CELLULAR CHOLESTEROL FLUX AND METABOLISM

在逆向胆固醇转运（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通量来研究。我们将通过测量ABCA1或SR-BI阳性和阴性细胞对脂蛋白通量的差异来探讨这些质膜蛋白的具体贡献，并与本项目的其他项目密切合作，研究ABCA1、SR-BI、载脂蛋白A-I、血清因子、RCT与动脉粥样硬化之间的关系。在Specific Aim 1中，我们将与Rader博士合作，将小鼠血清的通量电位与体内由腺病毒介导的脂质酶和转移蛋白的过表达调节的脂蛋白联系起来。在具体目标2中，我们将与博士合作。lundd - katz和Phillips在体外操纵胆固醇脂蛋白组成，以更好地测量单个成分对通量的影响。在Specific Aim 3中，我们将在Dr. Rader的预防心脏病诊所收集血清，通过血管造影检测个体的血流量与血清成分之间的关系，以记录动脉粥样硬化的存在/不存在。因此，abca1和SR-BI介导的胆固醇通量将首次与人类病变相关。在Specific Aim 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介导胆固醇团块去除的细胞途径和血清成分

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.