Cholesterol Ester Hydrolysis and cholesterol efflux

描述(由申请人提供)：含有胆固醇酯的巨噬细胞泡沫细胞是动脉粥样硬化病变最显著的特征之一。细胞外受体介导的游离胆固醇(FC)外流是细胞胆固醇清除的主要机制，在防止泡沫细胞形成和动脉粥样硬化病变发展中起着关键作用。然而，为了发生外排，必须首先在中性胆固醇酯水解酶(CEH)的介导下，将FC从储存的胆固醇酯(CE)中释放出来，这一步骤越来越被认为是FC外流的限速步骤。PI已经克隆和鉴定了人巨噬细胞CEH，并证明了CEH过表达后细胞CE的动员增加，通过调节细胞CE的水解来确定CEH在动脉粥样硬化形成中的作用是上一个资金周期的重点。在动脉粥样硬化易感的LDLR-/-小鼠中，巨噬细胞特异性的人巨噬细胞CEH的转基因表达不仅能显著降低饮食诱导的动脉粥样硬化和主动脉病变的胆固醇含量，而且还能减少病变坏死。我们的机制研究表明，增强CEH介导的巨噬细胞内CE的水解增加了胆固醇从巨噬细胞到肝脏的通量，最终随着胆汁酸进入胆汁和粪便而被清除，这为增强CEH介导的细胞内CE水解具有抗动脉粥样硬化作用提供了直接证据。目前提案的总体目标是在三个关键领域扩大我们关于CEH在调节巨噬细胞脂负荷中的作用的工作：描述巨噬细胞CEH表达增强对现有动脉粥样硬化病变进展的影响；确定CEH介导的降低巨噬细胞脂负荷的全身效应，并获得缺乏CEH将导致动脉粥样硬化的最终证据。因此，本研究的中心假设是：巨噬细胞CEH水平决定了CE在细胞内的积聚，从而影响动脉粥样硬化病变的发展以及与动脉壁相关巨噬细胞泡沫细胞的脂质负荷相关的全身性异常。目的1：确定巨噬细胞特异性CEH表达在减少LDLR-/-小鼠动脉粥样硬化病变进展中的作用；目的2：确定CEH介导的降低LDLR-/-CEHTg小鼠巨噬细胞胆固醇负荷的系统效应，并阐明其可能的机制和目的3：通过巨噬细胞特异性靶向干扰CEH在小鼠体内获得概念验证，并确定其对CE代谢和动脉粥样硬化的影响。巨噬细胞泡沫细胞的形成是动脉粥样硬化形成的初始事件之一。高密度脂蛋白介导的未酯化或游离胆固醇(FC)从这些泡沫细胞中储存的胆固醇酯(CE)中流出是去除细胞内胆固醇和泡沫细胞退化的主要机制。早期广泛的研究表明，在易患动脉粥样硬化的动物物种中，巨噬细胞胆固醇酯代谢异常。此外，这些研究还发现，中性胆固醇酯水解酶(CEH)催化的细胞内胆固醇酯水解酶是胆固醇外流过程中的一个限制性步骤。在这个项目的最后一个资金周期中，我们展示了巨噬细胞CEH在调节细胞CE水平、增加FC外流从而减轻饮食诱导的LDLR-/-小鼠动脉粥样硬化中的作用。拟议的研究将建立在这一基础上，并确定CEH不仅在防止现有斑块的进展方面发挥作用，而且由于降低巨噬细胞的脂质负荷而在改善全身炎症和胰岛素敏感性方面发挥作用。鉴于以肥胖和胰岛素抵抗为主要危险因素的动脉粥样硬化和冠状动脉疾病的流行，目前的发现可能具有重要的临床意义。

DESCRIPTION (provided by applicant): Presence of cholesteryl ester-laden macrophage foam cells is one of the most prominent characteristics of an atherosclerotic lesion. Extracellular acceptor-mediated free cholesterol (FC) efflux is the primary mechanism for the removal of cellular cholesterol and is critical in preventing foam cell formation and atherosclerotic lesion development. However, in order for efflux to occur, FC must first be released from stored cholesteryl esters (CE) by hydrolysis mediated by neutral cholesteryl ester hydrolase (CEH) and this step is increasingly being recognized as the rate-limiting step in FC efflux. The PI has cloned and characterized the human macrophage CEH and demonstrated increased cellular CE mobilization after CEH over-expression and establishing the role of CEH in atherogenesis by regulating cellular CE hydrolysis was the focus during the last funding cycle. Macrophage-specific transgenic expression of human macrophage CEH in athero-susceptible LDLR-/- mice not only led to a significant reduction in diet-induced atherosclerosis and cholesterol content of the aortic lesions but also reduced lesion necrosis. Our mechanistic studies showed that enhancing CEH-mediated hydrolysis of CE in macrophages enhanced the flux of cholesterol from macrophages to liver for the final elimination as bile acids into bile and feces providing direct evidence that enhanced CEH-mediated intracellular CE hydrolysis is anti-atherogenic. The overall objective of the current proposal is to expand on our work on the role of CEH in regulating macrophage lipid burden in three critical areas: to delineate the effect of enhancing macrophage CEH expression on progression of existing atherosclerotic lesions; to determine the systemic effects of CEH-mediated reduction in macrophage lipid burden and to obtain the final proof that lack of CEH will be atherogenic. Therefore, it is the central hypothesis of this proposal that: Macrophage CEH levels determine intracellular CE accumulation and thereby influence atherosclerotic lesion development as well as systemic aberrations associated with lipid burden of artery wall associated macrophage foam cells. The following three specific Aims are proposed: Aim 1: To determine the role of macrophage-specific CEH expression in reducing the progression of existing atherosclerotic lesions in LDLR-/- mice; Aim 2: To determine the systemic effects of CEH-mediated reduction in Macrophage cholesterol burden in LDLR-/-CEHTg mice and delineate the underlying mechanisms and Aim 3: To obtain in vivo proof of concept by macrophage-specific targeted disruption of CEH in mice and to determine its effect on intracellular CE metabolism and atherosclerosis. Formation of macrophage foam cells is one of the initial events in atherogenesis. HDL- mediated efflux of unesterified or free cholesterol (FC) from the stored cholesteryl esters (CE) in these foam cells is the primary mechanism for removal of intracellular cholesterol and foam cell regression. Extensive earlier research has indicated aberrations in macrophage cholesterol ester metabolism in animal species susceptible to atherosclerosis. Further, these studies have also identified intracellular cholesterol ester hydrolysis catalyzed by neutral cholesteryl ester hydrolase (CEH) as a limiting step in the process of cholesterol efflux. During the last funding cycle of this project we demonstrated the role of macrophage CEH in regulating cellular CE levels, enhancing FC efflux and thereby attenuating diet-induced atherosclerosis in LDLR-/- mice. The proposed studies will build on this foundation and establish the role of CEH in not only preventing progression of existing plaque but in improving systemic inflammation and insulin sensitivity as a result of decreasing macrophage lipid burden. Given the prevalence of atherosclerosis and coronary artery disease with obesity and insulin resistance as major risk factors, the current findings are likely to have important clinical relevance.