Impact of dyslipidemia on endothelial biomechanics

血脂异常对内皮生物力学的影响

• 批准号: 8656732
• 负责人: MICHAEL CHO
• 金额: $ 53.79万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-04 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8656732
• 关键词: Accounting; Actins; Address; Antiatherogenic; Aorta; Apolipoprotein E; Atherosclerosis; Atomic Force Microscopy; Belief; Biological Assay; Biomechanics; Blood Vessels; Cardiovascular Diseases; Caveolins; Cell membrane; Cell physiology; Cessation of life; Cholesterol; Complex; Deposition; Development; Disease; Dyslipidemias; Endothelial Cells; Endothelium; Environment; Event; Exposure to; Functional disorder; Funding; Goals; Grant; Guanosine Triphosphate Phosphohydrolases; Harvest; Image; Impairment; In Vitro; Individual; Inflammatory; Link; Lipids; Low-Density Lipoproteins; Mass Spectrum Analysis; Measures; Mechanics; Mediating; Membrane; Microscopy; Modeling; Modification; Morbidity - disease rate; Mus; Myosin ATPase; Myosin Light Chains; Pathway interactions; Permeability; Phosphorylation; Plasma; Play; Property; Regulation; Resistance; Rho-associated kinase; Risk Factors; Role; Scheme; Signal Pathway; Signal Transduction; Staging; Testing; Tissues; Woman; athero susceptible; base; biophysical techniques; caveolin 1; cell injury; disorder prevention; endothelial dysfunction; gain of function; hemodynamics; in vivo; insight; loss of function; low density lipoprotein inhibitor; men; mutant; myosin phosphatase; novel; oxidized lipid; oxidized low density lipoprotein; response; rho; rho GTP-Binding Proteins; shear stress; two-photon; uptake

DESCRIPTION (provided by applicant): Biomechanical properties of endothelial cells (ECs) are crucially important in regulation of multiple EC functions, such as mechanotransduction and the integrity of the EC barrier. We have recently discovered that oxidized modifications of LDL (oxLDL) induce significant EC stiffening indicating that dyslipidemia plays a major role in the regulation of EC mechanics. Our long term goal is to elucidate the mechanisms responsible for dyslipidemia-induced changes in EC biomechanics and to determine the contribution of these mechanisms to endothelial dysfunction. During the first funding period of this grant, we have provided the first mechanistic insights into oxLDL-induced EC stiffening and demonstrated that it may facilitate the sensitivity of endothelial cells to flow. In the current proposal, we extend thee studies to address three new goals: In Aim 1, we will identify specific oxidized lipids that induce EC stiffening and address the hypothesis that EC stiffening is mediated by the insertion of oxidized lipids into the plasma membranes of endothelial cells and disruption of lipid packing of membrane domains. To achieve this goal, we will perform Mass Spectrometry analysis of oxidized lipids found in both oxLDL complex and in the vascular walls of aortas isolated from dyslipidemic ApoE-/- mice. EC stiffness will be measured using a combination of two biophysical techniques, Microaspiration and Atomic Force Microscopy and lipid packing will be assayed by two-photon microscopy. In Aim 2, we will elucidate the downstream signaling pathways that are responsible for oxLDL-induced EC stiffening focusing on the roles of caveolin and Rho-GTPases. Specifically, we will address a hypothesis that oxLDL/oxidized lipids-induced disruption of cholesterol-rich membrane domains activate a signaling pathway that includes phosphorylation of caveolin-1, activation of Rho-GTPase and its major downstream target, ROCK, with subsequent changes in actin/myosin organization. This hypothesis will be addressed using an array of gain-of-function and loss-of-function mutants of caveolin, Rho and Rac- GTPases and ROCK. In Aim 3, we will determine the impact dyslipidemia-induced EC stiffening on endothelial permeability under different hemodynamic environments in vitro and in vivo. More specifically, first we will test the hypothesis that oxLDL-induced EC stiffening impairs EC barrier and augments an increase in EC permeability under disturbed pro-atherogenic flow environment in vitro. Finally, we will determine whether an increase in EC stiffness correlates with an increase in endothelial permeability in vivo in ApoE-/- mice and determine whether disruption of the endothelial barrier in ApoE-/- mice can be rescued by caveolin-1 deficiency and/or ROCK inhibition.

描述（由申请人提供）：内皮细胞（EC）的生物力学特性对于多种 EC 功能的调节至关重要，例如机械转导和 EC 屏障的完整性。我们最近发现 LDL 的氧化修饰 (oxLDL) 会引起显着的 EC 硬化，表明血脂异常在 EC 力学的调节中起着重要作用。我们的长期目标是阐明血脂异常引起的 EC 生物力学变化的机制，并确定这些机制对内皮功能障碍的贡献。在本次资助的第一个资助期内，我们对 oxLDL 诱导的 EC 硬化提供了第一个机制见解，并证明它可以提高内皮细胞对流动的敏感性。在当前的提案中，我们扩展了这些研究以解决三个新目标：在目标 1 中，我们将确定诱导 EC 硬化并解决了 EC 硬化是由氧化脂质插入内皮细胞质膜和膜域脂质堆积破坏介导的假设。为了实现这一目标，我们将对 oxLDL 复合物和从血脂异常 ApoE-/- 小鼠中分离出的主动脉血管壁中发现的氧化脂质进行质谱分析。 EC硬度将使用微抽吸和原子力显微镜这两种生物物理技术的组合进行测量，并且脂质堆积将通过双光子显微镜进行测定。在目标 2 中，我们将阐明负责 oxLDL 诱导 EC 硬化的下游信号通路，重点关注小窝蛋白和 Rho-GTP 酶的作用。具体来说，我们将提出一个假设，即 oxLDL/氧化脂质诱导的富含胆固醇膜结构域的破坏激活信号通路，包括 Caveolin-1 的磷酸化、Rho-GTPase 及其主要下游靶标 ROCK 的激活，以及肌动蛋白/肌球蛋白组织的后续变化。该假设将通过一系列 Caveolin、Rho 和 Rac-GTPase 以及 ROCK 的功能获得和功能丧失突变体得到解决。在目标 3 中，我们将确定在体外和体内不同血流动力学环境下血脂异常诱导的 EC 硬化对内皮通透性的影响。更具体地说，首先我们将检验以下假设：oxLDL 诱导的 EC 硬化会损害 EC 屏障并在体外受干扰的促动脉粥样化血流环境下增强 EC 通透性的增加。最后，我们将确定 ApoE-/- 小鼠体内 EC 硬度的增加是否与内皮细胞通透性的增加相关，并确定 ApoE-/- 小鼠内皮屏障的破坏是否可以通过 Caveolin-1 缺陷和/或 ROCK 抑制来挽救。