Abstract 14657: Regulation of Endothelial Nitric Oxide Synthase by NADPH Oxidase 4 Inhibition
The NADPH oxidase isoform NOX4 mainly produces H2O2. Previously we could show, that NOX4 is the major endothelial NOX isoform and constitutively active. Regulation of NOX4 and formation of reactive oxygen species is involved in NO release. The transcription factor NRF2 is a key mediator of cellular adaptation to redox stress. Therefore, regulation of NOX4 on the transcriptional level by NRF2 might be directly linked to NO release and endothelial function. Besides endothelial nitric oxide synthase (eNOS), a role of neuronal nitric oxide synthase (nNOS) in endothelial NO and H2O2 release has been proposed. In this study, endothelial cells (HUVEC) were constantly exposed to high laminar shear stress (24 h, 30 dyn/cm²). Application of shear stress stimulated NO formation and induced elongation of endothelial cells in the direction of flow. Overexpression of NOX4 strongly increased H2O2 release. Next, we transduced HUVEC with lentiviral particles containing shRNA against NOX4. Downregulation of NOX4 using shNOX4 inhibited the shear stress-dependent elongation of cell shape in response to flow. Furthermore, application of shear stress caused downregulation of NOX4, upregulation of eNOS and antioxidative responses via NRF2 and its target genes NQO-1 and HO-1. H2O2 can affect NO release by eNOS. We could show that downregulation of NOX4 leads to upregulation of eNOS mRNA expression and increased cytosolic protein expression under flow conditions. Determination of NO release confirmed these results. This supports a compensatory mechanism maintaining a stable NO release after NOX4 inhibition in response to flow. Attenuation of NRF2 by shNRF2 inhibited shear stress-dependent induction of NRF2 and its target genes. In addition, shNRF2 enhanced the shear stress-dependent downregulation of NOX4. Finally, we detected a stable mRNA expression of nNOS in endothelial cells which was not affected by flow. Interestingly, downregulation of NOX4 resulted in strong induction of nNOS mRNA expression. In conclusion, our data suggest a novel mechanism how H2O2 released by NADPH oxidase 4 might affect endothelial and neuronal nitric oxide synthase. This can play a role in maintaining a stable NO release in response to flow in endothelial cells.
- © 2012 by American Heart Association, Inc.