EFFECTS OF INTRACELLULAR FREE-CHOLESTEROL ACCUMULATION ON MACROPHAGE VIABILITY - A MODEL FOR FOAM CELL-DEATH

This study was designed to identify cellular responses associated with free cholesterol (FC) accumulation in model macrophage foam cells. Mo use peritoneal macrophages (MPMs) or J774 macrophages were loaded with cholesteryl esters using acetylated LDL and FC/phospholipid dispersio ns and were subsequently exposed to an acyl coenzyme A:cholesterol acy ltransferase (ACAT) inhibitor. This treatment produced a rapid accumul ation of cellular FC. The FC that accumulated due to ACAT inhibition w as more readily available for efflux to 2-hydroxypropyl-beta-cyclodest rin (which removes cholesterol from the plasma membrane) than FC in un treated control cells. After a 3-hour exposure to an ACAT inhibitor, a significant increase in phospholipid synthesis was seen, followed by the leakage of LDH after 12 hours of treatment. We also observed, by e lectron and fluorescence microscopy, morphological indications of both apoptosis and necrosis in cells treated with an ACAT inhibitor, In ad dition, inhibition of ACAT for 48 hours resulted in the formation of F C crystals in MPMs but not in J774 cells. If compound 3 beta-[2-(dieth ylamino)ethoxyl]androst-5-en-17-one (U18666A), which modulates intrace llular trafficking of cholesterol, was added together with the ACAT in hibitor, each of the metabolic changes elicited by the accumulation of excess FC was either diminished or eliminated. The protective affect of U18666A was not due to a decrease in cellular FC concentrations, be cause cells treated with an ACAT inhibitor accumulated similar amounts of FC in the presence or absence of U18666A. Thus, treatment with U18 666A results in the sequestering of FC in a pool that prevents it from causing various responses to FC deposition in macrophages. The metabo lic changes that were produced when these model foam cells were treate d with the ACAT inhibitor parallel the pathological events that have b een shown to occur in the developing atherosclerotic plaque.