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The flowing blood generates shear stress at the endothelial cell surface. In endothelial cells, NAD(P)H oxidase complexes have been identified as major sources of superoxide anion (·O 2 − ) formation. In this study, we analysed the effect of laminar shear stress on ·O 2 − formation by cytochrome c reduction assay and on NAD(P)H oxidase subunit expression by standard calibrated competitive reverse transcription-polymerase chain reaction and Western blot in human endothelial cells. Primary cultures of human umbilical vein endothelial cells were exposed to laminar shear stress in a cone-and-plate viscometer for up to 24 h. Short-term application of shear stress transiently induced ·O 2 − formation. This was inhibited by NAD(P)H oxidase inhibitor gp91ds-tat, but NAD(P)H oxidase subunit expression was unchanged. Long-term arterial laminar shear stress (30 dyne cm −2 , 24 h) down-regulated ·O 2 − formation, and mRNA and protein expression of NAD(P)H oxidase subunits Nox2/gp91 phox and p47 phox . In parallel, endothelial NO formation and eNOS, but not Cu/Zn SOD, protein expression was increased. Down-regulation of ·O 2 − formation, gp91 phox and p47 phox expression by long-term laminar shear stress was blocked by L-NAME. NO donor DETA-NO down-regulates ·O 2 − formation, gp91 phox and p47 phox expression in static cultures. In conclusion, our data suggest a transient activation of ·O 2 − formation by short-term shear stress, followed by a down-regulation of endothelial NAD(P)H oxidase in response to long-term laminar shear stress. NO-mediated down-regulation by shear stress preferentially affects the gp91 phox /p47 phox -containing NAD(P)H oxidase complex. This mechanism might contribute to the regulation of endothelial NO/·O 2 − balance and the vasoprotective potential of physiological levels of laminar shear stress.

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The endothelial cell layer plays a major role in the development and progression of atherosclerosis. Endothelial NO synthase (eNOS) produces nitric oxide (NO) from L-arginine. NO can rapidly react with reactive oxygen species to form peroxynitrite. This reduces NO availability, impairs vasodilatation, and mediates proinflammatory and prothrombotic processes such as leukocyte adhesion and platelet aggregation. In the vessel wall, specific NAD(P)H oxidase complexes are major sources of reactive oxygen species. These NAD(P)H oxidases can transfer electrons across membranes to oxygen and generate superoxide anions. The short-lived superoxide anion rapidly dismutates to hydrogen peroxide, which can further increase the production of reactive oxygen species. This can lead to uncoupling of eNOS switching enzymatic activity from NO to superoxide production. This review describes the structure and regulation of different NAD(P)H oxidase complexes. We will also focus on NO/superoxide anion balance as modulated by hemodynamic forces, vasoconstrictors, and oxidized low-density lipoprotein. We will then summarize the recent advances defining the role of nitric oxide and NAD(P)H oxidase-derived reactive oxygen species in the development and progression of atherosclerosis. In conclusion, novel mechanisms affecting the vascular NO/superoxide anion balance will allow the development of therapeutic strategies in the treatment of cardiovascular diseases.