Mitochondrial Oxidative Stress In Angiotensis II Induced Endothelial Dysfunction

描述(申请人提供)：美国有5000多万人受到影响，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。我们发现血管紧张素II通过依赖PKC激活NADPH氧化酶来诱导线粒体氧化应激。这导致线粒体激活mitoKATP通道，导致线粒体功能障碍，增加线粒体产生O27和H_2O_2。通过一个正反馈回路，线粒体增加的过氧化氢通过c-SCR途径进一步激活细胞内的NADPH氧化酶，导致细胞内O27的产生增加，降低了NO7的生物利用度。这种恶性循环导致血管O27的过度产生，内皮细胞NO7的减少，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的这种恶性循环。事实上，用线粒体靶向的SOD模拟物MitoTEMPO或抑制mitoKATP通道治疗内皮细胞可以减少线粒体氧化应激，改善线粒体呼吸，阻止血管紧张素转换酶诱导的内皮氧化应激，并恢复NO7。这项建议将研究线粒体ROS的上游和下游细胞调控。我们将使用siRNA和转基因技术，通过耗尽或过度表达特定的NOX亚型来研究NOX的作用。将研究线粒体PKC5在刺激线粒体ROS产生依赖于mitoKATP的过程中的作用。线粒体ROS在c-Src依赖的NADPH氧化酶激活的氧化还原调节中的下游作用将使用结构性活性突变体c-Src Y527F来研究。在这项提案中，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)的过表达抑制了血管紧张素转换酶诱导的内皮氧化应激，并恢复了NO7。我们认为线粒体功能障碍和线粒体氧化应激之间的相互作用可能构成线粒体损伤，从而导致内皮功能障碍。在这项研究中，我们将研究MitoTEMPO、SOD2缺失或过度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治疗高血压动物显著降低了血管氧化应激，增加了NO7，改善了内皮依赖松弛，并减轻了高血压，而相同剂量的非靶向抗氧化剂Tempoll则没有。这项建议的总体目标是明确了解线粒体氧化应激在内皮功能障碍和高血压中的作用，这可能为开发新的线粒体靶向治疗高血压的药物提供关键信息。 公共卫生相关性：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。我们的初步数据显示，抑制线粒体氧化应激显著改善内皮功能，减轻高血压。这项工作将研究线粒体氧化应激在内皮功能障碍中的作用，并为治疗和预防高血压提供新的治疗靶点。

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.