Crystallization of free cholesterol in model macrophage foam cells

The present study examined free cholesterol (FC) crystallization in macrophage foam cells. Model foam cells J774 or mouse peritoneal macrophages [MPMs] were incubated with acetylated low density lipoprotein and FC/phospholipid dispersions for 48 hours, resulting in the deposition of large stores of cytoplasmic cholesteryl esters (CEs). The model foam cells were then incubated for up to 5 days with an acyl-coenzyme A:cholesterol acyltransferase (ACAT) inhibitor (CP-113,818) in the absence of an extracellular FC acceptor to allow intracellular accumulation of PC. FC crystals of various shapes and sizes formed in the MPMs but not in the J774 macrophages. Examination of the MPM monolayers by microscopy indicated that the cryst;als were externalized rapidly after formation and thereafter continued to increase in size. Incubating J774 macrophages with 8-(4-chlorophenylthio)adenosine 3':5'-cyclic monophosphate (CPT-cAMP) in addition to CP-113,818 caused FC crystal formation as a consequence of CPT-cAMP stimulation of CE hydrolysis and inhibition of cell growth. In addition, 2 separate cholesterol: phases (liquid-crystalline and cholesterol monohydrate) in the plane of the membrane bilayer were detected after 31 hours of ACAT inhibition by the use of small-angle x-ray diffraction of J774 macrophage foam cells treated with CPT-cAMP. Other compounds reported to inhibit ACAT, namely progesterone (20 mu g/mL) and N-acetyl-D-sphingosine (c(2),-ceramide, 10 mu g/mL), induced cellular toxicity in J774 macrophage foam cells and PC crystallization when coincubated with CPT-cAMP. Addition of the extracellular PC accepters apolipoproteins (apo) E and A-I (50 mu g/mL) reduced FC crystal formation. In MPMs, lower cell density and frequent changes of medium were conducive to crystal formation. This may be due to "dilution" of apoE secreted by the MPMs and is consistent with our observation that the addition of exogenous apoE or apoA-I inhibits FC crystal formation in J774 macrophage foam cells cotreated with CP-113,818 plus CPT-cAMP. These data demonstrate that PC crystals can form from the hydrolysis of cytoplasmic stores of CEs in model foam cells. PC crystal formation can be modulated by the addition of extracellular PC accepters or by affecting the cellular rate of CE hydrolysis. This process may contribute to the formation of PC crystals in atherosclerotic plaques.