Cholesterol Ester Hydrolysis and Cholesterol Efflux

描述(申请人提供)：动脉内膜内富含脂质的巨噬细胞泡沫细胞的形成是动脉粥样硬化的标志。这些细胞的泡沫状外观是由于含有胆固醇酯(CE)的细胞质脂质包涵体的积累。胆固醇摄取和外流途径之间动态平衡的丧失是泡沫细胞形成的核心。虽然摄取天然或修饰的脂蛋白是胆固醇外流的途径，但将未酯化的胆固醇从这些泡沫细胞转移到高密度脂蛋白(高密度脂蛋白)等受体是唯一公认的胆固醇外流机制。通过高密度脂蛋白从泡沫细胞中去除胆固醇的必备第一步是将储存的CE水解，释放出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。这项研究的长期目标是确定CEH在人巨噬细胞中的调节机制，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/或退化。本研究的中心假设是：巨噬细胞CEH水平影响高密度脂蛋白介导的胆固醇从泡沫细胞外流，CEH的表达受影响细胞胆固醇水平及其代谢产物的过程的调节。目的1：检测人巨噬细胞CEH基因过表达对细胞内胆固醇酯代谢的影响：对脂滴动员和质膜胆固醇浓缩的影响。目的：研究人巨噬细胞CEH基因过表达对胆固醇外流的影响以及由此引起的细胞内胆固醇/胆固醇酯的变化，探讨高密度脂蛋白介导的CEH信号转导机制。目的3：探讨细胞内胆固醇/氧化甾醇水平调节CEH表达的机制。目的：通过在低密度脂蛋白受体基因敲除(LDLR-/-)小鼠体内导入人巨噬细胞CEH基因，减轻胆固醇摄入诱导的动脉粥样硬化，获得体内“概念证明”。虽然在转基因小鼠中产生所需的表型可能为未来的基因治疗提供基础，但所涉及的调控机制的描述将确定潜在的治疗干预靶点。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意义。

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.