Receptor Tyrosine Kinases in Mechanotransduction

描述（由申请人提供）：动脉粥样硬化是美国死亡和残疾的主要原因。血管中的生理性层流已被证明具有动脉粥样硬化保护作用，这在一定程度上是由层流刺激的内皮一氧化氮合酶（eNOS）激活和内皮细胞（EC）产生一氧化氮（NO）介导的。层流介导eNOS激活和动脉粥样硬化保护作用的分子机制尚不清楚，但可能与机械信号转导有关。在寻找eNOS激活潜在介质的蛋白质组学研究中，我们发现血管内皮生长因子受体2 （VEGFR2）参与层流诱导的eNOS激活。我实验室发表的数据表明，层流诱导的eNOS磷酸化和激活仅取决于VEGFR2的激活，因为高度特异性的VEGFR2激酶抑制剂完全消除了层流介导的eNOS激活和体外NO生成以及体内血管舒张。重要的是，层流激活VEGFR2是一种与配体无关的方式。众所周知，在EC中，VEGF作为一种天然配体刺激VEGFR2并部分通过NO的产生诱导炎症反应。相反，来自我实验室的新证据支持VEGFR2通过NO依赖和独立机制在层流的抗炎作用中发挥新作用。我的中心假设是，VEGFR2的特异性激活通过对接蛋白Gab1的独特信号模块的组装和激活，赋予层流的抗炎作用。这一全球假设将通过以下三个具体目标进行检验：目标1。研究层流激活VEGFR2中酪氨酸残基的磷酸化及其功能。目标2。确定Gab1在层流诱导的VEGFR2信号通路和功能中的作用。目标3。利用完整主动脉离体器官培养系统研究VEGFR2在层流抗炎作用中的作用。本文提出的这些研究将提供一个框架，开始定义将机械力应用与VEGFR2激活、eNOS激活和促炎基因抑制联系起来的机制途径。在这一途径中，VEGFR2可能作为层流动脉粥样硬化保护作用的中心介质出现，这将为预防动脉粥样硬化性血管疾病提供新的见解。

DESCRIPTION (provided by applicant): Atherosclerosis is the major cause of death and disability in the United States. Physiological laminar flow in blood vessels has been shown to be atheroprotective, which is, in part, mediated by laminar flow-stimulated endothelial nitric oxide synthase (eNOS) activation and nitric oxide (NO) production from endothelial cells (EC). The molecular mechanisms by which laminar flow mediates eNOS activation and atheroprotective effects are not clear but likely relate to mechanical signaling transduction. In a proteomic search for potential mediators of eNOS activation, we identified vascular endothelial growth factor receptor 2 (VEGFR2) as being involved in laminar flow-induced eNOS activation. The published data from my laboratory have demonstrated that laminar flow-induced eNOS phosphorylation and activation depends solely upon VEGFR2 activation, since highly specific VEGFR2 kinase inhibitors completely abolish laminar flow-mediated eNOS activation and NO production in vitro and vasodilation in vivo. Importantly, VEGFR2 activation by laminar flow occurs in a ligand-independent manner. It is well known that VEGF as a natural ligand stimulates VEGFR2 and induces an inflammatory response partially via NO production in EC. Conversely, emerging evidence from my laboratory support a new role of VEGFR2 in the anti-inflammatory effect of laminar flow via NO dependent and independent mechanisms. My central hypothesis is that specific activation of VEGFR2 confers the anti-inflammatory effect of laminar flow through assembly and activation of a distinct signaling module via docking protein Gab1. This global hypothesis will be tested via the following three specific aims: Aim 1. To study phosphorylation and function of tyrosine residues in VEGFR2 activated by laminar flow. Aim 2. To determine the role of Gab1 in VEGFR2 signaling and function induced by laminar flow. Aim 3. To examine the role of VEGFR2 in the anti-inflammatory effect of laminar flow using an ex vivo organ culture system of intact aorta. These studies proposed herein will provide a framework to begin defining a mechanistic pathway linking mechanical force application to VEGFR2 activation, eNOS activation and proinflammatory gene inhibition. In this pathway, VEGFR2 may emerge as a central mediator for laminar flow atheroprotective effects, which will provide new insight into the prevention of atherosclerotic vascular disease.