Rap1 in Endothelial Homeostasis

响应于流动血液的剪切应力，内皮细胞分泌许多因子，包括 一氧化氮（NO），其在调节血管稳态中起关键作用。NO释放中的缺陷导致 内皮功能障碍，并有助于心血管疾病，包括高血压，再狭窄和 动脉粥样硬化我们在上一个周期的开创性研究将Rap 1确定为一种新颖的，关键的 内皮细胞剪切感应和一氧化氮释放的调节剂。我们的生理意义 内皮细胞（EC）特异性Rap 1基因敲除小鼠的表型强调了这一发现， 包括内皮功能障碍和高血压。我们的初步研究还表明，Rap 1是必需的， 剪切应力信号从血小板内皮细胞粘附分子-1（PECAM-1）传递到 血管内皮生长因子2（VEGFR 2）的反式激活和下游信号传导， 内皮型一氧化氮合酶（eNOS）。 本提案的目标是研究Rap 1在转导所需剪切应力信号中的作用。 维持EC稳态。总体假设是Rap 1促进剪切应力诱导的 从机械感受受体PECAM-1经由其效应子Afadin向VEGFR 2发出信号， 到eNOS下游。此外，Rap 1功能的破坏促进了促炎性内皮细胞增殖。 表型并加剧动脉粥样硬化。这个假设将在三个目标中得到检验。 目的1将研究Rap 1激活响应剪切应力的分子机制。研究 利用表达PECAM-1突变体的永生化人内皮细胞将确定 Rap 1激活子（Rap 1 GEF）C3 G和Rap 1效应子Afadin参与信号传递， PECAM-1对VEGFR 2的激活和下游信号传导。 目的2将研究Rap 1在层流和扰动流信号转导中的作用。利用 体外Rap 1缺陷EC和内皮特异性Rap 1基因敲除小鼠的血管， 将检查剪切对急性信号传导和长期促炎基因表达的影响。 目的3将研究Rap 1信号传导被破坏作为一种加重内皮细胞损伤的因素的作用。 导致体内促炎状态的功能。这些研究将探讨内皮细胞的作用， Rap 1缺失对小鼠体内动脉粥样硬化进展的影响。 拟议的研究将揭示新的，以前意想不到的机制，管理EC的反应， 剪切，并可能导致一个新的方向，在恢复EC功能，通过控制Rap 1信号。

In response to shear stress of flowing blood, endothelial cells secrete a number of factors, including nitric oxide (NO), which play a key role in regulating vascular homeostasis. Defects in NO release lead to endothelial dysfunction and contribute to cardiovascular disease, including hypertension, restenosis and atherosclerosis. Our pioneering studies during the previous cycle identified Rap1 as a novel, critical regulator of endothelial cell shear sensing and nitric oxide release. The physiological significance of our finding is underscored by the phenotype of endothelial cell (EC)-specific Rap1 knockout mice, which include endothelial dysfunction and hypertension. Our pilot studies also suggest that Rap1 is required for transmission of shear stress signals from Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1) to Vascular Endothelial Growth Factor 2 (VEGFR2) transactivation and downstream signaling to endothelial NO Synthase (eNOS). The goal of this proposal is to examine the role of Rap1 in transducing shear stress signals required for maintaining EC homoeostasis. The overall hypothesis is that Rap1 promotes shear stress-induced signals from the mechanosensing receptor PECAM-1, via its effector, Afadin, to VEGFR2 and downstream to eNOS. Further, disruption of Rap1 function promotes pro-inflammatory endothelial phenotype and exacerbates atherosclerosis. The hypothesis will be tested in three aims. Aim 1 will examine molecular mechanisms of Rap1 activation in response to shear stress. Studies utilizing immortalized human endothelial cells expressing PECAM-1 mutants will determine the involvement of Rap1 activator (Rap1 GEF) C3G and Rap1 effector, Afadin, in transmission of signals from PECAM-1 to VEGFR2 activation and downstream signaling. Aim 2 will investigate the role of Rap1 in transducing laminar and disturbed flow signals. Utilizing Rap1-deficient ECs in vitro and vessels from endothelial-specific Rap1 knockout mice ex vivo, the effect of shear on acute signaling and long-term pro-inflammatory gene expression will be examined. Aim 3 will examine the effect of disrupted Rap1 signaling as a factor exacerbating endothelial function leading to a pro-inflammatory state in vivo. The studies will investigate the effect of endothelial Rap1 deletion on progression of atherosclerosis in a mouse model in vivo. Proposed studies will uncover novel, previously unexpected mechanisms governing EC responses to shear and may lead to a new direction in restoring EC function by controlling Rap1 signaling.