Differential Roles of Protein Complexes NOX1-NOXO1 and NOX2-p47phox in Mediating Endothelial Redox Responses to Oscillatory and Unidirectional Laminar Shear Stress
Differential Roles of Protein Complexes NOX1-NOXO1 and NOX2-p47phox in Mediating Endothelial Redox Responses to Oscillatory and Unidirectional Laminar Shear Stress
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DOI：
10.1074/jbc.m115.713149
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发表时间：
2016-04-15
影响因子：
4.8
通讯作者：
Cai, Hua
中科院分区：
文献类型：
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作者：
Siu, Kin Lung;Gao, Ling;Cai, Hua
内皮暴露于各种流动模式，例如血管保护性单向层流剪切应力（LSS）和致动脉粥样硬化振荡剪切应力（OSS）。使用具有平行室的软件控制、阀门操作的 OsciFlow 装置将 LSS 和 OSS 应用于内皮细胞。虽然 LSS 随着时间的推移抑制超氧化物，但 OSS 随时间依赖性地增加内皮细胞超氧化物的产生。免疫细胞化学染色显示，在静息状态下，p47phox 与 NOX2 共定位，而 NOXO1 与 NOX1 共定位。 p47phox 的 RNAi 对响应 OSS 的超氧化物或 NO 产生没有影响，但显着降低了 LSS 中的 NO 产生，这表明 p47phox 结合的 NADPH 氧化酶 (NOX) 介导基础 NO 产生。事实上，p47phox的RNAi抑制内皮一氧化氮合酶（eNOS）丝氨酸1179磷酸化，而细胞内过氧化氢的PEG-过氧化氢酶清除或NOX2的RNAi产生类似的结果，表明NOX2/p47phox衍生的过氧化氢在介导eNOS产生NO的基础活性中的作用。相比之下，NOXO1 的 RNAi 不会导致响应 LSS 的 NO 和超氧化物水平发生显着变化，但响应 OSS 显着减少超氧化物，同时增加 NO。此外，我们首次发现 OSS 解偶联 eNOS，并通过 NOXO1 的 RNAi 纠正。总之，LSS 激活 NOX2-p47phox 复合物来激活 eNOS 磷酸化和 NO 产生。 OSS 相反会激活 NOX1-NOXO1 复合物来解开 eNOS。这些结果证明了 NOX 在响应不同剪切应力而调节氧化还原态方面的不同作用，这可能促进新型治疗剂的开发，以模拟 LSS 的保护作用，同时抑制 OSS 的有害作用。
The endothelium is exposed to various flow patterns such as vasoprotective unidirectional laminar shear stress (LSS) and atherogenic oscillatory shear stress (OSS). A software-controlled, valve-operated OsciFlow device with parallel chambers was used to apply LSS and OSS to endothelial cells. Although LSS inhibited superoxide over time, OSS time-dependently increased superoxide production from endothelial cells. Immunocytochemical staining revealed that, at resting state, p47phox colocalizes with NOX2, whereas NOXO1 colocalizes with NOX1. RNAi of p47phox had no effects on superoxide or NO production in response to OSS but significantly reduced NO production in LSS, implicating a p47phox-bound NADPH oxidase (NOX) in mediating basal NO production. Indeed, RNAi of p47phox inhibited endothelial nitric oxide synthase (eNOS) serine 1179 phosphorylation, whereas PEG-catalase scavenging of intracellular hydrogen peroxide or RNAi of NOX2 produced similar results, indicating a role of NOX2/p47phox-derived hydrogen peroxide in mediating the basal activity of NO production from eNOS. In contrast, RNAi of NOXO1 resulted in no significant changes in NO and superoxide levels in response to LSS but significantly reduced superoxide while increasing NO in response to OSS. Furthermore, we identified, for the first time, that OSS uncouples eNOS, which was corrected by RNAi of NOXO1. In summary, LSS activates the NOX2-p47phox complex to activate eNOS phosphorylation and NO production. OSS instead activates the NOX1-NOXO1 complex to uncouple eNOS. These results demonstrate differential roles of NOXs in modulating the redox state in response to different shear stresses, which may promote the development of novel therapeutic agents to mimic the protective effects of LSS while inhibiting the injurious effects of OSS.