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

描述(由申请者提供)：该NRSA个人奖学金的目标是：1)促进研究技能的发展，使候选人能够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)中氧化剂诱导内皮细胞钙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，其特征是肺内皮细胞通透性增加，允许液体进入肺泡，导致无法为血液氧合。活性氧簇(ROS)是已知在包括ARDS在内的许多疾病中增加的氧源分子，也可导致细胞应激和损伤。除ROS外，细胞内异常高的钙离子水平也可导致内皮通透性增加。然而，ROS与Ca~(2+)进入肺内皮细胞的关系尚不清楚。这位候选人有初步数据表明，肺内皮细胞暴露于ROS后，钙水平会上升，而缺乏CD36蛋白质的细胞没有这种钙水平的上升。CD36是一种正常情况下与脂肪酸结合的受体。它已被证明参与免疫和神经细胞中的钙和ROS信号转导，但其在肺内皮细胞中的作用尚未被研究。本项目的重点是研究CD36如何影响肺内皮细胞内ROS对钙离子内流的影响。这项提议的长期目标是确定调节钙离子进入内皮细胞的新机制，这些机制可以被设计用于帮助ARDS患者以及内皮异常通透的其他疾病的药物靶向。在特定的目标1，使用荧光钙成像和肺内皮细胞上的siRNA，候选人将测试在候选人的初步研究中显示可能参与ROS诱导的钙内流的钙通道TRPV4是否是负责钙内流的通道。在特定的目标2中，通过对正常内皮细胞和CD36基因缺失的内皮细胞进行免疫印迹和钙离子成像，将评估ROS对CD36和另一种与CD36相关的蛋白质Fyn的影响。这一目标的目的将是确定ROS最终导致钙通道开放的途径。在具体目标3中，利用小鼠体内肺损伤模型和体内钙离子测定，将测试药物阻断连接ROS、CD36和TRPV4的通路的各个成员对钙内流和肺水肿发展的影响。这些实验的目的是了解ROS损伤的肺内皮细胞钙离子内流和随后的屏障功能丧失的机制。

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.