Cytoskeletal regulation of endothelial barrier function by WAVE2

描述（由申请人提供）：排列在血管上的内皮细胞是屏障功能的关键元素。它们调节血液和组织之间大分子、营养物质和废物的通道。炎症期间，内皮屏障功能降低，导致白细胞积聚，炎症分子渗漏到靶组织。虽然炎症是对病原体和组织损伤的必要反应，但当它变成慢性或失调时，它与组织损伤和严重疾病（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）有关。因此，控制炎症是许多临床干预的核心，逆转炎症期间内皮屏障功能的丧失具有重要的治疗价值。已经确定了几种增强内皮屏障功能的介质。例如，与Gs结合的受体的信号触发第二信使cAMP的增加。这通常会导致体外和体内屏障功能的改善，主要是通过调节内皮连接。在内皮细胞中，cAMP的主要效应物被认为是cAMP依赖性蛋白激酶（PKA）。最近，小GTPase Rap1的激活已被证明可以显着改善内皮屏障，引起肌动蛋白在连接处的聚合。连接肌动蛋白是粘合和紧密连接的重要组成部分。本研究将聚焦于内皮细胞连接处促进肌动蛋白丝成核的蛋白。初步研究表明，作为肌动蛋白成核促进因子WASP-WAVE家族成员之一的WAVE2可能与内皮连接处的Arp2/3复合物协同引起肌动蛋白聚合。基于这些观察，我们假设WAVE2位于cAMP信号的下游，并通过在连接处形成成核肌动蛋白聚合在调节内皮屏障功能中发挥关键作用。这一假设将在以下具体目标中进行检验：1。内皮细胞中WAVE2信号通路成分的识别和定位2. 通过多种策略干扰其信号传导，测量WAVE2对单层通透性的功能影响和结构对结组成的影响。这些研究将测试WAVE2在连接肌动蛋白聚合和内皮细胞屏障功能中的作用，以响应屏障增强和减弱的刺激。确定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以对抗其失调的必要步骤。公共卫生相关性：炎症是对病原体和组织损伤的必要反应，但当它变成慢性或失调时，它与组织损伤和严重疾病（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）有关。炎症期间，内皮屏障功能降低，导致白细胞积聚，炎症分子渗漏到靶组织。该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，并且是设计更具体的治疗方法以对抗其失调的必要步骤。

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.