Impact of dyslipidemia on endothelial biomechanics

描述(申请人提供)：内皮细胞(ECs)的生物力学特性在调节EC的机械转导、细胞与细胞间的相互作用、血管活性物质的分泌和血管重塑方面至关重要。我们最近的研究表明，内皮细胞的生物力学特性因细胞胆固醇的变化而显着改变，这表明在血脂异常的情况下，内皮细胞的力学机制受到损害。该项目的长期目标是阐明血脂异常导致内皮细胞机制受损的机制，并确定这些影响对内皮细胞功能的影响。我们的初步研究表明，氧化的低密度脂蛋白强烈增加内皮细胞的硬度，支持血脂异常对内皮细胞机制的调节很重要的假说。在本研究中，我们建议：(1)确定血脂异常条件对体外和体外动脉内皮细胞力学特性的影响。首先，我们将重点研究oxLDL及其成分对不同动脉床来源的人内皮细胞的影响。然后，我们将比较新鲜从正常和高胆固醇血症猪的动脉中分离的内皮细胞的机械性能。将结合三种生物物理方法：微吸管吸引法、原子力显微镜和牵引力显微镜，对EC力学进行综合分析。我们将结合3D成像和脂质生化进一步阐明EC力学、细胞骨架组织和细胞胆固醇水平之间的关系。(2)探讨血脂异常引起内皮细胞生物力学改变的分子机制。具体地说，我们将首先确定膜-细胞骨架复合体的机械性能是否取决于膜脂双层的物理性质和/或富含胆固醇的膜域和小凹的完整性。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者，负责胆固醇诱导的EC机制变化的假设。(3)阐明血脂异常与血流在内皮细胞力学和细胞骨架重塑调节中的相互作用。在这部分研究中，我们将首先探讨血脂异常与血流动力学条件在调节内皮细胞机械性能方面的关系。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.