Although HDL-cholesterol (HDL-C) is a recognized risk factor for premature cardiovascular disease, the mechanism(s) by which it is formed, remodeled and of removed from the plasma compartment are not known. Lipolysis of triglyceride-rich lipoproteins is thought to liberate surface components that are HDL precursors; HDL subfractions, with electrophoretic mobilities and sizes distinct from that of alpha-migrating HDL are thought to be precursors of the mature forms of HDL2 and HDL3. However, until recently one source of nascent HDL, the earliest form of this lipoprotein remained unknown. Two facts have implicated the ABC1 transporter protein in HDL formation. Mutations in human ABC1 transporter are associated with HDL-C deficiencies, including Tangier disease and certain other forms of hypoalphalipoproteinemia. In human monocyte derived macrophages and macrophage cell lines, ABC1 is associated with the transfer of cholesterol and phospholipids to the extracellular space. Early forms of HDL are remodeled by several plasma activities giving a form of HDL that is recognized and removed from the plasma compartment by a receptor. Recent evidence suggests that this receptor is scavenger receptor class B type 1 (SRB1), which belongs to the scavenger receptor family of proteins. Thus, ABC1 activity provides an early form of HDL that is remodeled to the mature form that is removed by SRB1. Hypothetically, the connections between ABC1 and SRB1 are formed by the remodeling activities of plasma. These include lecithin:cholesterol acyltransferase, cholesteryl ester transfer protein, phospholipid transfer protein, hepatic lipase, and lipoprotein lipase. The broad goal of this proposal is to identify the mechanism by which this process occurs and the conditions that optimize the transfer of lipids to SRB1. This will be achieved by studying the in vitro remodeling of lipoproteins secreted by macrophage cell lines with high level expression of ABC1 protein and activity as assessed by measurements of lipid efflux. The composition, structure and biological activity of the various remodeled HDL formed under various conditions of plasma transfer, esterification, and lipolysis will be determined. Composition will be determined by various enzymatic and chromatographic methods. Structure will be assessed on the basis of several physical methods including electrophoresis (charge and size), surface and core structure (fluorescence), and circular dichroism. Function will be determined by measuring the binding to SRB1 in ovarian cells and by measuring the turnover in rat plasma using a nontransferable label.
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