title: Anti-atherogenic effects of the acyl-CoA:cholesterol acyltransferase inhibitor, avasimibe (CI-1011), in cultured primary human macrophages.
abstract: Acyl-CoA:cholesterol acyltransferase (ACAT) inhibitors have been shown to reduce atherosclerotic lesions in animals; however, the mechanism(s) for this effect remains unclear. Therefore, we used cultured primary human monocyte-derived macrophages (HMMs) to examine the effect of the ACAT inhibitor, avasimibe (CI-1011), during foam cell formation and during cholesterol efflux from established foam cells. To examine the effect of CI-1011 on foam cell development, HMMs were incubated with aggregated acetylated LDL (ag-acLDL)+/-CI-1011 for 48 h. Total cholesterol (TC) was 29% lower in HMMs incubated with ag-acLDL and CI-1011 compared with ag-acLDL (P<0.05). To determine if TC reduction was due to reduced ag-acLDL uptake by CI-1011, 125I-acLDL binding at 4 degrees C for 4 h to HMMs preincubated with acLDL or ag-acLDL, CI-1011, acLDL+CI-1011, or ag-acLDL+CI-1011 for 48 h was measured. Specific binding was 40% lower in cells preincubated with acLDL+CI-1011, 52% lower in cells preincubated with ag-acLDL+CI-1011 and 49% lower in cells preincubated with CI-1011 compared with cells preincubated with acLDL (P<0.0003). Because CI-1011 appeared to directly affect acLDL binding, 125I-acLDL (3-80 microg protein/ml) binding was done in HMMs preincubated with CI-1011 (0-10 microg/ml) for 48 h. The calculated B(max) decreased in HMMs exposed to increasing concentrations of CI-1011, suggesting that CI-1011 altered scavenger receptor function and/or number. To examine the effects of CI-1011 on cholesterol efflux from established foam cells, we first examined whether CI-1011 was cytotoxic. HMMs were preincubated with ag-acLDL for 24 h, and then radiolabeled with [14C]adenine for 2 h (time zero). The radiolabeled cells were exposed to control RPMI medium or the same medium+HDL, CI-1011, or HDL+CI-1011 for 24 h. The release of [14C]adenine into the medium was not significantly different between cells exposed to RPMI, HDL, CI-1011, or HDL+CI-1011, suggesting that CI-1011 was not cytotoxic. Foam cells exposed to RPMI and CI-1011 (1-10 microg/ml) for 48 h showed time dependent reduction in cellular TC mass, with a corresponding increase in radiolabeled unesterified cholesterol into the medium. We then asked whether CI-1011 enhanced apoE mediated cholesterol efflux. Although cellular apoE increased between 2- and 7-fold in foam cells compared to control macrophages, apoE secreted into the medium was not significantly different between cells exposed to RPMI or CI-1011. Thus, CI-1011 exerted anti-atherogenic effects by reducing TC accumulation, inhibiting acLDL binding, and by limiting lipid storage in HMMs.
authors.affiliation: Departments of Medicine, Sinai Hospital of Baltimore, The Johns Hopkins University School of Medicine, Schapiro Research Building, Room 200, 2401 West Belvedere Avenue, Baltimore, MD 21215, USA. arodrigu@lifebridgehealth.com
authors.forename: Annabelle
authors.initials: A
authors.lastname: Rodriguez
authors.forename: David C
authors.initials: DC
authors.lastname: Usher
mesh_terms: D000081:Acetamides; D000085:Acetates / Q000494:pharmacology*; D001057:Apolipoproteins E / Q000378:metabolism; D002478:Cells, Cultured; D002784:Cholesterol / Q000378:metabolism; D006801:Humans; D008077:Lipoproteins, LDL / Q000378:metabolism; D008264:Macrophages / Q000187:drug effects*; D002785:Sterol O-Acyltransferase / Q000037:antagonists & inhibitors*; D013449:Sulfonamides; D013451:Sulfonic Acids / Q000494:pharmacology*
chemical_list: D000081:Acetamides; D000085:Acetates; D001057:Apolipoproteins E; D008077:Lipoproteins, LDL; D013449:Sulfonamides; D013451:Sulfonic Acids; C032629:acetyl-LDL; C423185:avasimibe; D002784:Cholesterol; D002785:Sterol O-Acyltransferase