WO2021203141A1 - Full-length cilp as a biomarker for cardiac fibrosis - Google Patents
Full-length cilp as a biomarker for cardiac fibrosis Download PDFInfo
- Publication number
- WO2021203141A1 WO2021203141A1 PCT/US2021/070349 US2021070349W WO2021203141A1 WO 2021203141 A1 WO2021203141 A1 WO 2021203141A1 US 2021070349 W US2021070349 W US 2021070349W WO 2021203141 A1 WO2021203141 A1 WO 2021203141A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- cilp
- fibrosis
- subject
- cardiac fibrosis
- heart failure
- Prior art date
Links
- 230000009787 cardiac fibrosis Effects 0.000 title claims abstract description 87
- 239000000090 biomarker Substances 0.000 title claims description 52
- 102100026622 Cartilage intermediate layer protein 1 Human genes 0.000 claims abstract description 153
- 206010019280 Heart failures Diseases 0.000 claims abstract description 74
- 238000000034 method Methods 0.000 claims abstract description 49
- 238000011282 treatment Methods 0.000 claims abstract description 47
- 206010016654 Fibrosis Diseases 0.000 claims abstract description 39
- 230000004761 fibrosis Effects 0.000 claims abstract description 39
- 239000002773 nucleotide Substances 0.000 claims abstract description 28
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 28
- 101710181683 Cartilage intermediate layer protein 1 Proteins 0.000 claims abstract description 21
- 239000011230 binding agent Substances 0.000 claims abstract description 21
- 239000002243 precursor Substances 0.000 claims abstract description 21
- 238000003776 cleavage reaction Methods 0.000 claims abstract description 17
- 230000007017 scission Effects 0.000 claims abstract description 17
- 238000012544 monitoring process Methods 0.000 claims abstract description 9
- 201000010099 disease Diseases 0.000 claims abstract description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 5
- 208000032170 Congenital Abnormalities Diseases 0.000 claims abstract description 4
- 208000031856 Haemosiderosis Diseases 0.000 claims abstract description 4
- 206010002022 amyloidosis Diseases 0.000 claims abstract description 4
- 208000022368 idiopathic cardiomyopathy Diseases 0.000 claims abstract description 4
- 208000028867 ischemia Diseases 0.000 claims abstract description 4
- 102100027473 Cartilage oligomeric matrix protein Human genes 0.000 claims description 59
- 101710176668 Cartilage oligomeric matrix protein Proteins 0.000 claims description 59
- 101001054649 Homo sapiens Latent-transforming growth factor beta-binding protein 2 Proteins 0.000 claims description 56
- 101001054646 Homo sapiens Latent-transforming growth factor beta-binding protein 3 Proteins 0.000 claims description 56
- 102100027017 Latent-transforming growth factor beta-binding protein 2 Human genes 0.000 claims description 56
- 239000000523 sample Substances 0.000 claims description 50
- 210000002966 serum Anatomy 0.000 claims description 38
- 230000003247 decreasing effect Effects 0.000 claims description 25
- 239000013074 reference sample Substances 0.000 claims description 22
- 102000015427 Angiotensins Human genes 0.000 claims description 8
- 108010064733 Angiotensins Proteins 0.000 claims description 8
- 108010001517 Galectin 3 Proteins 0.000 claims description 8
- 239000003814 drug Substances 0.000 claims description 8
- 229940079593 drug Drugs 0.000 claims description 8
- 101800000407 Brain natriuretic peptide 32 Proteins 0.000 claims description 7
- 102000004190 Enzymes Human genes 0.000 claims description 6
- 108090000790 Enzymes Proteins 0.000 claims description 6
- 239000002934 diuretic Substances 0.000 claims description 6
- 229940030606 diuretics Drugs 0.000 claims description 6
- 102100039558 Galectin-3 Human genes 0.000 claims description 5
- 102000004887 Transforming Growth Factor beta Human genes 0.000 claims description 5
- 108090001012 Transforming Growth Factor beta Proteins 0.000 claims description 5
- 239000002170 aldosterone antagonist Substances 0.000 claims description 5
- 229940083712 aldosterone antagonist Drugs 0.000 claims description 5
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 claims description 5
- 229940121710 HMGCoA reductase inhibitor Drugs 0.000 claims description 4
- 102400001263 NT-proBNP Human genes 0.000 claims description 4
- 108020004711 Nucleic Acid Probes Proteins 0.000 claims description 4
- 229960000074 biopharmaceutical Drugs 0.000 claims description 4
- 239000002471 hydroxymethylglutaryl coenzyme A reductase inhibitor Substances 0.000 claims description 4
- 238000002483 medication Methods 0.000 claims description 4
- 239000002853 nucleic acid probe Substances 0.000 claims description 4
- 108010008064 pro-brain natriuretic peptide (1-76) Proteins 0.000 claims description 4
- 239000002464 receptor antagonist Substances 0.000 claims description 4
- 229940044551 receptor antagonist Drugs 0.000 claims description 4
- 229940100334 sacubitril / valsartan Drugs 0.000 claims description 4
- 230000001629 suppression Effects 0.000 claims description 4
- 238000001514 detection method Methods 0.000 abstract description 13
- 101000913767 Homo sapiens Cartilage intermediate layer protein 1 Proteins 0.000 description 132
- 210000001054 cardiac fibroblast Anatomy 0.000 description 61
- 108090000623 proteins and genes Proteins 0.000 description 61
- 102000009618 Transforming Growth Factors Human genes 0.000 description 43
- 108010009583 Transforming Growth Factors Proteins 0.000 description 43
- 210000002216 heart Anatomy 0.000 description 43
- 230000014509 gene expression Effects 0.000 description 39
- 102000004169 proteins and genes Human genes 0.000 description 39
- 108020004414 DNA Proteins 0.000 description 21
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 20
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 20
- 241000699670 Mus sp. Species 0.000 description 19
- 230000003176 fibrotic effect Effects 0.000 description 18
- 210000002744 extracellular matrix Anatomy 0.000 description 15
- 238000001356 surgical procedure Methods 0.000 description 15
- 210000004027 cell Anatomy 0.000 description 14
- 230000000295 complement effect Effects 0.000 description 14
- 210000002950 fibroblast Anatomy 0.000 description 14
- 210000004165 myocardium Anatomy 0.000 description 14
- 238000010186 staining Methods 0.000 description 13
- 102000053602 DNA Human genes 0.000 description 12
- 108091034117 Oligonucleotide Proteins 0.000 description 12
- 239000003153 chemical reaction reagent Substances 0.000 description 12
- 238000002965 ELISA Methods 0.000 description 11
- 241001529936 Murinae Species 0.000 description 11
- 208000027418 Wounds and injury Diseases 0.000 description 11
- 230000006378 damage Effects 0.000 description 11
- 208000014674 injury Diseases 0.000 description 11
- 239000003550 marker Substances 0.000 description 11
- 238000003559 RNA-seq method Methods 0.000 description 10
- 239000012634 fragment Substances 0.000 description 10
- 230000000302 ischemic effect Effects 0.000 description 10
- 102100036723 Discoidin domain-containing receptor 2 Human genes 0.000 description 9
- 101710127786 Discoidin domain-containing receptor 2 Proteins 0.000 description 9
- 241000699666 Mus <mouse, genus> Species 0.000 description 9
- 206010028594 Myocardial fibrosis Diseases 0.000 description 9
- 238000003365 immunocytochemistry Methods 0.000 description 9
- 210000001519 tissue Anatomy 0.000 description 9
- 238000001262 western blot Methods 0.000 description 9
- 101150035562 LTBP2 gene Proteins 0.000 description 8
- 238000004458 analytical method Methods 0.000 description 8
- 238000011161 development Methods 0.000 description 8
- 230000018109 developmental process Effects 0.000 description 8
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 7
- 238000011746 C57BL/6J (JAX™ mouse strain) Methods 0.000 description 7
- 238000000338 in vitro Methods 0.000 description 7
- 210000004898 n-terminal fragment Anatomy 0.000 description 7
- 230000011664 signaling Effects 0.000 description 7
- 208000020446 Cardiac disease Diseases 0.000 description 6
- 230000004913 activation Effects 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 6
- 208000019622 heart disease Diseases 0.000 description 6
- 239000002953 phosphate buffered saline Substances 0.000 description 6
- 241001465754 Metazoa Species 0.000 description 5
- 238000010166 immunofluorescence Methods 0.000 description 5
- 238000001727 in vivo Methods 0.000 description 5
- 102000040430 polynucleotide Human genes 0.000 description 5
- 108091033319 polynucleotide Proteins 0.000 description 5
- 239000002157 polynucleotide Substances 0.000 description 5
- 238000007634 remodeling Methods 0.000 description 5
- 230000004044 response Effects 0.000 description 5
- 231100000241 scar Toxicity 0.000 description 5
- JWZZKOKVBUJMES-UHFFFAOYSA-N (+-)-Isoprenaline Chemical compound CC(C)NCC(O)C1=CC=C(O)C(O)=C1 JWZZKOKVBUJMES-UHFFFAOYSA-N 0.000 description 4
- 102000007469 Actins Human genes 0.000 description 4
- 108010085238 Actins Proteins 0.000 description 4
- 108010035532 Collagen Proteins 0.000 description 4
- 102000008186 Collagen Human genes 0.000 description 4
- 238000000692 Student's t-test Methods 0.000 description 4
- 238000003556 assay Methods 0.000 description 4
- 210000004900 c-terminal fragment Anatomy 0.000 description 4
- 210000004899 c-terminal region Anatomy 0.000 description 4
- 210000000845 cartilage Anatomy 0.000 description 4
- 230000001413 cellular effect Effects 0.000 description 4
- 229920001436 collagen Polymers 0.000 description 4
- 239000003636 conditioned culture medium Substances 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 230000006870 function Effects 0.000 description 4
- 230000004217 heart function Effects 0.000 description 4
- 238000003125 immunofluorescent labeling Methods 0.000 description 4
- 229940039009 isoproterenol Drugs 0.000 description 4
- 210000003205 muscle Anatomy 0.000 description 4
- 230000008458 response to injury Effects 0.000 description 4
- 230000003827 upregulation Effects 0.000 description 4
- 102000014914 Carrier Proteins Human genes 0.000 description 3
- 101150005987 Cilp gene Proteins 0.000 description 3
- 238000008157 ELISA kit Methods 0.000 description 3
- 102000000802 Galectin 3 Human genes 0.000 description 3
- 208000013875 Heart injury Diseases 0.000 description 3
- 108020005187 Oligonucleotide Probes Proteins 0.000 description 3
- 229930040373 Paraformaldehyde Natural products 0.000 description 3
- 206010063837 Reperfusion injury Diseases 0.000 description 3
- 108091008324 binding proteins Proteins 0.000 description 3
- 238000001574 biopsy Methods 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 238000002659 cell therapy Methods 0.000 description 3
- 230000004087 circulation Effects 0.000 description 3
- 239000003102 growth factor Substances 0.000 description 3
- 238000009396 hybridization Methods 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 230000002107 myocardial effect Effects 0.000 description 3
- 239000002751 oligonucleotide probe Substances 0.000 description 3
- 229920002866 paraformaldehyde Polymers 0.000 description 3
- 230000001575 pathological effect Effects 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000003753 real-time PCR Methods 0.000 description 3
- 230000002829 reductive effect Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 229920002477 rna polymer Polymers 0.000 description 3
- 230000036573 scar formation Effects 0.000 description 3
- 238000012353 t test Methods 0.000 description 3
- 239000005541 ACE inhibitor Substances 0.000 description 2
- 102000009027 Albumins Human genes 0.000 description 2
- 108010088751 Albumins Proteins 0.000 description 2
- 208000031229 Cardiomyopathies Diseases 0.000 description 2
- 102000004961 Furin Human genes 0.000 description 2
- 108090001126 Furin Proteins 0.000 description 2
- 101001054659 Homo sapiens Latent-transforming growth factor beta-binding protein 1 Proteins 0.000 description 2
- 201000009794 Idiopathic Pulmonary Fibrosis Diseases 0.000 description 2
- PIWKPBJCKXDKJR-UHFFFAOYSA-N Isoflurane Chemical compound FC(F)OC(Cl)C(F)(F)F PIWKPBJCKXDKJR-UHFFFAOYSA-N 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- 101710143111 Mothers against decapentaplegic homolog 3 Proteins 0.000 description 2
- 208000037273 Pathologic Processes Diseases 0.000 description 2
- 229920001213 Polysorbate 20 Polymers 0.000 description 2
- 102000049939 Smad3 Human genes 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 239000002333 angiotensin II receptor antagonist Substances 0.000 description 2
- 229940125364 angiotensin receptor blocker Drugs 0.000 description 2
- 229940044094 angiotensin-converting-enzyme inhibitor Drugs 0.000 description 2
- 230000006793 arrhythmia Effects 0.000 description 2
- 206010003119 arrhythmia Diseases 0.000 description 2
- 239000002876 beta blocker Substances 0.000 description 2
- 229940097320 beta blocking agent Drugs 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 230000000747 cardiac effect Effects 0.000 description 2
- 210000004413 cardiac myocyte Anatomy 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- MYSWGUAQZAJSOK-UHFFFAOYSA-N ciprofloxacin Chemical compound C12=CC(N3CCNCC3)=C(F)C=C2C(=O)C(C(=O)O)=CN1C1CC1 MYSWGUAQZAJSOK-UHFFFAOYSA-N 0.000 description 2
- 230000008045 co-localization Effects 0.000 description 2
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 2
- 239000003937 drug carrier Substances 0.000 description 2
- 239000000839 emulsion Substances 0.000 description 2
- 229940100321 entresto Drugs 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 2
- 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 2
- 238000012760 immunocytochemical staining Methods 0.000 description 2
- 230000001771 impaired effect Effects 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 208000036971 interstitial lung disease 2 Diseases 0.000 description 2
- 208000012947 ischemia reperfusion injury Diseases 0.000 description 2
- 229960002725 isoflurane Drugs 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 210000004379 membrane Anatomy 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 208000010125 myocardial infarction Diseases 0.000 description 2
- 208000031225 myocardial ischemia Diseases 0.000 description 2
- 230000009054 pathological process Effects 0.000 description 2
- 230000026731 phosphorylation Effects 0.000 description 2
- 238000006366 phosphorylation reaction Methods 0.000 description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 230000010410 reperfusion Effects 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 230000002441 reversible effect Effects 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 230000019491 signal transduction Effects 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 2
- 230000001131 transforming effect Effects 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- LCSKNASZPVZHEG-UHFFFAOYSA-N 3,6-dimethyl-1,4-dioxane-2,5-dione;1,4-dioxane-2,5-dione Chemical group O=C1COC(=O)CO1.CC1OC(=O)C(C)OC1=O LCSKNASZPVZHEG-UHFFFAOYSA-N 0.000 description 1
- WZRJTRPJURQBRM-UHFFFAOYSA-N 4-amino-n-(5-methyl-1,2-oxazol-3-yl)benzenesulfonamide;5-[(3,4,5-trimethoxyphenyl)methyl]pyrimidine-2,4-diamine Chemical compound O1C(C)=CC(NS(=O)(=O)C=2C=CC(N)=CC=2)=N1.COC1=C(OC)C(OC)=CC(CC=2C(=NC(N)=NC=2)N)=C1 WZRJTRPJURQBRM-UHFFFAOYSA-N 0.000 description 1
- 229930024421 Adenine Natural products 0.000 description 1
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 238000009020 BCA Protein Assay Kit Methods 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 206010056370 Congestive cardiomyopathy Diseases 0.000 description 1
- 101100447432 Danio rerio gapdh-2 gene Proteins 0.000 description 1
- 201000010046 Dilated cardiomyopathy Diseases 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 102100031509 Fibrillin-1 Human genes 0.000 description 1
- 108010030229 Fibrillin-1 Proteins 0.000 description 1
- 102100037362 Fibronectin Human genes 0.000 description 1
- 108010067306 Fibronectins Proteins 0.000 description 1
- 229920001917 Ficoll Polymers 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 101150112014 Gapdh gene Proteins 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 1
- 101000978212 Homo sapiens Latent-transforming growth factor beta-binding protein 4 Proteins 0.000 description 1
- 206010020880 Hypertrophy Diseases 0.000 description 1
- 206010048858 Ischaemic cardiomyopathy Diseases 0.000 description 1
- YQEZLKZALYSWHR-UHFFFAOYSA-N Ketamine Chemical compound C=1C=CC=C(Cl)C=1C1(NC)CCCCC1=O YQEZLKZALYSWHR-UHFFFAOYSA-N 0.000 description 1
- 102100027000 Latent-transforming growth factor beta-binding protein 1 Human genes 0.000 description 1
- 102100023757 Latent-transforming growth factor beta-binding protein 4 Human genes 0.000 description 1
- 108010052014 Liberase Proteins 0.000 description 1
- 101100137244 Mus musculus Postn gene Proteins 0.000 description 1
- 108020001621 Natriuretic Peptide Proteins 0.000 description 1
- 102000004571 Natriuretic peptide Human genes 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 239000002033 PVDF binder Substances 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 108091093037 Peptide nucleic acid Chemical group 0.000 description 1
- 102100037765 Periostin Human genes 0.000 description 1
- 101710199268 Periostin Proteins 0.000 description 1
- RVGRUAULSDPKGF-UHFFFAOYSA-N Poloxamer Chemical compound C1CO1.CC1CO1 RVGRUAULSDPKGF-UHFFFAOYSA-N 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 108091028664 Ribonucleotide Chemical group 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 108091081021 Sense strand Proteins 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 101150052863 THY1 gene Proteins 0.000 description 1
- 108010046722 Thrombospondin 1 Proteins 0.000 description 1
- 102100036034 Thrombospondin-1 Human genes 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 102400000700 Tumor necrosis factor, membrane form Human genes 0.000 description 1
- 101800000716 Tumor necrosis factor, membrane form Proteins 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 229960000643 adenine Drugs 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 239000000443 aerosol Substances 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000000692 anti-sense effect Effects 0.000 description 1
- 210000000709 aorta Anatomy 0.000 description 1
- 230000003126 arrythmogenic effect Effects 0.000 description 1
- 230000002238 attenuated effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000000091 biomarker candidate Substances 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- RMRJXGBAOAMLHD-IHFGGWKQSA-N buprenorphine Chemical compound C([C@]12[C@H]3OC=4C(O)=CC=C(C2=4)C[C@@H]2[C@]11CC[C@]3([C@H](C1)[C@](C)(O)C(C)(C)C)OC)CN2CC1CC1 RMRJXGBAOAMLHD-IHFGGWKQSA-N 0.000 description 1
- 229960001736 buprenorphine Drugs 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- IVUMCTKHWDRRMH-UHFFFAOYSA-N carprofen Chemical compound C1=CC(Cl)=C[C]2C3=CC=C(C(C(O)=O)C)C=C3N=C21 IVUMCTKHWDRRMH-UHFFFAOYSA-N 0.000 description 1
- 229960003184 carprofen Drugs 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000005754 cellular signaling Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 235000013330 chicken meat Nutrition 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 229960003405 ciprofloxacin Drugs 0.000 description 1
- 208000019425 cirrhosis of liver Diseases 0.000 description 1
- 230000017804 collagen fibril organization Effects 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 210000004351 coronary vessel Anatomy 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 229940104302 cytosine Drugs 0.000 description 1
- 230000002354 daily effect Effects 0.000 description 1
- 230000002939 deleterious effect Effects 0.000 description 1
- 239000005547 deoxyribonucleotide Substances 0.000 description 1
- 125000002637 deoxyribonucleotide group Chemical group 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 238000013401 experimental design Methods 0.000 description 1
- 102000008604 extracellular matrix structural constituent Human genes 0.000 description 1
- 108040002290 extracellular matrix structural constituent Proteins 0.000 description 1
- 210000001723 extracellular space Anatomy 0.000 description 1
- 230000008713 feedback mechanism Effects 0.000 description 1
- 238000001943 fluorescence-activated cell sorting Methods 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
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 210000005003 heart tissue Anatomy 0.000 description 1
- 229920000140 heteropolymer Polymers 0.000 description 1
- 230000003284 homeostatic effect Effects 0.000 description 1
- 230000013632 homeostatic process Effects 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000003601 intercostal effect Effects 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 229960003299 ketamine Drugs 0.000 description 1
- 238000011813 knockout mouse model Methods 0.000 description 1
- 210000005246 left atrium Anatomy 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000011177 media preparation Methods 0.000 description 1
- 238000002493 microarray Methods 0.000 description 1
- 230000004660 morphological change Effects 0.000 description 1
- 239000012120 mounting media Substances 0.000 description 1
- 239000000692 natriuretic peptide Substances 0.000 description 1
- 239000000101 novel biomarker Substances 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 229940100692 oral suspension Drugs 0.000 description 1
- 210000004789 organ system Anatomy 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 201000008482 osteoarthritis Diseases 0.000 description 1
- 238000007911 parenteral administration Methods 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 210000003516 pericardium Anatomy 0.000 description 1
- 230000000737 periodic effect Effects 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 210000002381 plasma Anatomy 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 230000004983 pleiotropic effect Effects 0.000 description 1
- 229960000502 poloxamer Drugs 0.000 description 1
- 229920001983 poloxamer Polymers 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 1
- 230000002206 pro-fibrotic effect Effects 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 238000002331 protein detection Methods 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000029058 respiratory gaseous exchange Effects 0.000 description 1
- 230000036387 respiratory rate Effects 0.000 description 1
- 230000020874 response to hypoxia Effects 0.000 description 1
- 239000002336 ribonucleotide Chemical group 0.000 description 1
- 125000002652 ribonucleotide group Chemical group 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000009919 sequestration Effects 0.000 description 1
- 239000004017 serum-free culture medium Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 229940006995 sulfamethoxazole and trimethoprim Drugs 0.000 description 1
- CCEKAJIANROZEO-UHFFFAOYSA-N sulfluramid Chemical group CCNS(=O)(=O)C(F)(F)C(F)(F)C(F)(F)C(F)(F)C(F)(F)C(F)(F)C(F)(F)C(F)(F)F CCEKAJIANROZEO-UHFFFAOYSA-N 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 210000002435 tendon Anatomy 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 229940113082 thymine Drugs 0.000 description 1
- 238000002627 tracheal intubation Methods 0.000 description 1
- 230000007306 turnover Effects 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
- BPICBUSOMSTKRF-UHFFFAOYSA-N xylazine Chemical compound CC1=CC=CC(C)=C1NC1=NCCCS1 BPICBUSOMSTKRF-UHFFFAOYSA-N 0.000 description 1
- 229960001600 xylazine Drugs 0.000 description 1
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
- G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- 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
- G01N2800/325—Heart failure or cardiac arrest, e.g. cardiomyopathy, congestive heart failure
Definitions
- Myocardial fibrosis is a pathological process associated with various forms of cardiac disease that contributes to impaired cardiac function, development of arrhythmias, and ultimately heart failure.
- Cardiac fibroblasts are the main participating cells in the development of myocardial fibrosis by their secretion of excess extracellular matrix proteins that contribute to scar tissue .
- therapies for heart failure are ineffective at preventing the formation of scar tissue.
- the methods described herein provide for the detection, monitoring, and treatment of cardiac fibrosis, progression of cardiac fibrosis, or heart failure.
- the methods provide a non- invasive clinical tool for determining the presence, extent, and progression of fibrosis in a subject.
- the method for detecting cardiac fibrosis, progression of cardiac fibrosis, or heart failure in a subject comprises: (a) contacting a sample obtained from the subject with a binding agent that binds a region of cartilage intermediate layer protein 1 (CILP) that spans the cleavage site of the CILP precursor or a nucleotide encoding same.
- the nucleotide is a ribonucleic acid molecule (RNA).
- the method further comprises: (b) measuring the amount of CILP in the sample; and (c) detecting cardiac fibrosis, progression of cardiac fibrosis, or heart failure in the subject when the amount of CILP in the sample is decreased relative to a reference sample.
- the method further comprises treating the subject for cardiac fibrosis, progression of cardiac fibrosis, or heart failure.
- the sample is a serum sample.
- the method for treating cardiac fibrosis, progression of cardiac fibrosis, or heart failure in a subject comprises: (a) contacting a sample, such as, for example, a serum sample, obtained from the subject with a binding agent that binds a region of CILP that spans the cleavage site of the CILP precursor or a nucleotide encoding same;
- the cardiac fibrosis is associated with one or more of: ischemia, congenital defect, familial fibrosis, infiltrative fibrosis, idiopathic fibrosis, amyloidosis, hemosiderosis, valvular disease, and other idiopathic cardiomyopathies.
- the method comprises: (a) contacting a serum sample obtained from the subject with a binding agent that binds a region of CILP that spans the cleavage site of the CILP precursor or a nucleotide encoding same; (b) measuring the amount of CILP in the serum sample; and (c) adjusting treatment of the subject for cardiac fibrosis or heart failure when the amount of CILP in the serum sample is decreased relative to a reference sample, and making a corresponding adjustment to treatment of the subject for cardiac fibrosis or heart failure when the amount of CILP is equal to or increased relative to the reference sample.
- adjusting treatment comprises changing classes of medication, switching medication, changing dosages (in some cases by increasing, and in some cases by decreasing).
- adjusting treatment comprises administering, changing, or discontinuing a treatment as described herein.
- the treatment comprises administering to the subject one or more of: angiotensin (AT)-converting enzymes, ATI receptor antagonists, b-blockers, Sacubitril/Valsartan, Aldosterone antagonists, statins, diuretics, or other medications/biologics (e.g., growth factor or cell therapy) effective for treating underlying heart failure and fibrosis.
- AT angiotensin
- ATI receptor antagonists ATI receptor antagonists
- b-blockers Sacubitril/Valsartan
- Aldosterone antagonists e.g., statins, diuretics
- other medications/biologics e.g., growth factor or cell therapy
- the binding agent is an antibody.
- the antibody binds to full-length CILP.
- the binding agent is a nucleic acid probe, such as for detection of RNA.
- the method further comprises measuring additional biomarkers.
- the additional biomarkers can include those described herein and/or other biomarkers of interest.
- the additional biomarkers are a combination of one, two, three, four, or five biomarkers selected from B-type natriuretic peptide (BNP; or its stable precursor, NT-proBNP), Galectin-3 (Gal-3), suppression of tumorigenicity 2 (ST2), latent transforming growth factor beta (LTBP2), and cartilage oligomeric matrix protein (COMP).
- BNP B-type natriuretic peptide
- NT-proBNP Galectin-3
- ST2 tumorigenicity 2
- LTBP2 latent transforming growth factor beta
- COMP cartilage oligomeric matrix protein
- up to 10 additional biomarkers are measured.
- up to 3, 4, 5, 6, 7, 8, or 9 additional biomarkers are measured.
- kits comprising reagents for use in detection of CILP, alone or together with additional biomarkers.
- the kit comprises reagents for use in detecting up to 3, 5, 10, or 15 biomarkers, including CILP.
- FIGS. 1A-1C Identification of potential biomarkers for cardiac fibrosis.
- (1C) RT-qPCR confirmation of upregulation of Ltbp2, Comp, and Cilp in cardiac fibroblasts after 7D TAC (n 3).
- TAC Transverse Aortic Constriction. P* ⁇ 0.050 (t-test).
- FIGS. 2A-2C LTBP2, COMP, and CILP are upregulated in cultured cardiac fibroblasts after TGF
- 3 treatment (n 4).
- FIGS. 3A-3B LTBP2, COMP, and CILP localize to the fibrotic myocardium.
- fibroblast marker DDR2 left
- activated fibroblast marker aSMA right
- Red channel (LTBP2, COMP, and CILP) images are shown separately for clarity.
- aSMA a-Smooth Muscle Actin.
- FIGS. 4A-4D LTBP2, COMP, and CILP are upregulated in human cardiac fibroblasts in response to TGF
- 4A RNA-sequencing data from a public database (GSE46224) show that expression of LTBP2, COMP, and CILP are upregulated in heart biopsies from ischemic heart failure (HF) patients compared to healthy patients.
- 4B Schematic of culture and TGFp treatment of human cardiac fibroblasts.
- FIGS. 5A-5B LTBP2, COMP, and CILP are upregulated in the myocardium of human heart failure patients.
- 5A Masson’s trichrome staining show extensive fibrosis in heart sections of heart failure patients. Insets are higher magnification images of boxed area.
- 5B LTBP2, COMP, and CILP (red) expression is significantly increased in hearts undergoing heart failure. DAPI stained for nuclei. Scale bar: 50pm.
- FIGS. 6A-6D Full length CILP is downregulated in the serum of heart failure patients.
- FIGS. 7A-7B LTBP2, COMP, and CILP are upregulated in Thy1+ sorted cardiac fibroblasts after TGF
- 7A Schematic of cardiac fibroblast sort, culture and TGF
- 7B Immunocytochemistry show that TGF
- 3 Transforming Growth Factor-b
- FIG. 8 Expression of LTBP2, COMP, and CILP are elevated in ischemia-reperfusion injury. Immunofluorescence staining of heart sections from mice that had undergone ischemia-reperfusion injury shows colocalization of LTBP2, COMP, and CILP with fibroblast marker DDR2 and activated fibroblast marker aSMA. DAPI stained for nuclei. Scale bar: 50pm. DDR2: Discoidin Domain Receptor Tyrosine Kinase 2. aSMA: a-Smooth Muscle Actin.
- FIGS. 9A-9C Levels of LTBP2, COMP, and CILP in conditioned media from cultured human CFbs. There were no statistically significant differences in levels of LTBP2 (9A), COMP (9B), and CILP (9C) in collected media from human CFbs treated with TGF
- 3 (n 3-5).
- FIG. 10 CILP levels are decreased in the serum of mice that have undergone TAC.
- the invention described herein is based on the surprising discovery that the full length CILP protein provides a circulating biomarker for cardiac fibrosis. Serum from heart failure patients exhibited significantly decreased levels of CILP compared to serum from healthy volunteers when measured by ELISA.
- the CILP gene encodes a precursor protein that undergoes cleavage into an N-terminal fragment of roughly 75kDa and a C-terminal fragment of about 55kDa(7). Both fragments were shown to inhibit Smad3 phosphorylation, which is normally promoted by active TGF
- the invention provides new methods for detection, monitoring, and treatment of cardiac fibrosis, progression of cardiac fibrosis, and heart failure.
- a “control” or “reference” sample means a sample that is representative of normal measures of the respective marker, such as would be obtained from normal, healthy control subjects, or a baseline amount of marker to be used for comparison. Typically, a baseline will be a measurement taken from the same subject or patient. The sample can be an actual sample used fortesting, or a reference level or range, based on known normal measurements of the corresponding marker.
- a “significant difference” means a difference that can be detected in a manner that is considered reliable by one skilled in the art, such as a statistically significant difference, or a difference that is of sufficient magnitude that, under the circumstances, can be detected with a reasonable level of reliability.
- an increase or decrease of 10% relative to a reference sample is a significant difference.
- an increase or decrease of 20%, 30%, 40%, or 50% relative to the reference sample is considered a significant difference.
- an increase of two-fold relative to a reference sample is considered significant.
- Nucleotide sequence refers to a heteropolymer of deoxyribonucleotides, ribonucleotides, or peptide-nucleic acid sequences that may be assembled from smaller fragments, isolated from larger fragments, or chemically synthesized de novo or partially synthesized by combining shorter oligonucleotide linkers, or from a series of oligonucleotides, to provide a sequence which is capable of expressing the encoded protein.
- primer means an oligonucleotide designed to flank a region of DNA to be amplified.
- one primer is complementary to nucleotides present on the sense strand at one end of a polynucleotide fragment to be amplified and another primer is complementary to nucleotides present on the antisense strand at the other end of the polynucleotide fragment to be amplified.
- a primer can have at least about 11 nucleotides, and preferably, at least about 16 nucleotides and no more than about 35 nucleotides.
- a primer has at least about 80% sequence identity, preferably at least about 90% sequence identity with a target polynucleotide to which the primer hybridizes.
- probe refers to an oligonucleotide, naturally or synthetically produced, via recombinant methods or by PCR amplification, that hybridizes to at least part of another oligonucleotide of interest.
- a probe can be single-stranded or double- stranded.
- active fragment refers to a substantial portion of an oligonucleotide that is capable of performing the same function of specifically hybridizing to a target polynucleotide.
- hybridizes means that the oligonucleotide forms a noncovalent interaction with the target DNA molecule under standard conditions.
- Standard hybridizing conditions are those conditions that allow an oligonucleotide probe or primer to hybridize to a target DNA molecule. Such conditions are readily determined for an oligonucleotide probe or primer and the target DNA molecule using techniques well known to those skilled in the art.
- the nucleotide sequence of a target polynucleotide is generally a sequence complementary to the oligonucleotide primer or probe.
- the hybridizing oligonucleotide may contain nonhybridizing nucleotides that do not interfere with forming the noncovalent interaction.
- the nonhybridizing nucleotides of an oligonucleotide primer or probe may be located at an end of the hybridizing oligonucleotide or within the hybridizing oligonucleotide.
- an oligonucleotide probe or primer does not have to be complementary to all the nucleotides of the target sequence as long as there is hybridization under standard hybridization conditions.
- complement and “complementary” as used herein, refers to the ability of two DNA molecules to base pair with each other, where an adenine on one DNA molecule will base pair to a guanine on a second DNA molecule and a cytosine on one DNA molecule will base pair to a thymine on a second DNA molecule.
- Two DNA molecules are complementary to each other when a nucleotide sequence in one DNA molecule can base pair with a nucleotide sequence in a second DNA molecule.
- the two DNA molecules 5'-ATGC and 5'-GCAT are complementary, and the complement of the DNA molecule 5'-ATGC is 5'-GCAT.
- complement and complementary also encompasses two DNA molecules where one DNA molecule contains at least one nucleotide that will not base pair to at least one nucleotide present on a second DNA molecule.
- the third nucleotide of each of the two DNA molecules 5'-ATTGC and 5'-GCTAT will not base pair, but these two DNA molecules are complementary as defined herein.
- two DNA molecules are complementary if they hybridize under the standard conditions referred to above.
- two DNA molecules are complementary if they have at least about 80% sequence identity, preferably at least about 90% sequence identity.
- pharmaceutically acceptable carrier includes any material which, when combined with an active ingredient, allows the ingredient to retain biological activity and is non-reactive with the subject's immune system.
- examples include, but are not limited to, any of the standard pharmaceutical carriers such as a phosphate buffered saline solution, water, emulsions such as oil/water emulsion, and various types of wetting agents.
- Preferred diluents for aerosol or parenteral administration are phosphate buffered saline or normal (0.9%) saline.
- compositions comprising such carriers are formulated by well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A.
- the term “subject” includes any human or non-human animal.
- the term “non-human animal” includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, horses, sheep, dogs, cows, pigs, chickens, and other veterinary subjects. In a typical embodiment, the subject is a human.
- the invention provides methods for the detection, monitoring, and treatment of cardiac fibrosis, optionally including progression of cardiac fibrosis, or heart failure.
- the method for detecting cardiac fibrosis, progression of cardiac fibrosis, or heart failure in a subject comprises: (a) contacting a sample obtained from the subject with a binding agent that binds a region of cartilage intermediate layer protein 1 (CILP; UniProt 075339; SEQ ID NO: 1) that spans the cleavage site of the CILP precursor or a nucleotide encoding same.
- the binding agent detects the presence of full-length CILP.
- the binding agent by binding a region of CILP that spans the cleavage site, is capable of distinguishing between the detection of full-length CILP and its 55 kDa C-terminal fragment.
- the two peptides that CILP is cleaved into are known as CILP protein 1 C1 and C2, and the cleavage site is at amino acid 724 of SEQ ID NO: 1.
- the binding agent targets Pro604 to His864 of SEQ ID NO: 1.
- the nucleotide is a ribonucleic acid molecule (RNA).
- the method further comprises: (b) measuring the amount of CILP in the sample; and (c) detecting cardiac fibrosis, progression of cardiac fibrosis, or heart failure in the subject when the amount of CILP (or RNA encoding same) in the sample is decreased relative to a reference sample.
- the method further comprises treating the subject for cardiac fibrosis, progression of cardiac fibrosis, or heart failure.
- the method for detecting cardiac fibrosis or heart failure in a subject comprises: (a) contacting a sample obtained from the subject with a binding agent that binds a region of CILP precursor.
- the binding agent detects the presence of full-length CILP.
- the method further comprises: (b) measuring the amount of CILP in the sample; and (c) detecting cardiac fibrosis, progression of cardiac fibrosis, or heart failure in the subject when the amount of CILP in the sample is decreased relative to a reference sample.
- the method for treating cardiac fibrosis, progression of cardiac fibrosis, or heart failure in a subject comprises: (a) contacting a sample obtained from the subject with a binding agent that binds a region of CILP that spans the cleavage site of the CILP precursor or a nucleotide encoding same; (b) measuring the amount of CILP in the sample; and (c) treating the subject for cardiac fibrosis, progression of cardiac fibrosis, or heart failure when the amount of CILP in the sample is decreased relative to a reference sample.
- the treatment comprises administering to the subject one or more of: angiotensin (AT)-converting enzymes, ATI receptor antagonists, b-blockers, Sacubitril/Valsartan, Aldosterone antagonists, statins, diuretics, or other medications/biologics (e.g., growth factor or cell therapy) effective for treating underlying heart failure and fibrosis.
- AT angiotensin
- ATI receptor antagonists e.g., ATI receptor antagonists
- b-blockers e.g., Sacubitril/Valsartan
- Aldosterone antagonists e.g., statins, diuretics
- other medications/biologics e.g., growth factor or cell therapy
- the cardiac fibrosis is associated with one or more of: ischemia, congenital defect, familial fibrosis, infiltrative fibrosis, idiopathic fibrosis, amyloidosis, hemosiderosis, valvular disease,
- the method comprises: (a) contacting a sample obtained from the subject with a binding agent that binds a region of CILP that spans the cleavage site of the CILP precursor or a nucleotide encoding same; (b) measuring the amount of CILP in the sample; and (c) adjusting treatment of the subject for cardiac fibrosis or heart failure when the amount of CILP in the sample is decreased relative to a reference sample, and making a corresponding adjustment to treatment of the subject for cardiac fibrosis or heart failure when the amount of CILP is equal to or increased relative to the reference sample.
- adjusting treatment comprises administering, changing, or discontinuing a treatment as described herein.
- adjusting treatment comprises changing classes of medication, switching medication, changing dosages (in some cases by increasing, and in some cases by decreasing).
- Representative examples of adjusting treatment include, but are not limited to, starting or increasing angiotensin (AT)-converting enzymes, ATI receptor antagonists, b-blockers, Sacubitril/Valsartan, Aldosterone antagonists, statins, diuretics, or other medications/biologics (e.g., growth factor or cell therapy).
- the reference sample is from a normal, healthy control subject. In some embodiments, the reference sample is from the same subject, such as a previously obtained sample from the subject. Such same-subject samples can be useful, for example, in monitoring a subject’s progress and ensuring recovery.
- sample include, but are not limited to, blood, plasma or serum, tissue, cell cultures, and other bodily fluids or tissue specimens.
- sample is serum.
- the binding agent is an antibody. In some embodiments, the antibody binds to full-length CILP. In some embodiments, the binding agent is a nucleic acid probe, such as for detection of RNA. [0044] In some embodiments, the method further comprises measuring additional biomarkers. In some embodiments, the additional biomarkers are serum biomarkers. In some embodiments, the additional biomarkers are suitable for use with tissue samples or biopsy material.
- the additional biomarkers include one or more of: 13- type natriuretic peptide (BNP; or its stable precursor, NT-proBNP), Galectin-3 (Gal-3), suppression of tumorigenicity 2 (ST2), latent transforming growth factor beta (LTBP2), and cartilage oligomeric matrix protein (COMP).
- BNP 13- type natriuretic peptide
- Gal-3 Galectin-3
- ST2 tumorigenicity 2
- LTBP2 latent transforming growth factor beta
- COMP cartilage oligomeric matrix protein
- up to 10 additional biomarkers are measured.
- up to 3, 4, 5, 6, 7, 8, or 9 additional biomarkers are measured.
- up to 15 or 20 additional biomarkers are measured.
- the invention provides a kit comprising reagents for use in detection of CILP, alone or together with additional biomarkers.
- the kit comprises reagents for use in detecting up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, or up to 25 biomarkers, including CILP.
- the additional biomarkers include one or more of: B-type natriuretic peptide (BNP; or its stable precursor, NT-proBNP), Galectin-3 (Gal-3), suppression of tumorigenicity 2 (ST2), latent transforming growth factor beta (LTBP2), and cartilage oligomeric matrix protein (COMP).
- kits comprising a set of reagents as described herein, such as antibodies that specifically bind one or more markers of the invention (including genes and their expression products), and optionally, one or more suitable containers containing reagents of the invention.
- Reagents include molecules that specifically bind and/or amplify and/or detect one or more markers of the invention. Such molecules can be provided in the form of a microarray or other article of manufacture for use in an assay described herein.
- a reagent is an antibody or nucleic acid probe that is specific for the marker(s).
- Another example includes probes (or primers) that selectively identify one or more genotypes described herein.
- Reagents can optionally include a detectable label.
- Labels can be fluorescent, luminescent, enzymatic, chromogenic, or radioactive.
- Kits of the invention optionally comprise an assay standard or a set of assay standards, either separately or together with other reagents.
- An assay standard can serve as a normal control by providing a reference level of normal expression for a given marker that is representative of a healthy individual.
- Kits can include probes for detection of alternative gene expression products in addition to antibodies for protein detection.
- the kit can optionally include a buffer.
- Reagents and standards can be provided in combinations reflecting the combinations of markers described herein as useful for detection.
- Example 1 Cardiac Fibrosis is Associated with Decreased Circulating Levels of Full-Length
- This Example identifies circulating biomarkers for cardiac fibrosis.
- This Example demonstrates that, upon in vitro stimulation or in vivo pressure overload injury, activated cardiac fibroblasts express LTBP2, COMP, and CILP. Further, in ischemic heart disease, LTBP2, COMP, and CILP localize to the fibrotic regions of the injured heart. The results show that circulating levels of full-length CILP protein are decreased in heart failure patients, supporting the use of this protein as a biomarker for the presence of cardiac fibrosis.
- Cardiac fibrosis leads to pathological remodeling that can deteriorate cardiac function. Heart failure arising from cardiac fibrosis is a debilitating syndrome, and there is a need to identify circulating biomarkers that can help diagnose the extent of fibrosis.
- LTBP2, COMP, and CILP were upregulated in murine and human cardiac fibroblasts after in vitro TGF
- the full-length CILP protein showed a significant decrease in circulating levels in heart failure patients compared to healthy volunteers.
- LTBP2 and COMP are additional markers that specifically localized to the fibrotic regions of the injured myocardium. Further studies are warranted to determine the functional contributions of these proteins to the development of cardiac fibrosis.
- ECM Extracellular Matrix
- TAC Transverse Aortic Constriction
- RNA Ribonucleic Acid
- LTBP2 Latent TGF
- 3 Transforming Growth Factor-Beta
- COMP Cartilage Oligomeric Matrix Protein
- CILP Cartilage Intermediate Layer Protein
- RT-qPCR Reverse Transcription-quantitative PCR
- ELISA Enzyme-linked immunosorbent assay
- Myocardial fibrosis is a pathological process associated with various forms of cardiac disease that contributes to impaired cardiac function, development of arrhythmias, and ultimately heart failure(1 ,2).
- the formation of fibrosis can be initiated by either an acute ischemic event to the heart, such as myocardial infarction, or through a chronic progression driven by increased cardiac load.
- Cardiac fibroblasts (CFbs) are the main participating cells in the development of myocardial fibrosis(3). Under homeostatic conditions, resident CFbs are responsible for maintaining the structural integrity of the heart by regulating extracellular matrix (ECM) production ⁇ ).
- ECM extracellular matrix
- CFbs become activated, proliferate, and secrete an excess amount of ECM proteins, contributing to scar tissue(3,4).
- This scar replaces healthy myocardium, renders the substrate arrhythmogenic, induces stiffening of the heart and leads to adverse remodeling.
- the sequela of fibrosis can have deleterious effects on the ability of the heart to pump blood effectively and hinders the recovery of cardiac function.
- therapies for heart failure are ineffective at preventing the formation of scartissue(5). It has been suggested that identifying diagnostic markers for fibrosis may provide prognostic value for clinicians(6).
- CFbs may release factors that could serve as promising biomarkers for cardiac fibrosis(7).
- Identification of circulating biomarkers would serve as a non-invasive clinical tool of determining the presence, extent, and progression of fibrosis in cardiac disease patients.
- TAC transverse aortic constriction
- sham operation we isolated CFbs from C57BL/6J mice that underwent transverse aortic constriction (TAC, a pressure overload injury model) or sham operation and performed RNA-sequencing to identify key upregulated genes in response to injury(8).
- Ltbp2 latent TGFp-binding protein 2
- Comp cartilage oligomeric matrix protein
- C//p cartilage intermediate layer protein 1
- Ltbp2 is part of the latent TGF8-binding protein family, which consists of key regulators of TGF8 signaling(9).
- Comp and Cilp are mainly known for their roles in the binding of specific ECM proteins, such as collagens, in cartilage(10,11).
- LTBP2, COMP, and CILP were upregulated in cultured murine CFbs and in the fibrotic regions of TAC hearts, suggesting that their expression is specific to the formation of scar.
- Cardiac fibroblasts were isolated from murine hearts, as previously described, for RNA-sequencing (8). This data is publicly available on GEO (GSE51620). Downstream analysis was conducted using the DESeq2, Enhanced Volcanoplot and gplots R packages(12,13). Detailed strategy for identifying potential gene targets is described in the Supplementary Methods presented in Example 2 below.
- mice were used for all experiments.
- mice were randomly assigned into sham, TAC, and ischemic reperfusion (IR) treatment groups. No phenotypic differences were observed between male and female mice. Details of surgery are described in the Example 2. All procedures were carried out with the approval of the University of California, Los Angeles (UCLA) Animal Research Committee or the Institutional Animal Care.
- UCLA University of California, Los Angeles
- mice were collected, digested, and plated as described in Example 2 below. Twelve hours after plating, the floating cells were removed, and the media was replaced. Media changes were done every other day until cells reached 80% confluency, at which point they were passaged and cultured in serum-free media for 24 hours prior to TGF
- RT-qPCR Reverse transcription quantitative-PCR
- Protocols for conditioned media and serum sample preparation are described in Example 2 below.
- ELISA kits were purchased from MyBiosource and the manufacturer’s protocol was followed.
- protein concentration was measured by a PierceTM BCA Protein assay kit (ThermoScientific) and 20pg was loaded into each well of 4-20% Mini- PROTEAN TGX Precast Protein gels (Bio-Rad). After transferring the gel onto a PVDF membrane, detection of CILP was conducted by incubating the membrane with primary antibody followed by secondary antibody conjugated with HRP (Table 2). The signal was developed using the Pierce® ECL Western Blotting Substrate (ThermoScientific).
- Murine cardiac fibroblasts express Ltbp2, Comp , and Cilp after injury
- the TGFp signaling pathway is a major component of injury response in cardiac fibroblasts(14).
- 3 activates and induces proliferation of cultured cells, imitating in vivo responses (15).
- 3 signaling pathway stimulates a robust increase in the expression of the identified genes.
- CFbs from uninjured C57BL/6J mice were cultured in media with or without TGF
- 3 treatment induced expression of fibroblast activation genes such as Periostin ( Postn ) and a-smooth muscle actin (Acfa2)(17), as well as Ltbp2, Comp, and Cilp (Figure 2B).
- fibroblast activation genes such as Periostin ( Postn ) and a-smooth muscle actin (Acfa2)(17), as well as Ltbp2, Comp, and Cilp (Figure 2B).
- immunocytochemistry (ICC) confirmed that expression of LTBP2, COMP, and CILP were increased at the protein level in cultured CFbs after exposure to TGF
- Figure 7 fluorescence-activated cell sorting
- LTBP2, COMP, and CILP expression appeared to colocalize with Discoidin domain-containing receptor 2 (DDR2), a marker for fibroblasts(19), and a-smooth muscle actin (aSMA)(20) within the fibrotic regions of the myocardium in TAC hearts ( Figure 3B). Areas of nonfibrotic myocardium in TAC hearts did not stain for any of the target proteins, indicating that expression of LTBP2, COMP, and CILP are expressed by activated cardiac fibroblasts and localized to regions of fibrosis.
- DDR2 Discoidin domain-containing receptor 2
- aSMA smooth muscle actin
- ICC staining confirmed the upregulation of LTBP2, COMP, and CILP in response to TGF
- 31 treatment did not show significant differences in the levels of LTBP2, an increasing trend of COMP levels, and decreased levels of CILP ( Figure 9).
- These results may be due to unknown mechanisms of protein secretion that affect the presence of these proteins in the context of our culture protocol.
- the results from the in vitro culture of human CFbs mirrored our data from mice, further supporting the potential of these proteins to be biomarkers for cardiac fibrosis.
- LTBP2, COMP, and CILP are potential biomarkers for cardiac fibrosis
- the CILP gene encodes a precursor protein that undergoes cleavage into an N- terminal fragment of roughly 75kDa and a C-terminal fragment of about 55kDa(23). Both of these fragments were shown to inhibit Smad3 phosphorylation, which is normally promoted by active TGF
- cardiac fibroblasts may be a source of novel biomarkers for myocardial fibrosis.
- Ltbp2, Comp , and Cilp were identified and identified Ltbp2, Comp , and Cilp to be upregulated in hearts after pressure overload injury. The expression of these proteins by CFbs in response to injury were validated by in vitro studies in both murine and human CFbs.
- LTBP2 and COMP expression is specific to fibrotic regions
- LTBP2 is a member of the latent TGF
- TGFp has diverse and pleiotropic effects on various cell types through its binding and activation of TGFp receptors(14,25).
- TGFp is secreted from cells as a multiplex form that is covalently bound to latent TGFp binding proteins LTBP1 , LTBP3, and LTBP4. These proteins target the latent complex to specific sites for storage within the ECM where it awaits activation. Matrix sequestration of latent TGFp may serve to regulate its immediate bioavailability while achieving critical threshold concentration at sites of intended function(9,25).
- LTBP2 does not bind to latent TGFp but may interact with other ECM proteins(9).
- Other studies have additionally reported on the competitive role of LTBP2 with LTBP1 for binding sites on fibrillin-1 within the ECM(26).
- Our data show strong support for increased expression of LTBP2 in response to injury and a strong localization of LTBP2 in activated fibroblasts within the fibrotic regions of the myocardium. Whether LTBP2 is merely a surrogate for cardiac fibrosis or is involved in its pathogenesis is not entirely known.
- COMP is another ECM protein that is mainly studied in the context of tendons and cartilage(27).
- the main function of COMP is to directly bind with other ECM components, including collagens and TGF
- COMP in pathological remodeling is less understood. Studies have shown that COMP is upregulated in the context of idiopathic pulmonary fibrosis(30) and liver fibrosis(31), although there have been conflicting reports as to whether it can serve as an accurate circulating marker for fibrosis in patients(30,32).
- LTBP2 and COMP both have a strong potential for being markers for cardiac fibrosis as the expression of these proteins are specific to scar formation.
- our data does not support their use as circulating biomarkers after cardiac injury.
- these proteins are known to be secreted, it is possible that they remain within the ECM and participate in the process of fibrosis and scar formation. Further research is warranted to investigate the specific functional contributions of LTBP2 and COMP to the development of cardiac fibrosis. Due to their known roles in other organ systems, it is possible that these proteins may be markers for general fibrosis and not specific to cardiac fibrosis(33).
- Decreased levels of CILP may be indicative of heart failure
- the CILP gene encodes for a precursor protein containing a furin cleavage site.
- the precursor is first synthesized and processed by furin proteases intracellularly prior to being secreted (23).
- the N-terminal fragment has been shown to directly interact with TGF
- NTPPHase porcine nucleotide pyrophosphohydrolase
- the functional role of full- length CILP protein has not been well-studied.
- the full-length CILP has been shown to inhibit TGF
- 3 TGF
- further studies to determine any functional differences between the N-terminal fragment and the full- length CILP are required.
- Our data specifically demonstrates that circulating levels of the full- length CILP are attenuated in heart failure patients but show an abundance of expression in the fibrotic myocardium.
- a possible mechanism for this paradox is that full-length CILP is sequestered to the ECM by its binding to TGF
- 3 is reduced(42,43). Due to the inhibitory role of full-length CILP in TGF
- this Example confirms the potential for LTBP2, COMP, and CILP as novel markers of cardiac fibrosis in both mouse and human heart failure models. Most notably, we discovered a significant reduction in serum levels of full-length CILP in patients with heart failure. Biomarkers for cardiac fibrosis will serve as a noninvasive method to gain diagnostic and prognostic information regarding patients with heart failure. Our study confirms the utility of several markers of cardiac fibrosis in mouse and human subjects.
- Example 1 describes further details of the methods employed in Example 1. In addition, Tables refenced in Example 1 can be found below.
- G0:0062023 collagen-containing extracellular matrix
- G0:0001666 response to hypoxia
- G0:0001968 fibronectin binding
- G0:0030199 collagen fibril organization
- G0:0005201 extracellular matrix structural constituent
- G0:0005615 extracellular space
- Aortic constriction was performed by tying a 7-0 nylon suture ligature against a 27-gauge blunt needle and then removing the needle to yield a constriction of roughly 0.4mm in diameter.
- left thoracotomy between ribs four and five was performed. The pericardium was opened, and a suture was placed around the left anterior descending coronary artery 1-2mm from the tip of the left atrium. The suture was tightened to occlude blood flow for 45 minutes and subsequently removed. Mice that underwent sham operations underwent the same procedure, excluding the constriction/occlusion.
- mice After the operation, the chest was closed in layers using 5-0 Vicryl sutures and the mice remained on the ventilators until sufficient spontaneous breathing was resumed, at which point the endotracheal tube was removed. The entire surgical procedure was performed under aseptic conditions. Buprenorphine (0.1 mg/kg) was administrated by subcutaneous injection immediately prior to surgery, followed by every 12 hours for 48 hours, and carprofen (5mg/Kg) was administrated post operation every 24 hours for 48 hours. Mice were additionally treated post-operatively with Sulfamethoxazole and Trimethoprim oral suspension (Aurbindo). Operators blinded to the experimental designs performed all animal surgeries and in vivo analyses.
- the cells were then passed through a 70pm filter, centrifuged, and the pellet was resuspended in DMEM containing 20% FBS, 1% Penicillin Streptomycin, and 0.1% Ciprofloxacin.
- the cells from one heart were plated in a single well of a 6-well plate that had been coated with 0.1% gelatin.
- the PCR conditions for RT-qPCR had the following steps: (1) Initial denaturation - 95°C - 2 minutes and 10 seconds; (2) Denaturation - 95°C - 15 seconds; (3) Annealing - 60°C - 30 seconds; (4) Extension - 72°C - 30 seconds; (5) Repeat steps 2-4 for a total of 39 cycles; (6) Final extension - 72°C - 10 minutes.
- the mean cycle threshold (Ct) values were taken from triplicate measurements to determine relative gene expression, as normalized to Gapdh/GAPDH expression.
- Table 3 Significantly up-regulated genes in mouse CFbs in TAC (Fold change > 4, p- value ⁇ 0.05)
- Non-iCMY Non-ischemic cardiomyopathy
- D Diuretics
- BB Beta blockers
- Ent Entresto
- ACE-I Ace-inhibitors
- ARB Angiotensin receptor blockers
- AA Aldosterone antagonists
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Organic Chemistry (AREA)
- Cell Biology (AREA)
- Toxicology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Zoology (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Methods for the detection, monitoring, and treatment of cardiac fibrosis, progression of cardiac fibrosis, or heart failure in a subject comprising: (a) contacting a sample obtained from the subject with a binding agent that binds a region of cartilage intermediate layer protein 1 (CILP) that spans the cleavage site of the CILP precursor or a nucleotide encoding same. The cardiac fibrosis may be associated with one or more of: ischemia, congenital defect, familial fibrosis, infiltrative fibrosis, idiopathic fibrosis, amyloidosis, hemosiderosis, valvular disease, and other idiopathic cardiomyopathies.
Description
FULL-LENGTH CILP AS A BIOMARKER FOR CARDIAC FIBROSIS
[0001] This application claims benefit of United States provisional patent application number 63/003,451 , filed April 1 , 2020, the entire contents of which are incorporated by reference into this application.
REFERENCE TO A SEQUENCE LISTING SUBMITTED VIA EFS-WEB
[0002] The content of the ASCII text file of the sequence listing named “UCLA280_seq” which is 15 kb in size was created on March 25, 2021 , and electronically submitted via EFS- Web herewith the application is incorporated herein by reference in its entirety.
ACKNOWLEDGEMENT OF GOVERNMENT SUPPORT
[0003] This invention was made with government support under Grant Number HL127728, awarded by the National Institutes of Health. The government has certain rights in the invention.
BACKGROUND
[0004] Myocardial fibrosis is a pathological process associated with various forms of cardiac disease that contributes to impaired cardiac function, development of arrhythmias, and ultimately heart failure. Cardiac fibroblasts are the main participating cells in the development of myocardial fibrosis by their secretion of excess extracellular matrix proteins that contribute to scar tissue . There are currently limited treatment options for the reversal of cardiac fibrosis, and available therapies for heart failure are ineffective at preventing the formation of scar tissue.
[0005] There remains a need for identification of circulating biomarkers that can serve as a non-invasive clinical tool for determining the presence, extent, and progression of fibrosis in cardiac disease patients. In addition, there remains a need for such biomarkers that can aid in the prognosis and monitoring of cardiac fibrosis as an aid in the treatment and management of cardiac disease.
SUMMARY
[0006] The methods described herein provide for the detection, monitoring, and treatment of cardiac fibrosis, progression of cardiac fibrosis, or heart failure. The methods provide a non- invasive clinical tool for determining the presence, extent, and progression of fibrosis in a subject. In some embodiments, the method for detecting cardiac fibrosis, progression of cardiac fibrosis, or heart failure in a subject comprises: (a) contacting a sample obtained from the subject with a binding agent that binds a region of cartilage intermediate layer
protein 1 (CILP) that spans the cleavage site of the CILP precursor or a nucleotide encoding same. In some embodiments, the nucleotide is a ribonucleic acid molecule (RNA). The method further comprises: (b) measuring the amount of CILP in the sample; and (c) detecting cardiac fibrosis, progression of cardiac fibrosis, or heart failure in the subject when the amount of CILP in the sample is decreased relative to a reference sample. In some embodiments, the method further comprises treating the subject for cardiac fibrosis, progression of cardiac fibrosis, or heart failure. In some embodiments, the sample is a serum sample.
[0007] In some embodiments, the method for treating cardiac fibrosis, progression of cardiac fibrosis, or heart failure in a subject comprises: (a) contacting a sample, such as, for example, a serum sample, obtained from the subject with a binding agent that binds a region of CILP that spans the cleavage site of the CILP precursor or a nucleotide encoding same;
(b) measuring the amount of CILP in the sample; and (c) treating the subject for cardiac fibrosis, progression of cardiac fibrosis, or heart failure when the amount of CILP in the sample is decreased relative to a reference sample. In some embodiments, the cardiac fibrosis is associated with one or more of: ischemia, congenital defect, familial fibrosis, infiltrative fibrosis, idiopathic fibrosis, amyloidosis, hemosiderosis, valvular disease, and other idiopathic cardiomyopathies.
[0008] Also provided herein is a method for monitoring the status of cardiac fibrosis or heart failure in a subject. In some embodiments, the method comprises: (a) contacting a serum sample obtained from the subject with a binding agent that binds a region of CILP that spans the cleavage site of the CILP precursor or a nucleotide encoding same; (b) measuring the amount of CILP in the serum sample; and (c) adjusting treatment of the subject for cardiac fibrosis or heart failure when the amount of CILP in the serum sample is decreased relative to a reference sample, and making a corresponding adjustment to treatment of the subject for cardiac fibrosis or heart failure when the amount of CILP is equal to or increased relative to the reference sample. In some embodiments, adjusting treatment comprises changing classes of medication, switching medication, changing dosages (in some cases by increasing, and in some cases by decreasing). In some embodiments, adjusting treatment comprises administering, changing, or discontinuing a treatment as described herein. In some embodiments, the treatment comprises administering to the subject one or more of: angiotensin (AT)-converting enzymes, ATI receptor antagonists, b-blockers, Sacubitril/Valsartan, Aldosterone antagonists, statins, diuretics, or other medications/biologics (e.g., growth factor or cell therapy) effective for treating underlying heart failure and fibrosis.
[0009] In some embodiments, the reference sample is from a normal, healthy control subject. In some embodiments, the reference sample is a previously obtained sample from the subject.
[0010] In some embodiments, the binding agent is an antibody. In some embodiments, the antibody binds to full-length CILP. In some embodiments, the binding agent is a nucleic acid probe, such as for detection of RNA.
[0011] In some embodiments, the method further comprises measuring additional biomarkers. The additional biomarkers can include those described herein and/or other biomarkers of interest. In some embodiments, the additional biomarkers are a combination of one, two, three, four, or five biomarkers selected from B-type natriuretic peptide (BNP; or its stable precursor, NT-proBNP), Galectin-3 (Gal-3), suppression of tumorigenicity 2 (ST2), latent transforming growth factor beta (LTBP2), and cartilage oligomeric matrix protein (COMP). In some embodiments, up to 10 additional biomarkers are measured. In some embodiments, up to 3, 4, 5, 6, 7, 8, or 9 additional biomarkers are measured. In some embodiments, up to 15 or 20 additional biomarkers are measured. Also provided is a kit comprising reagents for use in detection of CILP, alone or together with additional biomarkers. In some embodiments, the kit comprises reagents for use in detecting up to 3, 5, 10, or 15 biomarkers, including CILP.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIGS. 1A-1C. Identification of potential biomarkers for cardiac fibrosis. (1A) Volcano plot depicting highly differentially expressed genes in cardiac fibroblasts after TAC. (1B) Heatmap of pro-fibrotic genes that are upregulated in cardiac fibroblasts isolated from sham and TAC mouse hearts. Select genes ( Ltbp2 , Comp, and Cilp) are boxed. (1C) RT-qPCR confirmation of upregulation of Ltbp2, Comp, and Cilp in cardiac fibroblasts after 7D TAC (n=3). TAC: Transverse Aortic Constriction. P*<0.050 (t-test).
[0013] FIGS. 2A-2C. LTBP2, COMP, and CILP are upregulated in cultured cardiac fibroblasts after TGF|3 treatment. (2A) Schematic of cardiac fibroblast culture and TGF|3 treatment. (2B) RT-qPCR of cardiac fibroblasts treated with TGF|3 normalized to untreated controls (indicated by dotted line at y=1) (n=4). (2C) Immunocytochemistry for LTBP2, COMP, and CILP show significant increase in expression after TGF|3 treatment (n=4). DAPI stained for nuclei. Scale bar: 100pm. TGF|3: Transforming Growth Factor-b. P*<0.050 (t- test).
[0014] FIGS. 3A-3B. LTBP2, COMP, and CILP localize to the fibrotic myocardium. (3A) Mice that had undergone TAC surgery exhibited myocardial fibrosis as shown by Masson’s
trichrome staining. (3B) Immunofluorescence staining shows colocalization of LTBP2,
COMP, and CILP with fibroblast marker DDR2 (left) and activated fibroblast marker aSMA (right). Red channel (LTBP2, COMP, and CILP) images are shown separately for clarity. DAPI stained for nuclei. Scale bar: 50pm. DDR2: Discoidin Domain Receptor Tyrosine Kinase 2. aSMA: a-Smooth Muscle Actin.
[0015] FIGS. 4A-4D. LTBP2, COMP, and CILP are upregulated in human cardiac fibroblasts in response to TGF|3. (4A) RNA-sequencing data from a public database (GSE46224) show that expression of LTBP2, COMP, and CILP are upregulated in heart biopsies from ischemic heart failure (HF) patients compared to healthy patients. (4B) Schematic of culture and TGFp treatment of human cardiac fibroblasts. (4C) RT-qPCR of human cardiac fibroblasts treated with TGF|3 normalized to untreated controls (indicated by dotted line at y=1) (n=3). (4D) Immunocytochemistry shows increased expression of LTBP2, COMP, and CILP in cultured human cardiac fibroblasts after TGF|3 treatment. DAPI stained for nuclei (n=4). RPKM: Reads Per Kilobases Mapped. Scale bar: 100pm. *P<0.050 (t-test).
[0016] FIGS. 5A-5B. LTBP2, COMP, and CILP are upregulated in the myocardium of human heart failure patients. (5A) Masson’s trichrome staining show extensive fibrosis in heart sections of heart failure patients. Insets are higher magnification images of boxed area. (5B) LTBP2, COMP, and CILP (red) expression is significantly increased in hearts undergoing heart failure. DAPI stained for nuclei. Scale bar: 50pm.
[0017] FIGS. 6A-6D. Full length CILP is downregulated in the serum of heart failure patients. (6A) ELISA demonstrates non-significant changes in levels of COMP in the serum of heart failure patients compared to healthy individuals (Healthy n=23, Heart Failure n=22). (6B) Heart failure patients had a significant decrease in circulating CILP levels (Healthy n=23, Heart Failure n=22). (6C) Representative western blotting shows decreased levels of full length CILP (~133kDa) in heart failure patient serum. Quantified data is shown in (6D) (Healthy n= 5, Heart Failure n=5). a.u.: arbitrary units. *P<0.050 (t-test).
[0018] FIGS. 7A-7B. LTBP2, COMP, and CILP are upregulated in Thy1+ sorted cardiac fibroblasts after TGF|3 treatment. (7A) Schematic of cardiac fibroblast sort, culture and TGF|3 treatment. (7B) Immunocytochemistry show that TGF|3 treatment induces increased expression of LTBP2, COMP, and CILP. DAPI stained for nuclei. Scale bar: 100pm. TGF|3: Transforming Growth Factor-b
[0019] FIG. 8. Expression of LTBP2, COMP, and CILP are elevated in ischemia-reperfusion injury. Immunofluorescence staining of heart sections from mice that had undergone ischemia-reperfusion injury shows colocalization of LTBP2, COMP, and CILP with fibroblast marker DDR2 and activated fibroblast marker aSMA. DAPI stained for nuclei. Scale bar:
50pm. DDR2: Discoidin Domain Receptor Tyrosine Kinase 2. aSMA: a-Smooth Muscle Actin.
[0020] FIGS. 9A-9C. Levels of LTBP2, COMP, and CILP in conditioned media from cultured human CFbs. There were no statistically significant differences in levels of LTBP2 (9A), COMP (9B), and CILP (9C) in collected media from human CFbs treated with TGF|3 (n=3-5).
[0021] FIG. 10. CILP levels are decreased in the serum of mice that have undergone TAC. ELISA demonstrate a decrease in levels of CILP in the serum of mice 7 days after TAC surgery (n=4-6).
DETAILED DESCRIPTION
[0022] The invention described herein is based on the surprising discovery that the full length CILP protein provides a circulating biomarker for cardiac fibrosis. Serum from heart failure patients exhibited significantly decreased levels of CILP compared to serum from healthy volunteers when measured by ELISA. The CILP gene encodes a precursor protein that undergoes cleavage into an N-terminal fragment of roughly 75kDa and a C-terminal fragment of about 55kDa(7). Both fragments were shown to inhibit Smad3 phosphorylation, which is normally promoted by active TGF|3 signaling. While commercially available ELISA kits target the C-terminal region of CILP (hence detecting both the C-terminal and the full- length fragment), previous work showed that cardiac fibroblasts secrete the N-terminal fragment as well as the full-length CILP protein. As described herein, use of an antibody that spans the cleavage site of the CILP precursor and western blotting levels of circulating full- length CILP, the studies described herein showed that serum from heart failure patients had significantly decreased levels of full-length CILP in circulation.
[0023] The invention provides new methods for detection, monitoring, and treatment of cardiac fibrosis, progression of cardiac fibrosis, and heart failure.
Definitions
[0024] All scientific and technical terms used in this application have meanings commonly used in the art unless otherwise specified. As used in this application, the following words or phrases have the meanings specified.
[0025] As used herein, a “control” or “reference” sample means a sample that is representative of normal measures of the respective marker, such as would be obtained from normal, healthy control subjects, or a baseline amount of marker to be used for comparison. Typically, a baseline will be a measurement taken from the same subject or
patient. The sample can be an actual sample used fortesting, or a reference level or range, based on known normal measurements of the corresponding marker.
[0026] As used herein, a “significant difference” means a difference that can be detected in a manner that is considered reliable by one skilled in the art, such as a statistically significant difference, or a difference that is of sufficient magnitude that, under the circumstances, can be detected with a reasonable level of reliability. In one example, an increase or decrease of 10% relative to a reference sample is a significant difference. In other examples, an increase or decrease of 20%, 30%, 40%, or 50% relative to the reference sample is considered a significant difference. In yet another example, an increase of two-fold relative to a reference sample is considered significant.
[0027] “Nucleotide sequence” refers to a heteropolymer of deoxyribonucleotides, ribonucleotides, or peptide-nucleic acid sequences that may be assembled from smaller fragments, isolated from larger fragments, or chemically synthesized de novo or partially synthesized by combining shorter oligonucleotide linkers, or from a series of oligonucleotides, to provide a sequence which is capable of expressing the encoded protein.
[0028] The term "primer," as used herein, means an oligonucleotide designed to flank a region of DNA to be amplified. In a primer pair, one primer is complementary to nucleotides present on the sense strand at one end of a polynucleotide fragment to be amplified and another primer is complementary to nucleotides present on the antisense strand at the other end of the polynucleotide fragment to be amplified. A primer can have at least about 11 nucleotides, and preferably, at least about 16 nucleotides and no more than about 35 nucleotides. Typically, a primer has at least about 80% sequence identity, preferably at least about 90% sequence identity with a target polynucleotide to which the primer hybridizes.
[0029] As used herein, the term “probe” refers to an oligonucleotide, naturally or synthetically produced, via recombinant methods or by PCR amplification, that hybridizes to at least part of another oligonucleotide of interest. A probe can be single-stranded or double- stranded.
[0030] As used herein, the term “active fragment” refers to a substantial portion of an oligonucleotide that is capable of performing the same function of specifically hybridizing to a target polynucleotide.
[0031] As used herein, "hybridizes," "hybridizing," and "hybridization" means that the oligonucleotide forms a noncovalent interaction with the target DNA molecule under standard conditions. Standard hybridizing conditions are those conditions that allow an oligonucleotide probe or primer to hybridize to a target DNA molecule. Such conditions are readily determined for an oligonucleotide probe or primer and the target DNA molecule using
techniques well known to those skilled in the art. The nucleotide sequence of a target polynucleotide is generally a sequence complementary to the oligonucleotide primer or probe. The hybridizing oligonucleotide may contain nonhybridizing nucleotides that do not interfere with forming the noncovalent interaction. The nonhybridizing nucleotides of an oligonucleotide primer or probe may be located at an end of the hybridizing oligonucleotide or within the hybridizing oligonucleotide. Thus, an oligonucleotide probe or primer does not have to be complementary to all the nucleotides of the target sequence as long as there is hybridization under standard hybridization conditions.
[0032] The term "complement" and "complementary" as used herein, refers to the ability of two DNA molecules to base pair with each other, where an adenine on one DNA molecule will base pair to a guanine on a second DNA molecule and a cytosine on one DNA molecule will base pair to a thymine on a second DNA molecule. Two DNA molecules are complementary to each other when a nucleotide sequence in one DNA molecule can base pair with a nucleotide sequence in a second DNA molecule. For instance, the two DNA molecules 5'-ATGC and 5'-GCAT are complementary, and the complement of the DNA molecule 5'-ATGC is 5'-GCAT. The term complement and complementary also encompasses two DNA molecules where one DNA molecule contains at least one nucleotide that will not base pair to at least one nucleotide present on a second DNA molecule. For instance, the third nucleotide of each of the two DNA molecules 5'-ATTGC and 5'-GCTAT will not base pair, but these two DNA molecules are complementary as defined herein. Typically, two DNA molecules are complementary if they hybridize under the standard conditions referred to above. Typically, two DNA molecules are complementary if they have at least about 80% sequence identity, preferably at least about 90% sequence identity.
[0033] As used herein, "pharmaceutically acceptable carrier" or “excipient” includes any material which, when combined with an active ingredient, allows the ingredient to retain biological activity and is non-reactive with the subject's immune system. Examples include, but are not limited to, any of the standard pharmaceutical carriers such as a phosphate buffered saline solution, water, emulsions such as oil/water emulsion, and various types of wetting agents. Preferred diluents for aerosol or parenteral administration are phosphate buffered saline or normal (0.9%) saline.
[0034] Compositions comprising such carriers are formulated by well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A.
Gennaro, ed., Mack Publishing Co., Easton, PA, 1990).
[0035] As used herein, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, horses, sheep, dogs, cows, pigs, chickens, and other veterinary subjects. In a typical embodiment, the subject is a human.
[0036] As used herein, “a” or “an” means at least one, unless clearly indicated otherwise. Methods
[0037] The invention provides methods for the detection, monitoring, and treatment of cardiac fibrosis, optionally including progression of cardiac fibrosis, or heart failure. In some embodiments, the method for detecting cardiac fibrosis, progression of cardiac fibrosis, or heart failure in a subject comprises: (a) contacting a sample obtained from the subject with a binding agent that binds a region of cartilage intermediate layer protein 1 (CILP; UniProt 075339; SEQ ID NO: 1) that spans the cleavage site of the CILP precursor or a nucleotide encoding same. In some embodiments, the binding agent detects the presence of full-length CILP. The binding agent, by binding a region of CILP that spans the cleavage site, is capable of distinguishing between the detection of full-length CILP and its 55 kDa C-terminal fragment. The two peptides that CILP is cleaved into are known as CILP protein 1 C1 and C2, and the cleavage site is at amino acid 724 of SEQ ID NO: 1. In one embodiment, the binding agent targets Pro604 to His864 of SEQ ID NO: 1.
[0038] In some embodiments, the nucleotide is a ribonucleic acid molecule (RNA). The method further comprises: (b) measuring the amount of CILP in the sample; and (c) detecting cardiac fibrosis, progression of cardiac fibrosis, or heart failure in the subject when the amount of CILP (or RNA encoding same) in the sample is decreased relative to a reference sample. In some embodiments, the method further comprises treating the subject for cardiac fibrosis, progression of cardiac fibrosis, or heart failure. In some embodiments, the method for detecting cardiac fibrosis or heart failure in a subject comprises: (a) contacting a sample obtained from the subject with a binding agent that binds a region of CILP precursor. In some embodiments, the binding agent detects the presence of full-length CILP. The method further comprises: (b) measuring the amount of CILP in the sample; and (c) detecting cardiac fibrosis, progression of cardiac fibrosis, or heart failure in the subject when the amount of CILP in the sample is decreased relative to a reference sample.
[0039] In some embodiments, the method for treating cardiac fibrosis, progression of cardiac fibrosis, or heart failure in a subject comprises: (a) contacting a sample obtained from the subject with a binding agent that binds a region of CILP that spans the cleavage site of the CILP precursor or a nucleotide encoding same; (b) measuring the amount of CILP in the sample; and (c) treating the subject for cardiac fibrosis, progression of cardiac fibrosis,
or heart failure when the amount of CILP in the sample is decreased relative to a reference sample. In some embodiments, the treatment comprises administering to the subject one or more of: angiotensin (AT)-converting enzymes, ATI receptor antagonists, b-blockers, Sacubitril/Valsartan, Aldosterone antagonists, statins, diuretics, or other medications/biologics (e.g., growth factor or cell therapy) effective for treating underlying heart failure and fibrosis. In some embodiments, the cardiac fibrosis is associated with one or more of: ischemia, congenital defect, familial fibrosis, infiltrative fibrosis, idiopathic fibrosis, amyloidosis, hemosiderosis, valvular disease, and other idiopathic cardiomyopathies.
[0040] Also provided herein is a method for monitoring the status of cardiac fibrosis or heart failure in a subject. In some embodiments, the method comprises: (a) contacting a sample obtained from the subject with a binding agent that binds a region of CILP that spans the cleavage site of the CILP precursor or a nucleotide encoding same; (b) measuring the amount of CILP in the sample; and (c) adjusting treatment of the subject for cardiac fibrosis or heart failure when the amount of CILP in the sample is decreased relative to a reference sample, and making a corresponding adjustment to treatment of the subject for cardiac fibrosis or heart failure when the amount of CILP is equal to or increased relative to the reference sample. In some embodiments, adjusting treatment comprises administering, changing, or discontinuing a treatment as described herein. In some embodiments, adjusting treatment comprises changing classes of medication, switching medication, changing dosages (in some cases by increasing, and in some cases by decreasing). Representative examples of adjusting treatment include, but are not limited to, starting or increasing angiotensin (AT)-converting enzymes, ATI receptor antagonists, b-blockers, Sacubitril/Valsartan, Aldosterone antagonists, statins, diuretics, or other medications/biologics (e.g., growth factor or cell therapy).
[0041] In some embodiments, the reference sample is from a normal, healthy control subject. In some embodiments, the reference sample is from the same subject, such as a previously obtained sample from the subject. Such same-subject samples can be useful, for example, in monitoring a subject’s progress and ensuring recovery.
[0042] For use in the methods described herein, representative examples of the sample include, but are not limited to, blood, plasma or serum, tissue, cell cultures, and other bodily fluids or tissue specimens. In a typical embodiment, the sample is serum.
[0043] In some embodiments, the binding agent is an antibody. In some embodiments, the antibody binds to full-length CILP. In some embodiments, the binding agent is a nucleic acid probe, such as for detection of RNA.
[0044] In some embodiments, the method further comprises measuring additional biomarkers. In some embodiments, the additional biomarkers are serum biomarkers. In some embodiments, the additional biomarkers are suitable for use with tissue samples or biopsy material. In some embodiments, the additional biomarkers include one or more of: 13- type natriuretic peptide (BNP; or its stable precursor, NT-proBNP), Galectin-3 (Gal-3), suppression of tumorigenicity 2 (ST2), latent transforming growth factor beta (LTBP2), and cartilage oligomeric matrix protein (COMP). In some embodiments, up to 10 additional biomarkers are measured. In some embodiments, up to 3, 4, 5, 6, 7, 8, or 9 additional biomarkers are measured. In some embodiments, up to 15 or 20 additional biomarkers are measured.
[0045] The invention provides a kit comprising reagents for use in detection of CILP, alone or together with additional biomarkers. In some embodiments, the kit comprises reagents for use in detecting up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, or up to 25 biomarkers, including CILP. In some embodiments, the additional biomarkers include one or more of: B-type natriuretic peptide (BNP; or its stable precursor, NT-proBNP), Galectin-3 (Gal-3), suppression of tumorigenicity 2 (ST2), latent transforming growth factor beta (LTBP2), and cartilage oligomeric matrix protein (COMP).
[0046] Provided are kits comprising a set of reagents as described herein, such as antibodies that specifically bind one or more markers of the invention (including genes and their expression products), and optionally, one or more suitable containers containing reagents of the invention. Reagents include molecules that specifically bind and/or amplify and/or detect one or more markers of the invention. Such molecules can be provided in the form of a microarray or other article of manufacture for use in an assay described herein.
One example of a reagent is an antibody or nucleic acid probe that is specific for the marker(s). Another example includes probes (or primers) that selectively identify one or more genotypes described herein. Reagents can optionally include a detectable label.
Labels can be fluorescent, luminescent, enzymatic, chromogenic, or radioactive.
[0047] Kits of the invention optionally comprise an assay standard or a set of assay standards, either separately or together with other reagents. An assay standard can serve as a normal control by providing a reference level of normal expression for a given marker that is representative of a healthy individual.
[0048] Kits can include probes for detection of alternative gene expression products in addition to antibodies for protein detection. The kit can optionally include a buffer. Reagents
and standards can be provided in combinations reflecting the combinations of markers described herein as useful for detection.
EXAMPLES
[0049] The following examples are presented to illustrate the present invention and to assist one of ordinary skill in making and using the same. The examples are not intended in any way to otherwise limit the scope of the invention.
Example 1 : Cardiac Fibrosis is Associated with Decreased Circulating Levels of Full-Length
CILP in Heart Failure
[0050] This Example identifies circulating biomarkers for cardiac fibrosis. This Example demonstrates that, upon in vitro stimulation or in vivo pressure overload injury, activated cardiac fibroblasts express LTBP2, COMP, and CILP. Further, in ischemic heart disease, LTBP2, COMP, and CILP localize to the fibrotic regions of the injured heart. The results show that circulating levels of full-length CILP protein are decreased in heart failure patients, supporting the use of this protein as a biomarker for the presence of cardiac fibrosis.
[0051] Cardiac fibrosis leads to pathological remodeling that can deteriorate cardiac function. Heart failure arising from cardiac fibrosis is a debilitating syndrome, and there is a need to identify circulating biomarkers that can help diagnose the extent of fibrosis.
[0052] We performed experimental pressure overload injury in C57BL/6J mice by transverse aortic constriction (TAC) and isolated cardiac fibroblasts 7 days post injury or sham operation for RNA-sequencing. Potential biomarkers for cardiac fibrosis were identified and results confirmed by reverse transcription-qPCR. Expression of the biomarkers were measured in fibroblasts treated in vitro with TGF|3 by immunocytochemistry. Immunofluorescence staining confirmed expression in hearts from TAC murine hearts and human heart failure biopsies. Circulating protein levels were measured by ELISA and Western blotting of serum from human subjects.
[0053] The results showed that LTBP2, COMP, and CILP were upregulated in murine and human cardiac fibroblasts after in vitro TGF|3 treatment. All three proteins were found to be expressed specifically in the fibrotic regions of injured murine and human hearts.
Additionally, the full-length CILP protein showed a significant decrease in circulating levels in heart failure patients compared to healthy volunteers.
[0054] The full-length CILP protein thus provides a circulating biomarker for cardiac fibrosis. LTBP2 and COMP are additional markers that specifically localized to the fibrotic regions of the injured myocardium. Further studies are warranted to determine the functional contributions of these proteins to the development of cardiac fibrosis.
[0055] Abbreviations used:
[0056] CFb: Cardiac Fibroblasts
[0057] ECM: Extracellular Matrix
[0058] TAC: Transverse Aortic Constriction
[0059] RNA: Ribonucleic Acid
[0060] LTBP2: Latent TGF|3-binding Protein
[0061] TGF|3: Transforming Growth Factor-Beta
[0062] COMP: Cartilage Oligomeric Matrix Protein
[0063] CILP: Cartilage Intermediate Layer Protein
[0064] RT-qPCR: Reverse Transcription-quantitative PCR
[0065] IF: Immunofluorescence
[0066] ICC: Immunocytochemistry
[0067] ELISA: Enzyme-linked immunosorbent assay
[0068] Myocardial fibrosis is a pathological process associated with various forms of cardiac disease that contributes to impaired cardiac function, development of arrhythmias, and ultimately heart failure(1 ,2). The formation of fibrosis can be initiated by either an acute ischemic event to the heart, such as myocardial infarction, or through a chronic progression driven by increased cardiac load. Cardiac fibroblasts (CFbs) are the main participating cells in the development of myocardial fibrosis(3). Under homeostatic conditions, resident CFbs are responsible for maintaining the structural integrity of the heart by regulating extracellular matrix (ECM) production^). However, under pathological conditions, CFbs become activated, proliferate, and secrete an excess amount of ECM proteins, contributing to scar tissue(3,4). This scar replaces healthy myocardium, renders the substrate arrhythmogenic, induces stiffening of the heart and leads to adverse remodeling. Collectively, the sequela of fibrosis can have deleterious effects on the ability of the heart to pump blood effectively and hinders the recovery of cardiac function. There are currently limited treatment options for the reversal of cardiac fibrosis, and available therapies for heart failure are ineffective at preventing the formation of scartissue(5). It has been suggested that identifying diagnostic markers for fibrosis may provide prognostic value for clinicians(6). Considering the critical role of CFbs in myocardial fibrosis, we hypothesized that CFbs may release factors that could serve as promising biomarkers for cardiac fibrosis(7). Identification of circulating biomarkers would serve as a non-invasive clinical tool of determining the presence, extent, and progression of fibrosis in cardiac disease patients.
[0069] In the present Example, we isolated CFbs from C57BL/6J mice that underwent transverse aortic constriction (TAC, a pressure overload injury model) or sham operation and performed RNA-sequencing to identify key upregulated genes in response to injury(8). From this data, we identified three genes encoding secreted proteins that could be potential biomarkers for myocardial fibrosis: latent TGFp-binding protein 2 ( Ltbp2 ), cartilage oligomeric matrix protein (Comp), and cartilage intermediate layer protein 1 (C//p). Ltbp2 is part of the latent TGF8-binding protein family, which consists of key regulators of TGF8 signaling(9). Comp and Cilp are mainly known for their roles in the binding of specific ECM proteins, such as collagens, in cartilage(10,11). LTBP2, COMP, and CILP were upregulated in cultured murine CFbs and in the fibrotic regions of TAC hearts, suggesting that their expression is specific to the formation of scar. Furthermore, there was an increase in expression of these proteins in stimulated human CFbs and within the fibrotic regions of heart sections from heart failure patients, demonstrating their potential as clinical biomarkers for fibrosis. Finally, we show that CILP, specifically the full length CILP protein, demonstrated a significant difference in circulating levels in the serum of mice after TAC and heart failure patients. The findings in this study introduce potential markers for myocardial fibrosis and support the need to pursue studies on CILP as a possible circulating biomarker for the development of cardiac fibrosis.
[0070] METHODS
[0071] Study Approvals
[0072] All mouse surgery procedures were carried out with the approval of the University of California, Los Angeles (UCLA) Animal Research Committee or the Institutional Animal Care. The study was approved by an IRB (12-001164) and human participants gave written informed consent.
[0073] RNA-Sequencing and Analysis
[0074] Cardiac fibroblasts were isolated from murine hearts, as previously described, for RNA-sequencing (8). This data is publicly available on GEO (GSE51620). Downstream analysis was conducted using the DESeq2, Enhanced Volcanoplot and gplots R packages(12,13). Detailed strategy for identifying potential gene targets is described in the Supplementary Methods presented in Example 2 below.
[0075] Mice
[0076] Adult C57BL/6J mice (age 8-12 weeks) were used for all experiments. For the in vivo experiments, mice were randomly assigned into sham, TAC, and ischemic reperfusion (IR) treatment groups. No phenotypic differences were observed between male and female mice.
Details of surgery are described in the Example 2. All procedures were carried out with the approval of the University of California, Los Angeles (UCLA) Animal Research Committee or the Institutional Animal Care.
[0077] Cardiac fibroblast culture and TGF|3 treatment (murine and human)
[0078] For mouse explant fibroblast cultures, hearts were collected, digested, and plated as described in Example 2 below. Twelve hours after plating, the floating cells were removed, and the media was replaced. Media changes were done every other day until cells reached 80% confluency, at which point they were passaged and cultured in serum-free media for 24 hours prior to TGF|3 treatment (Cell Signaling, 50ng/mL). Throughout the TGF|3 treatment, the media was changed daily. Human fibroblasts were cultured according to the company’s instructions (Cell Applications) and similarly passaged for TGFp treatment (R&D Systems, 10ng/mL).
[0079] RNA Extraction and RT-qPCR
[0080] RNA was extracted from cells using TRIzol™ LS Reagent (ThermoFisher) and following the manufacturer’s instructions. RNA was quantified by NanoDrop, and cDNA was prepared using the iScript™ Reverse Transcription Supermix kit (Bio-Rad). Reverse transcription quantitative-PCR (RT-qPCR) reactions were prepared using SYBR Green (Bio- Rad) and primers (IDT) unique for each gene of interest (Table 1). The reactions were run on a CFX96™ Real-Time PCR Detection System and relative gene expression data was calculated by double delta CT analysis.
[0081] Immunocytochemical/lmmunofluorescence Staining
[0082] Cells were cultured on 8-well chamber slides (Falcon) and washed with PBS prior to fixation with 4% paraformaldehyde (PFA). For in vivo staining, murine hearts were isolated and fixed with 4% PFA overnight prior to being incubated in 30% sucrose and embedded in Optimal Cutting Temperature (OCT) compound (Fisher). Hearts were sectioned at a thickness of 8pm in a cryostat, mounted on Colorfrost Plus microscope slides (Fisher), and stored at -20°C until ready to stain. Detailed staining protocol is provided in Example 2 below. Slides were incubated with antibodies outlined in Table 2.
[0083] ELISA and Western Blot
[0084] Protocols for conditioned media and serum sample preparation are described in Example 2 below. ELISA kits were purchased from MyBiosource and the manufacturer’s protocol was followed. For western blot, protein concentration was measured by a Pierce™ BCA Protein assay kit (ThermoScientific) and 20pg was loaded into each well of 4-20% Mini- PROTEAN TGX Precast Protein gels (Bio-Rad). After transferring the gel onto a PVDF
membrane, detection of CILP was conducted by incubating the membrane with primary antibody followed by secondary antibody conjugated with HRP (Table 2). The signal was developed using the Pierce® ECL Western Blotting Substrate (ThermoScientific).
[0085] Statistical Analysis
[0086] Continuous data are presented using the mean ± standard error of the mean (SEM) and comparisons between groups were performed using Student's t-test. A p-value < 0.05 was considered statistically significant and data were analyzed using GraphPad Prism 6.
[0087] RESULTS
[0088] Murine cardiac fibroblasts express Ltbp2, Comp , and Cilp after injury
[0089] To identify secreted proteins expressed by CFbs in fibrotic hearts, we conducted RNA-sequencing on isolated CFbs from female C57BL/6J adult mice (8-12 weeks) that had undergone either sham or TAC surgery (n=3). CFbs were isolated seven days after surgery to observe gene expression changes in the early stages of fibrosis(8). After TAC, many genes were differentially expressed in CFbs (Figure 1A, Table 3). Specifically, CFbs from mice that had undergone TAC showed higher expression of various genes associated with fibrosis (Figure 1B). From these, we selected genes that encoded for secreted proteins and then further filtered the list to those that were novel in the context of heart failure and had previously reported roles in extracellular matrix formation/remodeling. We identified Ltbp2, Comp, and Cilp as potential candidate biomarkers. These results were further validated by RT-qPCR (Figure 1C).
[0090] The TGFp signaling pathway is a major component of injury response in cardiac fibroblasts(14). Treatment of fibroblasts in vitro with TGF|3 activates and induces proliferation of cultured cells, imitating in vivo responses (15). To confirm that the TGF|3 signaling pathway stimulates a robust increase in the expression of the identified genes, CFbs from uninjured C57BL/6J mice were cultured in media with or without TGF|3 for 72 hours (Figure 2A). CFbs were isolated by whole explant culture to encompass the entire CFb population in the heart, rather than a subpopulation(16). TGF|3 treatment induced expression of fibroblast activation genes, such as Periostin ( Postn ) and a-smooth muscle actin (Acfa2)(17), as well as Ltbp2, Comp, and Cilp (Figure 2B). Furthermore, immunocytochemistry (ICC) confirmed that expression of LTBP2, COMP, and CILP were increased at the protein level in cultured CFbs after exposure to TGF|3 (Figure 2C). We observed similar patterns of staining for these proteins when CFbs were isolated by fluorescence-activated cell sorting (Figure 7)(8). These data confirm that CFbs are a cellular source of LTBP2, COMP, and CILP under stimulatory conditions.
[0091] LTBP2, COMP, and CILP are localized to fibrotic regions
[0092] Although TAC surgery induces fibrosis, it also causes other cardiac pathologies, such as hypertrophy(18). In order to confirm that the increase in LTBP2, COMP, and CILP expression after injury was specific to scar formation, we analyzed the anatomic location of LTBP2, COMP, and CILP in the hearts of mice that had undergone TAC surgery. After seven days, there was visible perivascular and interstitial fibrosis in TAC hearts, compared to sham which exhibited no fibrosis (Figure 3A). Immunofluorescence (IF) staining showed minimal expression of the three proteins in sham hearts. In TAC hearts, LTBP2, COMP, and CILP expression appeared to colocalize with Discoidin domain-containing receptor 2 (DDR2), a marker for fibroblasts(19), and a-smooth muscle actin (aSMA)(20) within the fibrotic regions of the myocardium in TAC hearts (Figure 3B). Areas of nonfibrotic myocardium in TAC hearts did not stain for any of the target proteins, indicating that expression of LTBP2, COMP, and CILP are expressed by activated cardiac fibroblasts and localized to regions of fibrosis.
[0093] We next sought to determine whether the expression of these biomarkers is also observed in other types of cardiac fibrosis, such as replacement fibrosis after myocardial infarction. Our findings were further confirmed in an ischemic reperfusion (IR) injury model in which the hearts exhibited discrete areas of fibrosis, although not to the severity of TAC injury. LTBP2, COMP, and CILP were found to be specifically co-localized with DDR2 and aSMA in hearts that had undergone IR (Figure 8). Together, these data suggest that LTBP2, COMP and CILP are expressed by activated cardiac fibroblasts and are localized to regions of fibrosis.
[0094] Human cardiac fibroblasts have increased LTBP2, COMP, and CILP levels in response to TGF|31 treatment
[0095] To confirm the clinical utility of our identified proteins as biomarkers for cardiac fibrosis, we sought to assess their expression levels in human ischemic myocardial tissue. RNA-sequencing data of human cardiac tissue from ischemic heart failure patients in a publicly available database (GSE46224) demonstrated that LTBP2, COMP, and CILP are upregulated in ischemic hearts (Figure 4A)(21). We next cultured human CFbs and treated them with TGF|31 to stimulate their in vitro activation (Figure 4B). TGF|31 treatment led to morphological changes in human CFbs and induced expression of LTBP2, COMP, and CILP, along with fibroblast activation genes (Figure 4C). ICC staining confirmed the upregulation of LTBP2, COMP, and CILP in response to TGF|31 treatment, as seen in mouse CFbs (Figure 4D). Conditioned media from cells that had undergone TGF|31 treatment did not show significant differences in the levels of LTBP2, an increasing trend of
COMP levels, and decreased levels of CILP (Figure 9). These results may be due to unknown mechanisms of protein secretion that affect the presence of these proteins in the context of our culture protocol. The results from the in vitro culture of human CFbs mirrored our data from mice, further supporting the potential of these proteins to be biomarkers for cardiac fibrosis.
[0096] LTBP2, COMP, and CILP are potential biomarkers for cardiac fibrosis
[0097] We used IF staining to observe the expression of LTBP2, COMP, and CILP within the myocardium of heart failure patients compared to healthy hearts. Myocardial tissue from heart failure patients (with a documented diagnosis of ischemic cardiomyopathy) exhibited significant amounts of fibrosis compared to healthy hearts (Figure 5A). In healthy hearts, we observed no or minimal positive staining for the candidate markers throughout the myocardium (Figure 5B). However, sections from diseased hearts demonstrated a significant increase in expression of all three proteins (Figure 5B). Staining for these three proteins were localized to disarrayed regions of the myocardium, indicative of the specificity of these proteins for fibrotic areas.
[0098] Full length CILP is significantly decreased in serum from heart failure patients
[0099] In addition to increased expression within the fibrotic myocardium, we sought to determine the utility of LTBP2, COMP, and CILP as novel circulating biomarkers for cardiac fibrosis. We measured the protein levels in serum from healthy volunteers and heart failure patients by ELISA (Table 4). We observed no significant differences in the circulating levels of LTBP2(22) or COMP (Figure 6A). However, serum from heart failure patients exhibited significantly decreased levels of CILP (Figure 6B). Mice that had undergone TAC injury exhibited a similar trend in decreased levels of circulating CILP compared to sham mice (Figure 10). The CILP gene encodes a precursor protein that undergoes cleavage into an N- terminal fragment of roughly 75kDa and a C-terminal fragment of about 55kDa(23). Both of these fragments were shown to inhibit Smad3 phosphorylation, which is normally promoted by active TGF|3 signaling. While commercially available ELISA kits target the C-terminal region of CILP (hence detecting both the C-terminal and the full-length fragment), previous work discovered that CFbs secrete the N-terminal fragment as well as the full-length CILP protein (24). We specifically used an antibody that spans the cleavage site of the CILP precursor and performed western blotting to confirm levels of circulating full-length CILP. Our results showed that serum from heart failure patients had significantly decreased levels of full-length CILP in circulation (Figure 6C-D). Together, these data suggest while activated fibroblasts in the fibrotic regions of human myocardium express high levels of CILP, the
circulating level of CILP is significantly reduced when compared to healthy individuals with no evidence of cardiac fibrosis.
[0100] DISCUSSION
[0101] With the increasing prevalence of cardiac disease worldwide, there is significant value in identifying a robust biomarker panel to non-invasively measure the presence and progression of cardiac fibrosis. We hypothesized that, as key participants of the fibrotic response, cardiac fibroblasts may be a source of novel biomarkers for myocardial fibrosis. We performed RNA-sequencing of CFbs from TAC and sham murine hearts and identified Ltbp2, Comp , and Cilp to be upregulated in hearts after pressure overload injury. The expression of these proteins by CFbs in response to injury were validated by in vitro studies in both murine and human CFbs. Additionally, we demonstrated that these proteins localize in fibrotic regions in murine hearts after pressure overload and ischemic reperfusion injury. These findings were further confirmed by high levels of these three biomarkers in the fibrotic areas of human ischemic myocardial tissue. Notably, the circulating levels of full-length CILP protein were significantly reduced in the serum of ischemic heart failure patients compared to healthy individuals, indicating its potential to be a circulating biomarker.
[0102] LTBP2 and COMP expression is specific to fibrotic regions
[0103] LTBP2 is a member of the latent TGF|3-binding protein family, which consists of key regulators of TGF|3 signaling. TGFp has diverse and pleiotropic effects on various cell types through its binding and activation of TGFp receptors(14,25). TGFp is secreted from cells as a multiplex form that is covalently bound to latent TGFp binding proteins LTBP1 , LTBP3, and LTBP4. These proteins target the latent complex to specific sites for storage within the ECM where it awaits activation. Matrix sequestration of latent TGFp may serve to regulate its immediate bioavailability while achieving critical threshold concentration at sites of intended function(9,25). However, the functional role of LTBP2 is not well understood. Recent studies suggest that LTBP2 does not bind to latent TGFp but may interact with other ECM proteins(9). Other studies have additionally reported on the competitive role of LTBP2 with LTBP1 for binding sites on fibrillin-1 within the ECM(26). Our data show strong support for increased expression of LTBP2 in response to injury and a strong localization of LTBP2 in activated fibroblasts within the fibrotic regions of the myocardium. Whether LTBP2 is merely a surrogate for cardiac fibrosis or is involved in its pathogenesis is not entirely known.
[0104] COMP is another ECM protein that is mainly studied in the context of tendons and cartilage(27). The main function of COMP is to directly bind with other ECM components, including collagens and TGF|31, and to facilitate the stability of the ECM network by the formation of collagen fibrils(28). This role is crucial to maintaining homeostasis of the heart
as COMP-knockout mice have been shown to develop dilated cardiomyopathy(29).
However, the role of COMP in pathological remodeling is less understood. Studies have shown that COMP is upregulated in the context of idiopathic pulmonary fibrosis(30) and liver fibrosis(31), although there have been conflicting reports as to whether it can serve as an accurate circulating marker for fibrosis in patients(30,32).
[0105] Our results suggest that LTBP2 and COMP both have a strong potential for being markers for cardiac fibrosis as the expression of these proteins are specific to scar formation. However, our data does not support their use as circulating biomarkers after cardiac injury. Although these proteins are known to be secreted, it is possible that they remain within the ECM and participate in the process of fibrosis and scar formation. Further research is warranted to investigate the specific functional contributions of LTBP2 and COMP to the development of cardiac fibrosis. Due to their known roles in other organ systems, it is possible that these proteins may be markers for general fibrosis and not specific to cardiac fibrosis(33).
[0106] Decreased levels of CILP may be indicative of heart failure
[0107] The exact function of CILP within cartilage is still unknown, but it has been implicated in cartilage remodeling and maintenance of the ECM(11 ,34). The upregulation of CILP has been found in various disease models including osteoarthritis, idiopathic pulmonary fibrosis, and ischemic heart disease(34-37). However, the contribution of CILP to the development of cardiac fibrosis remains unknown. While most studies suggest that CFbs are the major source of CILP expression in the heart, a recent study has shown evidence of cardiomyocytes being another a major contributor(38,39). Although we did not explore the expression of CILP in cardiomyocytes, our data supports the claim that CFbs are a major cellular source of CILP. Several studies have reported that cardiac injury causes an upregulation of CILP in CFbs but the potential for CILP to be a potential biomarker for fibrosis had not been previously explored(24,39).
[0108] The CILP gene encodes for a precursor protein containing a furin cleavage site. The precursor is first synthesized and processed by furin proteases intracellularly prior to being secreted (23). The N-terminal fragment has been shown to directly interact with TGF|3, suppressing TGF|3 signaling in CFbs, while the C-terminal fragment is homologous to a porcine nucleotide pyrophosphohydrolase (NTPPHase) which has been reported to have limited enzymatic activity(23,40). In contrast to the two fragments, the functional role of full- length CILP protein has not been well-studied. The full-length CILP has been shown to inhibit TGF|3 signaling, similarly to the N-terminal fragment, most likely due to the common thrombospondin-1 domain which has been shown to bind to TGF|3(24). However, further
studies to determine any functional differences between the N-terminal fragment and the full- length CILP are required. Our data specifically demonstrates that circulating levels of the full- length CILP are attenuated in heart failure patients but show an abundance of expression in the fibrotic myocardium. A possible mechanism for this paradox is that full-length CILP is sequestered to the ECM by its binding to TGF|3, therefore reducing circulating levels.
Studies have reported that while injury induces increased expression of TGF|3 in the myocardium of heart failure patients(41), circulating TGF|3 is reduced(42,43). Due to the inhibitory role of full-length CILP in TGF|3 signaling, it is possible that increased levels of CILP may reside in the ECM and promote a negative feedback mechanism to suppress CFb activation(24). Further studies on the dynamics of CILP turnover in the ECM are required to elucidate the significance of both circulating and interstitial CILP.
[0109] In conclusion, this Example confirms the potential for LTBP2, COMP, and CILP as novel markers of cardiac fibrosis in both mouse and human heart failure models. Most notably, we discovered a significant reduction in serum levels of full-length CILP in patients with heart failure. Biomarkers for cardiac fibrosis will serve as a noninvasive method to gain diagnostic and prognostic information regarding patients with heart failure. Our study confirms the utility of several markers of cardiac fibrosis in mouse and human subjects.
[0110] REFERENCES
[0111] 1. Kong P., et al. Cell Mol Life Sci 2014;71(4):549-74..
[0112] 2. Khan R., Sheppard R. Immunology 2006; 118(1):10— 24.
[0113] 3. Travers JG., et al. Circ Res 2016;118(6): 1021-40.
[0114] 4. Fan D., et al. Fibrogenesis & Tissue Repair 2012;5(1 ) : 15.
[0115] 5. Park S., N et al. Translational Research 2019.
[0116] 6. Pitt Bertram, Zannad Faiez. Circulation: Cardiovascular Imaging 2012;5(1):9— 11. [0117] 7. Lajiness JD., Conway SJ. J Cardiovasc Transl Res 2012;5(6):739-48.
[0118] 8. Ali SR., et al. Circ Res 2014; 115(7):625— 35.
[0119] 9. Robertson IB., et al. Matrix Biology 2015;47:44-53.
[0120] 10. Acharya C., et al. Matrix Biology 2014;37:102-11.
[0121] 11. Lorenzo P., et al. J Biol Chem 1998;273(36):23463-8.
[0122] 12. Love Ml., et al. Genome Biology 2014; 15(12):550.
[0123] 13. Blighe K. EnhancedVolcano: Publication-ready volcano plots with enhanced colouring and labeling. R Package Version 1.2.02019. Available at: github.com/kevinblighe/EnhancedVolcano.
[0124] 14. Meng X-M., et al. Nat Rev Nephrol 2016 ; 12(6) : 325-38.
[0125] 15. Clark RA., et al. J Cell Physiol 1997; 170(1):69-80.
[0126] 16. Lynch MD., Watt FM. J Clin Invest 2018;128(1):26-35.
[0127] 17. Snider Paige, et al. Circulation Research 2009; 105(10):934-47.
[0128] 18. Patten Richard D., Hall-Porter Monica R. Circulation: Heart Failure 2009;2(2): 138-44.
[0129] 19. Goldsmith EC., et al. Dev Dyn 2004;230(4):787-94.
[0130] 20. Leslie KO., et al. Am J Pathol 1991 ; 139(1 ):207-16.
[0131] 21. Yang K-C., et al. Circulation 2014; 129(9) : 1009-21.
[0132] 22. Park S., et al. Circulation 2018;138:1224-1235.
[0133] 23. Lorenzo P., et al. J Biol Chem 1998;273(36):23469-75.
[0134] 24. Shindo K., et al. International Journal of Gerontology 2017;11(2):67-74. [0135] 25. Dobaczewski M., et al. J Mol Cell Cardiol 2011 ;51(4):600-6.
[0136] 26. Hirani R., et al. Matrix Biol 2007;26(4):213-23.
[0137] 27. Hedbom E., et al. J Biol Chem 1992;267(9):6132-6.
[0138] 28. Rosenberg K., et al. J Biol Chem 1998;273(32):20397-403.
[0139] 29. Huang Y., et al. Basic Res Cardiol 2013;108(5):374.
[0140] 30. Vuga LJ., et al. PLoS ONE 2013;8(12):e83120.
[0141] 31. Magdaleno F., et al. J Hepatol 2016;65(5):963-71.
[0142] 32. Hesselstrand R., et al. Rheumatology (Oxford) 2012;51(5):915-20.
[0143] 33. Zachou K., et al. Eur J Intern Med 2017;38:83-8.
[0144] 34. Bernardo BC., et al. J Biol Chem 2011 ;286(43):37758-67.
[0145] 35. Luzina IG., et al. Cell Immunol 2018;325:1-13.
[0146] 36. Sanders YY„ et al. Am J Respir Crit Care Med 2012;186(6):525-35.
[0147] 37. Barallobre-Barreiro, J. et al. Circulation 2012;125(6):789-802.
[0148] 38. Zhang C-L, et al. Journal of Molecular and Cellular Cardiology 2018; 116: 135— 44.
[0149] 39. Nieuwenhoven FA van., et al. Scientific Reports 2017;7(1):16042.
[0150] 40. Seki S., et al. Nature Genetics 2005;37(6):607-12.
[0151] 41. Edgley AJ., et al. Cardiovascular Therapeutics 2012;30(1):e30-40.
[0152] 42. Aukrust P., et al. The American Journal of Cardiology 1999;83(3):376-82.
[0153] 43. Kapur Navin K. Circulation: Heart Failure 2011 ;4(1):5— 7.
Example 2: Additional Methods and Tables
[0154] This Example describes further details of the methods employed in Example 1. In addition, Tables refenced in Example 1 can be found below.
[0155] RNA-Sequencing and Analysis
[0156] To identify potential candidates for secreted biomarkers of cardiac fibrosis, we first identified genes that were differentially expressed in cardiac fibroblasts after transverse aortic constriction compared to sham operation (GSE51620) (p<0.05). We selected for genes that exhibited a minimum fold-change increase of 4 to select for significantly upregulated genes in TAC. We then identified genes that were associated with fibrosis through the literature and screening for genes associated with the following Gene Ontology classes: G0:0062023 (collagen-containing extracellular matrix), G0:0001666 (response to hypoxia), G0:0001968 (fibronectin binding), G0:0030199 (collagen fibril organization), G0:0005201 (extracellular matrix structural constituent) and/or G0:0005615 (extracellular space). From this list, we further selected for genes that encoded for at least one protein product that was reportedly secreted. This was identified using online resources such as Uniprot, the Human Protein Atlas, and previously published literature. This process allowed for the selection of only secreted proteins that may mark the presence of cardiac fibrosis.
[0157] Validation of secreted proteins that were identified by our RNA sequencing was conducted using a different cardiac injury, treatment with isoproterenol (ISO). C57BL/6J mice were implanted with ALZET osmotic pumps in the abdominal cavity while under anesthesia with isoflurane. The pumps were filled with saline or ISO (Sigma) at a concentration to treat the mice at a rate of 30mg/kg body weight/day for a treatment period of 21 days. Mouse serum was collected at the end of the treatment period and prepared for analysis as described in the section titled “Serum Preparation”. Western blot and ELISA were used to screen through potential secreted proteins. Proteins that demonstrated a
notable difference between saline- and ISO-treated samples were further selected for experiments.
[0158] Transverse Aortic Constriction Surgery, Ischemic-Reperfusion Surgery, and Aftercare
[0159] Adult C57BL/6J mice were anesthetized by intraperitoneal injection of ketamine/xylazine (100mg/10mg/kg). Endotracheal intubation was performed using a blunt 20-gauge needle connected to a volume-cycled rodent ventilator (SAR-830/P; OWE, Inc.) with a tidal volume of 0.2 ml and a respiratory rate of 120/min. For transverse aortic constriction surgery, the chest was opened to expose the transverse aorta, located through the second intercostal space. Aortic constriction was performed by tying a 7-0 nylon suture ligature against a 27-gauge blunt needle and then removing the needle to yield a constriction of roughly 0.4mm in diameter. For ischemic-reperfusion surgery, left thoracotomy between ribs four and five was performed. The pericardium was opened, and a suture was placed around the left anterior descending coronary artery 1-2mm from the tip of the left atrium. The suture was tightened to occlude blood flow for 45 minutes and subsequently removed. Mice that underwent sham operations underwent the same procedure, excluding the constriction/occlusion. After the operation, the chest was closed in layers using 5-0 Vicryl sutures and the mice remained on the ventilators until sufficient spontaneous breathing was resumed, at which point the endotracheal tube was removed. The entire surgical procedure was performed under aseptic conditions. Buprenorphine (0.1 mg/kg) was administrated by subcutaneous injection immediately prior to surgery, followed by every 12 hours for 48 hours, and carprofen (5mg/Kg) was administrated post operation every 24 hours for 48 hours. Mice were additionally treated post-operatively with Sulfamethoxazole and Trimethoprim oral suspension (Aurbindo). Operators blinded to the experimental designs performed all animal surgeries and in vivo analyses.
[0160] Mouse Heart Explant Fibroblast Culture
[0161] Adult C57BL/6J mice were sacrificed by isoflurane followed by cervical dislocation. Hearts were dissected from the mice and cannulated with a blunt syringe. The hearts were perfused with 20-30ml PBS, subsequently chopped into small pieces and incubated in 7- 10ml enzyme at 37°C on a rotator for an hour with periodic pipetting to digest larger pieces. The enzyme mix consisted of TH and TM liberases (Roche), Dnase (Invitrogen), and Poloxamer. The cells were then passed through a 70pm filter, centrifuged, and the pellet was resuspended in DMEM containing 20% FBS, 1% Penicillin Streptomycin, and 0.1% Ciprofloxacin. The cells from one heart were plated in a single well of a 6-well plate that had been coated with 0.1% gelatin.
[0162] Immunocytochemical/lmmunofluorescence Staining Detailed Protocol
[0163] For immunocytochemical staining, the fixed cells were blocked with blocking buffer (10% NGS, PBS-0.1%Tween 20) for 1 hour at room temperature and then incubated in diluted primary antibodies (Table 2) overnight at 4°C. The slides were then washed 3 times with PBS-0.1%Tween and then incubated in diluted secondary antibodies (Table 2) for an hour at room temperature. After another round of washing with PBS-0.1%Tween 20, the coverslips were mounted using mounting medium containing DAPI (Vector Laboratories).
For immunofluorescence staining, slides were incubated at room temperature for 10 minutes prior to 3 washes with PBS. The sections were treated with 0.25% Triton X-100 in PBS to permeabilize for 10 minutes. The subsequent blocking and staining protocol mirrored immunocytochemical staining as described. Imaging was done on either a Zeiss confocal microscope (LSM880) or a Leica fluorescent microscope (LEICACTR6500). Image processing and analysis was done through either ZEN 2 (blue edition) or LAS AF Lite.
[0164] RT-qPCR Conditions
[0165] The PCR conditions for RT-qPCR had the following steps: (1) Initial denaturation - 95°C - 2 minutes and 10 seconds; (2) Denaturation - 95°C - 15 seconds; (3) Annealing - 60°C - 30 seconds; (4) Extension - 72°C - 30 seconds; (5) Repeat steps 2-4 for a total of 39 cycles; (6) Final extension - 72°C - 10 minutes. The mean cycle threshold (Ct) values were taken from triplicate measurements to determine relative gene expression, as normalized to Gapdh/GAPDH expression.
[0166] Conditioned Media Preparation
[0167] Cells were cultured in 6-well plates and culture media was collected every day during the treatment period for a total of three times. The collected media was centrifuged for 10,000rpm for 10 minutes at 4°C and the supernatant was transferred to a separate tube to remove any cellular contamination. The media was stored at -80°C until ready for analysis. For ELISA, the media from each day was pooled into a single sample per well. The media was concentrated by Amicon® Ultra Centrifugal Filters and the final concentrated volume was noted for total concentration calculations after ELISA.
[0168] Serum Preparation
[0169] Healthy human samples were purchased from Equitech Enterprises. Blood samples from heart failure patients were collected with informed consent (Table 4). Serum was isolated using Ficoll and stored at -80°C until enough samples were collected for experiments. For western blots, albumin was removed from the samples with the AlbuSorb™ Albumin Depletion Kit (Biotech Support Group).
[0170] Table 1 : RT-qPCR Primers
[0172] Table 3: Significantly up-regulated genes in mouse CFbs in TAC (Fold change > 4, p- value < 0.05)
[0174] ‘Abbreviations: Non-iCMY: Non-ischemic cardiomyopathy; D: Diuretics; BB: Beta blockers; Ent: Entresto; ACE-I: Ace-inhibitors; ARB: Angiotensin receptor blockers; AA: Aldosterone antagonists
[0175] Throughout this application various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to describe more fully the state of the art to which this invention pertains.
[0176] Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims.
Claims
1. A method for detecting cardiac fibrosis, progression of cardiac fibrosis, or heart failure in a subject, the method comprising:
(a) contacting a serum sample obtained from the subject with a binding agent that binds a region of cartilage intermediate layer protein 1 (CILP) that spans the cleavage site of the CILP precursor or a nucleotide encoding same;
(b) measuring the amount of CILP in the serum sample; and
(c) detecting cardiac fibrosis, progression of cardiac fibrosis, or heart failure in the subject when the amount of CILP in the serum sample is decreased relative to a reference sample.
2. A method for treating cardiac fibrosis, progression of cardiac fibrosis, or heart failure in a subject, the method comprising:
(a) contacting a serum sample obtained from the subject with a binding agent that binds a region of CILP that spans the cleavage site of the CILP precursor or a nucleotide encoding same;
(b) measuring the amount of CILP in the serum sample; and
(c) treating the subject for cardiac fibrosis, progression of cardiac fibrosis, or heart failure when the amount of CILP in the serum sample is decreased relative to a reference sample.
3. A method for monitoring the status of cardiac fibrosis or heart failure in a subject, the method comprising:
(a) contacting a serum sample obtained from the subject with a binding agent that binds a region of CILP that spans the cleavage site of the CILP precursor or a nucleotide encoding same;
(b) measuring the amount of CILP in the serum sample; and
(c) increasing treatment of the subject for cardiac fibrosis or heart failure when the amount of CILP in the serum sample is decreased relative to a reference sample, and decreasing treatment of the subject for cardiac fibrosis or heart failure when the amount of CILP is equal to or increased relative to the reference sample.
4. The method of claim 1 , further comprising treating the subject for cardiac fibrosis or heart failure.
5. The method of claim 2, 3 or 4, wherein the treatment comprises administering to the subject one or more of: angiotensin (AT)-converting enzymes, ATI receptor antagonists, b-blockers, Sacubitril/Valsartan, Aldosterone antagonists, statins, diuretics, or other medications/biologics with the purpose of treating underlying heart failure and fibrosis.
6. The method of any of claims 1-5, wherein the reference sample is from a normal, healthy control subject.
7. The method of any of claims 1-5, wherein the reference sample is a previously obtained sample from the subject.
8. The method of any of the preceding claims, wherein the binding agent is an antibody.
9. The method of claim 8, wherein the antibody binds to full-length CILP.
10. The method of any of the preceding claims, wherein the binding agent is a nucleic acid probe.
11. The method of any of the preceding claims, further comprising measuring up to 10 additional biomarkers.
12. The method of claim 11 , wherein the additional biomarkers are selected from B-type natriuretic peptide (BNP; or its stable precursor, NT-proBNP), Galectin-3 (Gal-3), suppression of tumorigenicity 2 (ST2), latent transforming growth factor beta (LTBP2), and cartilage oligomeric matrix protein (COMP).
13. The method of any of the preceding claims, wherein the cardiac fibrosis is associated with one or more of: ischemia, congenital defect, familial fibrosis, infiltrative fibrosis, idiopathic fibrosis, amyloidosis, hemosiderosis, valvular disease, and other idiopathic cardiomyopathies.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US17/995,266 US20230152331A1 (en) | 2020-04-02 | 2021-04-01 | Full-length cilp as a biomarker for cardiac fibrosis |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US202063003451P | 2020-04-01 | 2020-04-01 | |
US63/003,451 | 2020-04-01 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2021203141A1 true WO2021203141A1 (en) | 2021-10-07 |
Family
ID=77929187
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2021/070349 WO2021203141A1 (en) | 2020-04-01 | 2021-04-01 | Full-length cilp as a biomarker for cardiac fibrosis |
Country Status (1)
Country | Link |
---|---|
WO (1) | WO2021203141A1 (en) |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7211649B1 (en) * | 1999-07-02 | 2007-05-01 | Ana Mar Medical Ab | Cartilage intermediate layer protein |
US20080241945A1 (en) * | 2007-03-27 | 2008-10-02 | Pilar Lorenzo | Peptide for differentiating osteoarthritis from rheumatoid arthritis and non-disease conditions |
-
2021
- 2021-04-01 WO PCT/US2021/070349 patent/WO2021203141A1/en active Application Filing
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7211649B1 (en) * | 1999-07-02 | 2007-05-01 | Ana Mar Medical Ab | Cartilage intermediate layer protein |
US20080241945A1 (en) * | 2007-03-27 | 2008-10-02 | Pilar Lorenzo | Peptide for differentiating osteoarthritis from rheumatoid arthritis and non-disease conditions |
Non-Patent Citations (3)
Title |
---|
LEHR STEFAN, HARTWIG SONJA, LAMERS DANIELA, FAMULLA SUSANNE, MÜLLER STEFAN, HANISCH FRANZ-GEORG, CUVELIER CLAUDE, RUIGE JOHANNES, : "Identification and validation of novel adipokines released from primary human adipocytes", MOLECULAR AND CELLULAR PROTEOMICS, vol. 11, no. 1, January 2012 (2012-01-01), pages 1 - 13, XP055927653, DOI: 10.1074/mcp.M111.010504 * |
SHINDO, K ET AL.: "Cartilage Intermediate Layer Protein 1 Suppresses TGF-beta Signaling in Cardiac Fibroblasts", INTERNATIONAL JOURNAL OF GERONTOLOGY, vol. 11, no. 2, June 2017 (2017-06-01), pages 67 - 74, XP085077085, DOI: 10.1016/j.ijge.2017.01.002 * |
VAN NIEUWENHOVEN FRANS A., MUNTS CHANTAL, OP’T VELD ROEL C., GONZÁLEZ ARANTXA, DÍEZ JAVIER, HEYMANS STEPHANE, SCHROEN BLANCHE, VAN: "Cartilage intermediate layer protein 1 (CILP1): A novel mediator of cardiac extracellular matrix remodelling", SCIENTIFIC REPORTS, vol. 7, no. 1, 2 November 2017 (2017-11-02), pages 1 - 9, XP055748475, DOI: 10.1038/s41598-017-16201-y * |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Mura et al. | Osteopontin lung gene expression is a marker of disease severity in pulmonary arterial hypertension | |
Schaefer et al. | Small proteoglycans in human diabetic nephropathy: discrepancy between glomerular expression and protein accumulation of decorin, biglycan, lumican, and fibromodulin | |
Park et al. | Cardiac fibrosis is associated with decreased circulating levels of full-length CILP in heart failure | |
Chen et al. | WWP2 regulates pathological cardiac fibrosis by modulating SMAD2 signaling | |
EP1730181A2 (en) | Novel brain natriuretic peptide variants and methods of use thereof | |
EP2565275B1 (en) | Method of treatment of vascular complications using modulators of TRX and TRXNIP | |
JP5686730B2 (en) | Methods and pharmaceutical compositions for inhibiting, delaying and / or preventing cardiac hypertrophy | |
Vergara et al. | The direct effect of fibroblast growth factor 23 on vascular smooth muscle cell phenotype and function | |
WO2021203141A1 (en) | Full-length cilp as a biomarker for cardiac fibrosis | |
Nordquist et al. | Tgfβ1-cthrc1 signaling plays an important role in the short-term reparative response to heart valve endothelial injury | |
US20230152331A1 (en) | Full-length cilp as a biomarker for cardiac fibrosis | |
JP2008507261A (en) | Novel nucleotide and amino acid sequences for lung cancer diagnosis, and assays and methods of use thereof | |
EP4242661A1 (en) | Composition for regulating osteoclast differentiation, and use thereof | |
CN110699442B (en) | Application of LncRNA PEBP1P2, kit for diagnosing heart diseases and medicine for treating heart diseases | |
WO2015061534A1 (en) | Methods for diagnosing, screening, identifying, monitoring, and treating adverse local tissue reactions in orthopedic implants | |
CN111154863B (en) | Application of lncRNA in preparation of product for diagnosing and/or treating osteoarthritis | |
CN111118143B (en) | Reagent for detecting and targeting RP11-754B17.1 and application thereof in arthritis | |
US20220251569A1 (en) | Treatment and prevention of disease mediated by wwp2 | |
JP2007525213A (en) | Novel variant of brain natriuretic peptide and method of use thereof | |
WO2017026733A1 (en) | Composition for diagnosing, preventing, or treating vascular smooth muscle cell proliferative diseases using fgf12 | |
WO2010084668A1 (en) | Diagnostic method for nephrotic syndrome, prophylactic or therapeutic agent for nephrotic syndrome, and method for screening the prophylactic or therapeutic agent | |
KR20160062517A (en) | A pharmaceutical composition for inhibition of vascular calcification comprising dpp-4 inhibitor as an effective component | |
CN111235263B (en) | Target gene for diagnosis and treatment of osteoarthritis | |
WO2023120612A1 (en) | Therapeutic or prophylactic agent for heart attack, heart fibrosis, or heart failure, where htra3 is therapeutic target | |
KR102436301B1 (en) | A composition for diagnosing or treating fibrosis |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 21779588 Country of ref document: EP Kind code of ref document: A1 |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
122 | Ep: pct application non-entry in european phase |
Ref document number: 21779588 Country of ref document: EP Kind code of ref document: A1 |