Mechanoregulation of endothelial mitochondrial function

描述（由申请人提供）：内皮线粒体功能的机械调节众所周知，一氧化氮（NO）通过其与电子传递链（ETC）组分的相互作用，不仅可以调节细胞呼吸，还可以增加线粒体活性氧（ROS）的产生，从而触发细胞存活或死亡的机制。缺血(I)/再灌注（RP）后，NO和超氧化物（O27）的产生均增加，并形成过氧亚硝酸盐（ONOO）。ONOO在多个位点使ETC失活，导致ROS生成增强、蛋白质硝化、脂质过氧化和心肌损伤。冠状动脉内皮细胞（ECs）在心脏I/RP后受损。虽然从缺氧(H)/再氧化（RO）模型中获得了一些知识，但导致RP时EC功能障碍的分子机制尚不清楚。内皮细胞调节血管张力和血栓形成，因此，保护冠状动脉内皮细胞免受I/RP损伤是一个重要的目标。我们最近发现，暴露于稳定的层流剪切应力下的培养EC可诱导线粒体中ONOO的形成，从而在多个位点抑制ETC。由于在常压O2浓度（21% O2）的流动条件下，剪切诱导的NO负责线粒体氧化应激，因此我们建议在暴露于剪切应力和氧张力（PO2）控制水平的环境中，对培养的人冠状动脉内皮细胞的线粒体（dys）功能进行表征。更具体地说，我们建议：目的1：定量表征暴露于：(a)生理性PO2 （10% O2）下剪切应力的冠状动脉内皮细胞的线粒体功能；(b) I (H，无葡萄糖，酸性pH)/RP （10% O2剪切）。在相对“高氧”21% O2下剪切的ECs中，线粒体O27的形成可能增加，导致ONOO形成增强和线粒体功能障碍。线粒体功能将通过测量O27水平、ETC复合体活性、蛋白质硝化和膜电位来表征；通过测量NO生成、O2消耗、ATP水平、脂质过氧化和细胞凋亡来测定EC功能。目的2：研究线粒体靶向抗氧化肽和缺血后适应治疗是否能保护缺血后心肌免受RP损伤，也能保护冠状动脉内皮细胞。抗氧化肽可能清除线粒体ONOO，导致RP过程中蛋白质硝化和脂质过氧化减少。缺血后适应可减少RP时NO的形成，导致线粒体O27和ONOO的生成减少，并保持细胞功能。如果时间允许，还将检查冠状动脉ECs在I/RP之前在10% O2下预处理过夜的血流。这个提议是新颖的（因为它是基于一个新的发现，即EC线粒体功能是由剪切诱导的NO调节的），并且是探索性的（因为我们提议探索PO2，先验I和潜在的治疗策略对剪切EC线粒体功能的影响），使其适合R21类别。这一建议的目标是更好地了解导致心肌缺血/再灌注（RP）后冠状动脉内皮细胞（ECs）线粒体和细胞功能障碍的分子机制。冠状动脉内皮是I/RP损伤的重要靶点。继发于急性心肌梗死的缺血性心脏病是世界上最普遍的健康问题之一，也是发病率和死亡率的主要原因。

DESCRIPTION (provided by applicant): Mechanoregulation of Endothelial Mitochondrial Function It is known that nitric oxide (NO), through its interaction with electron transport chain (ETC) components, may function not only as a regulator of cell respiration, but also to augment the generation of reactive oxygen species (ROS) by mitochondria and thereby trigger mechanisms of cell survival or death. Following ischemia (I)/reperfusion (RP), the production of both NO and superoxide (O27 ) is increased and peroxynitrite (ONOO ) is formed. ONOO inactivates the ETC at several sites leading to enhanced ROS generation, protein nitration, lipid peroxidation and myocardial damage. Coronary endothelial cells (ECs) are injured following cardiac I/RP. Although some knowledge was gained from hypoxia (H)/reoxygenation (RO) models, the molecular me- chanisms leading to the EC dysfunction upon RP are not well understood. ECs regulate the vascular tone and thrombosis, and, hence, protection of coronary ECs from I/RP injury is an important goal. We recently showed that cultured EC exposure to steady laminar shear stress induces ONOO formation in the mitochondria leading to inhibition of ETC at multiple sites. Since, under flow conditions at atmos- pheric O2 concentrations (21% O2), the shear-induced NO is responsible for mitochondrial oxidative stress, we propose to characterize the mitochondrial (dys)function in cultured human coronary arterial ECs exposed to environments with controlled levels of shear stress and oxygen tension (PO2). More specifically, we propose to: Aim 1: Quantitatively characterize the mitochondrial function in coronary ECs exposed to: (a) shear stress under physiologic PO2 (10% O2) and (b) I (H, no glucose, acidotic pH)/RP (shear at 10% O2). In ECs sheared under the relatively `hyperoxic' 21% O2, mitochondrial O27 formation may be increased lead- ing to enhanced ONOO formation and mitochondrial dysfunction. Mitochondrial function will be characteri- zed by measuring O27 levels, ETC complex activities, protein nitration and membrane potential; EC function by measuring NO production, O2 consumption, ATP levels, lipid peroxidation and apoptosis. Aim 2: Examine if mitochondria-targeted antioxidant peptides and ischemic postconditioning, treatments shown to protect the postischemic myocardium from RP injury, also protect the coronary ECs. The antioxidant peptides may scavenge mitochondrial ONOO leading to decreased protein nitration and lipid peroxidation upon RP. Ischemic postconditioning may reduce NO formation upon RP leading to decreased ge- neration of mitochondrial O27 and ONOO , and preservation of cell function. If time permits, coronary ECs flow-preconditioned overnight at 10% O2 prior to I/RP will also be examined. This proposal is novel (since it is based on a new discovery that EC mitochondrial function is modulated by shear-induced NO) and is exploratory (since we propose to explore the effect of PO2, prior I and potential therapeutic strategies on the mitochondrial function in sheared ECs), making it suitable for the R21 category.Relevance to Public Health This goal of this proposal is to better understand the molecular mechanisms that lead to mitochondrial and cell dysfunction of coronary endothelial cells (ECs) following cardiac ischemia (I)/reperfusion (RP). The coronary endothelium is a critical target of I/RP injury. Ischemic heart disease secondary to acute myocardial infarction is among the most prevalent health problems in the world and a major cause of morbidity and mortality.