Combined deficiency of ABCA1 and ABCG1 promotes foam cell accumulation and accelerates atherosclerosis in mice (original) (raw)
Massive myocardial foam cell accumulation in Abca1–/–Abcg1–/– BM recipients. We bred mice with single or combined KO of Abca1 and Abcg1, transplanted BM from these mice into irradiated Ldlr+/– mice, and, after a recovery period, placed the recipients on a high-cholesterol diet for 12 weeks. Upon sacrificing the mice, we noticed a striking phenotype that was seen in all recipients of Abca1–/–Abcg1–/– BM (n = 9) but not in mice receiving WT, Abca1–/–, or Abcg1–/– BM. In the Abca1–/–Abcg1–/– BM recipients, the hearts were smaller and were pale compared with mice of the other genotypes (Figure 1A). Pallor was also observed in the small intestine, and the spleen was enlarged, in Abca1–/–Abcg1–/– recipients. Microscopic examination of the hearts of Abca1–/–Abcg1–/– mice revealed massive infiltration with cells containing foamy cytoplasm that stained positively with Mac-3, indicating that they were macrophage foam cells (Figure 1, B and C). In addition, these infiltrates contained empty spaces of similar appearance to the cholesterol clefts commonly seen in atherosclerotic lesions (Figure 1C). There were also a moderate number of neutrophils in the infiltrate as shown by MCA771G staining (Figure 1D). TUNEL staining revealed clusters of apoptotic cells in the midst of the inflammatory infiltrate and this was observed only in Abca1–/–Abcg1–/– BM recipients (Figure 1E). Previous studies have shown accumulation of foam cells and other inflammatory cells in the lungs of Abcg1–/– mice (23). We also found foci of foam cells and other inflammatory cells including neutrophils in the lungs of Abcg1–/– BM recipients; interestingly, this phenotype was not increased in Abca1–/–Abcg1–/– recipients beyond what was seen in Abcg1–/– recipients (Figure 1F). In Abca1–/–Abcg1–/– recipients, the spleen also showed an increase in foam cells compared with control and single-KO recipients (Figure 1G).
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.
Accelerated atherosclerosis in Abca1–/–Abcg1–/– BM recipients. After 12 weeks on the high-cholesterol diet, control mice receiving WT BM had developed only sparse, foam cell–containing atherosclerotic lesions in their proximal aortas (Figure 2B). Atherosclerotic lesion development was similar to control mice in Abcg1–/– BM recipients and was moderately increased in recipients of Abca1–/– BM (Figure 2A). In contrast, atherosclerosis was more dramatically increased in mice receiving Abca1–/–Abcg1–/– BM (Figure 2). Further characterization of these atherosclerotic lesions revealed complex plaques containing macrophages, necrotic cores, fibrous caps (Figure 2), and small numbers of neutrophils and TUNEL-positive apoptotic cells (6–8 cells per high-power field; data not shown). TUNEL-positive cells were rarely observed in lesions from mice of the other genotypes.
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.
Plasma lipoprotein profiles. We next carried out studies to elucidate potential mechanisms responsible for increased macrophage foam cell accumulation in arteries, myocardium, and other tissues in mice receiving Abca1–/–Abcg1–/– BM. The atherogenic diet resulted in moderate hyperlipidemia in all groups of mice (Supplemental Table 1; supplemental material available online with this article; doi:10.1172/JCI33372DS1). Plasma lipoprotein analysis by fast protein liquid chromatography revealed slightly increased VLDL and LDL cholesterol levels (less than 30%) in mice receiving Abca1–/–Abcg1–/– BM (Supplemental Figure 1A) and no major difference in the lipoprotein profile of animals receiving Abca1–/– or Abcg1–/– compared with WT BM (data not shown). Decreased macrophage apoE secretion (see below) could explain the slightly higher VLDL and LDL levels (27, 28) and lower levels of apoE in these fractions in Abca1–/–Abcg1–/– BM recipients (Supplemental Figure 1B). There were no changes in HDL-cholesterol levels, most likely because after 12 weeks on the atherogenic diet, mice showed a redistribution of apoE among plasma lipoproteins and less than 10% associated with the HDL fraction (29). Although the increase in VLDL and LDL could have contributed to increased atherosclerosis, it is unlikely to have been the major causative factor.
Defective cholesterol efflux and foam cell formation in Abca1–/–Abcg1–/– macrophages. To test the hypothesis of a defect in cholesterol efflux as the underlying cause of increased atherosclerosis, we carried out cholesterol efflux studies and measured the cholesterol content of freshly isolated peritoneal macrophages. Abcg1–/– cells displayed a decrease in cholesterol efflux to serum, polyethyleneglycol-HDL (PEG-HDL), and human HDL isolated by ultracentrifugation (20%, 30%, and 30% decreases, respectively), while in Abca1–/–Abcg1–/– macrophages there was a more profound decrease (more than 2-fold) in the ability of serum and HDL to promote cholesterol efflux compared with single-KO and control macrophages (Figure 3). Cholesterol efflux to HDL was slightly increased in Abca1–/– cells (Figure 3C), likely reflecting a compensatory induction of ABCG1 (see below). Cholesterol efflux studies were also performed using media devoid of acceptors or containing lipid-poor apoA-1 or apoE (Figure 3D). This showed a profound defect in cholesterol efflux under all 3 conditions in Abca1–/–Abcg1–/– macrophages, even below the level observed for Abca1–/– cells. Defective cholesterol efflux led to a marked increase in cholesterol content primarily in the form of cholesteryl esters in peritoneal macrophages isolated from Abca1–/–Abcg1–/– mice fed standard chow (Figure 4A) or high-cholesterol diet (Figure 4B). In contrast, single-KO mice did not show increased cholesterol content when fed standard chow and only modestly increased cholesterol content compared with controls when fed high-cholesterol diet (Figure 4, A and B). These studies suggest synergistic roles of ABCA1 and ABCG1 in promoting macrophage cholesterol efflux, consistent with recent in vivo studies of macrophage reverse cholesterol transport showing additive effects of deficiencies of ABCG1 and ABCA1 in macrophages (30). Together these data indicate that a major defect in cholesterol efflux in Abca1–/–Abcg1–/– macrophages is the underlying mechanism responsible for increased atherosclerosis.
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. Χ_P_ < 0.05 versus single-KOs.
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. Χ_P_ < 0.05 versus single-KOs.
Decreased apoE secretion in Abca1–/–Abcg1–/– macrophages. Next we assessed apoE secretion in transporter-deficient macrophages. Previous studies have suggested that ABCA1 deficiency impairs apoE secretion most likely secondary to the role of ABCA1 in adding phospholipids and cholesterol to apoE (25, 31, 32) and have shown increased apoE secretion in Abcg1–/– macrophages associated with upregulation of ABCA1 (25). Macrophage apoE accumulation in media was measured in freshly isolated peritoneal macrophages or following loading of macrophages with acLDL plus liver X receptor (LXR) activator (T0901317) to maximally upregulate apoE expression and secretion (33). ABCG1 protein was increased in Abca1–/– cells, while ABCA1 protein was increased in Abcg1–/– cells (Figure 5), reflecting compensatory induction of their cognate mRNAs (data not shown). There was a small decrease in apoE secretion from _Abca1_–/– cells under basal conditions and an increase in apoE secretion in Abcg1–/– cells (Figure 5), as expected (25, 32). ApoE secretion was profoundly decreased in Abca1–/–Abcg1–/– cells compared with control and Abcg1–/– cells and was even significantly lower than in Abca1–/– cells (Figure 5). These effects were posttranscriptional: apoE mRNA was not changed in Abcg1–/– cells and slightly increased in Abca1–/– and Abca1–/–Abcg1–/– macrophages compared with WT cells (data not shown). Cell lysates from Abca1–/– and Abca1–/–Abcg1–/– macrophages showed amounts of apoE protein similar to those of WT cells, consistent with a defect in secretion rather than intracellular apoE degradation as the underlying cause (Figure 5). These findings suggest that the increase in apoE secretion in Abcg1–/– cells is secondary to induction of ABCA1 and indicate a possible role of ABCG1 in supporting residual apoE secretion in Abca1–/– cells. The profound defect in apoE secretion in Abca1–/–Abcg1–/– macrophages likely contributes to the increased atherosclerosis in Abca1–/–Abcg1–/– BM recipients.
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. Χ_P < 0.05 versus single KOs.
Increased secretion of inflammatory cytokines and chemokines by Abca1–/–Abcg1–/– macrophages. In order to understand the increased infiltration of inflammatory cells in the tissues of Abca1–/–Abcg1–/– mice, we measured the secretion of inflammatory cytokines and chemokines into media by freshly isolated peritoneal macrophages not treated with exogenous inflammatory stimuli during 6 h in cell culture. This showed increased secretion of a variety of inflammatory cytokines and chemokines in Abca1–/–Abcg1–/– macrophages, notably TNF-α, IL-6, IL-1β, and IL-12 as well as MIP-1α, MIP-2, growth-regulated oncogene α, and, to a lesser extent, MCP-1 (Figure 6). There was also significantly increased secretion of some inflammatory cytokines and chemokines in Abcg1–/– cells (i.e., TNF-α, IL-1β, IL-12, MIP-1α, MIP-2, and MCP-1), but, except for a slight increase in MIP-2 and IL-12, no major difference in _Abca1_–/– cells (Figure 6). The increased secretion of inflammatory cytokines and chemokines paralleled mRNA expression changes as determined by real-time PCR (data not shown) and likely explains the increased tissue inflammatory responses in Abca1–/–Abcg1–/– BM recipients.
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. Χ_P_ < 0.05 versus single-KOs.
Increased apoptosis after loading with free cholesterol or oxidized LDL in Abca1–/–Abcg1–/– macrophages. The Abca1–/–Abcg1–/– BM recipients showed increased numbers of apoptotic macrophages in the heart and atherosclerotic lesions. Apoptosis of macrophages in atherosclerotic lesions involves different mechanisms and may be brought about by accumulation of oxysterols derived from oxidized LDL or by accumulation of free cholesterol related to uptake of modified LDL and a failure of ACAT-mediated cholesterol esterification (34, 35). In order to understand the accumulation of apoptotic cells in heart and atherosclerotic lesions, we compared the apoptotic responses of macrophages of the 4 different genotypes using oxidized LDL or a standardized free cholesterol loading procedure (acLDL plus ACAT inhibitor 58035) in the presence or absence of HDL (Figure 7). Free cholesterol–induced apoptosis was dramatically increased in Abca1–/–Abcg1–/– macrophages compared with cells of the other genotypes (Figure 7A). Apoptosis induced by oxidized LDL was also markedly increased in Abca1–/–Abcg1–/– and Abcg1–/– cells, but not in cells of the other genotypes (Figure 7B). Treatment with HDL ameliorated apoptotic responses induced by oxidized LDL in WT and Abca1–/– cells, but not in Abcg1–/– or Abca1–/–Abcg1–/– macrophages. The enhanced susceptibility of Abca1–/–Abcg1–/– macrophages to cholesterol- and oxysterol-induced apoptosis likely explains the in vivo findings of increased apoptotic cells in the inflammatory infiltrate in the heart and atherosclerotic lesions.
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.