Mitochondrial Oxidative Stress In Angiotensis II Induced Endothelial Dysfunction

描述（由申请人提供）：美国高血压患者超过5000万人，在高血压治疗方面面临严峻挑战。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。我们发现血管紧张素II通过pkc依赖的NADPH氧化酶激活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H2O2的增加。通过一个正反馈回路，增加的线粒体H2O2通过c-Scr途径导致细胞NADPH氧化酶进一步激活，导致细胞O27产量增加，NO7生物利用度降低。这种恶性循环导致血管O27过量产生，内皮细胞NO7减少，并导致内皮功能障碍。我们建议我们可以通过使用线粒体靶向抗氧化剂抑制线粒体氧化应激来中断内皮细胞中的这种恶性循环。事实上，用线粒体靶向SOD模拟mitoTEMPO或抑制mitoKATP通道处理内皮细胞可减少线粒体氧化应激，改善线粒体呼吸，阻断血管诱导的内皮氧化应激并恢复NO7。本提案将研究线粒体ROS的上游和下游细胞调控。我们将使用siRNA和转染技术通过消耗或过表达特定的NOX异构体来研究NOX的作用。我们将研究线粒体PKC5在刺激mitoKATP依赖性线粒体ROS产生中的作用。我们将使用组成型活性突变体c-Src Y527F研究线粒体ROS在c-Src依赖性NADPH氧化酶激活的氧化还原调控中的下游作用。在本提案中，我们将研究线粒体损伤在内皮功能障碍中的作用。我们的初步数据显示，线粒体超氧化物歧化酶（SOD2）的过表达抑制血管内皮氧化应激，恢复一氧化氮。我们认为线粒体功能障碍和线粒体氧化应激之间的交叉对话可能构成线粒体损伤，从而驱动内皮功能障碍。在本研究中，我们将研究mitoTEMPO、SOD2缺失或过表达对线粒体和内皮功能的影响。最后，我们将利用C57Blk/6、tgSOD2和SOD2（）小鼠研究线粒体ROS在Ang II -和doca -盐诱导的高血压中的作用。我们首次发现用mitoTEMPO治疗高血压动物可显著降低血管氧化应激，增加NO7，改善内皮依赖性松弛和减轻高血压，而相同剂量的非靶向抗氧化剂TEMPOL则没有。本研究的总体目标是明确线粒体氧化应激在内皮功能障碍和高血压中的作用，这可能为开发新的线粒体靶向治疗高血压的药物提供重要信息。

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. PUBLIC HEALTH RELEVANCE: Oxidative stress is strongly implicated in the pathogenesis of hypertension; however, the role of mitochondrial oxidative stress is not clear. Our preliminary data showed that inhibition of mitochondrial oxidative stress significantly improves endothelial function and attenuates hypertension. This work will study the role of mitochondrial oxidative stress in endothelial dysfunction and provide a novel therapeutic target to treat and prevent hypertension.