De Keulenaer G W, Chappell D C, Ishizaka N, Nerem R M, Alexander R W, Griendling K K
Division of Cardiology, Emory University School of Medicine, Atlanta, GA 30322, USA.
Circ Res. 1998 Jun 1;82(10):1094-101. doi: 10.1161/01.res.82.10.1094.
Atherosclerotic lesions are found opposite vascular flow dividers at sites of low shear stress and oscillatory flow. Since endothelial proinflammatory genes prominent in lesions are regulated by oxidation-sensitive transcriptional control mechanisms, we examined the redox state of cultured human umbilical vein endothelial cells after either oscillatory or steady laminar fluid shear stress. Endothelial oxidative stress was assessed by measuring activity of the superoxide (O2.- )-producing NADH oxidase (a major source of reactive oxygen species in vascular cells), intracellular O2.- levels, induction of the redox-sensitive gene heme oxygenase-1 (HO-1), and abundance of Cu/Zn superoxide dismutase (Cu/Zn SOD), an antioxidant defense enzyme whose level of expression adapts to changes in oxidative stress. When cells were exposed to oscillatory shear (+/-5 dyne/cm2, 1 Hz) for 1, 5, and 24 hours, NADH oxidase activity and the amount of HO-1 progressively increased up to 174+/-16% (P<0.05) and 505+/-111% (P<0.05) versus static conditions, respectively, whereas levels of Cu/Zn SOD remained unchanged. This upregulation of HO-1 was completely blocked by the antioxidant N-acetylcysteine (NAC, 20 mmol/L). In contrast, steady laminar shear (5 dyne/cm2) induced NADH oxidase activity and NAC-sensitive HO-1 mRNA expression only at 1 and 5 hours, a transient response that returned toward baseline at 24 hours. Levels of Cu/Zn SOD mRNA and protein were increased after 24 hours of steady laminar shear. Furthermore, intracellular O2.-, as measured by dihydroethidium fluorescence, was higher in cells exposed to oscillatory than to laminar shear. These data are consistent with the hypothesis that continuous oscillatory shear causes a sustained activation of pro-oxidant processes resulting in redox-sensitive gene expression in human endothelial cells. Steady laminar shear stress initially activates these processes but appears to induce compensatory antioxidant defenses. We speculate that differences in endothelial redox state, orchestrated by different regimens of shear stress, may contribute to the focal nature of atherosclerosis.
动脉粥样硬化病变出现在低剪切应力和振荡血流部位的血管分流器对面。由于病变中突出的内皮促炎基因受氧化敏感转录控制机制调节，我们检测了振荡或稳定层流剪切应力作用后培养的人脐静脉内皮细胞的氧化还原状态。通过测量产生超氧化物（O2.-）的NADH氧化酶（血管细胞中活性氧的主要来源）的活性、细胞内O2.-水平、氧化还原敏感基因血红素加氧酶-1（HO-1）的诱导情况以及铜/锌超氧化物歧化酶（Cu/Zn SOD）的丰度来评估内皮氧化应激，Cu/Zn SOD是一种抗氧化防御酶，其表达水平会适应氧化应激的变化。当细胞暴露于振荡剪切（±5达因/平方厘米，1赫兹）1、5和24小时时，与静态条件相比，NADH氧化酶活性和HO-1量分别逐渐增加至174±16%（P<0.05）和505±111%（P<0.05），而Cu/Zn SOD水平保持不变。HO-1的这种上调被抗氧化剂N-乙酰半胱氨酸（NAC，20毫摩尔/升）完全阻断。相比之下，稳定层流剪切（5达因/平方厘米）仅在1和5小时诱导NADH氧化酶活性和NAC敏感的HO-1 mRNA表达，这是一种短暂反应，在24小时时恢复到基线水平。稳定层流剪切24小时后，Cu/Zn SOD mRNA和蛋白水平升高。此外，通过二氢乙锭荧光测量，暴露于振荡剪切的细胞内O2.-高于层流剪切的细胞。这些数据与以下假设一致：持续的振荡剪切导致促氧化过程的持续激活，从而导致人内皮细胞中氧化还原敏感基因的表达。稳定层流剪切应力最初激活这些过程，但似乎诱导了代偿性抗氧化防御。我们推测，由不同剪切应力方案精心编排的内皮氧化还原状态差异可能导致动脉粥样硬化的局灶性。
