Combined deficiency of ABCA1 and ABCG1 promotes foam cell accumulation and accelerates atherosclerosis in mice

Abstract

HDLs protect against the development of atherosclerosis, but the underlying mechanisms are poorly understood. HDL and its apolipoproteins can promote cholesterol efflux from macrophage foam cells via the ATP-binding cassette transporters ABCA1 and ABCG1. Experiments addressing the individual roles of ABCA1 and ABCG1 in the development of atherosclerosis have produced mixed results, perhaps because of compensatory upregulation in the individual KO models. To clarify the role of transporter-mediated sterol efflux in this disease process, we transplanted BM from Abca1(-/-)Abcg1(-/-) mice into LDL receptor-deficient mice and administered a high-cholesterol diet. Compared with control and single-KO BM recipients, Abca1(-/-)Abcg1(-/-) BM recipients showed accelerated atherosclerosis and extensive infiltration of the myocardium and spleen with macrophage foam cells. In experiments with isolated macrophages, combined ABCA1 and ABCG1 deficiency resulted in impaired cholesterol efflux to HDL or apoA-1, profoundly decreased apoE secretion, and increased secretion of inflammatory cytokines and chemokines. In addition, these cells showed increased apoptosis when challenged with free cholesterol or oxidized LDL loading. These results suggest that the combined effects of ABCA1 and ABCG1 in mediating macrophage sterol efflux are central to the antiatherogenic properties of HDL.

Figure 1. Foam cell infiltration of the myocardium in Abca1^–/–Abcg1^–/– BM recipients but not WT or single-KO recipients.

(A) Representative hearts obtained from Ldlr^+/– mice transplanted with BM derived from WT, Abcg1^–/–, Abca1^–/–, or Abca1^–/–Abcg1^–/– mice and fed a high-cholesterol diet for 12 weeks. Hearts from all Abca1^–/–Abcg1^–/– recipients were visibly smaller and pale compared with WT and single-KO controls. Scale bar: 1 cm. (B–G) Paraffin-embedded serial sections obtained from the myocardium surrounding the proximal aorta, lung, and spleen of mice transplanted with BM of all 4 genotypes. Original magnification, ×200. (B) H&E staining revealed an extensive cellular infiltrate in the myocardium of Abca1^–/–Abcg1^–/– recipients (9 of 9) but not controls (WT, 0 of 12; Abcg1^–/–, 0 of 8; Abca1^–/–, 0 of 16). The infiltrate resembled foam cell lesions of the vessel wall, including apparent cholesterol clefts (arrows). (C) Mac-3 immunostaining (dark brown) confirmed that the majority of cells were macrophages. (D) MCA771G immunostaining (dark brown) revealed a moderate number of neutrophils (arrows) as well. (E) TUNEL staining revealed pockets of apoptotic cells (red) in the infiltrated regions of myocardium from Abca1^–/–Abcg1^–/– recipients (7 of 9) only. (F and G) H&E staining revealed an extensive cellular infiltrate in the lungs and spleens of Abca1^–/–Abcg1^–/–recipients as well as in the lungs of Abcg1^–/– recipients. Arrows indicate foam cells.

Figure 2. Atherosclerotic lesion development in the proximal aorta.

(A) Quantification of proximal aortic root lesion area by morphometric analysis of H&E-stained sections in Ldlr^+/– mice transplanted with WT (n = 12), Abcg1^–/– (n = 8), Abca1^–/– (n = 16), or Abca1^–/–Abcg1^–/– (n = 9) BM after 12 weeks of high-cholesterol diet. Values for individual mice are shown as open circles, representing the average of 5 sections per mouse. Horizontal bars indicate the group medians: control, 3,994 μm²/section; Abcg1^–/–, 3,724 μm²/section; Abca1^–/–, 40,070 μm²/section; Abca1^–/–Abcg1^–/–, 186,182 μm²/section. Mean lesion areas (mean ± SEM) were as follows: control, 9,090 ± 3,872 μm²/section; Abcg1^–/–, 15,412 ± 1,640 μm²/section; Abca1^–/–, 72,675 ± 28,879 μm²/section; Abca1^–/–Abcg1^–/–, 174,958 ± 36,269 μm²/section. ANOVA was performed with square root–transformed data. (B) Representative H&E-stained proximal aortas from mice receiving BM of all the 4 genotypes after 12 weeks of high-cholesterol diet. Original magnification, ×100.

Figure 3. Cholesterol efflux from peritoneal macrophages isolated from WT, Abca1^–/–, Abcg1^–/–, and Abca1^–/–Abcg1^–/– mice.

(A, B, and D) Thioglycollate-elicited macrophages were cultured for 24 h in DMEM plus 0.2% BSA containing 50 μg/ml acLDL, 3 μmol/l T0901317, and 2 μCi/ml [³H]-cholesterol. Then, a pool of human serum (2.5%; A), a pool of human serum devoid of apoB-containing particles (2.5%; B), and media devoid of acceptors or containing lipid-poor apoA-1 or apoE (D) were added as acceptor and incubated for 6 h before the media and cells were collected for analysis. (C) Cholesterol efflux was also confirmed by cholesterol mass analysis. Cells were cultured overnight with 50 μg/ml acLDL and 3 μmol/l T0901317 and then incubated for 6 hours with 50 μg/ml human HDL isolated by ultracentrifugation. The HDL-mediated net cholesterol efflux (ΔTC) was calculated as the difference of cholesterol mass of the medium with and without cells. Values are mean ± SEM. *P < 0.05 versus WT. ^ΧP < 0.05 versus single-KOs.

Figure 4. Cellular cholesterol mass in peritoneal macrophages from WT, Abcg1^–/–, Abca1^–/–, and Abca1^–/–Abcg1^–/– macrophages.

Thioglycollate-elicited macrophages were harvested from WT, Abcg1^–/–, Abca1^–/–, and Abca1^–/–Abcg1^–/– mice fed standard chow (A) or high-cholesterol diet for 2 weeks (B). After a 1-hour incubation at 37°C, nonadherent cells were removed and adherent cells consisting of macrophages were directly used to estimate cellular cholesterol and cholesteryl ester mass content by gas-liquid chromatography. TC, total cholesterol; FC, free cholesterol; CE, cholesteryl esters. Values are mean ± SEM. *P < 0.05 versus WT. ^ΧP < 0.05 versus single-KOs.

Figure 5. Western blots showing ApoE protein in media and cell lysates of peritoneal macrophages derived from WT, Abcg1^–/–, Abca1^–/–, and Abca1^–/–Abcg1^–/–mice.

Thioglycollate-elicited peritoneal macrophages were loaded with 50 μg/ml acLDL and treated with LXR activator (3 μmol/l T0901317) for 24 h and then incubated for 16 h in DMEM plus 0.2% BSA. Equivalent volumes of media normalized for cellular protein levels and equal amounts of cellular proteins (20 μg) from each sample were used for western blot analysis. Numbers below blots indicate change in KO cells compared with loaded control cells from 3 independent experiments. *P < 0.05 versus WT. ^ΧP < 0.05 versus single KOs.

Figure 6. Inflammatory and chemokine gene expression in WT, Abcg1^–/–, Abca1^–/–, and Abca1^–/–Abcg1^–/– macrophages.

Peritoneal macrophages were harvested from WT, Abcg1^–/–, Abca1^–/–, and Abca1^–/–Abcg1^–/– mice fed high-cholesterol diet for 2 weeks. After a 1-h incubation at 37°C, nonadherent cells were removed and adherent cells consisting of macrophages were incubated in 0.2% BSA DMEM. After 6 h, media were used for secretion analysis. (A) Secretion of chemokines MIP-1α, MIP-2, and MCP-1. KC, growth-regulated oncogene α. (B) Secretion of inflammatory cytokines IL-6, TNF-α, and IL-1β. Secretion levels were normalized to cellular protein amount and expressed as a percentage of WT. Values are mean ± SEM. *P < 0.05 versus WT. ^ΧP < 0.05 versus single-KOs.

Figure 7. Apoptosis of peritoneal macrophages after loading with free cholesterol loading or oxidized LDL.

Peritoneal macrophages were cultured in DMEM plus 0.2% BSA containing 100 μg/ml acLDL plus ACAT inhibitor (compound 58035; A) or 100 μg/ml Cu-oxidized LDL (oxLDL; B) for 24 h in the presence or absence of 100 μg/ml human HDL. Apoptosis of macrophages was determined by annexin V staining. Results are mean ± SEM. *P < 0.05, genotype effect. ^#P < 0.05, HDL effect.