High-density lipoprotein protects macrophages from oxidized low-density lipoprotein-induced apoptosis by promoting efflux of 7-ketocholesterol via ABCG1

Abstract

Oxidized sterols consumed in the diet or formed on low-density lipoprotein (LDL) are toxic to endothelial cells and macrophages and are thought to have a central role in promoting atherogenesis. The ATP-binding cassette transporter ABCG1 was recently shown to promote efflux of cholesterol from macrophages to high-denisty lipoprotein (HDL). We show that HDL protects macrophages from apoptosis induced by loading with free cholesterol or oxidized LDL. The protective effect of HDL was reduced in Abcg1(-/-) macrophages, especially after loading with oxidized LDL. Similarly, HDL exerted a protective effect against apoptosis induced by 7-ketocholesterol, the major oxysterol present in oxidized LDL and atherosclerotic lesions, in Abcg1(+/+), but not in Abcg1(-/-) macrophages. In transfected 293 cells, efflux of 7-ketocholesterol and related oxysterols was completely dependent on expression of ABCG1 and the presence of HDL in media. In contrast, ABCA1 and apoA-1 did not stimulate the efflux of 7-ketocholesterol into media. HDL stimulated the efflux of 7-ketocholesterol from Abcg1(+/+), but not from Abcg1(-/-) macrophages. In Abcg1(-/-) mice fed a high-cholesterol diet, plasma levels of 7-ketocholesterol were reduced, whereas their macrophages accumulated 7-ketocholesterol. These findings indicate a specific role for ABCG1 in promoting efflux of 7-ketocholesterol and related oxysterols from macrophages onto HDL and in protecting these cells from oxysterol-induced cytotoxicity.

Fig. 1.

ABCG1 deficiency increases susceptibility to oxLDL-induced apoptosis. (A) Peritoneal macrophages from Abcg1^+/+ or Abcg1^−/− mice were cultured in DMEM plus 5% LPDS containing AcLDL (100 μg/ml) plus 58035 (10 μg/ml) or oxLDL (100 μg/ml) with or without HDL (100 μg/ml) for 24 h. Apoptosis of macrophage was determined by annexin V staining and is expressed as a percentage of the total number of cells in at least three separate fields (containing ≈1,000 cells) from duplicate wells. (B and C) Concentration response of HDL (0–200 μg/ml) on apoptosis in response to FC loading (B) or oxLDL loading (C). Data are represented as the mean ± SE of three separate experiments. *, P < 0.05.

Fig. 2.

ABCG1 deficiency increases susceptibility to 7-ketocholesterol-induced apoptosis. (A) Peritoneal macrophages from Abcg1^+/+ or Abcg1^−/− mice were cultured in DMEM plus 5% LPDS containing 7-ketocholesterol (10–40 μg/ml) with or without HDL (100 μg/ml) for 24 h. Apoptosis of macrophage was determined by annexin V staining and is expressed as a percentage of the total number of cells in at least three separate fields (containing ≈1,000 cells) from duplicate wells. (B) Concentration response of HDL (0–200 μg/ml) on apoptosis in response to 7-ketocholesterol. Data are represented as mean ± SE of three separate experiments. *, P < 0.05.

Fig. 3.

7-Ketocholesterol is selectively exported to HDL in ABCG1-transfected 293 cells. (A) HEK293 cells were transiently transfected with plasmid constructs expressing ABCA1/ABCG1 or empty vector and incubated with a cholesterol and 7-ketocholesterol mixture (each 10 μg/ml) for 17 h. Transfected HEK293 cells were washed with PBS and incubated with DMEM plus 5% LPDS containing apoA-1 (10 μg/ml) or HDL (100 μg/ml) for 8 h. (B) Concentration response of apoA-1 (0–25 μg/ml) or HDL (0–200 μg/ml) for 8 h. Data are represented as the mean ± SE of an experiment performed in triplicate. Similar data were obtained in two separate experiments.

Fig. 4.

7α- and 7β-hydroxycholesterol is selectively exported to HDL in ABCG1-transfected 293 cells. HEK293 cells were transiently transfected with plasmid constructs expressing ABCA1/ABCG1 or empty vector and incubated with 10 μg/ml of cholesterol (Chol), 7α-hydroxycholesterol (7αOH), 7β-hydroxycholesterol (7βOH), 7-ketocholesterol (7K), or 25-hydroxycholesterol (25OH) for 17 h. Transfected HEK293 cells were washed with PBS and incubated with DMEM plus 5% LPDS containing apoA-1 (10 μg/ml) (A) or HDL (100 μg/ml) (B) for 8 h. Data are represented as the mean ± SE of an experiment performed in triplicate. Similar data were obtained in two separate experiments.

Fig. 5.

Mass sterol efflux to apoA-1 or HDL in Abcg1^+/+ or Abcg1^−/− macrophages. Peritoneal macrophages from Abcg1^+/+ or Abcg1^−/− mice were incubated with 50 μg/ml of oxLDL for 17 h. Macrophages were washed with PBS and incubated in DMEM plus 5% LPDS with apoA-1 (10 μg/ml) or HDL (50 μg/ml) for 8 h. Cholesterol (A) and 7-ketocholesterol (B) efflux were determined. Data are represented as the mean ± SE of an experiment performed in triplicate. *, P < 0.05 as compared with control.

Fig. 6.

7-Ketocholesterol concentrations in Abcg1^−/− macrophages and lipoproteins in vivo. (A and B) Cellular sterol mass in Abcg1^+/+ or Abcg1^−/− mice from a bone marrow transplantation (Abcg1^+/+ or Abcg1^−/− → LDLR^−/−). Abcg1^+/+ or Abcg1^−/− macrophages were harvested from LDLR^−/− mice transplanted with Abcg1^+/+ or Abcg1^−/− bone marrow and were fed a Western diet for 12 weeks. Pooled Abcg1^+/+ or Abcg1^−/− macrophages were incubated in DMEM plus 10% FBS for 2 h. Then, cells were washed with PBS, and cellular lipids were extracted. (A and B) Cellular cholesterol (A) and 7-ketocholesterol (B) mass were determined. Data are represented as the mean ± SE of an experiment performed in triplicate. (C and D) Abcg1^+/+ or Abcg1^−/− mice fed a high-fat/cholesterol diet for 4 weeks. Lipoprotein cholesterol (C) and 7-ketocholesterol (D) concentrations were determined. Data are represented as the mean ± SE (n = 4). *, P < 0.05 as compared with Abcg1^+/+.