Mitochondrial Oxidative Stress In Angiotensis II Induced Endothelial Dysfunction

描述（由申请人提供）：美国有超过5000万人受影响，在治疗高血压方面面临着严峻的挑战。氧化应激与高血压的发病机制密切相关;然而，线粒体氧化应激的作用尚不清楚。我们已经发现，血管紧张素II通过PKC依赖性激活NADPH氧化酶诱导线粒体氧化应激。这导致mitoKATP通道的激活，导致线粒体功能障碍和线粒体产生O27和H2 O2的增加。通过一个正反馈回路，线粒体H2 O2增加导致细胞NADPH氧化酶通过c-Scr途径进一步激活，导致细胞O27产生增加和NO 7生物利用度降低。这种恶性循环导致血管O27的过度产生，内皮NO 7的减少，并导致内皮功能障碍。我们认为，我们可以中断这种恶性循环，在内皮细胞的线粒体氧化应激的抑制，使用靶向的抗氧化剂。事实上，用线粒体靶向的SOD模拟物mitoTEMPO或mitoKATP通道的抑制处理内皮细胞降低了线粒体氧化应激，改善了线粒体呼吸，阻断了AngII诱导的内皮氧化应激并恢复了NO 7。本研究将探讨线粒体活性氧的上下游调控机制。我们将通过使用siRNA和转染技术耗尽或过度表达特定的NOX亚型来研究NOX的作用。将检查线粒体PKC 5在刺激线粒体ROS的mitoKATP依赖性产生中的作用。将使用组成型活性突变体c-Src Y 527 F研究线粒体ROS在c-Src依赖的NADPH氧化酶活化的氧化还原调节中的下游作用。在这个提议中，我们将研究线粒体损伤在内皮功能障碍中的作用。我们的初步数据表明，线粒体超氧化物歧化酶（SOD 2）的过表达抑制血管紧张素II诱导的内皮细胞氧化应激和恢复NO 7。我们认为，线粒体功能障碍和线粒体氧化应激之间的串扰可能构成线粒体损伤，从而驱动内皮功能障碍。在这个提议中，我们将研究mitoTEMPO，SOD 2耗竭或过表达对线粒体和内皮功能的影响。最后，我们将使用C57 Blk/6、tgSOD 2和SOD 2（）小鼠研究线粒体ROS在Ang II和DOCA盐诱导的高血压中的作用。我们第一次发现，用mitoTEMPO治疗高血压动物显著降低了血管氧化应激，增加了NO 7，改善了内皮依赖性舒张和减轻了高血压，而相同剂量的非靶向抗氧化剂TEMPOL则没有。本提案的总体目标是清楚地了解线粒体氧化应激在内皮功能障碍和高血压中的作用，这可能为开发新的靶向治疗高血压的药物提供关键信息。

DESCRIPTION (provided by applicant): With more than 50 million individuals affected, the United States is facing a serious challenge in the treatment of hypertension. Oxidative stress is strongly implicated in the pathogenesis of hypertension; however, the role of mitochondrial oxidative stress is not clear. We have found that angiotensin II induces mitochondrial oxidative stress via PKC-dependent activation of NADPH oxidases. This result in activation of mitoKATP channels leading to mitochondrial dysfunction and increase in O27 and H2O2 production by mitochondria. Through a positive feedback loop, the increased mitochondrial H2O2 lead to further activation of cellular NADPH oxidases via c-Scr pathway, resulting in increased cellular O27 production and diminished NO7 bioavailability. This vicious cycle is responsible for overproduction of vascular O27, diminished endothelial NO7 and result in endothelial dysfunction. We suggest that we can interrupt this vicious cycle in endothelial cells by inhibition of mitochondrial oxidative stress using mitochondria-targeted antioxidants. Indeed, treatment of endothelial cells with mitochondria- targeted SOD mimetic mitoTEMPO or inhibition of mitoKATP channels reduced mitochondrial oxidative stress, improved mitochondrial respiration, blocked AngII-induced endothelial oxidative stress and restored NO7. This proposal will investigate the upstream and downstream cellular regulations of mitochondrial ROS. We will examine the role of NOX by depletion or overexpression of specific NOX isoforms using siRNA and transfection techniques. The role of mitochondrial PKC5 in stimulation of mitoKATP dependent production of mitochondrial ROS will be examined. The downstream effect of mitochondrial ROS in redox regulation of c-Src dependent activation of NADPH oxidase will be studied using constitutively active mutant c-Src Y527F. In this proposal we will examine the role of mitochondrial impairment in endothelial dysfunction. Our preliminary data showed that overexpression of mitochondrial superoxide dismutase (SOD2) inhibited AngII-induced endothelial oxidative stress and restored NO7. We suggest that a cross-talk between mitochondrial dysfunction and mitochondrial oxidative stress may constitute a mitochondrial impairment, which drives endothelial dysfunction. In this proposal we will study the effect of mitoTEMPO, SOD2 depletion or overexpression on mitochondrial and endothelial functions. Finally, we will investigate the role of mitochondrial ROS in Ang II - and DOCA-salt induced hypertension using C57Blk/6, tgSOD2 and SOD2() mice. For the first time we have found that treatment of hypertensive animals with mitoTEMPO significantly reduced vascular oxidative stress, increased NO7, improved endothelial dependent relaxation and attenuated hypertension, while the same dose of non-targeted antioxidant TEMPOL did not. The overall objective of this proposal is to gain a clear understanding of the role of mitochondrial oxidative stress in endothelial dysfunction and hypertension, which may provide critical information for the development of new mitochondria-targeted therapeutic agents to treat hypertension.