Ultimate understanding of the anti-atherogenic role of plasma HDL depends, in large part, on an appreciation of how HDL structure and metabolism affect intravascular and extravascular cholesterol redistribution. Specificity of HDL metabolism and function is closely linked to their apolipoprotein composition and two major apolipoprotein-specific populations have recently been described. The present proposal is directed to investigation, by use of model and native lipoprotein particles, of the physical-chemical and metabolic bases for speciation, remodeling and function in cholesterol redistribution of nascent and plasma apolipoprotein-specific HDL populations. In view of the important linkages between triglyceride (TG) metabolism and plasma HDL, our research emphasizes studies on understanding how lipolysis=derived products of metabolism of TG-rich particles influence nascent and plasma apolipoprotein-specific HDL metabolism and function in cholesterol transport. Since as phosphatidylethanolamine (PE) and lysoPE, as well as other lipolysis derived products, we will investigate their influence on structure and metabolism of the model nascent apolipoprotein-specific HDL subpopulations. Reassembly of single and hybrid apolipoprotien analogs of nascent HDL will be performed mainly by the cholate-dialysis method. Apolipoprotein-specific model and plasma HDL particles will be isolated by immunoaffinity chromatography and further fractionated by FPLC or gel filtration chromatography. Purified preparations of lecithin:cholesterol acyltransferase, cholesteryl ester and phospholipid transfer proteins and hepatic lipase will be used in in vitro metabolic studies. Control particles and conversion products will be analyzed by methods including gradient gel electrophoresis, electron microscopy, circular dichroism, immunoassay of apoAI epitope exposure, apolipoprotein immunoassays and lipid assays by gas-liquid chromatography and standard techniques. Our rationale for emphasizing the model system approach is that it provides ample, well-defined material, together with tight control of experimental variables, and has already proven highly informative in delineating origins of plasma apoAI without apoAII HDL subpopulations. Lastly, the proposal will provide new information on differential behavior of HDL particles at the cellular level. The macrophage-like cell line THP-1 and primary human monocyte-derived macrophage cells will be used to investigate compositional determinants of apolipoprotein-specific HDL in effecting cholesterol efflux from cholesterol laden macrophage cells.
