Role of xanthine oxidoreductase and NAD(P)H oxidase in endothelial superoxide production in response to oscillatory shear stress
Role of xanthine oxidoreductase and NAD(P)H oxidase in endothelial superoxide production in response to oscillatory shear stress
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DOI：
10.1152/ajpheart.00515.2003
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发表时间：
2003-12-01
影响因子：
4.8
通讯作者：
Harrison, DG
中科院分区：
文献类型：
--
作者：
McNally, JS;Davis, ME;Harrison, DG
振荡剪切应力发生在易受动脉粥样硬化影响的循环部位。由于氧化应激有助于动脉粥样硬化，我们试图确定是否振荡剪切应力增加内皮细胞产生的活性氧，并确定酶负责这种现象。将牛主动脉内皮细胞暴露于静态、层流（15 dyn/cm（2））和振荡剪切应力（+/-15 dyn/cm（2））。振荡剪切增加了超氧化物（O2（圈））生产超过三倍的静态和层流条件下，使用电子自旋共振（ESR）检测。氧嘌呤醇和含钨的内皮细胞培养物可抑制O-2（圈-）的增加，但其他酶源抑制剂则不能抑制O-2的增加。通过二氯荧光素二乙酸酯和Amplex Red荧光测定，羟嘌呤醇还阻止了响应于振荡剪切应力的H2 O2产生。黄嘌呤依赖的O-2（圈）生产增加的内皮细胞匀浆暴露于振荡剪切应力。这与黄嘌呤脱氢酶（XDH）蛋白水平和酶活性降低有关，导致黄嘌呤氧化酶（XO）与XDH的比率升高。我们还研究了缺乏NAD（P）H氧化酶p47（phox）亚单位的内皮细胞。这些细胞表现出显着抑制O-2（环）的生产，并有最小的XO蛋白和活动。用p47（phox）转染这些细胞恢复XO蛋白水平。最后，在牛主动脉内皮细胞，长期抑制NAD（P）H氧化酶与夹竹桃黄素降低XO蛋白水平，并防止内皮细胞刺激O-2（环）生产响应振荡剪切应力。这些数据表明，NAD（P）H氧化酶维持内皮细胞XO水平，XO是负责增加活性氧产生响应振荡剪切应力。
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/cm(2)), and oscillatory shear stress (+/-15 dyn/cm(2)). Oscillatory shear increased superoxide (O2(circle-)) production by more than threefold over static and laminar conditions as detected using electron spin resonance (ESR). This increase in O-2(circle-) 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 O-2(circle-) 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 p47(phox) subunit of the NAD(P) H oxidase. These cells exhibited dramatically depressed O-2(circle-) production and had minimal XO protein and activity. Transfection of these cells with p47(phox) 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 O-2(circle-) 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.