Impact of dyslipidemia on endothelial biomechanics

描述（由申请人提供）：内皮细胞（EC）的生物力学特性在多种EC功能的调节中至关重要，例如机械转导和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）诱导显着的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负责血脂异常引起的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。在该补助金的第一个资助期内，我们提供了第一个机制的见解oxLDL诱导EC硬化，并证明它可能有助于内皮细胞流动的敏感性。在目前的建议中，我们将这些研究扩展到三个新的目标：在目标1中，我们将确定诱导细胞凋亡的特定氧化脂质。 EC硬化和地址的假设，EC硬化介导的氧化脂质插入到内皮细胞的质膜和膜结构域的脂质包装的破坏。为了实现这一目标，我们将对oxLDL复合物和从血脂异常ApoE-/-小鼠中分离的血管壁中发现的氧化脂质进行质谱分析。将使用两种生物物理技术（微抽吸和原子力显微镜）的组合测量EC硬度，并通过双光子显微镜测定脂质堆积。在目标2中，我们将阐明下游信号通路，负责oxLDL诱导的EC硬化的小窝蛋白和Rho-GTP酶的作用为重点。具体来说，我们将解决一个假设，oxLDL/氧化脂质诱导的破坏富含胆固醇的膜结构域激活的信号通路，包括磷酸化的小窝蛋白-1，激活Rho-GTdR及其主要下游目标，ROCK，与随后的肌动蛋白/肌球蛋白组织的变化。将使用小窝蛋白、Rho和Rac-GTP酶和ROCK的功能获得和功能丧失突变体的阵列来解决该假设。在目标3中，我们将在体外和体内确定在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oxLDL诱导的EC硬化损害 EC屏障和增强EC渗透性的增加，在干扰促动脉粥样硬化的流动环境下，在体外。最后，我们将确定EC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-/-小鼠中内皮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。

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.