Shear Stress, AMPK, and Endothelial Functions

描述（由申请人提供）：动脉树的弯曲和分支中动脉粥样硬化病变的普遍分布证明了血流模式在动脉壁易发生动脉粥样硬化中的关键作用。具有高剪切应力的稳定流动，即作用于内皮的血流动力学力的切向分量，构成了动脉粥样硬化保护流的波形。相反，具有低剪切应力、振荡和反向流动的流动模式则容易发生动脉粥样硬化。流动通道已被用作体外模型系统，以证明剪切应力调节参与血管功能的基因的表达。在研究机械转导机制时，我们最近发现amp激活的蛋白激酶（AMPK）在血管内皮细胞（ECs）中受到剪切应力的调节。此外，krf2是一种锌指转录因子，整合多种内皮功能，通过AMPK调节剪切应力。因此，我们假设动脉粥样硬化保护性血流调节AMPKK的活性[即钙调素依赖性蛋白激酶（CaMKK）和LKB1]，进而激活AMPK。除了快速激活eNOS外，AMPK还增强了klf2基因的转录激活，从而有利于ec依赖性血管功能。激活的AMPK-KLF2也有助于血流的动脉粥样硬化保护作用。为了验证我们的假设，提出了三个具体目标。特异性目标1将剖析与动脉粥样硬化保护血流相关的关键剪切应力参数，该血流可激活内皮细胞中的AMPKK-AMPK级联。Specific Aim 2将阐明AMPK调控KLF2转录激活的分子机制，进而调控KLF2靶向基因。特异性目的3将研究血流激活AMPK和KLF2在血管张力和动脉粥样硬化保护中的作用。具体来说，我们将评估AMPK敲除（AMPK -/-）小鼠ec依赖性血管扩张和NO生物利用度。此外，ampk-/-小鼠将与apoE-/-小鼠杂交。与apoE-/-的小鼠相比，这些双基因敲除小鼠的动脉粥样硬化病变预计会增强。本研究旨在研究AMPK在剪切胁迫下EC的翻译后调控和转录调控，为进一步了解流动调控EC生物学的机制和分子基础建立框架。

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.