Receptor Tyrosine Kinases in Mechanotransduction

描述(申请人提供)：动脉粥样硬化是美国人死亡和残疾的主要原因。生理性层流在血管中具有动脉粥样硬化保护作用，其部分机制是通过层流刺激内皮型一氧化氮合酶(ENOS)激活和内皮细胞(EC)产生一氧化氮(NO)来实现的。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)参与了层流诱导的eNOS激活。本实验室已发表的数据表明，层流诱导的eNOS磷酸化和激活完全依赖于VEGFR2的激活，因为高度特异性的VEGFR2激酶抑制剂完全取消了层流介导的eNOS激活和体内NO的产生和体内血管扩张。重要的是，VEGFR2的层流激活是以不依赖于配体的方式发生的。众所周知，血管内皮生长因子作为一种天然配体刺激血管内皮细胞生长因子受体2，并通过在内皮细胞中产生NO部分地诱导炎症反应。相反，来自我的实验室的新证据支持VEGFR2通过不依赖和独立的机制在层流的抗炎作用中发挥新的作用。我的中心假设是，VEGFR2的特异性激活通过组装和激活一个不同的信号模块，通过对接蛋白GAB1赋予层流的抗炎作用。这一全球假说将通过以下三个具体目标来验证：目的1.研究层流激活的VEGFR2中酪氨酸残基的磷酸化及其功能。目的2.探讨GAB1在层流诱导的血管内皮细胞生长因子受体(VEGFR2)信号转导和功能中的作用。目的3.利用体外培养的完整主动脉器官系统，研究血管内皮生长因子受体2在层流抗炎作用中的作用。这些研究将提供一个框架，以开始确定机械力作用于VEGFR2激活、eNOS激活和致炎基因抑制之间的机制途径。在这一途径中，VEGFR2可能成为层流动脉粥样硬化保护作用的中枢介质，这将为动脉粥样硬化性血管疾病的预防提供新的视角。

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.