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

项目总结 血管功能和发育在很大程度上是由血管内皮细胞(VECs)介导的。 它们排列在血管内壁上。血流产生的流体剪应力(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。动脉健康区的血管内皮细胞处于单向层流状态，其中 它们沿着流动的方向排列，并激活抗炎途径，从而 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 在流型中形成扰动。这些扰动的流动模式无法使血管内皮细胞和 激活炎症通路，与动脉粥样硬化形成的易感性相关 斑块。与细胞排列有关的FSS方向也调节炎症信号 输出，这表明细胞的极性和流动方向的感知对差异很重要 动脉粥样硬化保护和动脉粥样硬化反应。因此，VEC如何感知和响应流 方向是关系到人类健康的重要基础科学问题，但其机理 目前还不清楚。由VE-Cahderin、PECAM1和 血管内皮细胞生长因子是整合内皮细胞流动反应的关键。我们实验室最近发现 与VE-Cahderin直接结合的极性适配蛋白LGN对正常的 内皮细胞排列。由于LGN直接与依赖流动的机械敏感剂相互作用 复杂，并具有调节细胞骨架动力学的既定作用，我假设LGN是 对于流向检测很重要。我计划使用以下具体的方法来解决这一假设 目的：目标1：通过在LGN的功能域中产生突变来表征其机制 以确定哪些位点在介导内皮细胞流依赖的信号转导中起重要作用。我 我将同样研究已知的LGN相互作用的影响，如果它们被证明对 流介导的信令。目的2：研究细胞极性在流信号中的作用。我会这么做的 首先通过跟踪LGN在细胞内的定位来响应流动和使用图案化 底物分别限制细胞和细胞骨架的极性，以确定其中的哪一种 变量对LGN的极性和炎症与抗炎信号很重要。目标3： 通过分析LGN内皮细胞缺失的小鼠来确定LGN在体内的作用 阐述LGN在体内血管内皮细胞排列、炎症和由此导致的动脉粥样硬化中的作用。 总之，这些目标将揭示血管内皮细胞血流感知的新机制 炎症和动脉粥样硬化性疾病。

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.