title: MMP mediated degradation of type VI collagen is highly associated with liver fibrosis--identification and validation of a novel biochemical marker assay.
abstract: OBJECTIVE
  During fibrogenesis, in which excessive remodeling of the extracellular matrix occurs, both the quantity of type VI collagen and levels of matrix metalloproteinases, including MMP-2 and MMP-9, increase significantly. Proteolytic degradation of type VI collagen into small fragments, so-called neo-epitopes, may be specific biochemical marker of liver fibrosis. The aim of this study was to develop an ELISA detecting a fragment of type VI collagen generated by MMP-2 and MMP-9, and evaluate this assay in two preclinical models of liver fibrosis.
  
  
  METHODS
  Mass spectrometric analysis of cleaved type VI collagen revealed a large number of protease-generated neo-epitopes. A fragment unique to type VI collagen generated by MMP-2 and MMP-9 was selected for ELISA development. The CO6-MMP assay was evaluated in two rat models of liver fibrosis: bile duct ligation (BDL) and carbon tetrachloride (CCl4)-treated rats.
  
  
  RESULTS
  Intra- and inter-assay variation was 4.1% and 10.1% respectively. CO6-MMP levels were significantly elevated in CCl(4)-treated rats compared to vehicle-treated rats at weeks 12 (mean 30.9 ng/mL vs. 12.8 ng/mL, p = 0.002); week 16 (mean 34.0 ng/mL vs. 13.7 ng/mL, p = 0.0018); and week 20 (mean 35.3 ng/mL vs. 13.3 ng/mL, p = 0.0033) with a tight correlation between hepatic collagen content and serum levels of CO6-MMP (R(2) = 0.58, p<0.0001) in CCl(4)- treated rats. In BDL rats, serum levels of CO6-MMP were significantly elevated compared to the levels in sham-operated animals both at 2 weeks (mean 29.5 ng/mL vs. 14.2 ng/mL, p = 0.0001) and 4 weeks (mean 33.0 ng/mLvs. 11.8 ng/mL, p = 0.0003).
  
  
  CONCLUSIONS
  This novel ELISA is the first assay enabling assessment of MMP degraded type VI collagen, allowing quantification of type VI collagen degradation, which would be relevant for different pathologies. The marker was highly associated with liver fibrosis in two liver fibrosis animal models, suggesting type VI turnover to be a central player in fibrogenesis.
authors.affiliation: Nordic Bioscience A/S, Herlev, Denmark. ssv@nordicbioscience.com
authors.forename: Sanne Skovgård
authors.initials: SS
authors.lastname: Veidal
authors.forename: Morten Asser
authors.initials: MA
authors.lastname: Karsdal
authors.forename: Efstathios
authors.initials: E
authors.lastname: Vassiliadis
authors.forename: Arkadiusz
authors.initials: A
authors.lastname: Nawrocki
authors.forename: Martin Røssel
authors.initials: MR
authors.lastname: Larsen
authors.forename: Quoc Hai Trieu
authors.initials: QH
authors.lastname: Nguyen
authors.forename: Per
authors.initials: P
authors.lastname: Hägglund
authors.forename: Yunyun
authors.initials: Y
authors.lastname: Luo
authors.forename: Qinlong
authors.initials: Q
authors.lastname: Zheng
authors.forename: Ben
authors.initials: B
authors.lastname: Vainer
authors.forename: Diana Julie
authors.initials: DJ
authors.lastname: Leeming
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000032:analysis; D002251:Carbon Tetrachloride / Q000633:toxicity; D024142:Collagen Type VI / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D008103:Liver Cirrhosis / Q000139:chemically induced / Q000378:metabolism*; D008297:Male; D013058:Mass Spectrometry; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism*; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism*; D051379:Mice; D051381:Rats; D017207:Rats, Sprague-Dawley; D017208:Rats, Wistar
chemical_list: D015415:Biomarkers; D024142:Collagen Type VI; D002251:Carbon Tetrachloride; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9