title: Assessment of proteolytic degradation of the basement membrane: a fragment of type IV collagen as a biochemical marker for liver fibrosis.
abstract: BACKGROUND
  Collagen deposition and an altered matrix metalloproteinase (MMP) expression profile are hallmarks of fibrosis. Type IV collagen is the most abundant structural basement membrane component of tissue, which increases 14-fold during fibrogenesis in the liver. Proteolytic degradation of collagens by proteases produces small fragments, so-called neoepitopes, which are released systemically. Technologies investigating MMP-generated fragments of collagens may provide more useful information than traditional serological assays that crudely measure total protein. In the present study, we developed an ELISA for the quantification of a neoepitope generated by MMP degradation of type IV collagen and evaluated the association of this neoepitope with liver fibrosis in two animal models.
  
  
  METHODS
  Type IV collagen was degraded in vitro by a variety of proteases. Mass spectrometric analysis revealed more than 200 different degradation fragments. A specific peptide sequence, 1438'GTPSVDHGFL'1447 (CO4-MMP), in the α1 chain of type IV collagen generated by MMP-9 was selected for ELISA development. ELISA was used to determine serum levels of the CO4-MMP neoepitope in two rat models of liver fibrosis: inhalation of carbon tetrachloride (CCl4) and bile duct ligation (BDL). The levels were correlated to histological findings using Sirius red staining.
  
  
  RESULTS
  A technically robust assay was produced that is specific to the type IV degradation fragment, GTPSVDHGFL. CO4-MMP serum levels increased significantly in all BDL groups compared to baseline, with a maximum increase of 248% seen two weeks after BDL. There were no changes in CO4-MMP levels in sham-operated rats. In the CCl4 model, levels of CO4-MMP were significantly elevated at weeks 12, 16 and 20 compared to baseline levels, with a maximum increase of 88% after 20 weeks. CO4-MMP levels correlated to Sirius red staining results.
  
  
  CONCLUSIONS
  This ELISA is the first assay developed for assessment of proteolytic degraded type IV collagen, which, by enabling quantification of basement membrane degradation, could be relevant in investigating various fibrogenic pathologies. The CO4-MMP degradation fragment was highly associated with liver fibrosis in the two animal models studied.
authors.affiliation: Nordic Bioscience A/S, Herlev Hovedgade 207, DK-2730 Herlev, Denmark. ssv@nordicbioscience.com.
authors.forename: Sanne S
authors.initials: SS
authors.lastname: Veidal
authors.forename: Morten A
authors.initials: MA
authors.lastname: Karsdal
authors.forename: Arkadiusz
authors.initials: A
authors.lastname: Nawrocki
authors.forename: Martin R
authors.initials: MR
authors.lastname: Larsen
authors.forename: Yueqin
authors.initials: Y
authors.lastname: Dai
authors.forename: Qinlong
authors.initials: Q
authors.lastname: Zheng
authors.forename: Per
authors.initials: P
authors.lastname: Hägglund
authors.forename: Ben
authors.initials: B
authors.lastname: Vainer
authors.forename: Helene
authors.initials: H
authors.lastname: Skjøt-Arkil
authors.forename: Diana J
authors.initials: DJ
authors.lastname: Leeming