CELLULAR CHOLESTEROL FLUX AND METABOLISM

细胞胆固醇通量和代谢

• 批准号: 6925493
• 负责人: GEORGE H ROTHBLAT
• 金额: $ 30.68万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6925493
• 关键词: angiography; apolipoproteins; blood lipoprotein metabolism; blood proteins; cell cell interaction; cholesterol; high density lipoproteins; human data; human tissue; intracellular transport; laboratory mouse; lipid metabolism; lipid transport; macrophage; membrane activity; membrane lipids; membrane reconstitution /synthesis; protein protein interaction; steroid metabolism; tissue /cell culture

The first step in reverse cholesterol transport (RCT) will be investigated by asking how SR-BI and ABCA1 mediate cholesterol flux between cells and serum components. The specific contribution of each of these plasma membrane proteins will be probed by measuring the difference in flux to lipoproteins between ABCA1 or SR-BI positive and negative cells, In close interaction with other projects in this Program Project, the relationships between ABCA1, SR-BI, apo A-I, serum factors, RCT and atherosclerosis will be studied. In Specific Aim 1, we will collaborate with Dr. Rader to correlate the flux potential of mouse serum to lipoproteins modulated in vivo by adenovirus-mediated overexpression of lipid enzymes and transfer proteins. In Specific Aim 2, we will collaborate with Drs. Lund-Katz and Phillips to manipulate cholesterol lipoprotein composition in vitro to obtain a better measure of the impact of individual components on flux. In Specific Aim 3, with serum collected in Dr. Rader's Preventive Cardiology Clinic we will correlate flux to serum composition in individuals tested by angiography to document the presence/absence of atheroscterosis. Thus, for the first time, ABCA1-and SR-BI mediated cholesterol flux will be correlated to lesions in humans. In Specific Aim 4, we will identify the cellular pathways and serum components that mediate cholesterol mass removal via either SR-BI or ABCA1 using the THP1 human macrophage foam cell model. These studies will utilize mouse and human sera supplied by Dr. Rader's laboratory and mutant apolipoproteins developed in Project 2. In Specific Aim 5, we will probe the interaction between SR-BI and ABCA1. We will characterize the nascent particles produced upon incubation of apo A-t with J774 cells expressing ABCA1. Nascent particles obtained with both cholesterol-enriched/normal J774 cells will be characterized and used to measure cholesterol flux when incubated with SR-BI-expressing cells. Conversely, SR-BI-modified HDL will be obtained, characterized, and used to measure ABCA1-mediated flux. The results from these studies will provide fundamental information on the formation of nascent HDL and the flux of cholesterol in RCT.

反向胆固醇转运 (RCT) 的第一步将通过询问 SR-BI 和 ABCA1 如何介导细胞和血清成分之间的胆固醇流动来进行研究。将通过测量 ABCA1 或 SR-BI 阳性和阴性细胞之间脂蛋白通量的差异来探讨每种质膜蛋白的具体贡献。在与本计划项目中其他项目的密切互动中，将研究 ABCA1、SR-BI、apo A-I、血清因子、RCT 和动脉粥样硬化之间的关系。在具体目标 1 中，我们将与 Rader 博士合作，将小鼠血清的通量潜力与体内通过腺病毒介导的脂质酶和转移蛋白过度表达调节的脂蛋白相关联。在具体目标 2 中，我们将与博士合作。 Lund-Katz 和 Phillips 在体外操纵胆固醇脂蛋白成分，以便更好地测量各个成分对通量的影响。在具体目标 3 中，利用在 Rader 博士的预防心脏病学诊所收集的血清，我们将通过血管造影测试将通量与个体的血清成分相关联，以记录动脉粥样硬化的存在/不存在。因此，ABCA1 和 SR-BI 介导的胆固醇通量首次与人类病变相关。在具体目标 4 中，我们将使用 THP1 人巨噬细胞泡沫细胞确定通过 SR-BI 或 ABCA1 介导胆固醇质量去除的细胞途径和血清成分 模型。这些研究将利用 Rader 博士实验室提供的小鼠和人类血清以及项目 2 中开发的突变载脂蛋白。在具体目标 5 中，我们将探讨 SR-BI 和 ABCA1 之间的相互作用。我们将表征 apo A-t 与表达 ABCA1 的 J774 细胞一起孵育时产生的新生颗粒。用富含胆固醇/正常 J774 细胞获得的新生颗粒将被表征，并用于测量与 SR-BI 表达细胞一起孵育时的胆固醇通量。相反，将获得 SR-BI 修饰的 HDL、表征并用于测量 ABCA1 介导的通量。这些研究的结果将提供有关新生 HDL 的形成和通量的基本信息 随机对照试验中的胆固醇。