Cholesterol Ester Hydrolysis and cholesterol efflux

描述（由申请人提供）：胆固醇酯负载巨噬细胞泡沫细胞的存在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）外排是去除细胞胆固醇的主要机制，在防止泡沫细胞形成和动脉粥样硬化病变发展中至关重要。然而，为了发生外排，FC必须首先通过中性胆固醇酯水解酶（CEH）介导的水解从储存的胆固醇酯（CE）中释放出来，这一步越来越被认为是FC外排的限速步骤。PI已经克隆并表征了人巨噬细胞CEH，并证明CEH过表达后细胞CE动员增加，并且通过调节细胞CE水解来确定CEH在动脉粥样硬化中的作用是上一个资助周期的重点。巨噬细胞特异性转基因表达人巨噬细胞CEH在动脉粥样硬化易感的LDLR-/-小鼠中，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，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CE水解增强了胆固醇从巨噬细胞到肝脏的通量，并最终以胆汁酸的形式消除到胆汁和粪便中，这直接证明增强ceh介导的细胞内CE水解具有抗动脉粥样硬化的作用。当前提案的总体目标是在三个关键领域扩展CEH在调节巨噬细胞脂质负担中的作用：描述增强巨噬细胞CEH表达对现有动脉粥样硬化病变进展的影响；以确定CEH介导的巨噬细胞脂质负荷减少的全身性影响，并获得CEH缺乏会导致动脉粥样硬化的最终证据。因此，本研究的核心假设是：巨噬细胞CEH水平决定细胞内CE积累，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脂质负荷相关的系统性畸变。提出以下三个具体目的：目的1：确定巨噬细胞特异性CEH表达在减少LDLR-/-小鼠现有动脉粥样硬化病变进展中的作用；目的2：确定CEH介导的巨噬细胞胆固醇负荷降低对LDLR-/- cehtg小鼠的全身影响，并描述其潜在机制。目的3：通过巨噬细胞特异性靶向破坏小鼠CEH，获得体内概念证明，并确定其对细胞内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发生的初始事件之一。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）外排是去除细胞内胆固醇和泡沫细胞退化的主要机制。早期的大量研究表明，在动脉粥样硬化易感的动物物种中，巨噬细胞胆固醇酯代谢存在异常。此外，这些研究还确定了中性胆固醇酯水解酶（CEH）催化的细胞内胆固醇酯水解是胆固醇外排过程中的一个限制步骤。在本项目的最后一个资助周期中，我们证明了巨噬细胞CEH在调节细胞CE水平，增强FC外溢，从而减轻LDLR-/-小鼠饮食诱导的动脉粥样硬化中的作用。拟议的研究将建立在此基础上，并确定CEH不仅在预防现有斑块进展方面的作用，而且在减少巨噬细胞脂质负担的结果下改善全身炎症和胰岛素敏感性方面的作用。鉴于动脉粥样硬化和冠状动脉疾病的患病率，肥胖和胰岛素抵抗是主要的危险因素，目前的研究结果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.