Cytotoxic cholesterol is generated by the hydrolysis of cytoplasmic cholesteryl ester and transported to the plasma membrane

The present study examines the fate and effects of free cholesterol (FC) ge nerated by the hydrolysis of cytoplasmic cholesteryl esters (CE) in model m acrophage foam cells. J774 or elicited mouse peritoneal macrophages (MPM) w ere enriched with CE by incubating with acetylated low density lipoprotein (acLDL) and FC/phospholipid dispersions, thus creating model foam cells. Tr eatment of the foam cells with the acyl coenzyme-A:cholesterol acyltransfer ase (ACAT) inhibitor, CP-113,818, in the absence of any extracellular chole sterol accepters, resulted in cellular toxicity. This was accompanied by an increase in the amount of FC available for oxidation by an exogenous chole sterol oxidase. Furthermore, cellular toxicity was proportional to the size of the oxidase susceptible pool of FC over time. Morphological analysis an d in situ DNA fragmentation assay demonstrated the occurrence of apoptosis in the ACAT inhibited cells. Go-treatment with the hydrophobic amine U18666 A, an intracellular cholesterol transport inhibitor, led to a dose dependen t reduction in cytotoxicity and apoptosis, and blocked the movement of FC i nto the oxidase susceptible pool. In addition, treating model foam cells wi th CP-113,818 plus chloroquine, a compound that inhibits the function of ac idic vesicles, also diminished cellular toxicity. Staining with the cholest erol binding dye filipin revealed that the macrophages treated with CP-113, 818 contained a cholesterol oxidase accessible pool of FC in the plasma mem brane. These results suggest that FC generated by the hydrolysis of cytopla smic CE is transported through acidic vesicles to the plasma membrane, and accumulation of FC in this pool triggers cell death by necrosis and apoptos is. (C) 1999 Elsevier Science II eland Ltd. All rights reserved.