OBJECTIVETo investigate the role of acyl-coenzyme A: cholesterol acyltransferase inhibitor (ACATI) in apoptosis induced by lipids and whether lipids-induced apoptosis is accompanied by increase of free cholesterol in endoplasmic reticulum (ER), in order to further understand the mechanism of lipids-induced apoptosis in advanced atherosclerosis.

METHODSHuman vascular smooth muscle cells (VSMCs) and phorbol 12-myristate 13-acetate (PMA) differentiated THP-1 macrophages were used. Tritiated thymidine incorporation was applied to detect cell proliferation. Cytotoxicity was assessed by lactate dehydrogenase (LDH) release. 4',6-diamidino-2-phenylindole (DAPI) staining, caspase-3, -7 assay, and Annexin-V/propidium iodide (PI) staining were used to detect apoptosis. High performance liquid chromatography was used in intracellular free cholesterol and cholesterol ester assay. ER free cholesterol was quantified.

RESULTSDifferent lipids had different effects on proliferation and cytotoxicity of VSMCs. 25-hydroxycholesterol (25OHC) had biphasic effects on the proliferation of VSMCs. At low concentration, it stimulated cell proliferation, but turned to proliferation inhibition as concentration reached 15 mug/mL. 25OHC and acetylated low density lipoprotein (AcLDL) could respectively induce apoptosis in human VSMCs and PMA differentiated THP-1 macrophages, which was aggravated by ACATI, accompanied by increase of intracellular free cholesterol content. There was also an increase of cholesterol content in ER with AcLDL-induced apoptosis in THP-1 macrophages.

CONCLUSIONSLipids could induce apoptosis, accompanied by increase of intracellular free cholesterol content, which could be augmented by ACATI, suggesting that insults resulting in ER free cholesterol rise might be the initiator of apoptosis.

Apoptosis ; drug effects ; physiology ; Cells, Cultured ; Enzyme Inhibitors ; pharmacology ; Humans ; Lipids ; physiology ; Sterol O-Acyltransferase ; antagonists & inhibitors

We investigated the mechanism of spontaneous cholesterol efflux induced by acyl-coenzyme A:cholesterol acyltransferase (ACAT) inhibition, and how an alteration of cholesterol metabolism in macrophages impacts on that in HepG2 cells. Oleic acid anilide (OAA), a known ACAT inhibitor reduced lipid storage substantially by promotion of cholesterol catabolism and repression of cholesteryl ester accumulation without further increase of cytotoxicity in acetylated low-density lipoprotein-loaded THP-1 macrophages. Analysis of expressed mRNA and protein revealed that cholesterol 7alpha-hydroxylase (CYP7A1), oxysterol 7alpha- hydroxylase (CYP7B1), and cholesterol 27-hydroxylase (CYP27) were highly induced by ACAT inhibition. The presence of a functional cytochrome P450 pathway was confirmed by quantification of the biliary cholesterol mass in cell monolayers and extracelluar medium. Notably, massively secreted biliary cholesterol from macrophages suppressed the expression of CYP7 proteins in a farnesoid X receptor (FXR)-dependent manner in HepG2 cells. The findings reported here provide new insight into mechanisms of spontaneous cholesterol efflux, and suggest that ACAT inhibition may stimulate cholesterol-catabolic (cytochrome P450) pathway in lesion-macrophages, in contrast, suppress it in hepatocyte via FXR induced by biliary cholesterol (BC).
Anilides/*pharmacology ; Bile/metabolism ; Cells, Cultured ; Cholesterol/*metabolism ; Cholesterol Esters/metabolism ; DNA-Binding Proteins/agonists/*metabolism ; Enzyme Inhibitors/pharmacology ; Gene Expression Regulation, Enzymologic/drug effects ; Hepatocytes/*drug effects/metabolism ; Humans ; Lipid Metabolism/drug effects/genetics ; Macrophages/*drug effects/metabolism ; Models, Biological ; Oleic Acids/*pharmacology ; Receptors, Cytoplasmic and Nuclear/agonists/*metabolism ; Sterol O-Acyltransferase/*antagonists & inhibitors/physiology ; Transcription Factors/agonists/*metabolism