Crystallization of free cholesterol in model macrophage foam cells

The present study examined free cholesterol (FC) crystallization in macroph age foam cells. Model foam cells J774 or mouse peritoneal macrophages [MPMs ] were incubated with acetylated low density lipoprotein and FC/phospholipi d dispersions for 48 hours, resulting in the deposition of large stores of cytoplasmic cholesteryl esters (CEs). The model foam cells were then incuba ted for up to 5 days with an acyl-coenzyme A:cholesterol acyltransferase (A CAT) inhibitor (CP-113,818) in the absence of an extracellular FC acceptor to allow intracellular accumulation of PC. FC crystals of various shapes an d sizes formed in the MPMs but not in the J774 macrophages. Examination of the MPM monolayers by microscopy indicated that the cryst;als were external ized rapidly after formation and thereafter continued to increase in size. Incubating J774 macrophages with 8-(4-chlorophenylthio)adenosine 3':5'-cycl ic monophosphate (CPT-cAMP) in addition to CP-113,818 caused FC crystal for mation as a consequence of CPT-cAMP stimulation of CE hydrolysis and inhibi tion of cell growth. In addition, 2 separate cholesterol: phases (liquid-cr ystalline 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. Othe r 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 toxic ity in J774 macrophage foam cells and PC crystallization when coincubated w ith CPT-cAMP. Addition of the extracellular PC accepters apolipoproteins (a po) E and A-I (50 mu g/mL) reduced FC crystal formation. In MPMs, lower cel l density and frequent changes of medium were conducive to crystal formatio n. This may be due to "dilution" of apoE secreted by the MPMs and is consis tent with our observation that the addition of exogenous apoE or apoA-I inh ibits FC crystal formation in J774 macrophage foam cells cotreated with CP- 113,818 plus CPT-cAMP. These data demonstrate that PC crystals can form fro m the hydrolysis of cytoplasmic stores of CEs in model foam cells. PC cryst al formation can be modulated by the addition of extracellular PC accepters or by affecting the cellular rate of CE hydrolysis. This process may contr ibute to the formation of PC crystals in atherosclerotic plaques.