title: The effect of protease inhibitors on the induction of osteoarthritis-related biomarkers in bovine full-depth cartilage explants.
abstract: OBJECTIVE
  The specific degradation of type II collagen and aggrecan by matrix metalloproteinase (MMP)-9, -13 and ADAMTS-4 and -5 (aggrecanase-1 and -2) in the cartilage matrix is a critical step in pathology of osteoarthritis (OA). The aims of this study were: i) To investigate the relative contribution of ADAMTS-4 and ADAMTS-5 to cartilage degradation upon catabolic stimulation; ii) To investigate the effect of regulating the activities of key enzymes by mean of broad-spectrum inhibitors.
  
  
  METHODS
  Bovine full-depth cartilage explants stimulated with tumor necrosis factor alpha (TNF-α) and Oncostatin M (OSM) were cultured for 21 days with or without a number of inhibitors targeting different types of proteases. Monoclonal antibodies were raised against the active sites of ADAMTS-4, -5, MMP-9 and -13, and 4 ELISAs were developed and technically validated. In addition, the established AGNxI (ADAMTS-degraded aggrecan), AGNxII (MMP-degraded aggrecan), and CTX-II (MMP-derived type II collagen) were quantified in the explants-conditioned media.
  
  
  RESULTS
  We found that: i) Active ADAMTS-4, MMP-9, -13 were released in the late stage of TNF-α/ OSM stimulation, whereas no significant active ADAMTS-5 was detected in either extracts or supernatants; ii) Active ADAMTS-4 was primarily responsible for E373-374A bond cleavage in aggrecan in this setting; and iii) The compensatory mechanism could be triggered following the blockage of the enzyme caused by inhibitors.
  
  
  CONCLUSIONS
  ADAMTS-4 appeared to be the major protease for the generation of 374ARGS aggrecan fragment in the TNF-α/OSM stimulated bovine cartilage explants. This study addresses the need to determine the roles of ADAMTS-4 and ADAMTS-5 in human articular degradation in OA and hence identify the attractive target for slowing down human cartilage breakdown.
authors.affiliation: NordicBioscience, Herlev, Danmark.
authors.forename: Yi
authors.initials: Y
authors.lastname: He
authors.affiliation: NordicBioscience, Herlev, Danmark.
authors.forename: Qinlong
authors.initials: Q
authors.lastname: Zheng
authors.affiliation: NordicBioscience China, Beijing, P.R. China.
authors.forename: MengMeng
authors.initials: M
authors.lastname: Jiang
authors.affiliation: NordicBioscience, Herlev, Danmark.
authors.forename: Shu
authors.initials: S
authors.lastname: Sun
authors.affiliation: Orthopedic Department, Gentofte University Hospital, Hellerup, Denmark.
authors.forename: Thorbjørn G
authors.initials: TG
authors.lastname: Christiansen
authors.affiliation: Molecular Endocrinology Laboratory (KMEB), Odense University Hospital, Odense, Denmark.
authors.forename: Moustapha
authors.initials: M
authors.lastname: Kassem
authors.affiliation: NordicBioscience, Herlev, Danmark.
authors.forename: Morten A
authors.initials: MA
authors.lastname: Karsdal
authors.affiliation: NordicBioscience, Herlev, Danmark.
authors.forename: Anne C
authors.initials: AC
authors.lastname: Bay-Jensen
mesh_terms: D051722:ADAM Proteins / Q000276:immunology / Q000378:metabolism; D000071121:ADAMTS4 Protein; D053674:Aggrecans / Q000378:metabolism; D000818:Animals; D019540:Area Under Curve; D001323:Autoantibodies / Q000276:immunology; D015415:Biomarkers; D002358:Cartilage, Articular / Q000378:metabolism* / Q000473:pathology; D002417:Cattle; D003094:Collagen / Q000378:metabolism; D004195:Disease Models, Animal; D020782:Matrix Metalloproteinases / Q000276:immunology / Q000378:metabolism; D053683:Oncostatin M / Q000378:metabolism; D010003:Osteoarthritis / Q000276:immunology / Q000378:metabolism* / Q000628:therapy; D011348:Procollagen N-Endopeptidase / Q000276:immunology / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology*; D059748:Proteolysis; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D053674:Aggrecans; D001323:Autoantibodies; D015415:Biomarkers; D011480:Protease Inhibitors; D014409:Tumor Necrosis Factor-alpha; D053683:Oncostatin M; D003094:Collagen; D051722:ADAM Proteins; D020782:Matrix Metalloproteinases; D011348:Procollagen N-Endopeptidase; D000071121:ADAMTS4 Protein; C000603918:ADAMTS4 protein, human