apoE, arterial biomechanics, and cardiovascular disease

描述（由申请人提供）：动脉硬化是心血管疾病的风险因素，但动脉如何保持柔软以及动脉硬化如何导致疾病尚不清楚。我们的初步研究表明，动脉弹性是由载脂蛋白E（apoE）和载脂蛋白E的HDL通过抑制细胞外基质基因的表达。ApoE中断了机械驱动的前馈回路，该回路增加了胶原蛋白I、纤连蛋白和赖氨酰氧化酶的表达以响应基质硬化。这些作用与apoE脂质结合结构域无关。apoE基因敲除小鼠的动脉硬化增加，这种硬化可以通过施用赖氨酰氧化酶抑制剂BAPN来减少，并且BAPN治疗减弱动脉粥样硬化，尽管胆固醇高度升高。体内BAPN减少了病变中巨噬细胞的丰度，体外基质软化减少了单核细胞/巨噬细胞的粘附。从机制上讲，我们表明，载脂蛋白E和载脂蛋白E的HDL抑制Rho-GTP活性，降低细胞内的力量在VSMCs。VSMC力学的这些变化然后影响ECM基因表达。最后，我们表明，除了调节（纤维状）胶原蛋白-I，载脂蛋白E和载脂蛋白E-HDL抑制胶原蛋白-VIII，非纤维状胶原蛋白，有深远的影响VSMC功能和动脉粥样硬化的表达，但在很大程度上是未开发的机械性能和机械效果。总的来说，我们的数据描述了一个全新的apoE和apoE-HDL的作用，是独立的血浆胆固醇水平，密切相关的细胞和组织的机械生物学，因果关系联系到动脉粥样硬化的保护。我们现在提出了三个具体的目标来描述载脂蛋白E，细胞内力，基质重塑和动脉粥样硬化保护之间的关系。目的一是利用一种新型的VSMC微组织微加工平台，研究VIII型胶原对VSMC Rho活性、收缩性 ECM基因表达和3D组织硬度。我们还将使用该系统来确定胶原蛋白VIII如何控制apoE的机械反应。这些体外研究将通过从WT和胶原蛋白VIII缺陷小鼠分离的apoE+/+和apoE-/-动脉中的动脉硬度的离体分析进行补充。在目标2中，我们研究了硬度控制动脉粥样硬化病变发展的机制，特别是确定机械敏感性粘附受体，这些受体负责单核细胞和巨噬细胞与内皮下ECM蛋白的硬度依赖性粘附。如在目标1中，使用现有小鼠模型的补充体内实验将测试动脉硬度对体内单核细胞丰度的影响。目标3将通过开发一种新的小鼠模型将前两个目标中的结果联系起来，该模型可以从VSMCs中删除RhoA，并在体内建立细胞内力降低对ECM基因表达、动脉僵硬度和动脉粥样硬化的影响。这项工作汇集了一个由三名PI（Assoian、Chen和Bendeck）组成的团队，他们具有互补的专业知识和共同出版的既定记录，他们将共同确定apoE对ECM和VSMC机制的这种新型调节如何提供针对心血管疾病的胆固醇独立保护。

DESCRIPTION (provided by applicant): Arterial stiffening is a risk factor for cardiovascular disease, but how arteries stay supple and how arterial stiffness contributes to disease are unknown. Our preliminary studies show that arterial elasticity is maintained by Apo lipoprotein E (apoE) and apoE-containing HDL through a suppressive effect on the expression of extracellular matrix genes. ApoE interrupts a mechanically driven feed-forward loop that increases the expression of collagen-I, fibronectin, and lysyl oxidase in response to substratum stiffening. These effects are independent of the apoE lipid-binding domain. Arterial stiffness is increased in apoE-null mice, this stiffening can be reduced by administration of the lysyl oxidase inhibitor, BAPN, and BAPN treatment attenuates atherosclerosis despite highly elevated cholesterol. Macrophage abundance in lesions is reduced by BAPN in vivo, and monocyte/macrophage adhesion is reduced by substratum softening in vitro. Mechanistically, we show that apoE and apoE-containing HDL inhibit Rho-GTP activity and reduce intracellular force in VSMCs. These changes in VSMC mechanics then affect ECM gene expression. Finally, we show that in addition to regulating (fibrillar) collagen-I, apoE and apoE-HDL inhibit the expression of collagen-VIII, a non-fibrillar collagen that has profound effects on VSMC function and atherosclerosis, yet is largely unexplored in terms of its mechanical properties and mechanistic effects. Overall, our data describe a completely new role for apoE and apoE-HDL that is independent of plasma cholesterol levels, intimately connected to cell and tissue mechanobiology, and causally linked to protection from atherosclerosis. We now propose three specific aims to characterize the relationships between apoE, intracellular force, matrix remodeling, and protection from atherosclerosis. In Aim 1, we will use a new micro fabrication platform of VSMC micro-tissues to study the effect of collagen-VIII on Rho-activity, contractility, ECM gene expression, and tissues stiffness in 3D. We will also use this system to determine how collagen-VIII controls the mechanical response to apoE. These in vitro studies will be complemented with an ex vivo analysis of arterial stiffness in apoE+/+ and apoE-/- arteries isolated from WT and collagen-VIII deficient mice. In Aim 2, we examine the mechanism by which stiffness controls atherosclerotic lesion development, with the particular goal of identifyin mechano-sensitive adhesion receptors that account for stiffness-dependent attachment of monocytes and macrophages to sub endothelial ECM protein. As in Aim 1, complementary in vivo experiments with existing mouse models will test the effect of arterial stiffness on monocyte abundance in vivo. Aim 3 will link the results in the first two aims by developing a new mouse model that can delete RhoA from VSMCs and establish the effect of reduced intracellular force on ECM gene expression, arterial stiffness, and atherosclerosis in vivo. This work brings together a team of three PI's (Assoian, Chen and Bendeck) with complementary expertise and an established track record of co- publication who, jointly, will establish how this novel regulatin of the ECM and VSMC mechanics by apoE provides cholesterol-independent protection against cardiovascular disease.