Effect of hypoxia/reoxygenation on the cytokine-induced production of nitric oxide and superoxide anion in cultured osteoarthritic synoviocytes

Objective: Hypoxia/reoxygenation (H/R) is an important feature in the osteoarthritis (OA) physiopathology. Nitric oxide ((NO)-N-center dot) is a significant proinflammatory mediator in the inflamed synovium. The purpose of this study was to investigate the effects of H/R on inducible NO synthase (iNOS) activity and expression in OA synoviocytes. In addition we studied the relationship between nitrosative stress and NADPH oxidase (NOX) in such conditions. Methods: Human cultured synoviocytes from OA patients were treated for 24 h with interleukin 1-beta (IL-1 beta), tumour necrosis factor alpha (TNF-alpha) or neither; for the last 6 h, they were submitted to either normoxia or three periods of 1-h of hypoxia followed by 1-h of reoxygenation. (NO)-N-center dot metabolism (iNOS expression, nitrite and peroxynitrite measurements) was investigated. Furthermore, superoxide anion O-2(center dot-) production, NOX subunit expression and nitrosylation were also assessed. Results: iNOS expression and nitrite (but not peroxynitrite) production were significantly increased under H/R conditions when compared with to normoxia (P < 0.05). H/R conditions decreased O-2(center dot-) production from similar to 0.20 to similar to 0.12 nmol min(-1) mg proteins(-1) (P < 0.05), while NOXs' subunit expression and p47-phox phosphorylation were increased. NOXs and p47-phox were dramatically nitrosylated under H/R conditions (P < 0.05 vs normoxia). Using NOS inhibitors under H/R conditions, p47-phox nitrosylation was prevented and O-2(center dot-) production was restored at normoxic levels (0.21 nmol min(-1) mg of proteins(-1)). Conclusions: Our results provide evidence for an up-regulation of iNOS activity in OA synoviocytes under H/R conditions, associated to a down-regulation of NOX activity through nitrosylation. These findings highlight the importance of radical production to OA pathogenesis, and appraise the metabolic modifications of synovial cells under hypoxia. (C) 2013 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.