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。血管中的生理层流已被证明具有动脉粥样硬化保护作用，其部分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，但可能与机械信号传导有关。在蛋白质组学研究eNOS激活的潜在介质中，我们确定血管内皮生长因子受体2（VEGFR2）参与层流诱导的eNOS激活。从我的实验室发表的数据表明，层流诱导的eNOS磷酸化和激活完全依赖于VEGFR2激活，因为高度特异性的VEGFR2激酶抑制剂完全消除层流介导的eNOS激活和NO生产在体外和血管舒张在体内。重要的是，VEGFR2通过层流的活化以配体非依赖性方式发生。众所周知，VEGF作为天然配体刺激VEGFR2并部分通过EC中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抗炎作用中的新作用。我的中心假设是，VEGFR2的特异性激活通过对接蛋白Gab1组装和激活一个不同的信号模块，从而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。目的研究层流激活VEGFR2酪氨酸残基的磷酸化及其功能。目标2.探讨Gab1在层流诱导的VEGFR2信号转导和功能中的作用。目标3。采用离体完整主动脉器官培养系统检测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，以开始定义将机械力应用与VEGFR2活化、eNOS活化和促炎基因抑制联系起来的机制途径。在这一通路中，VEGFR2可能成为层流动脉粥样硬化保护作用的中心介质，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.