title: Effect of hypoxia/reoxygenation on the cytokine-induced production of nitric oxide and superoxide anion in cultured osteoarthritic synoviocytes.
abstract: OBJECTIVE
  Hypoxia/reoxygenation (H/R) is an important feature in the osteoarthritis (OA) physiopathology. Nitric oxide (NO) 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-β (IL-1β), tumour necrosis factor α (TNF-α) 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 metabolism (iNOS expression, nitrite and peroxynitrite measurements) was investigated. Furthermore, superoxide anion O2(·-) production, NOX subunit expression and nitrosylation were also assessed.
  
  
  RESULTS
  iNOS expression and nitrite (but not peroxynitrite) production were ~0.20 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 O2(·-) 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.
authors.affiliation: Laboratoire de Biochimie, Groupe Hospitalier Cochin-Broca-Hôtel Dieu, Assistance Publique-Hôpitaux de Paris (AP-HP), Paris, France. camille.gobeaux@cch.aphp.fr
authors.forename: C
authors.initials: C
authors.lastname: Chenevier-Gobeaux
authors.forename: C
authors.initials: C
authors.lastname: Simonneau
authors.forename: H
authors.initials: H
authors.lastname: Lemarechal
authors.forename: D
authors.initials: D
authors.lastname: Bonnefont-Rousselot
authors.forename: S
authors.initials: S
authors.lastname: Poiraudeau
authors.forename: F
authors.initials: F
authors.lastname: Rannou
authors.forename: O G
authors.initials: OG
authors.lastname: Ekindjian
authors.forename: P
authors.initials: P
authors.lastname: Anract
authors.forename: D
authors.initials: D
authors.lastname: Borderie
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D005260:Female; D006801:Humans; D000860:Hypoxia / Q000150:complications; D053583:Interleukin-1beta / Q000494:pharmacology; D008297:Male; D019255:NADPH Oxidases / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D009573:Nitrites / Q000378:metabolism; D020370:Osteoarthritis, Knee / Q000378:metabolism*; D010100:Oxygen / Q000494:pharmacology; D013481:Superoxides / Q000378:metabolism*; D013583:Synovial Membrane / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D053583:Interleukin-1beta; D009573:Nitrites; D014409:Tumor Necrosis Factor-alpha; D013481:Superoxides; D009569:Nitric Oxide; D019255:NADPH Oxidases; D010100:Oxygen