Contribution of Endothelial Planar Cell Polarity pathways in Blood Flow Direction Sensing

项目摘要 血管的功能和发育主要由血管内皮细胞（VECs）介导 排列在血管内壁上。血流产生的流体剪切应力（FSS）是一种 它们的功能和表型的主要决定因素，在发育，生理， 和疾病动脉的健康区域中的VEC处于单向层流下，其中 它们在流动方向上排列并激活抗炎通路， 抗动脉粥样硬化。相比之下，动脉弯曲或分支区域的血管内皮细胞 发展出流动模式的紊乱。这些扰动流型无法使VEC对齐， 激活炎症通路，这与形成动脉粥样硬化的易感性相关 斑块细胞排列的FSS方向也调节炎症信号传导 输出，这表明细胞极性和流动方向感测对于差异很重要 动脉粥样硬化保护和动脉粥样硬化酮反应。因此，血管内皮细胞如何感知和响应流 方向是与人类健康有关的重要基础科学问题，但机制 还不清楚一种连接性流动依赖性复合物，其包含VE-钙调蛋白、PECAM 1和 VEGF是整合内皮细胞流动反应的关键。我们的实验室最近发现， 极性衔接蛋白LGN直接与VE-Cahderin结合，对于正确的 内皮细胞排列由于LGN直接与流动依赖性机械敏感性 LGN是一个复杂的，并具有调节细胞骨架动力学的既定作用，我假设LGN是 对于流动方向感测是重要的。我计划使用以下具体的 目的：目的1：通过在LGN的功能结构域中产生突变来表征机制 以确定哪些位点对于介导内皮细胞流动依赖性信号传导是重要的。我 将同样检查已知的LGN相互作用的影响，如果他们被证明是至关重要的， 流动介导的信号传导。目的2：检查细胞极性在流动信号中的作用。我既这样行 首先通过跟踪LGN响应于流动的细胞内定位， 底物分别限制细胞和细胞骨架的极性，以确定这些 变量对于LGN极性和炎症与抗炎信号传导是重要的。目标3： 通过分析具有LGN内皮缺失的小鼠来确定LGN在体内的作用， 阐明LGN在体内VEC排列、炎症和导致的动脉粥样硬化中的作用。 总之，这些目标将揭示内皮细胞流量传感、血管内皮细胞和血管内皮细胞的新机制。 炎症和动脉粥样硬化疾病。

PROJECT SUMMARY Vascular function and development are largely mediated by vascular endothelial cells (VECs) that line the inner wall of blood vessels. Fluid shear stress (FSS) generated by blood flow is a major determinant of their function and phenotype with major roles in development, physiology, and disease. VECs in healthy regions of arteries are under unidirectional laminar flow, where they align in the direction of flow and activate anti-inflammatory pathways which confers resistance to atherosclerosis. By contrast, VECs in curved or branched regions of arteries develop disturbances in flow patterns. These disturbed flow patterns fail to align VECs and activate inflammatory pathways, which correlates with susceptibility to form atherosclerotic plaque. FSS direction with respect to cell alignment also regulates inflammatory signaling outputs, which suggests cell polarity and flow direction sensing is important for the differential atheroprotective and atheroprone responses. Thus, how VECs sense and respond to flow direction is an important basic science question pertinent to human health, but the mechanism is unclear. A junctional flow-dependent complex comprising of VE-Cahderin, PECAM1, and VEGF is critical for integrating endothelial cell flow responses. Our lab recently discovered that the polarity adaptor protein, LGN, which binds directly to VE-Cahderin is important for proper endothelial cell alignment. Since LGN directly interacts with a flow dependent mechanosensitive complex and has an established role regulating cytoskeletal dynamics, I hypothesize LGN is important for flow direction sensing. I plan to address this hypothesis using the following specific aims: Aim 1: Characterize the mechanism by generating mutations in LGN’s functional domains to determine which sites are important for mediating endothelial cell flow dependent signaling. I will similarly examine the effects of known LGN interactors if they are shown to be crucial for flow mediated signaling. Aim 2: Examine the role of cell polarity in flow signaling. I will do this first by tracking the intracellular localization of LGN in response to flow and use patterned substrates to separately constrain cell and cytoskeletal polarity to determine which of these variables is important for LGN polarity and inflammatory vs. anti-inflammatory signaling. Aim 3: Determine the role of LGN in vivo by analyzing mice with endothelial deletion of LGN which will address the role of LGN in VEC alignment, inflammation, and resulting atherosclerosis in vivo. Together, these aims will reveal new mechanisms for endothelial flow sensing, vascular inflammation, and atherosclerotic disease.