title: Malondialdehyde oxidation of cartilage collagen by chondrocytes.
abstract: OBJECTIVE
  The damage to cartilage collagen is a central event in the pathogenesis of cartilage aging and osteoarthritis (OA). We have previously developed an in vitro model of cartilage degradation which shows that chondrocyte-dependent lipid peroxidation mediates cartilage collagen degradation. The goal of our study was to investigate the role of vitamin C in this degradation model and to investigate effect of chondrocyte-dependent lipid peroxidation in the oxidation of cartilage collagen.
  
  
  METHODS
  We studied primary articular chondrocytes. Effect of vitamin C was investigated in the previously described model. Serum-free stimulated and unstimulated chondrocyte-matrix extracts were subjected to SDS-PAGE and immunoblot analysis. Malondialdehyde (MDA)-protein oxidation of cartilage proteins was demonstrated by the reactivity of chondrocyte extracts to a monoclonal antibody, MDA2, which detects MDA-lysine adducts.
  
  
  RESULTS
  Vitamin C treatment of chondrocyte cultures resulted in significant enhanced incorporation of 3H-proline label in cell-matrix. Cells treated with vitamin C, as compared to control untreated cells showed decreased spontaneous release of labeled matrix. Vitamin C treated or not treated chondrocytes responded comparably to stimulation with the agonist calcium ionophore A23187. The serum-free in vitro culture of chondrocytes resulted in MDA-protein oxidation. The treatment of chondrocytes with A23187 resulted in the enhancement of MDA-protein oxidation. The immunoblot reactivity pattern of extracts to MDA2 antibody and to polyclonal anti-type II collagen antibody was somewhat similar, which suggests that these two different types of antisera exhibit a crossreaction to chondrocyte proteins. Chondrocyte extracts were pretreated both with and without pure collagenase, and then subjected to immunoblot analysis. Only collagenase treated extracts showed a disappearance, or significant reduction, of larger than 60 kDa size MDA2 immunoreactive proteins. This suggests that the proteins that disappeared after the enzyme treatment were collagen proteins and which had also been modified by MDA oxidation.
  
  
  CONCLUSIONS
  These observations suggest that collagen hydroxylation of matrix by vitamin C does not play a role in this model of chondrocyte-dependent collagen degradation. Also, this study demonstrates that chondrocyte-derived lipid peroxidation product MDA mediates oxidation of cartilage collagens. Oxidative modification of cartilage collagen in vivo could result in alteration of biochemical and biophysical properties of cartilage collagen fibrils, making them prone to degradation, thus initiating the changes observed in aging and OA.
authors.affiliation: Department of Medicine, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, New Brunswick, NJ 08903-0019, USA. tikuml@umdnj.edu
authors.forename: M L
authors.initials: ML
authors.lastname: Tiku
authors.forename: G T
authors.initials: GT
authors.lastname: Allison
authors.forename: Karishma
authors.initials: K
authors.lastname: Naik
authors.forename: S K
authors.initials: SK
authors.lastname: Karry
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis; D001205:Ascorbic Acid / Q000494:pharmacology; D000001:Calcimycin / Q000494:pharmacology; D002358:Cartilage, Articular / Q000187:drug effects / Q000378:metabolism*; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects / Q000378:metabolism*; D003094:Collagen / Q000378:metabolism*; D024043:Collagen Type II / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005109:Extracellular Matrix / Q000378:metabolism; D015227:Lipid Peroxidation; D008315:Malondialdehyde / Q000378:metabolism*; D010084:Oxidation-Reduction; D011506:Proteins / Q000378:metabolism; D011817:Rabbits
chemical_list: D000911:Antibodies, Monoclonal; D024043:Collagen Type II; D011506:Proteins; D000001:Calcimycin; D008315:Malondialdehyde; D003094:Collagen; D001205:Ascorbic Acid