Hepatic Cholesteryl Ester Metabolism and Cholesterol Elimination

描述（由申请方提供）：胆固醇逆向转运（RCT）是胆固醇从外周组织（包括与动脉壁相关的巨噬细胞）转运至肝脏最终转化为胆汁酸或直接分泌至胆汁的主要机制。在细胞内或在与脂蛋白相关的血流中运输时，胆固醇主要以胆固醇酯（CE）的形式存在。因此，RCT的强制性第一和限速步骤是外周组织中储存的细胞内CE的细胞内水解，例如，巨噬细胞泡沫细胞，并且该反应由中性胆固醇酯水解酶（CEH）催化。被细胞外胆固醇受体（如HDL）去除的游离或未酯化胆固醇（FC）被血清LCAT重新酯化，并作为CE携带至肝脏，在肝脏中通过清道夫受体BI（SR-BI）选择性摄取递送。CE再次水解是随后将释放的FC转化为胆汁酸或直接分泌到胆汁中所必需的。已经确定了人巨噬细胞CEH在调节FC、RCT流出中的作用，从而减弱LDLR-/-小鼠中饮食诱导的动脉粥样硬化，PI最近报告了人肝脏CEH的克隆和表征。这种酶的过度表达导致细胞内CE动员和胆汁酸合成增加。腺病毒介导的CEH在小鼠中的过表达导致体内RCT的显著增加和作为分泌胆汁酸的胆固醇的消除增加，并且该过程需要HDL受体SR-BI的存在。因此，CEH介导的CE水解代表了调节RCT第一步（巨噬细胞中游离胆固醇的产生用于外排）以及最后一步（游离胆固醇的产生用于胆汁酸合成或胆汁胆固醇分泌）的关键事件。本研究项目的中心假设是：肝脏CEH通过调节细胞内胆固醇酯（内源性合成或通过SR-BI从HDL选择性摄取递送）的水解来影响RCT，从而提供游离胆固醇以胆汁酸形式消除或直接分泌到胆汁中，因此具有潜在的抗动脉粥样硬化作用。这一假设将通过以下四个具体目标进行检验：目标1：通过开发肝脏特异性CEH转基因小鼠来建立肝脏CEH的抗动脉粥样硬化作用。目标二：描述肝脏CEH介导的RCT调节的潜在机制：CEH在水解通过清道夫受体BI（SR-BI）或SR-BII介导的HDL相关CE摄入递送至肝脏的CE中的作用。目的3：确定肝CEH在调节FC的可用性为胆汁酸合成的中性或酸性途径中的作用。目标4：通过肝脏特异性靶向破坏小鼠CEH获得体内“概念验证”，并确定其对细胞内CE代谢和动脉粥样硬化的影响。 公共卫生相关性：肝脏是唯一负责将胆固醇以游离胆固醇（FC）或胆汁酸的形式从体内最终消除的器官，这些胆固醇通过粪便排泄。与过量未修饰的LDL相关的胆固醇以及通过HDL从外周器官（包括动脉壁相关的巨噬细胞泡沫细胞）去除的胆固醇被肝脏摄取。这两种脂蛋白都以胆固醇酯（CE）的形式递送胆固醇，并且在肝脏内，这些CE的水解对于提供FC用于胆汁酸合成或直接消除到胆汁中至关重要。这种水解在溶酶体外区室中通过中性胆固醇酯水解酶（CEH）进行。我们已经确定了人肝CEH，并证明了其在增加胆汁酸分泌以及增强胆固醇从巨噬细胞到胆汁的流量中的作用，这表明肝CEH具有抗动脉粥样硬化功能。拟议的研究将建立在这一基础上，并建立肝CEH的作用，不仅增加胆固醇从体内清除胆汁酸，而且在衰减饮食诱导的动脉粥样硬化。鉴于动脉粥样硬化和冠状动脉疾病的患病率，目前的研究结果可能具有重要的临床意义。

DESCRIPTION (provided by applicant): Reverse cholesterol transport (RCT) is the major mechanism by which cholesterol is transported from the peripheral tissues including macrophages associated with the artery wall, to liver for the ultimate conversion into bile acids or direct secretion into the bile. Within the cells or while being transported in the blood stream associated with the lipoproteins, cholesterol is mainly present as cholesteryl esters (CE). Thus, the obligatory first and rate-limiting step of RCT is the intracellular hydrolysis of the stored intracellular CE in the peripheral tissues, e.g., macrophage foam cells, and this reaction is catalyzed by a neutral cholesteryl ester hydrolase (CEH). Free or unesterified cholesterol (FC) that is removed by extra-cellular cholesterol acceptors such as HDL is re-esterified by serum LCAT and carried as CE to the liver where it is delivered by selective uptake via scavenger receptor BI (SR-BI). Hydrolysis of CE once again is obligatory to subsequent conversion of released FC to bile acids or direct secretion into bile. Having established the role of human macrophage CEH in regulating the efflux of FC, RCT and thus attenuating diet-induced atherosclerosis in LDLR-/- mice, the PI has recently reported the cloning and characterization of human liver CEH. Over-expression of this enzyme results in intracellular CE mobilization and an increase in bile acid synthesis. Adenovirus- mediated over-expression of CEH in mice led to significant increase in in vivo RCT and increased elimination of cholesterol as secreted bile acids, and this process required the presence of HDL- receptor SR-BI. CEH-mediated CE hydrolysis, therefore, represents a key event regulating the first step in RCT (generation of free cholesterol in macrophage for efflux) as well as the final step (generating free cholesterol for bile acid synthesis or biliary cholesterol secretion). The central hypothesis of this research project is: Hepatic CEH affects RCT by regulating the hydrolysis of intracellular cholesterol esters (endogenously synthesized or delivered via selective uptake from HDL through SR-BI) thereby providing free cholesterol for elimination as bile acids or direct secretion into the bile and is, therefore, potentially anti-atherosclerotic. This hypothesis will be tested by the following four specific aims: Aim 1: To establish the anti-atherosclerotic role of hepatic CEH by developing liver specific CEH transgenic mice. Aim 2: To delineate the mechanism(s) underlying hepatic CEH-mediated regulation of RCT: role of CEH in hydrolyzing CE delivered to liver by scavenger receptor BI (SR-BI) or SR-BII mediated uptake of HDL-associated CE. Aim 3: To determine the role of hepatic CEH in regulating FC availability for neutral or acidic pathways for bile acid synthesis. Aim 4: To obtain in vivo "proof of concept" by liver-specific targeted disruption of CEH in mice and to determine its effect on intracellular CE metabolism and atherosclerosis. PUBLIC HEALTH RELEVANCE: Liver is the only organ responsible for the ultimate elimination of cholesterol from the body as free cholesterol (FC) or bile acids that are excreted in the feces. Cholesterol associated with excess unmodified LDL as well as the cholesterol removed from the peripheral organs including artery wall- associated macrophage foam cells by HDL is taken up by the liver. Both these lipoproteins deliver cholesterol in the form of cholesterol esters (CE) and within liver hydrolysis of these CEs is essential to provide FC for bile acid synthesis or for direct elimination into the bile. This hydrolysis is carried out in the extra-lysosomal compartment by a neutral cholesteryl ester hydrolase (CEH). We have identified the human hepatic CEH and demonstrated its role in increasing bile acid secretion as well as in enhancing the flux of cholesterol from macrophages to bile suggesting an anti-atherosclerotic function of hepatic CEH. The proposed studies will build on this foundation and establish the role of hepatic CEH in not only increasing cholesterol elimination from the body as bile acids but also in attenuating diet-induced atherosclerosis. Given the prevalence of atherosclerosis and coronary artery disease, the current findings are likely to have important clinical relevance.