CELLULAR CHOLESTEROL FLUX AND METABOLISM

反向胆固醇转运(RCT)的第一步将通过询问SR-BI和ABCA1如何调节细胞和血清成分之间的胆固醇流动来进行研究。通过测量ABCA1或SR-BI阳性和阴性细胞对脂蛋白的通透性差异来探讨这些质膜蛋白的具体贡献，并与本项目中的其他项目密切合作，研究ABCA1、SR-BI、apo A-I、血清因子、RCT与动脉粥样硬化的关系。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在体受腺病毒介导的脂酶和转移蛋白过表达调节的脂蛋白相关联。在具体目标2中，我们将与Lund-Katz博士和Phillips博士合作，在体外操纵胆固醇脂蛋白成分，以更好地衡量个别成分对通量的影响。在具体目标3中，利用在Rader博士的预防心脏病诊所收集的血清，我们将通过血管造影术将血液流量与血清成分相关联，以证明动脉粥样硬化的存在/不存在。因此，ABCA1和SR-BI介导的胆固醇流量将第一次与人类的损伤相关。在特定目标4中，我们将使用THP1人巨噬细胞泡沫细胞鉴定通过SR-BI或ABCA1介导胆固醇质量清除的细胞途径和血清成分 模特。这些研究将利用Rader博士实验室提供的小鼠和人血清以及项目2中开发的突变载脂蛋白。在特定的目标5，我们将探索SR-BI和ABCA1之间的相互作用。我们将表征载脂蛋白A-t与表达ABCA1的J774细胞孵育时产生的新生颗粒。从胆固醇丰富/正常的J774细胞中获得的新生颗粒将被表征，并在与SR-BI表达细胞孵育时用于测量胆固醇通量。相反，将获得SR-BI修饰的高密度脂蛋白，并对其进行表征，并用于测量ABCA1介导的通量。这些研究的结果将为新生高密度脂蛋白的形成和通量提供基本信息。 RCT中胆固醇的含量。

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.