title: MMP and non-MMP-mediated release of aggrecan and its fragments from articular cartilage: a comparative study of three different aggrecan and glycosaminoglycan assays.
abstract: OBJECTIVE
  Aggrecan is the major proteoglycan in articular cartilage and is known to be degraded by various proteases, including matrix metalloproteinases (MMPs). The present study was undertaken to develop immunoassays detecting aggrecan and its fragments generated by MMP and non-MMP-mediated proteolysis.
  
  
  METHODS
  Two immunoassays were developed: (1) the G1/G2 sandwich assay employing a monoclonal antibody (F-78) both as a capturing and a detecting antibody, and (2) the 342-G2 sandwich assay substituting the capturing antibody in the G1/G2 test with a monoclonal antibody, AF-28 recognizing the 342FFGVG neo-epitope generated by MMP cleavage. These assays were compared to the commercially available glycosaminoglycan (GAG) assay.
  
  
  RESULTS
  In supernatants of Oncostatin M and Tumor Necrosis Factor alpha (OSM/TNFalpha) stimulated explants, high levels of G1/G2 fragments and GAGs were released in the initial phase (days 2-5), followed by low levels in the intermediate (days 9-12) and late phase (days 12-21). MMP-generated fragments were detected in the late phase only. In the presence of the general MMP inhibitor GM6001, 342-G2 was not detected, whereas the G1/G2 profile remained virtually unchanged. In patients with rheumatoid arthritis (RA), the release of G1/G2 molecules was decreased (27.3%), and that of the 342-G2 fragments increased compared to healthy controls (33.3%).
  
  
  CONCLUSIONS
  The stimulation of bovine articular cartilage explants with OSM/TNFalpha released aggrecan fragments both in an MMP and non-MMP-mediated route. These immunoassays carry a potential as diagnostic tools for the quantitative assessment of the cartilage turnover in RA patients in addition to their utility in ex vivo explant cultures.
authors.affiliation: Nordic Bioscience, Herlev, Denmark. eus@nordicbioscience.com
authors.forename: E U
authors.initials: EU
authors.lastname: Sumer
authors.forename: B C
authors.initials: BC
authors.lastname: Sondergaard
authors.forename: J C
authors.initials: JC
authors.lastname: Rousseau
authors.forename: P D
authors.initials: PD
authors.lastname: Delmas
authors.forename: A J
authors.initials: AJ
authors.lastname: Fosang
authors.forename: M A
authors.initials: MA
authors.lastname: Karsdal
authors.forename: C
authors.initials: C
authors.lastname: Christiansen
authors.forename: P
authors.initials: P
authors.lastname: Qvist
mesh_terms: D053674:Aggrecans / Q000378:metabolism*; D000818:Animals; D002358:Cartilage, Articular / Q000378:metabolism*; D002417:Cattle; D005260:Female; D006025:Glycosaminoglycans / Q000378:metabolism*; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D020782:Matrix Metalloproteinases / Q000378:metabolism*; D051379:Mice; D008875:Middle Aged
chemical_list: D053674:Aggrecans; D006025:Glycosaminoglycans; D020782:Matrix Metalloproteinases