Question: 

Endothelial dysfunction and reactive oxygen species play an important role in hypoxia-induced vascular disorders. Interestingly, NOX4 is the only isoform of NADPH oxidases producing H₂O₂ and might affect endothelial function. We could previously show that NOX4 is the main NOX isoform in endothelial cells. However, the impact of hypoxia on NOX4 and the underlying mechanisms remains to be elucidated.

Materials and results: 

Endothelial cells (HUVEC) exposed to 24h of hypoxia (1% O₂) strongly upregulated hypoxia-inducible factor (HIF) 1a, NOX4 expression and H₂O₂ release. Lentiviral downregulation using shNOX4 restored the hydrogen peroxide levels to normoxic values. To investigate this regulation on transcriptional level, we cloned several NOX4 promoter deletion constructs and performed dual-luciferase reporter assays. All NOX4 promoter constructs showed a comparable activity under hypoxic and normoxic conditions, suggesting that the recently discovered hypoxia responsive element (HRE) in the NOX4 promoter may be non-functional in endothelial cells. As positive controls VEGF-A promoter and a hypoxia responsive element (HRE3x) containing promoter were induced by hypoxia. We overexpressed HIF-1a in HEK 293T cells and stabilized HIF-1a under normoxic conditions by treatment with DMOG (24 h, 10 mM) in HUVEC. In addition, we blocked the HIF-pathway by adenoviral overexpression of a HIF-2a dominant-negative mutant in HUVEC. None of these experiments resulted in a significant change of NOX4 on the mRNA or promoter level. In contrast, stimulation of endothelial cells with actinomycin D indicated increased stability of the NOX4 and VEGF mRNA under hypoxic conditions.

Conclusion: 

Our data strongly suggest a HIF-independent increase in mRNA stability as major mechanism of NOX4 upregulation under hypoxic conditions in human endothelial cells.