Cholesterol and oxysterol metabolism and subcellular distribution in macrophage foam cells: accumulation of oxidized esters in lysosomes

Cholesterol- and cholesteryl ester-rich macrophage foam cells, characterist ic of atherosclerotic lesions, are often generated in vitro using oxidized low density lipoprotein (OxLDL), However, relatively little is known of the nature and extent of sterol deposition in these cells or of its relationsh ip to the foam cells formed in atherosclerotic lesions. The purpose of this study was to examine the content and cellular processing of sterols in OxL DL-loaded macrophages, and to compare this with macrophages loaded with ace tylated LDL (AcLDL; cholesteryl ester-loaded cells containing no oxidized l ipids) or 7-ketocholesterol-enriched acetylated LDL (7KCAcLDL; cholesteryl ester-loaded cells selectively supplemented with 7-ketocholesterol (7KC), t he major oxysterol present in OxLDL), Both cholesterol and 7KC and their es ters were measured in macrophages after uptake of these modified lipoprotei ns. Oxysterols comprised up to 50% of total sterol content of OxLDL-loaded cells. Unesterified 7KC and cholesterol partitioned into cell membranes, wi th no evidence of retention of either free sterol within lysosomes, The cel ls also contained cytosolic, ACAT-derived, cholesteryl and 7-ketocholestery l esters, The proportion of free cholesterol and 7KC esterified by ACAT was 10-fold less in OxLDL-loaded cells than in AcLDL or 7KCAcLDL-loaded cells. This poor esterification rate in OxLDL-loaded cells was partly caused by f atty acid limitation. OxLDL-loaded macrophages also contained large (simila r to 40-50% total cell sterol content) pools of oxidized esters, containing cholesterol or 7KC esterified to oxidized fatty acids. These were insensit ive to ACAT inhibition, very stable and located in lysosomes, indicating re sistance to lysosomal esterases. Macrophages loaded with OxLDL do not accum ulate free sterols in their lysosomal compartment, but do accumulate lysoso mal deposits of OxLDL-derived cholesterol and 7-ketocholesterol esterified to oxidized fatty acids. The presence of similar deposits in lesion foam ce lls would represent a pool of sterols that is particularly resistant to rem oval.