Abstract 676: Hypoxia Upregulates NADPH Oxidase 4-Mediated Hydrogen Peroxide Release by a HIF-Independent Mechanism in Human Endothelial Cells

Abstract

NADPH oxidases are important sources of reactive oxygen species in the vascular wall. Recent evidence supports a vasoprotective role of H 2 O 2 produced by the main endothelial isoform Nox4. The impact of hypoxia on NOX4 expression in human endothelial cells and the underlying mechanism remains to be elucidated. In this study, we show that NOX4 mRNA and protein expression was upregulated by hypoxia (1 % O 2 ) in human umbilical vein endothelial cells (HUVEC). Correspondingly, H 2 O 2 production was 2-fold elevated in HUVEC after hypoxia. In contrast to rotenone and oxypurinol, lentiviral downregulation via shNOX4 abolished the elevated hypoxic hydrogen peroxide levels to normoxic values. Hypoxia stabilized the hypoxia-inducible factor (HIF)-1α protein in endothelial cells. Furthermore, VEGF promoter activity and a control promoter containing 3 hypoxia-responsive elements (HRE) were induced by hypoxia. NOX4 promoter deletions up to -119/+239 had an increased basal activity compared to control vector. A full-length and a terminally deleted NOX4 promoter construct missing a putative HRE showed a comparable activity under hypoxic and normoxic conditions, suggesting that NOX4 is not induced on the transcriptional level by hypoxia in endothelial cells. In addition, stabilization of HIF-1α protein under normoxic conditions using DMOG did not change NOX4 mRNA expression in HUVEC. Furthermore, overexpression of HIF-1α did not alter NOX4 promoter activity. Blockade of active transcription by actinomycin D revealed an increased stability of the NOX4 mRNA under hypoxic conditions. In conclusion, this study demonstrates an HIF-independent upregulation of NOX4 as major source of endothelial hydrogen peroxide generation in response to hypoxia. Our data support as a novel mechanism an increased NOX4 mRNA stability under hypoxic conditions in human endothelial cells.