title: MMP mediated degradation of type IV collagen alpha 1 and alpha 3 chains reflects basement membrane remodeling in experimental and clinical fibrosis--validation of two novel biomarker assays.
abstract: OBJECTIVE
  Fibrosis is characterized by excessive tissue remodeling resulting from altered expression of various growth factors, cytokines and proteases. We hypothesized that matrix metalloproteinase (MMP) mediated degradation of type IV collagen, a main component of the basement membrane, will release peptide fragments (neo-epitopes) into the circulation. Here we present the development of two competitive enzyme-linked immunosorbent assays (ELISAs) for assessing the levels of specific fragments of type IV collagen α1 (C4M12a1) and α3 (C4M12a3) chains in serum as indicators of fibrosis.
  
  
  METHODS
  Fragments of type IV collagen cleaved in vitro by MMP-12 were identified by mass spectrometry, and two were chosen for ELISA development due to their unique sequences. The assays were evaluated using samples from a carbon tetrachloride (CCl₄) rat model of liver fibrosis and from patients with idiopathic pulmonary fibrosis (IPF) or chronic obstructive pulmonary disease (COPD).
  
  
  RESULTS
  Two technically robust ELISAs were produced using neo-epitope specific monoclonal antibodies. Mean serum C4M12a1 levels were significantly elevated in CCl₄-treated rats compared with controls in weeks 12, 16, and 20, with a maximum increase of 102% at week 16 (p < 0.0001). Further, C4M12a1 levels correlated with the total collagen content of the liver in CCl₄-treated rats (r = 0.43, p = 0.003). Mean serum C4M12a3 levels were significantly elevated in patients with mild, moderate, and severe IPF, and COPD relative to healthy controls, with a maximum increase of 321% in COPD (p < 0.0001).
  
  
  CONCLUSIONS
  Two assays measuring C4M12a1 and C4M12a3 enabled quantification of MMP mediated degradation of type IV collagen in serum. C4M12a1 was elevated in a pre-clinical model of liver fibrosis, and C4M12a3 was elevated in IPF and COPD patients. This suggests the use of these assays to investigate pathological remodeling of the basement membrane in different organs. However, validations in larger clinical settings are needed.
authors.affiliation: Fibrosis Biology and Biomarkers, Nordic, Bioscience, Herlev, Denmark.
authors.forename: Jannie Marie
authors.initials: JM
authors.lastname: Sand
authors.affiliation: Fibrosis Biology and Biomarkers, Nordic, Bioscience, Herlev, Denmark.
authors.forename: Lise
authors.initials: L
authors.lastname: Larsen
authors.affiliation: Department of Pathology, University of Michigan, Ann Arbor, Michigan, United States of America.
authors.forename: Cory
authors.initials: C
authors.lastname: Hogaboam
authors.affiliation: Division of Pulmonary and Critical Care Medicine, University of Michigan, Ann Arbor, Michigan, United States of America.
authors.forename: Fernando
authors.initials: F
authors.lastname: Martinez
authors.affiliation: Division of Pulmonary and Critical Care Medicine, University of Michigan, Ann Arbor, Michigan, United States of America.
authors.forename: Meilan
authors.initials: M
authors.lastname: Han
authors.affiliation: Faculty of Health Science, University of Southern Denmark, Odense, Denmark.
authors.forename: Martin
authors.initials: M
authors.lastname: Røssel Larsen
authors.affiliation: Faculty of Health Science, University of Southern Denmark, Odense, Denmark.
authors.forename: Arkadiusz
authors.initials: A
authors.lastname: Nawrocki
authors.affiliation: Nordic Bioscience Beijing, Beijing, China.
authors.forename: Qinlong
authors.initials: Q
authors.lastname: Zheng
authors.affiliation: Fibrosis Biology and Biomarkers, Nordic, Bioscience, Herlev, Denmark.
authors.forename: Morten Asser
authors.initials: MA
authors.lastname: Karsdal
authors.affiliation: Fibrosis Biology and Biomarkers, Nordic, Bioscience, Herlev, Denmark.
authors.forename: Diana Julie
authors.initials: DJ
authors.lastname: Leeming
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D001324:Autoantigens / Q000378:metabolism*; D001485:Basement Membrane / Q000503:physiopathology*; D002251:Carbon Tetrachloride / Q000633:toxicity; D024141:Collagen Type IV / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008103:Liver Cirrhosis / Q000139:chemically induced / Q000378:metabolism*; D013058:Mass Spectrometry; D053508:Matrix Metalloproteinase 12 / Q000378:metabolism*; D029424:Pulmonary Disease, Chronic Obstructive / Q000378:metabolism*; D011658:Pulmonary Fibrosis / Q000378:metabolism*; D051381:Rats
chemical_list: D001324:Autoantigens; D024141:Collagen Type IV; C057112:type IV collagen alpha3 chain; D002251:Carbon Tetrachloride; D053508:Matrix Metalloproteinase 12