Cholesterol Ester Hydrolysis and Cholesterol Efflux

胆固醇酯水解和胆固醇流出

• 批准号: 7092624
• 负责人: SHOBHA GHOSH
• 金额: $ 29.3万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7092624
• 关键词: animal genetic material tag; atherosclerosis; cholesterol; cholesterol esters; complementary DNA; dietary constituent; esterase; gel mobility shift assay; gene expression; genetically modified animals; high density lipoproteins; human genetic material tag; hydrolysis; intracellular; laboratory mouse; lipid metabolism; macrophage; steroid metabolism; tissue /cell culture; transcription factor; transfection; western blottings

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.

描述（由申请人提供）：动脉内膜内负载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) 的细胞质脂质内含物的积累所致。胆固醇摄取和流出途径之间稳态平衡的丧失对于泡沫细胞的形成至关重要。虽然天然或修饰脂蛋白的摄取构成了流入途径，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（HDL）等受体是唯一公认的胆固醇流出机制。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，释放游离或未酯化的胆固醇 (UC)。然后UC移动到质膜以转移到受体分子。中性胆固醇酯水解酶 (CEH) 催化储存胆固醇酯的水解。本研究的长期目标是确定人巨噬细胞中 CEH 调节所涉及的机制，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。该提议的中心假设是：巨噬细胞CEH水平影响HDL介导的泡沫细胞胆固醇流出，并且CEH的表达受到影响细胞胆固醇及其代谢物水平的过程的调节。提出了以下四个具体目标： 目标 1：确定人巨噬细胞 CEH cDNA 过度表达对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。目标 2：通过人巨噬细胞 CEH cDNA 的过度表达来确定胆固醇流出的变化以及由此导致的细胞胆固醇/胆固醇酯的变化，并检查 HDL 介导的信号传导对 CEH 的调节。目标 3：确定细胞内胆固醇/氧甾醇水平调节 CEH 表达的机制。目标 4：通过在 LDL 受体敲除 (LDLR-/-) 小鼠中引入人巨噬细胞 CEH 转基因并减轻胆固醇喂养引起的动脉粥样硬化，获得体内“概念验证”。虽然转基因小鼠中所需表型的产生可能为未来的基因治疗提供基础，但所涉及的调节机制的描述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普遍存在，目前的研究结果可能具有重要的临床意义。