The specific aims of the proposed research are to examine in greater detail the regulation of sterol metabolism by lipoproteins and the impact of such regulatory effects on lymphocyte function and to determine whether abnormalities of lipoprotein metabolism predisposing to the development of atherosclerosis can be detected and characterized using lymphocyte studies. The binding and uptake of lipoproteins by lumphocytes and subsequent regulation of endogenous sterol metabolism will be investigated. Lipoprotein characteristics required for lymphocyte receptor interaction will be determined by examining various lipoprotein classes including LDL, by chemical modification of lipoproteins to prevent binding of the apolipoproteins B and E (apo-B and apo-E) to the receptor and by the use of monoclonal antibodies directed against the receptor binding sites on apo-B and apo-E that block apoprotein-receptor interaction. The role of LDL receptors will be studied by comparing normal lymphocytes with those obtained from LDL receptor-negative familial hypercholesterolemia (FH) patients and by the use of a blocking monoclonal antibody directed against the apoprotein binding site of the LDL receptor. Using these techniques, lipoprotein cholesterol-dependent lymphocyte proliferation will be analyzed by culturing cells in lipoprotein-deficient medium, blocking endogenous sterol synthesis with specific inhibitors of the rate-limiting enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and providing cholesterol as limiting concentrations of specific lipoproteins. Apoprotein and receptor requirements for lipoprotein regulation of lymphocyte sterol synthesis will be investigated by measuring (1-14C)acetate incorporation into digitonin-precipitable sterols and the activity of HMG-CoA reductase. Apo-B mediated binding and uptake of lipoproteins and regulation of LDL receptor expression by lipoproteins will be examined using fluorescent-labeled LDL and analysis by flow cytometry and quantification of LDL receptors using monoclonal antibodies. Molecular events in the regulation of LDL receptor expression and sterol biosynthesis and the interrelationship of lymphocyte activation and sterol metabolism will be studied by measurements of specific mRNA for LDL receptors, HMG-CoA reductase and the mRNAs for lymphocyte activation antigens including the interleukin-2 receptor and the transferrin receptor. The information generated from these studies of normal and homozygous FH lymphocytes will be used to develop techniques to detect and characterize abnormalities in heterozygous FH, other possible lipoprotein receptor defects and lipoprotein abnormalities.
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