Emerging Functions of Mitochondrial Fission in Postischemic Endothelial Cells

描述（由申请人提供）：缺血后内皮细胞中线粒体分裂的新功能缺血（I）后早期再灌注（RP）时的内皮细胞（EC）功能障碍被认为是由于内源性氧化应激，特别是由于线粒体超氧化物（O27-）/活性氧（ROS）的产生而发生的。生物可利用的一氧化氮（NO）的下降损害了冠状动脉血管的EC依赖性扩张。已知NO本身和通过过氧亚硝酸盐形成促进线粒体O27-产生。培养EC暴露于剪切应力也被证明导致NO介导的线粒体O27-生产。 氧化应激被认为诱导线粒体通透性转换孔（mtPTP）的开放，导致细胞凋亡的线粒体途径的激活。然而，最近的文献表明，线粒体凋亡的诱导与线粒体分裂相关，并且所得的去极化线粒体通过自噬降解，这可以导致细胞存活、凋亡或自噬性细胞死亡。我们发现，静态或剪切EC维持其线粒体网络。缺氧（H）/复氧（RO）暴露的EC经历线粒体形态学的变化，但分裂显着低于在体外暴露于I/RP（I模拟为H; RP模拟为RO与流量）的EC。抗氧化剂或NO合酶抑制剂可抑制I/RP暴露的EC中的裂变，并伴有裂变蛋白动力蛋白相关蛋白1（Drp 1）的磷酸化和自噬增加。为了了解I/RP诱导的EC线粒体形态学变化以及这些变化是否决定了细胞命运，我们建议：（a）评估H/RO和I/RP对培养EC线粒体动力学的不同影响，并描绘导致线粒体分裂增加的细胞内信号传导途径。用在线粒体中表达绿色荧光蛋白的慢病毒转染将用于分析它们在静态、剪切、H/RO或I/RP期间的动态行为。我们将研究异常的线粒体动力学是否伴随着融合/裂变蛋白的变化，重点是通过Drp 1水平/活性进行裂变调节，并将描述ROS，NO和mtPTP在Drp 1激活和线粒体裂变中的作用。(b)检查是否阻断由于I/RP引起的广泛分裂将保护内皮线粒体功能并抑制细胞凋亡，以及分裂对细胞功能的影响是否至少部分由自噬介导。在药物Drp 1抑制剂存在下或在显性负性Drp 1形式或融合蛋白线粒体融合蛋白2过表达后，将EC暴露于I/RP，并测量线粒体功能、自噬和凋亡。后者也将在存在自噬抑制剂的情况下测量。这项研究是基于新的假设，线粒体网络动力学的变化可能是负责EC功能障碍后RP。我们的目标是，通过更好地了解EC功能障碍（心脏I/RP损伤的第一个关键步骤），开发新的治疗策略，靶向线粒体分裂。 公共卫生相关性：虽然急性心肌梗死后早期冠状动脉再灌注是改善患者预后的主要临床干预措施，但再灌注本身（主要通过产生自由基）可能导致进一步的组织损伤。冠状动脉内皮细胞是缺血/再灌注损伤的早期关键靶点。该提案旨在更好地了解线粒体片段化/分裂的作用及其对缺血后内皮细胞存活的影响，并提出针对内皮线粒体动力学的新治疗策略。

DESCRIPTION (provided by applicant): Emerging Functions of Mitochondrial Fission in Postischemic Endothelial Cells Endothelial cell (EC) dysfunction upon early reperfusion (RP) following ischemia (I) is thought to occur due to endogenous oxidative stress, and, specifically, due to mitochondrial superoxide (O27-)/reactive oxygen species (ROS) generation. The decline in bioavailable nitric oxide (NO) impairs the EC-dependent dilation in coronary vessels. NO, both by itself and via peroxynitrite formation, is known to promote mitochondrial O27- production. Cultured EC exposure to shear stress was also shown to result in NO-mediated mitochondrial O27- production. Oxidative stress is thought to induce the opening of the mitochondrial permeability transition pore (mtPTP) leading to activation of the mitochondrial pathway of apoptosis. However, recent literature suggests that induction of mitochondrial apoptosis correlates with mitochondrial fission, and the resultant depolarized mitochondria are degraded via autophagy, which can lead to cell survival, apoptosis or autophagic cell death. We found that static or sheared ECs maintain their mitochondrial network. Hypoxia (H)/reoxygenation (RO)- exposed ECs undergo mitochondrial morphology changes, but fission is significantly less compared to that in ECs exposed to in vitro I/RP (I is simulated as H; RP is simulated as RO with the addition of flow). Fission in I/RP-exposed ECs is inhibited by antioxidants or NO synthase inhibitors, and is accompanied by phosphorylation of the fission protein dynamin-related protein 1 (Drp1) and increased autophagy. In order to understand the I/RP-induced EC mitochondrial morphology changes and whether these dictate the cell fate, we propose to: (a) Assess the differential effects of H/RO and I/RP on cultured EC mitochondrial dynamics, and delineate the intracellular signaling pathways that lead to increased mitochondrial fission. Transfection with a lentivirus that expresses green fluorescent protein in mitochondria will be used to analyze their dynamic behavior during static, shear, H/RO or I/RP. We will examine if abnormal mitochondrial dynamics are accompanied by fusion/fission protein changes, with a focus on fission regulation by Drp1 levels/activity, and will also delineate the roles of ROS, NO, and mtPTP in Drp1 activation and mitochondrial fission. (b) Examine if blocking the extensive fission due to I/RP will preserve the endothelial mitochondrial function and suppress apoptosis, and if the effect of fission on cell function is, at least in part, mediated by autophagy. ECs, in the presence of a pharmacological Drp1 inhibitor or following overexpression of either a dominant negative Drp1 form or the fusion protein mitofusin 2, will be exposed to I/RP, and mitochondrial function, autophagy and apoptosis will be measured. The latter will also be measured in the presence of autophagy inhibitors. This study is based on the novel hypothesis that changes in mitochondrial network dynamics may be responsible for the EC dysfunction upon RP. Our goal is, via better understanding of the EC dysfunction (the first critical step in cardiac I/RP injury), to develop new therapeutic strategies that will target mitochondrial fission. PUBLIC HEALTH RELEVANCE: Although early coronary reperfusion following acute myocardial infarction is the primary clinical intervention for improving patient outcome, reperfusion itself, primarily via generation of free radicals, may cause further tissue damage. The endothelium in coronary arteries is an early critical target of ischemia/reperfusion injury. This proposal aims to better understand the role of mitochondrial fragmentation/fission and its repercussions on postischemic endothelial cell survival, and to propose new therapeutic strategies that target the endothelial mitochondrial dynamics.