Rap1 in Endothelial Homeostasis

为了应对血液流动的剪切力，内皮细胞分泌一系列因子，包括 一氧化氮(NO)，在调节血管内稳态中起着关键作用。NO版本中的缺陷导致 内皮功能障碍并导致心血管疾病，包括高血压、再狭窄和 动脉硬化。我们在前一个周期的开创性研究确定了Rap1是一种新的、关键的 内皮细胞剪切感和一氧化氮释放的调节因子。我们的生理意义 内皮细胞(EC)特异性RAP1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，RAP1是 血小板内皮细胞黏附分子-1(PECAM-1)的剪切力信号在细胞内的传递 血管内皮生长因子2(VEGFR2)反式激活及其下游信号转导 内皮型一氧化氮合酶(ENOS)。 这项建议的目的是研究Rap1在传递所需剪应力信号中的作用。 用于维持EC的动态平衡。总体假设是Rap1促进了剪应力诱导的 从机械感觉受体PECAM-1，通过其效应器Afadin，到VEGFR2和 向下游到埃诺斯。此外，Rap1功能的破坏促进了促炎内皮细胞的形成 表型和加重动脉粥样硬化。这一假设将在三个目标上得到检验。 目的1将研究RAP1在剪应力作用下激活的分子机制。研究 利用表达PECAM-1突变体的永生化人内皮细胞将确定 Rap1激活子(Rap1 Gef)C3G和Rap1效应子Afadin参与信号的传递 PECAM-1到VEGFR2的激活和下游信号转导。 目的2研究Rap1在层流和扰动流动信号传递中的作用。利用 RAP1基因敲除小鼠血管和体外培养的RAP1基因缺陷内皮细胞的作用 剪切力对急性信号和长期促炎基因表达的影响将被检测。 AIM 3将研究RAP1信号转导中断作为加剧内皮细胞病变的因素的作用 在体内导致促炎状态的功能。这些研究将调查内皮细胞的作用 RAP1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颖的、以前意想不到的管理欧共体对 通过调控Rap1信号通路，可能为恢复EC功能提供一个新的方向。

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.