title: Role of NADPH oxidase-mediated superoxide production in the regulation of E-selectin expression by endothelial cells subjected to anoxia/reoxygenation.
abstract: OBJECTIVE
  Anoxia followed by reoxygenation (A/R) increases endothelial cell superoxide (O2-) generation which is implicated in E-selectin overexpression. The mechanisms which govern these processes are not fully understood and therefore the goal of our study was to determine the functional importance of NADPH oxidase in the regulation of E-selectin expression in human umbilical veins endothelial cells (HUVECs) submitted to A/R.
  
  
  METHODS
  O2- production was estimated using lucigenin chemiluminescence and formazan accumulation. NADPH oxidase expression in HUVECs was studied by RT-PCR and Western blot and E-selectin by Northern blot analysis. NFkappaB activation was assessed by electrophoretic mobility shift assay.
  
  
  RESULTS
  A/R caused an increased O2- production which was inhibited by the superoxide dismutase mimetic M40403 (50 micromol/l), the protein kinase C inhibitor chelerythrine (10 micromol/l), the NADPH oxidase inhibitor diphenyleneiodonium (DPI, 10 micromol/l) and the NADPH oxidase assembly blocker apocynin (600 micromol/l). At the end of the anoxic period, the mRNA expression and the protein p47phox was increased as compared to normoxic HUVECs. NFkappaB activation of anoxic HUVECs was maximal after 1 h of reoxygenation and returned to basal normoxic levels after 2 h of reoxygenation. Apocynin reduced the NFkappaB activation at 1 h of reoxygenation. E-selectin mRNA expression was increased after 3 h of reoxygenation of anoxic HUVECs and the SOD mimetic M40403 as well as apocynin prevented this overexpression.
  
  
  CONCLUSIONS
  Activated NADPH oxidase is a critical enzyme in E-selectin overexpression after A/R of HUVECs. Moreover, A/R increased expression of membranous and cytosolic NADPH oxidase subunits as well as the protein p47phox. Strategies aimed at preventing endothelial NADPH oxidase activation and/or activity may be useful in controlling leukocyte adhesion during ischemia/reperfusion.
authors.affiliation: Division of Angiology, Servier Research Institute, 11 rue des Moulineaux, Suresnes 92150, France.
authors.forename: Alain
authors.initials: A
authors.lastname: Rupin
authors.forename: Jérôme
authors.initials: J
authors.lastname: Paysant
authors.forename: Patricia
authors.initials: P
authors.lastname: Sansilvestri-Morel
authors.forename: Nathalie
authors.initials: N
authors.lastname: Lembrez
authors.forename: Jean-Michel
authors.initials: JM
authors.lastname: Lacoste
authors.forename: Alex
authors.initials: A
authors.lastname: Cordi
authors.forename: Tony J
authors.initials: TJ
authors.lastname: Verbeuren
mesh_terms: D015139:Blotting, Southern / Q000379:methods; D015153:Blotting, Western / Q000379:methods; D002478:Cells, Cultured; D019040:E-Selectin / Q000378:metabolism*; D042783:Endothelial Cells / Q000378:metabolism*; D006801:Humans; D000860:Hypoxia / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000235:genetics; D009252:NADPH Dehydrogenase / Q000235:genetics; D019255:NADPH Oxidases / Q000378:metabolism*; D010750:Phosphoproteins / Q000032:analysis / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013481:Superoxides / Q000378:metabolism*
chemical_list: D019040:E-Selectin; D026901:Membrane Transport Proteins; D010750:Phosphoproteins; D012333:RNA, Messenger; C079789:neutrophil cytosol factor 67K; D013481:Superoxides; D019255:NADPH Oxidases; C086986:CYBA protein, human; C060505:neutrophil cytosolic factor 1; D009252:NADPH Dehydrogenase