Role of NADPH oxidase-mediated superoxide production in the regulation of E-selectin expression by endothelial cells subjected to anoxia/reoxygenation

Cardiovascular Research
Alain RupinTony J Verbeuren

Abstract

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. 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. 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...Continue Reading

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