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Flow-Related Reduction in Oxidative Stress from Nox4 and AP-1 in Endothelial Cells

C. Goettsch1, W. Goettsch1, G. Müller1, N. Duerrschmidt2, A. Wagner3, H. Morawietz1

1Vascular Endothelium and Microcirculation, University of Technology Dresden, Dresden, Germany; 2Cardiac Surgery, University of Leipzig, Leipzig, Germany; 3Institute of Physiology and Pathophysiology, University Heidelberg, Heidelberg, Germany;

Oxidative stress is considered to be a cardiovascular risk factor. The main sources of oxidative stress in the vessel wall are nicotine adenine dinucleotide phosphate (NADPH) oxidase complexes. The endothelial cells comprising the inner layer of the vessel wall are constantly exposed to shear stress from blood. However, the impact of shear stress on oxidative stress and expression of novel NADPH oxidase isoforms in endothelial cells is not well-understood. We have used DNA microarray, real-time PCR and Western blot to demonstrate that the NADPH oxidase subunit Nox4 is the major Nox isoform in human endothelial cells. Nox4 was localized by laser scanning confocal microscopy in the perinuclear space within endothelial cells. Nox4 overexpression enhanced endothelial superoxide anion formation. Long-term application of arterial laminar shear stress using a cone-and-plate viscometer reduced superoxide anion formation and Nox4 expression. Cloning and functional analysis of human Nox4 promoter revealed a region between -1200 and -1071 responsible for flow-dependent downregulation. Mutation of an activator protein-1 (AP-1)-binding site at position -1137 abolished shear stress-dependent downregulation of Nox4. Furthermore, DNA binding activity of a c-jun-containing AP-1 transcription factor was reduced in response to long-term laminar shear stress. This is the first report to describe a functional promoter analysis for the major human endothelial NADPH oxidase subunit, Nox4. An AP-1-binding site was found to be essential for downregulation of Nox4 by shear stress. This novel mechanism might be involved in the vasoprotective downregulation of endothelial superoxide anion formation in response to shear stress.

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