Shear Stress, AMPK, and Endothelial Functions

描述（由申请人提供）：动脉树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诱发动脉壁动脉粥样硬化中的关键作用。具有高剪切应力的稳定流动，即作用于内皮的血液动力学力的切向分量，构成动脉粥样硬化保护性流动的波形。相反，具有低剪切应力、振荡和反向流动的流动模式是动脉粥样硬化的。流动通道已被用作体外模型系统，以证明剪切应力调节涉及血管功能的基因的表达。在研究机械力信号转导机制的过程中，我们发现剪切力对血管内皮细胞中AMP激活蛋白激酶（AMPK）的活性有明显的调节作用。此外，Kr ppel样因子2（KLF 2）是一种整合多种内皮功能的锌指转录因子，在剪切应力下通过AMPK调节。因此，我们假设动脉粥样硬化保护性血流调节AMPKK的活性[即，钙调蛋白依赖性蛋白激酶激酶（CaMKK）和LKB 1]，其反过来激活AMPK。除了快速激活eNOS外，AMPK还增强了klf 2基因的转录激活，从而有利于EC依赖性血管功能。AMPK-KLF 2的激活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。具体目标1将剖析与动脉粥样硬化保护流相关的临界剪切应力参数，该动脉粥样硬化保护流激活EC中的AMPKK-AMPK级联。具体目标2将阐明AMPK调节KLF 2转录激活的分子机制，这反过来又调节KLF 2靶向基因。特定目标3将研究血流激活的AMPK和KLF 2在血管张力和动脉粥样硬化保护中的作用。具体而言，我们将评估EC依赖性血管扩张和NO生物利用度在AMPK敲除（ampk-/-）小鼠。此外，ampk-/-小鼠将与apoE-/-小鼠杂交。与apoE-/-同窝小鼠相比，预期这些双敲除小鼠中的动脉粥样硬化病变增强。本研究旨在探讨AMPK在切应力作用下对内皮细胞的翻译后和转录调控，为进一步理解流动调控内皮细胞生物学的机制和分子基础奠定基础。

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.