Impact of dyslipidemia on endothelial biomechanics

描述（由申请人提供）：内皮细胞（EC）的生物力学特性在EC机械转导、细胞-细胞相互作用、血管活性剂分泌和血管重塑的调节中至关重要。我们最近的研究表明，EC的生物力学特性显着改变细胞胆固醇的变化，表明EC力学受损血脂异常的条件下。本项目的长期目标是阐明血脂异常导致EC力学受损的机制，并确定这些效应对EC功能的影响。我们的初步研究表明，氧化低密度脂蛋白强烈增加EC刚度支持的假设，血脂异常是重要的调节EC力学。本研究的主要目的是：（1）研究血脂异常对离体动脉内皮细胞力学性能的影响。首先，我们将集中于确定oxLDL及其组分对来自不同动脉床的人EC的影响。此后，我们将比较从正常和高胆固醇血症猪的动脉新鲜分离的EC的机械特性。结合三种生物物理方法：微管抽吸，原子力和牵引力显微镜将用于EC力学的综合分析。我们将进一步阐明EC力学，细胞骨架组织和细胞胆固醇水平之间的关系，使用三维成像和脂质生物化学的组合。(2)探讨血脂异常引起EC生物力学变化的分子机制。具体而言，我们将首先确定膜-细胞骨架复合物的机械性质是否取决于膜脂质双层的物理性质和/或富含胆固醇的膜结构域和小窝的完整性。接下来，我们将检验Rho-GTP酶和/或PI（4，5）P3（膜-细胞骨架相互作用的主要调节剂）负责胆固醇诱导的EC力学变化的假设。(3)阐明血脂异常和血流在EC力学调节和细胞骨架重塑中的相互作用。在这部分的研究中，我们将首先调查血脂异常和血流动力学条件之间的关系，在调节EC的机械性能。最后，我们将扩展这些研究，以确定血脂异常对流动诱导的细胞骨架重塑和NO产生的影响。

DESCRIPTION (provided by applicant): Biomechanical properties of endothelial cells (ECs) are crucially important in regulation of EC mechanotransduction, cell-cell interactions, secretion of vasoactive agents, and vascular remodeling. Our recent studies have shown that EC biomechanical properties are significantly altered by changes in cellular cholesterol suggesting that EC mechanics is impaired under dyslipidemic conditions. The long-term goal of this project is to elucidate the mechanisms responsible for impairment of EC mechanics by dyslipidemia and to determine the impact of these effects on EC function. Our preliminary studies show that oxidized LDL strongly increases EC stiffness supporting the hypothesis that dyslipidemia is important for regulation of EC mechanics. In this study, we propose: (1) To determine the impact of dyslipidemic conditions on mechanical properties of arterial ECs in vitro and ex vivo. First, we will focus on determining the effects of oxLDL and its components on human ECs derived from different arterial beds. Thereafter, we will compare mechanical properties of ECs freshly-isolated from the arteries of normal and hypercholesterolemic pigs. A combination of three biophysical approaches: micropipette aspiration, atomic force and traction force microscopy will be used for comprehensive analysis of EC mechanics. We will further elucidate the relationship between EC mechanics, cytoskeleton organization and cellular cholesterol level using a combination of 3D imaging and lipid biochemistry. (2) To investigate molecular mechanisms responsible for dyslipidemia-induced changes in EC biomechanics. Specifically, we will first determine whether mechanical properties of membrane- cytoskeleton complex depend on the physical properties of the membrane lipid bilayer and/or on the integrity of cholesterol-rich membrane domains and caveolae. Next, we will test the hypothesis that Rho-GTPases and/or PI(4,5)P3, major regulators of membrane-cytoskeleton interactions, are responsible for cholesterol- induced changes in EC mechanics. (3) To elucidate the interplay between dyslipidemia and flow in the regulation of EC mechanics and cytoskeleton remodeling. In this part of the study, we will first investigate the relationship between dyslipidemia and hemodynamic conditions in regulating EC mechanical properties. Finally, we will extend these studies to determine the impact of dyslipidemia on flow-induced cytoskeleton remodeling and NO production.