Formation of atherogenic lipoproteins and their stimulation of cellular cholesterol esterification: Insights into macrophage-foam cell development

Arterial-wall macrophages become cholesteryl ester loaded through uptake of atherogenically modified lipoproteins followed by intracellular esterification of cholesterol by the enzyme acyl-coenzyme A:cholesterol O-acyltransferase (ACAT). Self-aggregated low density lipoprotein (LDL) is one atherogenic form of LDL prominent in vivo. The mechanisms causing LDL to aggregate in the arterial wall and the mechanisms by which atherogenic lipoproteins stimulate cholesterol esterification in macrophages remain unknown. In this thesis, I address two important questions regarding these events. First, I asked if the enzyme sphingomyelinase, which causes LDL to aggregate in vitro, is a plausible mechanism of LDL aggregation in vivo. Towards this goal, I show that SMase-treated LDL aggregates due to increases in particle ceramide content; and, remarkably, aggregated LDL extracted from human atherosclerotic lesions is enriched 10-50 fold in ceramide compared with plasma LDL. Furthermore, strips of rabbit aorta ex vivo can hydrolyze the sphingomyelin of retained LDL. Most important, I show that many cell types, including macrophages, secrete a zinc-dependent acid SMase (Zn-SMase) that, surprisingly, is a product of the same gene which codes for the classic cation-independent lysosomal-SMase. My results support a role for arterial SMases, and Zn-SMase in particular, in LDL aggregation in vivo.;Second, given the central role of intracellular cysteine proteases in other cholesterol regulated pathways, I asked if cysteine proteases play a role in regulating lipoprotein-induced cholesterol esterification. Herein, I show that cysteine protease inhibitors block the ability of atherogenic lipoproteins to stimulate cholesterol esterification in macrophages. Cysteine protease inhibitors, however, do not block the stimulation of cholesterol esterification induced by 25-hydroxycholesterol or sphingomyelinase. Furthermore, when added after the protein synthesis inhibitor cycloheximide, cysteine protease inhibitors do not block cycloheximide-induced cholesterol esterification. These data are consistent with a novel model in which an intracellular proteolytic reaction mediates the stimulation of cholesterol esterification by expanded cellular cholesterol pools, perhaps by cleaving an endogenous inhibitor of the interaction of these expanded pools with ACAT.