胞激活蛋白在小鼠心脏组织中时程表达变化及内肽酶活性测定", 中国分子心脏病学杂志, no. 03, pages 60 - 63 * |
| 袁翔天等: "急性心肌缺血猝死心肌中FAPα和TGF-β1的表达", 法医学杂志, vol. 28, no. 1, pages 18 - 20 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114606307B (en) | 2023-11-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Beninati et al. | The transglutaminase family: an overview: minireview article | |
| Devi et al. | Aggravated postinfarct heart failure in type 2 diabetes is associated with impaired mitophagy and exaggerated inflammasome activation | |
| Ueda et al. | Administration of pigment epithelium-derived factor inhibits left ventricular remodeling and improves cardiac function in rats with acute myocardial infarction | |
| RU2756313C2 (en) | Modified rna encoding vegf-a polypeptides, compositions containing it, and their application methods | |
| US20150329867A1 (en) | Pharmaceutical Compositions and Diagnostic Methods for Inflammatory Skin Diseases, Disorders or Conditions | |
| Mani et al. | Calpain inhibition preserves myocardial structure and function following myocardial infarction | |
| Christakopoulos et al. | Proteomics reveals a set of highly enriched proteins in epiretinal membrane compared with inner limiting membrane | |
| Wang et al. | MicroRNA-29b upregulation improves myocardial fibrosis and cardiac function in myocardial infarction rats through targeting SH2B3. | |
| Lodhi et al. | Relevance and perspectives of experimental wound models in wound healing research | |
| Zhao et al. | Effect of lncRNA GAS5 on the apoptosis of neurons via the notch1 signaling pathway in rats with cerebral infarction. | |
| CN112472690B (en) | Method for preparing compound or biological medicine for enhancing CNPase activity for treating heart diseases | |
| Gambichler et al. | A brief literature update on mid-dermal elastolysis with an emphasis on pathogenetic and therapeutic aspects | |
| Ma et al. | Cardiac extracellular matrix tenascin-C deposition during fibronectin degradation | |
| CN109223818A (en) | Application of the miR-3158-3p in preparation diagnosis, prevention and/or treatment atopic dermatitis product | |
| CN117563017A (en) | Application of NSun2 gene or coded protein thereof in treating pressure-loaded heart failure | |
| CN114606307A (en) | Application of fibroblast activation protein-alpha as drug target | |
| Cui et al. | Platelet endothelial aggregation receptor-1 (PEAR1) is involved in C2C12 myoblast differentiation | |
| Tatsukawa et al. | Distribution of transglutaminase family members in mouse whole body sections | |
| Yin et al. | Knocking down PFL can improve myocardial ischemia/reperfusion injury in rats by up-regulating heat shock protein-20. | |
| CN111214660B (en) | Application of PAX4 gene expression inhibitor in preparation of medicine for inhibiting fibrosis | |
| Yu et al. | Piezo1 is the cardiac mechanosensor that initiates the hypertrophic response to pressure overload | |
| CN109810981A (en) | Polynucleotide and its application in cardiovascular disease diagnosis and treatment | |
| WO2019051117A1 (en) | Hippo pathway deficiency reverses systolic heart failure post-infarction | |
| Li et al. | The effect of YiQiFuMai on ischemic heart failure by improve myocardial microcirculation and increase eNOS and VEGF expression | |
| Foote et al. | Oxidative DNA damage promotes vascular ageing associated with changes in extracellular matrix-regulating proteins |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |