Cytoskeletal regulation of endothelial barrier function by WAVE2

描述(由申请人提供)：排列血管的内皮细胞是屏障功能的关键要素。它们调节血液和组织之间的大分子、营养物质和废物的通道。在炎症过程中，内皮屏障功能降低，导致白细胞聚集，炎症分子泄漏到靶组织。虽然炎症是对病原体和组织损伤的必要反应，但当它变得慢性或调节失调时，它与组织损伤和严重疾病有关，如动脉粥样硬化、哮喘、糖尿病、感染性休克和自身免疫性疾病。因此，控制炎症是许多临床干预的核心，逆转炎症过程中内皮屏障功能的丧失具有重要的治疗价值。已经确定了几种可以增强内皮屏障功能的介体。例如，向与Gs偶联的受体发出的信号会触发第二信使阵营的增加。这在很大程度上是通过调节内皮细胞连接，在体外和体内改善屏障功能。在内皮细胞中，cAMP的主要效应因子被认为是cAMP依赖的蛋白激酶(PKA)。最近，小GTP酶Rap1的激活被证明可以显著改善内皮屏障，导致连接处肌动蛋白的聚合。连接肌动蛋白是粘连和紧密连接的重要组成部分。这项建议将重点放在促进内皮连接处肌动蛋白细丝成核的蛋白质上。初步研究表明，WAVE2是肌动蛋白成核促进因子WASP-WAVE家族中的一员，它可能与内皮连接处的Arp2/3复合体协同作用，引起肌动蛋白聚合。根据这些观察结果，推测WAVE2位于cAMP信号的下游，通过在连接处形成肌动蛋白聚合，在调节内皮屏障功能中发挥关键作用。这一假说将在以下特定目标中得到验证：1.在内皮细胞中识别和定位WAVE2信号通路的组成部分；2.使用各种干扰WAVE2信号通路的策略，测量WAVE2对单层通透性的功能影响以及结构对连接组成的影响。这些研究将测试WAVE2在内皮细胞连接肌动蛋白聚合和屏障功能中的作用，以响应屏障增强和减弱的刺激。识别调节内皮屏障功能的分子机制将有助于更好地理解炎症过程，并且是设计更具体的治疗方法来对抗内皮屏障功能失调的必要步骤。公共卫生相关性：炎症是对病原体和组织损伤的必要反应，但当它变成慢性或调节失调时，它与组织损伤和严重疾病有关，如动脉粥样硬化、哮喘、糖尿病、感染性休克和自身免疫性疾病。在炎症过程中，内皮屏障功能降低，导致白细胞聚集，炎症分子泄漏到靶组织。这项建议将确定调节内皮屏障功能的分子机制之一，这将有助于更好地理解炎症过程，并是朝着设计更具体的治疗方法对抗其调节失调的必要步骤。

DESCRIPTION (provided by applicant): Endothelial cells which line blood vessels are a critical element in barrier function. They regulate the passage between blood and tissues of macromolecules, nutrients and waste products. During inflammation, endothelial barrier function is reduced, leading to accumulation of leukocytes and leakage of inflammatory molecules into target tissue. While inflammation is a necessary response to pathogens and tissue damage, when it becomes chronic or dysregulated it is associated with tissue injury and serious diseases such as atherosclerosis, asthma, diabetes, septic shock and autoimmune diseases. Control of inflammation is therefore central to many clinical interventions, and reversing the loss of endothelial barrier function during inflammation has significant therapeutic value. Several mediators have been identified that augment endothelial barrier function. For example, signals to receptors which couple to Gs trigger an increase in the second messenger cAMP. This typically results in improved barrier function in vitro and in vivo, largely through regulation of endothelial junctions. In endothelium, the major effector for cAMP was thought to be cAMP dependent protein kinase (PKA). Recently, activation of the small GTPase Rap1 has been shown to dramatically improve the endothelial barrier, causing polymerization of actin at the junctions. Junctional actin is an essential component of adherence and tight junctions. This proposal will focus on proteins that promote actin filament nucleation at endothelial junctions. Preliminary studies indicate that WAVE2, a member of the WASP-WAVE family of actin nucleation promoting factors, may cooperate with the Arp2/3 complex at endothelial junctions to cause actin polymerization. Based on these observations it is hypothesized that WAVE2 lies downstream of cAMP signaling, and plays a critical role in regulating endothelial barrier function by nucleating actin polymerization at the junctions. This hypothesis will be tested in the following specific aims: 1. Identify and localize components of the WAVE2 signaling pathway in endothelial cells; 2. Measure the functional effect of WAVE2 on monolayer permeability and the structural effect on junctional composition using a variety of strategies to interfere with its signaling. These studies will test the role for WAVE2 in polymerization of junctional actin and barrier function in endothelial cells in response to barrier strengthening and weakening stimuli. Identification of the molecular mechanisms that regulate endothelial barrier function will lead to better understanding of the inflammatory process, and is a necessary step towards design of more specific therapies to combat it dysregulation. PUBLIC HEALTH RELEVANCE: Inflammation is a necessary response to pathogens and tissue damage, but when it becomes chronic or dysregulated it is associated with tissue injury and serious diseases such as atherosclerosis, asthma, diabetes, septic shock and autoimmune diseases. During inflammation, endothelial barrier function is reduced, leading to accumulation of leukocytes and leakage of inflammatory molecules into target tissue. This proposal will identify one of the molecular mechanisms that regulate endothelial barrier function which will lead to better understanding of the inflammatory process, and is a necessary step towards design of more specific therapies to combat its dysregulation.