Year of Publication

2013

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Medicine

Department

Molecular and Cellular Biochemistry

First Advisor

Dr. Deneys van der Westhuyzen

Abstract

Atherosclerosis is a disease characterized by cholesterol-rich plaques within the intima of medium and large arteries. Cholesterol deposition is thought to occur by infiltration of low-density lipoprotein (LDL) into lesions followed by uptake into macrophages, generating lipid-loaded “foam cells.” Foam cells can also be generated in vitro by treatment of macrophages with LDL or oxidized LDL (oxLDL). The purpose of the current investigation was to determine the contribution of selective cholesteryl ester (CE) uptake versus whole-particle uptake during LDL-induced foam cell formation in cultured macrophages. Murine bone marrow-derived macrophages (BMMs) exhibited significant cholesterol accumulation when treated with LDL as indicated by quantification of cellular cholesterol and visualization of Oil Red-O-stained neutral lipid droplets. Uptake of LDL cholesterol was determined by measuring uptake of 3H and 125I into BMMs during treatment with [3H]CE/125I-LDL. [3H]CE uptake was linearly related to the LDL concentration at the concentrations used and was much larger than 125I uptake, indicating that the majority of LDL-cholesterol was acquired by nonsaturable, selective CE uptake. This pathway was demonstrated to be independent of whole-particle uptake by showing that inhibition of actin polymerization blocked LDL particle uptake but not selective CE uptake. Analysis by thin-layer chromatography (TLC) indicated that following uptake, [3H]CE was rapidly hydrolyzed into [3H]cholesterol by cells and largely effluxed into the culture medium. In contrast to LDL, studies of [3H]CE/125I-oxLDL uptake demonstrated that CE was acquired from oxLDL by whole-particle uptake with little or no selective CE uptake. Using a series of ten different [3H]CE/125I-oxLDLs oxidized for 0-24 hours, selective [3H]CE uptake was shown to be progressively impaired by LDL oxidation, while 125I-LDL particle uptake was increased as expected. Interestingly, the impairment of selective CE uptake occurred very early in LDL oxidation and this minimally oxidized LDL induced significantly less cholesterol accumulation in BMMs compared to native LDL. Together, these results demonstrate that selective CE uptake is the primary mode of cholesterol uptake from LDL but not oxidized LDL, a finding that has important implications for cholesterol metabolism in atherosclerotic lesions. Future studies seek to identify the molecular components that participate in the macrophage selective CE uptake mechanism.

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