CELLULAR CHOLESTEROL FLUX AND METABOLISM

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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.