Plasma high density lipoprotein cholesterol (HDL-C) is an important but poorly understood negative risk factor for cardiovascular disease. It is thought that HDL transfers cholesterol from peripheral tissue, such as its site of synthesis in the arterial wall, to the liver for degradation or recycling. This process, reverse cholesterol transport, has been the subject of numerous investigations. An important part of the reverse cholesterol transport pathway involves the remodeling of HDL. The broad goals of this project are to improve our understanding of how HDL particles are remodeled by the major HDL-modifying proteins, which include cholesteryl ester transfer protein (CETP), lecithin:cholesterol acyltransferase (LCAT), phospholipid transfer protein (PLTP), and hepatic lipase (HL). These studies will investigate the role of triglyceride-rich lipoproteins and the interaction of each of the HDL-modifying proteins with model reassembled HDL (R-HDL) and native HDL subfractions with known composition, charge, and size. The following specific goals will be pursued as part of the broader topic of understanding HDL-remodeling: 1] determination of the acyl chain and molecular species specificity of CETP and PLTP by measuring the kinetics of lipid transfer from HDL and R-HDL to single bilayer vesicles in the presence and absence of purified CETP and PLTP; 2] measurement of the PLTP- and CETP-mediated rates of transfer of (3H)phosphatidylcholine from donors of native and model HDL to single bilayer vesicles as a function of surface structure and charge of the donor and acceptor; 3] quantification of the contributions of CETP, PLTP, and LCAT to the maturation of preBeta-HDL into HDL2 and HDL3; 4] comparison of the lipid and the fatty acid compositions of the phosphatidylcholines of the plasma membranes of fibroblasts with those of pre-Beta-HDL that are formed during the incubation of plasma with fibroblasts; and 5] use of purified CETP and PLTP to determine the effects of PLTP on the CETP-mediated transfer of very low density lipoprotein-triglycerides to HDL2 and HDL3. Key analytical methods involved are molecular sieve chromatography, reverse phase HPLC, gradient gel electrophoresis, and ultracentrifugation.
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