Cholesterol Ester Hydrolysis and Cholesterol Efflux

描述（由申请人提供）：动脉内膜内脂质巨噬细胞泡沫细胞的形成是动脉粥样硬化的标志。这些细胞的泡沫外观是由于含有胆固醇酯（CE）的细胞质脂包涵体的积累。胆固醇摄取和外排途径之间的稳态平衡的丧失是泡沫细胞形成的核心。虽然天然或修饰脂蛋白的摄取构成了内流途径，但未酯化胆固醇从这些泡沫细胞转移到高密度脂蛋白（HDL）等受体是唯一公认的胆固醇外排机制。通过HDL从泡沫细胞中去除胆固醇的第一步是水解储存的CE以释放游离或未酯化的胆固醇（UC）。UC然后移动到质膜转移到受体分子。中性胆固醇酯水解酶（CEH）催化储存的胆固醇酯的水解。本研究的长期目标是确定人巨噬细胞CEH调节的机制，以及如何操纵这些调节过程来影响巨噬细胞泡沫细胞的形成和/或消退。这一建议的中心假设是：巨噬细胞CEH水平影响高密度脂蛋白介导的泡沫细胞胆固醇外排，CEH的表达受影响细胞胆固醇及其代谢产物水平的过程调节。本文提出以下四个具体目的：目的1：通过过表达人巨噬细胞CEH cDNA来确定细胞内胆固醇酯代谢的变化：对脂滴动员和质膜胆固醇富集的影响。目的2：通过过度表达人巨噬细胞CEH cDNA来确定胆固醇外排的变化和由此引起的细胞胆固醇/胆固醇酯的变化，并研究hdl介导的信号传导对CEH的调节。目的3：确定细胞内胆固醇/氧甾醇水平调节CEH表达的机制。目的4：通过在低密度脂蛋白受体敲除（LDLR-/-）小鼠中引入人巨噬细胞CEH转基因，减轻胆固醇摄食诱导的动脉粥样硬化，获得体内“概念证明”。然而，在转基因小鼠中产生所需表型可能为未来的基因治疗提供基础，描述所涉及的调节机制将确定治疗干预的潜在靶点。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相关性。

DESCRIPTION (provided by applicant): The formation of lipid-laden macrophage foam cells within the arterial intima is a hallmark of atherosclerosis. The foamy appearance of these cells is due to the accumulation of cytoplasmic lipid inclusions containing cholesterol esters (CE). Loss of homeostatic balance between cholesterol uptake and efflux pathways is central to foam cell formation. While uptake of native or modified lipoproteins constitute the influx pathways, transfer of unesterified cholesterol from these foam cells to acceptors like high-density lipoproteins (HDL) is the only recognized mechanism for cholesterol efflux. The obligatory first step for the removal of cholesterol from foam cells via HDL is the hydrolysis of stored CE to release free or unesterified cholesterol (UC). UC then moves to the plasma membrane for transfer to the acceptor molecule. Neutral Cholesterol ester hydrolase (CEH) catalyzes the hydrolysis of stored cholesterol esters. The long-term objective of this research is to determine the mechanisms involved in the regulation of CEH in human macrophage and how these regulatory processes can be manipulated to affect macrophage foam cell formation and/or regression. It is the central hypothesis of this proposal that: Macrophage CEH levels affect HDL-mediated cholesterol efflux from foam cells and that expression of CEH is modulated by processes affecting the levels of cellular cholesterol and its metabolites. The following four Specific Aims are proposed: Aim 1: To determine changes in intracellular cholesterol ester metabolism by over-expression of human macrophage CEH cDNA: Effect on lipid droplet mobilization and cholesterol enrichment of plasma membrane. Aim 2: To determine changes in cholesterol efflux and resulting changes in cellular cholesterol/cholesterol esters by over-expression of human macrophage CEH cDNA and to examine the regulation of CEH by HDL-mediated signaling. Aim 3: To determine the mechanisms whereby intracellular cholesterol/oxysterols levels regulate CEH expression. Aim 4: To obtain in vivo "proof of concept" by introducing human macrophage CEH transgene in LDL-receptor knockout (LDLR-/-) mice and attenuating cholesterol-feeding induced atherosclerosis. While, the production of the desired phenotype in the transgenic mice may provide basis for future gene therapy, delineation of the regulatory mechanisms involved would identify potential targets for therapeutic intervention. Given the prevalence of atherosclerosis and coronary artery disease, the current findings are likely to have important clinical relevance.