Oscillatory shear stress occurs at sites of the circulation that are vulnerable to atherosclerosis. Because oxidative stress contributes to atherosclerosis, we sought to determine whether oscillatory shear stress increases endothelial production of reactive oxygen species and to define the enzymes responsible for this phenomenon. Bovine aortic endothelial cells were exposed to static, laminar (15 dyn/cm2), and oscillatory shear stress (+/-15 dyn/cm2). Oscillatory shear increased superoxide (O2.-) production by more than threefold over static and laminar conditions as detected using electron spin resonance (ESR). This increase in O2*- was inhibited by oxypurinol and culture of endothelial cells with tungsten but not by inhibitors of other enzymatic sources. Oxypurinol also prevented H2O2 production in response to oscillatory shear stress as measured by dichlorofluorescin diacetate and Amplex Red fluorescence. Xanthine-dependent O2*- production was increased in homogenates of endothelial cells exposed to oscillatory shear stress. This was associated with decreased xanthine dehydrogenase (XDH) protein levels and enzymatic activity resulting in an elevated ratio of xanthine oxidase (XO) to XDH. We also studied endothelial cells lacking the p47phox subunit of the NAD(P)H oxidase. These cells exhibited dramatically depressed O2*- production and had minimal XO protein and activity. Transfection of these cells with p47phox restored XO protein levels. Finally, in bovine aortic endothelial cells, prolonged inhibition of the NAD(P)H oxidase with apocynin decreased XO protein levels and prevented endothelial cell stimulation of O2*- production in response to oscillatory shear stress. These data suggest that the NAD(P)H oxidase maintains endothelial cell XO levels and that XO is responsible for increased reactive oxygen species production in response to oscillatory shear stress.

Arterial regions exposed to oscillatory shear (OS) in branched arteries are lesion-prone sites of atherosclerosis, whereas those of laminar shear (LS) are relatively well protected. Here, we examined the hypothesis that OS and LS differentially regulate production of O 2 ؊ from the endothelial NAD(P)H oxidase, which, in turn, is responsible for their opposite effects on a critical atherogenic event, monocyte adhesion. We used aortic endothelial cells obtained from C57BL/6 (MAE-C57) and p47 phox؊/؊ (MAE-p47 ؊/؊ ) mice, which lack a component of NAD(P)H oxidase. O 2 ؊ production was determined by dihydroethidium staining and an electron spin resonance using an electron spin trap methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine. Chronic exposure (18 h) to an arterial level of OS (؎ 5 dynes/cm 2 ) increased O 2 ؊ (2-fold) and monocyte adhesion (3-fold) in MAE-C57 cells, whereas chronic LS (15 dynes/cm 2 , 18 h) significantly decreased both monocyte adhesion and O 2 ؊ compared with static conditions. In contrast, neither LS nor OS were able to induce O 2 ؊ production and monocyte adhesion to MAE-p47 ؊/؊ . Treating MAE-C57 with a cellpermeable superoxide dismutase compound, polyethylene glycol-superoxide dismutase, also inhibited OS-induced monocyte adhesion. In addition, over-expressing p47 phox in MAE-p47 ؊/؊ restored OS-induced O 2 ؊ production and monocyte adhesion. These results suggest that chronic exposure of endothelial cells to OS stimulates O 2 ؊ and/or its derivatives produced from p47 phox -dependent NAD(P)H oxidase, which, in turn, leads to monocyte adhesion, an early and critical atherogenic event.Fluid shear stress, the frictional force generated by blood flow over the vascular endothelium, is a major factor in atherogenesis. The importance of shear stress in vascular biology and pathophysiology has been highlighted by the focal development patterns of atherosclerosis in hemodynamically defined regions. For example, regions of branched and curved arteries experience disturbed blood flow patterns, including oscillatory shear stress (OS), 1 typically ranging Ϯ 5 dynes/cm 2 (Ϯ indicates changes in flow directions) (1, 2). These disturbed shear regions correspond to "lesion-prone" areas that develop early forms of atherosclerotic lesions (1-3). In contrast, relatively straight arteries, which are exposed to steady uni-directional laminar shear stress (typically ranging from 5-25 dynes/cm 2 ), are usually protected from early atherosclerotic plaque development (1, 2). The mechanisms by which laminar shear (LS) acts as atheroprotective force whereas OS initiates or contributes to atherogenesis have been the subject of intense investigation by many researchers.The vascular endothelium is in direct contact with blood flow and acts as a mechanotransducer by sensing and transducing the changes in local mechanical forces into cellular signals. Endothelial function, shape, physiology, and pathophysiology are greatly regulated by the types (uni-directional laminar or disturbed flow conditions) and magnitudes (high or...

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