Shear Stress, AMPK, and Endothelial Functions

描述(由申请人提供)：动脉粥样硬化病变在动脉树的曲率和分支中的普遍分布表明了血流模式在动脉壁易患动脉粥样硬化中的关键作用。具有高切应力的恒定流是作用于血管内皮细胞的血流动力的切向分量，构成了动脉粥样硬化保护流的波形。相反，具有低剪切力、振荡和反向流动的流型是阿司匹林。血流通道已被用作体外模型系统，以证明剪切力调节与血管功能有关的基因的表达。在对其机制的研究中，我们最近发现，AMP激活的蛋白激酶(AMPK)在血管内皮细胞(ECs)中受到切应力的调节。此外，作为一种整合多种血管内皮细胞功能的锌指转录因子，Kr？ppel-like factor2(KLF2)受AMPK调节，以响应切应力。因此，我们假设动脉粥样硬化保护流调节AMPKK[即钙调蛋白依赖的蛋白激酶(CaMKK)和LKB1]的活性，进而激活AMPK。除了快速激活eNOS外，AMPK还能增强KLF2基因的转录激活，从而有利于EC依赖的血管功能。活化的AMPK-KLF2也可能参与FLOW的动脉粥样硬化保护作用。为了验证我们的假设，我们提出了三个具体目标。具体目标1将剖析与激活内皮细胞中AMPKK-AMPK级联的动脉粥样硬化保护流相关的临界剪应力参数。特异性目标2将阐明AMPK调节KLF2转录激活的分子机制，KLF2转录激活进而调节KLF2靶向基因。具体目标3将研究流动激活的AMPK和KLF2在血管张力和动脉粥样硬化保护中的作用。具体地说，我们将评估AMPK基因敲除(AMPK-/-)小鼠的EC依赖性血管扩张和无生物利用度。此外，AMPK-/-小鼠将与apoE-/-小鼠杂交。与apoE-/-基因敲除小鼠相比，这些双基因敲除小鼠的动脉粥样硬化病变有望得到加强。本研究旨在研究AMPK对切应力作用下内皮细胞的翻译后调控和转录调控，为进一步了解血流调控内皮细胞生物学的机制和分子基础奠定基础。 公共卫生相关性：动脉粥样硬化呈现局灶性分布模式，这主要是由动脉树中不同位置的不同血流模式造成的。本提案中的研究涉及动脉粥样硬化保护作用与动脉粥样硬化调节血管内皮细胞生物学的机制和分子基础问题。这项提议将显著提高我们对血流模式与高脂血症在动脉粥样硬化形成中相互作用的机制的理解，这可能有助于为其预防和/或治疗提供新的治疗方法。

DESCRIPTION (provided by applicant): The prevalent distribution of atherosclerotic lesions in the curvature and branches of arterial trees demonstrates the critical role of flow patterns in predisposing the arterial wall to atherosclerosis. Steady flows with a high magnitude of shear stress, the tangential component of hemodynamic forces acting on the endothelium, constitute the waveform of atheroprotective flow. In contrast, flow patterns with a low magnitude of shear stress, oscillation, and reverse flow, are atheroprone. Flow channels have been used as in vitro model systems to demonstrate that shear stress regulates the expression of genes involved in vascular functions. In investigating the mechanotransduction mechanisms, we recently found that AMP-activated protein kinase (AMPK) is modulated by shear stress in vascular endothelial cells (ECs). Moreover, Kr¿ppel-like factor 2 (KLF2), a zinc finger transcription factor integrating multiple endothelial functions, is regulated via AMPK in response to shear stress. We thus hypothesize that atheroprotective flow modulates the activity of AMPKK [i.e., calmodulin-dependent protein kinase kinase (CaMKK) and LKB1], which in turn activates AMPK. In addition to rapidly activating eNOS, AMPK augments the transcriptional activation of the klf2 gene to benefit the EC-dependent vascular functions. The activated AMPK-KLF2 would also contribute to the atheroprotective effect of flow. To test our hypothesis, three Specific Aims are proposed. Specific Aim 1 will dissect the critical shear stress parameters associated with the atheroprotective flow that activates the AMPKK-AMPK cascade in ECs. Specific Aim 2 will elucidate the molecular mechanism by which AMPK regulates the transcriptional activation of KLF2, which in turn modulates KLF2-targeted genes. Specific Aim 3 will investigate the role of flow-activated AMPK and KLF2 in vascular tone and atheroprotection. Specifically, we will assess the EC-dependent vessel dilation and NO bioavailability in AMPK knockout (ampk-/-) mice. Further, ampk-/- mice will be crossbred with apoE-/- mice. Atherosclerotic lesions are expected to be enhanced in these double knockout mice, as compared with the apoE-/- littermates. Aiming at studying post-translational and transcriptional regulation by AMPK in ECs exposed to shear stress, we hope this research will establish a framework to further understand the mechano and molecular basis of EC biology regulated by flow. PUBLIC HEALTH RELEVANCE: Atherosclerosis shows a focal pattern of distribution, which is mainly contributed by the distinct flow patterns depending on the locations in the arterial tree. The studies in this proposal address questions regarding the mechano and molecular basis of atheroprotective versus atheroprone flows in modulating the vascular endothelial biology. The proposal will significantly increase our understanding of mechanisms by which blood flow patterns interplay with hyperlipidemia in atherogenesis, which are likely to contribute to novel therapies for its prevention and/or treatment.