Epoxycholesterol Impairs Cholesteryl Ester Hydrolysis in Macrophage Foam Cells, Resulting in Decreased Cholesterol Efflux

Abstract

Objective-Strategies to inhibit or reverse cholesterol accumulation in macrophages have been shown to be atheroprotective. Notably, the administration of LXR agonists upregulates key players in the reverse cholesterol transport pathway, including the ABCA1 and ABCG1 transporters. However, the effects of natural LXR activators, oxysterols, on lipid-laden macrophages remains elusive. Methods and Results-We assessed the ability of 2 oxysterols, 22(R)-hydroxycholesterol (22-OH) and 24(S), 25-epoxycholesterol (epoxycholesterol), to promote cholesterol efflux to apoA-I from LDL-and modified LDL-labeled and loaded macrophages and thus rescue the phenotype associated with the accumulation of cellular cholesterol in these cells. In macrophages labeled with LDL-derived cholesterol, epoxycholesterol treatment enhances ABCA1-mediated cholesterol efflux. In contrast, in AcLDL-loaded macrophages, epoxycholesterol treatment decreases cholesterol efflux to apoA-I, despite a dramatic increase in the expression of ABCA1 in response to epoxycholesterol treatment. We show that the decreased efflux is attributable to impaired cholesterol mobilization from lipid droplets, resulting from decreased cholesteryl ester hydrolase activity. W hereas low density lipoprotein (LDL) endocytosis is a regulated process, the uptake of modified LDL via macrophage scavenger receptors 1 or via macropinocytosis 2 lacks feedback inhibition by intracellular cholesterol accumulation. The internalization of modified LDL, such as oxidized LDL (OxLDL), acetylated LDL (AcLDL), or aggregated LDL (AgLDL), leads to the formation of foam cells, 3,4 which are a major component of atherosclerotic lesions. Conclusion-EpoxycholesterolThe liver X receptors (LXRs) ␣ and ␤, members of the nuclear receptor family of transcription factors, are key regulators of whole body lipid homeostasis. LXR-␤ is expressed ubiquitously whereas LXR-␣ is predominantly expressed in tissues important in lipid metabolism including liver, adipose tissue, and macrophages. 5,6 The naturally occurring oxysterols 22(R)-hydroxycholesterol (22-HC) and 24(S),25-epoxycholesterol (epoxycholesterol) are endogenous LXR ligands and have been shown to be potent physiological activators of both LXRs. 7,8 LXRs function by forming an obligate heterodimer with the retinoid X receptor (RXR), and this complex drives LXR-dependent transactivation of multiple genes involved in cellular cholesterol homeostasis, including ABCA1, ABCG1, and apoE in the lipid efflux pathway, which normally prevent cholesterol accumulation as intracellular lipid droplets. 9 Given that ABCA1-mediated cholesterol efflux to lipidpoor apolipoprotein A-I (apoA-I) is the preferred efflux pathway of the macrophage foam cell, 10 we sought to promote this pathway in cholesterol-loaded cells using 22-HC and epoxycholesterol. Oxysterols have been shown to be beneficial for enhancing cholesterol efflux 6,11-13 and preventing foam cell formation, 14 but it is not established whether they are similarly effective when macrophages become cholestero...

Resveratrol prevents the impairment of advanced glycosylation end products (AGE) on macrophage lipid homeostasis by suppressing the receptor for AGE via peroxisome proliferator-activated receptor γ activation

“…Cellular cholesterol analysis. Cellular cholesterol and cholesterol ester (CE) were extracted and measured using a fluorometric assay (Cholesterol/CE Quantitation Kit, BioVision, USA), according to the manufacturer's instructions (23). Cells were counted with a counting plate before subjected to chloroform/methanol extraction to isolate cellular lipids.…”

Cathepsin G deficiency decreases complexity of atherosclerotic lesions in apolipoprotein E-deficient mice

“…Bone marrow-derived macrophages (BMDM) were washed, and medium was replaced by serum-free RPMI 1640 for 2 days, either without acetylated low-density lipoprotein (ac-LDL) or with ac-LDL (Biomedical Technologies, Stoughton, MA) at a cholesterol concentration of 50 g/ml (22). Cell media were collected in 15-ml tubes containing 10% of total volume EDTA-Na 2 and 1,10-phenantroline (inhibitor cocktail) (Sigma).…”

Role of renin-angiotensin system in activation of macrophages by modified lipoproteins

“…To differentiate these cells into macrophages, the monocytes were seeded in six-well plates at a density of 2-2.5 ϫ 10 6 cells/well in the same medium containing 100 nm phorbol 12-myristate 13-acetate (PMA) (Sigma, St Louis, MO) and incubated for 24 h. Differentiated macrophages were incubated for 24, 48, and 96 h in serum-free medium containing native LDL (n-LDL) or acetylated LDL (ac-LDL) (Biomedical Technologies, Stoughton, MA) at 50 g/ml (26). Cholesteryl ester formation was also assessed in differentiated THP-1 cells treated with 50 g/ml oxidized LDL (ox-LDL) (Biomedical Technologies) (27). To estimate the contribution of the scavenger receptor A (SR-A) to cholesteryl ester accumulation in ac-LDL-treated THP-1 cells, THP-1 cells were exposed to ac-LDL in the presence of 50 g·ml Ϫ1 ·day Ϫ1 of the SR-A ligands, dextran sulfate, or fucoidan (Sigma) (31).…”

“…Cells were differentiated to macrophages after 7 days incubation at 37°C. On day 7, cells were washed and medium was replaced by serum-free RPMI 1640 for lipoprotein treatment (27).…”