McNally J Scott, Davis Michael E, Giddens Don P, Saha Aniket, Hwang Jinah, Dikalov Sergey, Jo Hanjoong, Harrison David G
Division of Cardiology and Molecular and Systems Pharmacology Program, Emory University, Atlanta, GA 30322, USA.
Am J Physiol Heart Circ Physiol. 2003 Dec;285(6):H2290-7. doi: 10.1152/ajpheart.00515.2003. Epub 2003 Sep 4.
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.
振荡剪切应力发生在易患动脉粥样硬化的循环部位。由于氧化应激会导致动脉粥样硬化，我们试图确定振荡剪切应力是否会增加内皮细胞活性氧的产生，并确定负责这一现象的酶。将牛主动脉内皮细胞暴露于静态、层流（15达因/平方厘米）和振荡剪切应力（±15达因/平方厘米）下。使用电子自旋共振（ESR）检测发现，与静态和层流条件相比，振荡剪切使超氧化物（O2.-）的产生增加了三倍多。这种O2*-的增加被氧嘌呤醇和用钨培养内皮细胞所抑制，但不受其他酶源抑制剂的抑制。氧嘌呤醇还能防止二氯荧光素二乙酸酯和Amplex Red荧光检测到的振荡剪切应力诱导的H2O2产生。暴露于振荡剪切应力的内皮细胞匀浆中，黄嘌呤依赖性O2*-的产生增加。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的比例升高。我们还研究了缺乏NAD（P）H氧化酶p47phox亚基的内皮细胞。这些细胞的O2*-产生显著降低，XO蛋白和活性极低。用p47phox转染这些细胞可恢复XO蛋白水平。最后，在牛主动脉内皮细胞中，用夹竹桃麻素长期抑制NAD（P）H氧化酶可降低XO蛋白水平，并防止内皮细胞在振荡剪切应力刺激下产生O2*-。这些数据表明，NAD（P）H氧化酶维持内皮细胞XO水平，并且XO负责响应振荡剪切应力而增加活性氧的产生。
