Role of CD36 in ROS induced Ca influx in lung microvascular endothelial cells

描述（由申请人提供）：NRSA个人奖学金的目标是：1)促进研究技能的发展，使候选人成为一名有效的内科科学家；2)研究急性呼吸窘迫综合征（ARDS）中氧化诱导内皮Ca2+内流和肺通透性背后的机制。ARDS是一种常见的，有时是致命的肺部疾病，其特征是肺内皮通透性增加，使液体进入肺泡，导致血液无法供氧。活性氧（Reactive oxygen species， ROS）是一种氧源性分子，在包括ARDS在内的许多疾病中都会增加，也会引起细胞应激和损伤。除ROS外，细胞内异常高的Ca2+水平已被证明可导致内皮通透性增加。然而，ROS与Ca2+进入肺内皮之间的关系尚不清楚。候选人的初步数据显示，肺内皮细胞中的Ca2+水平在暴露于ROS后上升，而Ca2+的上升在缺乏CD36蛋白的细胞中是不存在的。CD36是一种通常与脂肪酸结合的受体。已有研究表明它参与免疫细胞和神经细胞中的钙和ROS信号传导，但其在肺内皮细胞中的作用尚未得到证实。本项目的重点是研究CD36如何影响Ca2+内流以响应肺内皮中的ROS。这项建议的长期目标是确定Ca2+进入内皮细胞的调节新机制，可以通过药物来帮助急性呼吸窘迫综合征患者以及其他内皮异常通透性疾病的患者。在Specific Aim 1中，使用肺内皮细胞上的荧光Ca2+成像和siRNA，候选人将测试TRPV4是否是负责Ca2+内流的通道，该通道在候选人的初步研究中显示可能参与ros诱导的Ca2+进入。在特异性目标2中，使用免疫印迹和Ca2+成像对正常内皮细胞和CD36基因缺失的内皮细胞，将评估ROS对CD36和另一种与CD36相关的蛋白Fyn的影响。这一目标的目标将是确定ROS最终导致Ca2+通道开放的途径。在Specific Aim 3中，通过小鼠体内肺损伤模型和体内Ca2+的测量，将测试药物阻断ROS， CD36和TRPV4连接途径的各种成员对钙进入和肺水肿发展的影响。这些实验的目的是了解背后的机制Ca2+进入和随后的屏障功能丧失在肺内皮细胞损伤的ROS。

DESCRIPTION (provided by applicant): The objectives of this NRSA individual fellowship are to 1) facilitate the development of research skills that will allow the candidate to become an effective physician-scientist and 2) investigate the mechanisms behind oxidant induced endothelial Ca2+ influx and lung permeability in Acute Respiratory Distress Syndrome (ARDS). ARDS is a common, sometimes fatal lung condition characterized by increased permeability of the lung endothelium, allowing for fluid to enter the alveoli, resulting in an inability to oxygenae the blood. Reactive oxygen species (ROS) are oxygen derived molecules known to be increased in many diseases including ARDS, and also known to cause cellular stress and injury. In addition to ROS, abnormally high intracellular Ca2+ levels have been shown to lead to increased endothelial permeability. However, the relationship between ROS and Ca2+ entry into the lung endothelium remains unclear. The candidate has preliminary data that shows that Ca2+ levels in lung endothelial cells rise after they have been exposed to ROS, and that this rise in Ca2+ is absent in cells that are lacking a protein called CD36. CD36 is a receptor that normally binds fatty acids. It has been shown by others to participate in calcium and ROS signaling in immune and neuronal cells, but its role in lung endothelial cells has not been examined. The focus of this project is to examine how CD36 influences Ca2+ influx in response to ROS in the lung endothelium. The long-term goal of this proposal is to identify novel mechanisms of regulation of Ca2+ entry into endothelial cells that can be targeted by drugs designed to help patients in ARDS as well as other diseases where the endothelium is abnormally permeable. In Specific Aim 1, using fluorescent Ca2+ imaging and siRNA on lung endothelial cells, the candidate will test whether TRPV4, a Ca2+ channel shown in the candidate's preliminary studies to be possibly involved in ROS-induced Ca2+ entry, is the channel that is responsible for Ca2+ influx. In Specific Aim 2, using immunoblots and Ca2+ imaging on both normal endothelial cells and endothelial cells with genetic deletion of CD36, the effect of ROS on CD36 and another protein associated with CD36 called Fyn will be assessed. The goal of this aim will be to determine the pathway by which ROS eventually causes opening of Ca2+ channels. In Specific Aim 3, using a model of in vivo lung injury and measurement of in vivo Ca2+ in mice, the effect of drugs blocking various members of the proposed pathway linking ROS, CD36 and TRPV4 on calcium entry and development of lung edema will be tested. The goal of these experiments is to understand the mechanisms behind Ca2+ entry and subsequent loss of barrier function in lung endothelial cells injured by ROS.