Hepatic Cholesteryl Ester Metabolism and Cholesterol Elimination

描述（由申请人提供）：胆固醇逆向转运（RCT）是胆固醇从与动脉壁相关的外周组织（包括巨噬细胞）转运到肝脏并最终转化为胆汁酸或直接分泌到胆汁中的主要机制。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的强制性第一步和限速步骤是外周组织（如巨噬细胞泡沫细胞）中储存的细胞内CE的细胞内水解，该反应由中性胆固醇酯水解酶（CEH）催化。游离或未酯化胆固醇（FC）被细胞外胆固醇受体（如HDL）去除，由血清LCAT重新酯化，并作为CE携带到肝脏，通过清除率受体BI （SR-BI）选择性摄取。CE的水解对于随后释放的FC转化为胆汁酸或直接分泌成胆汁是必需的。在确定了人巨噬细胞CEH在调节FC、RCT外排从而减轻LDLR-/-小鼠饮食诱导的动脉粥样硬化中的作用后，PI最近报道了人肝脏CEH的克隆和表征。这种酶的过度表达导致细胞内CE的动员和胆汁酸合成的增加。腺病毒介导的小鼠CEH过表达导致体内RCT显著增加，胆固醇作为分泌胆汁酸的消除增加，而这一过程需要HDL-受体SR-BI的存在。因此，ceh介导的CE水解是调控RCT第一步（巨噬细胞产生游离胆固醇用于外排）和最后一步（产生游离胆固醇用于胆汁酸合成或胆道胆固醇分泌）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的水解（内源性合成或通过SR-BI选择性摄取HDL传递）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来验证：目的1：通过培养肝脏特异性CEH转基因小鼠，建立肝脏CEH抗动脉粥样硬化的作用。目的2：描述肝脏CEH介导的RCT调节的机制：CEH在通过清除率受体BI （SR-BI）或SR-BII介导的hdl相关CE摄取水解CE到肝脏中的作用。目的3：确定肝CEH在调节胆汁酸合成中性或酸性途径FC可用性中的作用。目的4：通过肝脏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。

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.