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(申请人提供)：内皮细胞(ECs)的生物力学特性在多种EC功能的调节中至关重要，如机械转导和EC屏障的完整性。我们最近发现，氧化修饰的低密度脂蛋白(OxLDL)导致显着的EC硬化，这表明血脂异常在EC机制的调节中起着重要作用。我们的长期目标是阐明血脂异常导致内皮细胞生物力学改变的机制，并确定这些机制在内皮功能障碍中的作用。在这笔赠款的第一个资助期，我们首次提供了对oxLDL诱导的EC硬化的机械性见解，并证明了它可能促进内皮细胞对流动的敏感性。在目前的提案中，我们扩展了您的研究以解决三个新的目标：在目标1中，我们将确定特定的氧化脂质导致 EC硬化，并提出EC硬化是通过将氧化的脂质插入内皮细胞的质膜和破坏膜区的脂质堆积而介导的假说。为了实现这一目标，我们将对oxLDL复合体和从载脂蛋白E-/-小鼠分离的主动脉血管壁中发现的氧化脂质进行质谱分析。内皮细胞的硬度将使用两种生物物理技术的组合来测量，显微吸入法和原子力显微镜，而脂肪堆积将通过双光子显微镜进行分析。在目标2中，我们将阐明导致oxLDL诱导的EC硬化的下游信号通路，重点是小窝蛋白和Rho-GTP酶的作用。具体地说，我们将解决一个假说，即oxLDL/氧化脂质诱导的富含胆固醇的膜域的破坏激活了一条信号通路，包括小窝蛋白-1的磷酸化，Rho-GTP酶及其主要下游靶标ROCK的激活，从而导致肌动蛋白/肌球蛋白组织的变化。这一假说将通过一系列小窝蛋白、Rho和Rac-GTP酶和ROCK的功能获得和功能丧失突变来解决。在目标3中，我们将在体外和体内测定不同血流动力学环境下血脂异常引起的内皮细胞僵硬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在受扰的促动脉粥样硬化血流环境下具有阻隔作用，并增加EC的通透性。最后，我们将确定内皮细胞硬度的增加是否与ApoE-/-小鼠体内内皮通透性的增加相关，并确定ApoE-/-小鼠内皮细胞屏障的破坏是否可以通过小窝蛋白-1缺乏和/或ROCK抑制来挽救。