Mechanoregulation of endothelial mitochondrial function

说明书(申请人提供)：内皮线粒体功能的机械调节已知，一氧化氮(NO)通过与电子传递链(ETC)成分的相互作用，不仅起到调节细胞呼吸的作用，而且还能增加线粒体产生的活性氧物种(ROS)，从而触发细胞生存或死亡的机制。缺血(I)/再灌注(RP)后，NO和超氧化物歧化酶(O27)的产生均增加，并形成过氧亚硝酸盐(ONOO)。ONOO在几个部位使ETC失活，导致ROS生成增加，蛋白质硝化，脂质过氧化和心肌损伤。心脏I/RP后冠脉内皮细胞(ECs)受到损伤。虽然从低氧(H)/复氧(RO)模型中获得了一些知识，但导致内皮细胞功能障碍对RP的分子机制还不是很清楚。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，培养的EC暴露在稳定的层流切应力下，诱导线粒体中ONOO的形成，从而在多个位置抑制ETC。由于在常压O2浓度(21%O2)的流动条件下，剪切力诱导的NO是线粒体氧化应激的罪魁祸首，我们建议对暴露于剪切力和氧分压(PO2)控制水平的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，我们提出：目的1：定量描述在生理PO2(10%O2)和(B)I(H，无葡萄糖，酸性pH)/RP(10%O2剪切)下的冠状动脉内皮细胞线粒体功能。在相对“高氧”的21%O2下剪切的内皮细胞中，线粒体O27的形成可能增加，从而导致ONOO的形成增强和线粒体功能障碍。线粒体功能将通过检测O27水平、复合体活性、蛋白质硝化和膜电位等来表征；EC功能通过检测NO产生、O2消耗、ATP水平、脂质过氧化和细胞凋亡来表征。目的2：检测线粒体靶向抗氧化肽和缺血后处理是否也能保护冠脉内皮细胞，保护缺血后心肌免受RP损伤。抗氧化肽可清除线粒体ONOO，降低蛋白质硝化和RP时的脂质过氧化作用。缺血后处理可减少RP时NO的生成，导致线粒体O27和ONOO的生成减少，从而保护细胞功能。如果时间允许，冠脉内皮细胞在I/RP前在10%O2中进行通宵血流预适应也将被检查。这一建议是新颖的(因为它是基于一项新的发现，即EC线粒体功能受剪切诱导的NO调节)和探索性(因为我们建议探索PO2、Premior I和潜在的治疗策略对剪切内皮细胞线粒体功能的影响)，使其适用于R21类别。本建议的目标是更好地了解导致心肌缺血(I)/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。冠脉内皮细胞是I/RP损伤的重要靶点。急性心肌梗死后继发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.