Impaired Development of Atherosclerosis in Hyperlipidemic Ldlr−/− and ApoE−/− Mice Transplanted With Abcg1−/− Bone Marrow
Objective— The lungs of Abcg1−/− mice accumulate macrophage foam cells that contain high levels of unesterified and esterified cholesterol, consistent with a role for ABCG1 in facilitating the efflux of cholesterol from macrophages to high-density lipoprotein (HDL) and other exogenous sterol acceptors. Based on these observations, we investigated whether loss of ABCG1 affects foam cell deposition in the artery wall and the development of atherosclerosis.
Methods and Results— Bone marrow from wild-type or Abcg1−/− mice was transplanted into Ldlr−/− or ApoE−/− mice. After administration of a high-fat/high-cholesterol diet, plasma and tissue lipid levels and atherosclerotic lesion size were quantified and compared. Surprisingly, transplantation of Abcg1−/− bone marrow cells resulted in a significant reduction in lesion size in both mouse models, despite the fact that lipid levels increased in the lung, spleen, and kidney. Lesions of Ldlr−/− mice transplanted with Abcg1−/− cells contained increased numbers of apoptotic cells. Consistent with this observation, in vitro studies demonstrated that Abcg1−/− macrophages were more susceptible to oxidized low-density lipoprotein (ox-LDL)-dependent apoptosis than Abcg1+/+ cells.
Conclusions— Diet-induced atherosclerosis is impaired when atherosclerotic-susceptible mice are transplanted with Abcg1−/− bone marrow. The demonstration that Abcg1−/− macrophages undergo accelerated apoptosis provides a mechanism to explain the decrease in the atherosclerotic lesions.
Elevated blood levels of low-density lipoprotein (LDL) result in enhanced entry of LDL into the arterial subendothelial space, where it can become trapped and oxidized to generate oxidized LDL (ox-LDL) and aggregated ox-LDL.1–3 Ox-LDL contains bioactive lipids that stimulate endothelial cells and macrophages to secrete numerous cytokines that promote the entry of circulating monocytes into the subendothelial space where they differentiate into macrophages and take up oxidized and/or aggregated LDL.1–3 These lipid-loaded “foam cells,” containing multiple cytoplasmic cholesterol ester lipid droplets, are found in both early fatty streaks and more advanced atherosclerotic lesions.1–3 The latter often contain necrotic cores, extracellular lipids that include cholesterol crystals, and even calcified tissue.1–3
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Numerous studies have led to the concept that the liver-X-receptor/retinoid-X-receptor (LXR/RXR) heterodimeric nuclear receptor complex functions as a “cellular sterol sensor.”4–6 In addition, oxysterols present in macrophage foam cells have been shown to activate LXR and alter gene expression. LXR target genes that are induced affect many pathways including apoptosis (AIM)7 and lipid homeostasis (SREBP-1c, FAS, ApoE, Apo-CII, CETP, LPL, ABCA1, and ABCG1).4–6
ABCG1 and ABCA1, two LXR target genes that are highly induced as macrophages convert to foam cells,8–10 are members of the ATP-binding cassette (ABC) superfamily of transmembrane transporters.11 Both transporters promote the efflux of lipids to specific exogenous acceptors; in vitro studies that used cells that either overexpressed ABCG19,12–14 or lacked ABCG1,15 led to the proposal that ABCG1 stimulates the efflux of cellular cholesterol to high-density lipoprotein (HDL), phospholipid vesicles, or phospholipid/apo-AI complexes, whereas ABCA1 mediates the efflux of cellular phospholipids and cholesterol to lipid-poor apoproteins (apo-AI, apoE, apo-CII). Whether these two transporters function in independent pathways or whether they function in concert to mediate cholesterol efflux is currently unknown. Nonetheless, the cumulative evidence suggests that both ABCA1 and ABCG1 play essential roles in macrophage lipid metabolism.
Interestingly, atherosclerotic lesions were not observed when Abca1−/− mice were fed chow or an atherogenic diet for many months.16,17 In addition, compared with the control mice, lesion size was not increased in ApoE−/−/Abca1−/− or Ldlr−/−/Abca1−/− mice.16 Nonetheless, support for a role of ABCA1 in the development of atherosclerosis has come from bone marrow transplantation studies; transplantation of Abca1−/− or ApoE−/−/ Abca1−/− cells into Ldlr−/−, or ApoE−/− mice, respectively, followed by administration of a high-fat diet resulted in a 50% to 60% increase in atherosclerotic lesions.16,18
In contrast to ABCA1, the physiological importance and function of ABCG1 is not well understood and its role in atherosclerosis has not been reported. Studies with both Abcg1−/−/LacZ knock-in mice and human ABCG1 transgenic mice revealed the critical importance of ABCG1 in controlling intracellular lipid homeostasis.15 These studies demonstrated that administration of a high-fat/high-cholesterol diet to Abcg1−/− mice resulted in the accumulation of both unesterified and esterified cholesterol in the liver and lung. Pulmonary macrophages were particularly sensitive to loss of ABCG1.15
Based on these previous studies with Abcg1−/− mice, we hypothesized that deletion of Abcg1 would result in the accumulation of macrophage foam cells in the artery wall and increased atherosclerosis. We report here studies that were performed to test this hypothesis.
Animals and Bone Marrow Transplants
Female C57Bl/6 ApoE−/− and Lldr−/− mice were obtained from Jackson Laboratories (Bar Harbor, Me). Abcg1−/−/lacZ knock-in mice, on a C57Bl/6 background, were generated and maintained as described.15 Abcg1−/− mice and their wild-type littermates were fed a 17% fat, 1.25% cholesterol and 0.5% cholate diet (Harlan Teklad #TD90221) for 19 weeks. For bone marrow transplantation studies, recipient ApoE−/− and Lldr−/− mice (10 week old) were γ-irradiated with 900 rads before transplantation with cells (3×106) from 8- to 10-week-old donor male wild-type or Abcg1−/− animals via tail vein injection. After a 4 week recovery period, ApoE−/− mice were fed a 21% fat and 0.2% cholesterol diet (Research Diets #D12079B) for 12 weeks. Ldlr−/− mice received a 21% fat and 1.25% cholesterol diet (Research Diets #D12108) for 16 weeks.
Histological Analysis and Atherosclerosis Studies
Preparation and staining of frozen and paraffin-embedded sections from multiple tissues were as described.7,15 Atherosclerosis in aortic roots and descending aortas (en face) was quantified as described.19
Lipids were extracted from tissues following Folch’s method.20 Chloroform extracts were dried under nitrogen, resolubilized in water, and lipid content determined enzymatically as described.15 Plasma lipids and lipoprotein distribution of cholesterol were determined as described.15,21
RNA was isolated and analyzed by real-time quantitative polymerase chain reaction (PCR) (RT-qPCR).15 Each qPCR assay was performed in duplicate using cDNA samples isolated from individual mice (n=4/genotype). Primer sets are available on request. Values were normalized to GAPDH and calculated using the comparative Ct method.
Thioglycollate-elicited peritoneal macrophages were recovered from wild-type or Abcg1−/−mice, plated in poly-l-Lysine-coated coverslips in 12-well plates (0.5×106 cells/well), and cultured in DMEM/10% fetal bovine serum for 16 hours. The cells were then incubated in duplicate in media (DMEM+0.2% bovine serum albumin) supplemented with ox-LDL (50 μg/mL of protein) for 6 or 24 hours. Human ox-LDL was prepared as described.22 Cells were fixed in 4% paraformaldehyde and DNA fragmentation (apoptosis) determined using the deadend fluorometric terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) System (Promega), following the manufacturer’s recommendations. Apoptosis in paraffin-embedded sections of the aortic roots of Ldlr−/− mice were performed as described.7
Results were analyzed by unpaired Student t test.
Inactivation of the Mouse Abcg1 Gene Does Not Affect the Development of Atherosclerosis
The finding that cholesterol-loaded macrophage foam cells accumulate in the lungs of Abcg1−/− mice15 suggested that loss of ABCG1 expression might also result in both the accumulation of foam cells in the artery wall and accelerated atherosclerosis. To test this hypothesis, Abcg1−/−/LacZ knock-in mice and their wild-type C57Bl/6 litter mates were fed an atherosclerotic diet (21% fat, 1.25% cholesterol, 0.5% cholate) for 19 weeks. All mice exhibited similar increases in plasma lipid levels and in lipid deposition in the liver (data not shown). However, because none of the mice developed significant atherosclerotic lesions in the aortic arch (data not shown), we concluded that loss of ABCG1 per se is not sufficient to promote atherosclerosis.
As an alternative approach to assess the role of ABCG1 in the development of atherosclerosis, we performed bone marrow transplant into genetically engineered mice (Ldlr−/− and ApoE−/−) that display altered lipoprotein profiles and accelerated atherosclerosis in response to a high-fat high-cholesterol (HF/HC) diet.
Bone Marrow Transplants into Ldlr−/− Mice
Irradiated Ldlr−/− mice were transfused with bone marrow cells derived from either wild-type or Abcg1−/− mice. After a 4-week recovery period, mice were fed a HF/HC diet for 16 weeks. At the conclusion of the experiment, no significant differences in body weights were observed between mice transplanted with wild-type or Abcg1−/− bone marrow cells (Table 1). As expected, the HF/HC diet resulted in severe hypercholesterolemia and hypertriglyceridemia (Table 1). However, although total plasma lipid levels in Ldlr−/− mice were independent of the genotype of the transplanted bone marrow (Table 1), HDL cholesterol levels were decreased ≈20% in mice that had received Abcg1−/− cells (Table 1). The reduced HDL levels were unexpected as previous studies have shown that plasma HDL levels do not differ between wild-type and Abcg1−/− mice.15 The mechanism by which Abcg1−/− bone marrow cells promote a decrease in plasma HDL is unknown at this time.
Necropsy of the HF/HC-fed recipient Ldlr−/− mice revealed that, as compared with wild-type→Ldlr−/−, the lungs, spleen and, to a lesser extent, liver and kidney of the Abcg1−/−→Ldlr−/− mice were paler in appearance and/or contained pale foci (Figure 1). Quantification of tissue lipid levels revealed that unesterified and/or esterified cholesterol accumulated to higher levels in the lungs, spleens, and kidneys of Ldlr−/− mice receiving Abcg1−/−, as compared with wild-type bone marrow (Table 2). The lungs of the mice transplanted with Abcg1−/− cells also exhibited an altered architecture, subpleural macrophage accumulation, the presence of numerous multinucleated giant cells, lymphocytic infiltration, and oil red O-positive lipid droplets (Figure 2F versus 2E; 2H versus 2G). The extrafollicular regions of the spleen and secretory tubules of the kidney also stained positive with oil red O after Abcg1−/−→Ldlr−/− transplantation (Figure 2L versus 2K; 2P versus 2O). Analysis of hematoxylin and eosin (H&E) stained spleen and kidney sections indicated that these tissues are histologically normal (Figure 2I versus 2J; 2M versus 2N). In contrast, the livers of all Ldlr−/− mice showed signs of steatosis, as judged both by staining with H&E (Figure 2A and 2B) or oil red O (Figure 2C and 2D), consistent with the elevated levels of cholesterol esters and triglycerides (Table 2). We conclude that lipid homeostasis of many tissues in hyperlipidemic mice is dependent on ABCG1 expression in bone marrow-derived cells.
To demonstrate that Abcg1−/−/LacZ bone marrow donor cells repopulate multiple tissues, we stained frozen tissue sections for β-galactosidase activity; LacZ-positive (and thus, Abcg1−/−) cells were identified in the liver (Kupffer cells), lung (macrophages and lymphocytes), spleen (macrophages and lymphocytes) and kidney (macrophages in glomeruli and distal collecting tubules) of Ldlr−/− recipient mice (supplemental Figure I, available online at http://atvb.ahajournals.org). As expected, lacZ-positive cells were not observed in tissues obtained from mice transplanted with wild-type bone marrow (supplemental Figure I).
The increased lipid content of several tissues of the Ldlr−/− mice (Table 2; Figures 1 and 2⇑) prompted us to examine the pattern of expression of genes involved in lipid metabolism; supplemental Figure II shows that mRNA levels encoding Srebp-1c, Fas, Scd-1, and Hmgcr are decreased in the lungs of Abcg1−/−→Ldlr−/− mice, as compared with those mice that received wild-type cells. Srebp-1c and Hmgcr levels were also decreased in the livers of the Abcg1−/−→Ldlr−/− animals (supplemental Figure II). Expression of these same genes in the spleen and kidneys of the recipient Ldlr−/− mice were not affected by the genotype of the donor cells (data not shown). Thus, the results of Figures 1 and 2⇑ and supplemental Figure II demonstrate that ABCG1 expression in bone marrow-derived cells is critical for the maintenance of lipid homeostasis in a number of tissues in mice that exhibit hyperlipidemia.
Finally, we quantified atherosclerotic lesions in Ldlr−/− mice. The extent of atherosclerosis was determined using oil red O-stained tissue sections of the aortic root, and en face analysis along the length of the aorta. Surprisingly, atherosclerotic lesions were reduced significantly in Ldlr−/− mice transplanted with Abcg1−/− bone marrow cells; lesion size in the aortic root and in the descending aorta of Abcg1−/−→Ldlr−/− mice were decreased 40% and 35%, respectively (Figure 3A through 3D). The reduction in lesions in Abcg1−/−→Ldlr−/− mice is unlikely a consequence of a decreased macrophage migration into the subendothelial space of the blood vessels because the aortic roots of Ldlr−/− mice infused with Abcg1−/− cells contained large and intensely oil red O-stained, LacZ-positive (Abcg1−/−) macrophage foam cells (Figure 3D, compare panels d versus c; h versus g).
Bone Marrow Transplants into ApoE−/−Mice
Previous studies have shown that both the size of atherosclerotic lesions and the level of hyperlipidemia in ApoE−/− mice are significantly attenuated when these mice are recipients of ApoE+/+ bone marrow transplantation.23–25 Nonetheless, infusion of ApoE+/+/Lxrαβ−/− double knockout cells into ApoE−/− mice increased atherosclerosis ≈2-fold.19 Whether this increase in lesion size was caused by low levels of ApoE expression in the Lxrαβ−/− macrophages or to reduced expression of other LXR target genes such as Abcg119 is unknown. These latter data suggested that loss of ABCG1 per se might increase atherosclerosis in ApoE−/− mice. To test this hypothesis, we infused ApoE−/− mice with either wild-type or Abcg1−/−-derived bone marrow cells and quantified lesion development.
The data of Table 1 indicate that body weight and plasma lipid levels were similar in ApoE−/− mice receiving either wild-type or Abcg1−/− bone marrow. Plasma lipoprotein profiles, as determined by fast protein liquid, were superimposable (data not shown). The level of hyperlipidemia in transplanted ApoE−/− mice after 12 weeks on a HF/HC diet was relatively mild (Table 1), likely a result of a functional ApoE gene in the donor cells. Nonetheless, cholesterol and cholesterol ester levels were increased significantly in the lungs and spleens of ApoE−/− mice that had been infused with Abcg1−/− bone marrow, as compared with Abcg1+/+ cells (Table 2). As expected, LacZ-positive cells were observed in the liver, lung and spleen of mice receiving Abcg1−/− cells (data not shown).
Lipid deposition and/or histological architectural changes in the lungs and spleens of ApoE−/− mice were also observed after transplantation with Abcg1−/− cells; the lungs contained oil red O-positive macrophages, giant cells, and lymphocytic infiltrates, consistent with elevated lipid levels (Table 2 and data not shown). No such changes were observed in the lungs of mice transfused with wild-type cells (data not shown). In contrast, the livers of all HF/HC-fed transplanted ApoE−/− mice accumulated lipid (Table 2).
As expected, the atherosclerotic lesions in both the aortic root and descending aorta of ApoE−/− mice were much smaller than those present in Ldlr−/− mice (Figure 3E, and 3F versus 3A and 3B). This result is likely a consequence of the much lower plasma cholesterol levels in the ApoE−/−, as compared with Ldlr−/− mice (Table 1; ≈125 versus ≈1050 mg/dL of cholesterol). Nonetheless, en face analysis of the descending aorta demonstrated that lesions were reduced significantly, by ≈60%, when ApoE−/− mice received Abcg1−/−, as compared with Abcg1+/+ bone marrow (Figure 3F; P<0.001). Although we also noted a decrease in lesion size in the aortic root of mice receiving Abcg1−/− as compared with wild-type cells, the values did not reach statistical significance (Figure 3E).
Taken together, the data from both Ldlr−/− and ApoE−/− mice demonstrate that Abcg1−/− monocytes retain the ability to both enter the subendothelial space and differentiate into macrophage foam cells. However, the decreased lesions of Abcg1−/−→Ldlr−/− and Abcg1−/−→ApoE−/− mice suggest that Abcg1−/− macrophages may either exit the lesion or undergo some form of cell death.
Abcg1−/− Macrophages Undergo Increased Apoptosis In Vivo and In Vitro
The presence of enlarged oil red O-positive Abcg1−/− macrophages in atherosclerotic lesions (Figure 3C and 3D) is consistent with cholesterol ester accumulation, noted previously in pulmonary Abcg1−/− macrophages.15 We hypothesized that the decrease in atherosclerotic lesions in mice infused with Abcg1−/− bone marrow cells may result from increased apoptosis of the macrophages in response to altered lipid metabolism.
To test this hypothesis, paraffin-embedded sections taken from atherosclerotic lesions from four individual wild-type→Ldlr−/− and Abcg1−/−→Ldlr−/− mice were stained for TUNEL activity. The data of Figure 4A and 4B demonstrate unequivocally that lesions in the aortic roots of Ldlr−/− mice receiving Abcg1−/− bone marrow contained a 3.9-fold increase in the TUNEL-positive cells as compared with mice receiving Abcg1+/+ cells. In contrast, the smooth muscle cell rich media lacks LacZ-positive (Abcg1−/−) (Figure 3D) and apoptotic cells (Figure 4A).
To address the important question of whether the increase in apoptotic cells observed in Abcg1−/−→Ldlr−/− lesions was a macrophage-autonomous response, we isolated peritoneal macrophages from wild-type and Abcg1−/− mice and incubated them in the presence of ox-LDL. After 6 hours, ≈1% of the Abcg1+/+ or Abcg1−/− macrophages were positive for TUNEL staining (data not shown). However, after 24 hours, there was a 70% increase in the number of Abcg1−/− cells undergoing apoptosis as compared with wild-type cells (22.0±1.6 versus 13.1±0.8%; P≤0.001) (Figure 4C).
Based on the data of both in vivo and in vitro studies (Figure 4A through 4C), we conclude that Abcg1−/− macrophages are more susceptible than wild-type macrophages to apoptosis after exposure to lipid/cholesterol-rich lipoproteins.
We recently reported that administration of a HF/HC diet to Abcg1−/− mice resulted in the accumulation of cholesterol and cholesterol esters in pulmonary macrophages, hepatocytes, and Kupffer cells.15 In addition, in vitro studies have shown that ABCG1 promotes the efflux of cellular cholesterol to various exogenous lipid acceptors.9,12–15 These data suggested that Abcg1−/− mice might exhibit accelerated atherosclerosis as a result of the accumulation of cholesterol-loaded foam cells in the aorta and/or aortic root. The results of experiments to test this hypothesis were quite unexpected.
First, administration of a high-fat/high-cholesterol/cholic acid diet to Abcg1−/− and wild-type mice resulted in similar levels of hyperlipidemia but to no difference in atherosclerotic lesions. Second, bone marrow transplant studies showed that Abcg1−/− donor cells resulted in a significant decrease in lesion size in both Ldlr−/− and ApoE−/− hyperlipidemic mice (Figure 3). These latter results were unexpected because the Abcg1−/− donor bone marrow cells repopulated many tissues, including the aortic wall, aortic root as well as lung, liver, and spleen, and transplanted Abcg1−/− macrophages in the atherosclerotic lesions stained with oil red O, consistent with the accumulation of neutral lipid (Figure 3). Together, these data indicate that Abcg1−/− monocytes retain the capacity to enter multiple tissues and convert to lipid-engorged macrophages.
The finding that lesions of Ldlr−/− mice transplanted with Abcg1−/− cells contained increased numbers of apoptotic cells and that Abcg1−/− macrophages are more susceptible to apoptosis in vitro following incubation with ox-LDL (Figure 4) provides a mechanism to account for the decrease in atherosclerotic lesions. Key evidence for the importance of apoptosis in lesion development was recently provided by Arai et al on studies of the anti-apoptotic gene AIM.7 Expression of AIM is largely restricted to macrophages.7 Importantly, AIM−/−/Ldlr−/− double knock-out mice showed a >80% decrease in atherosclerotic lesions as a result of enhanced apoptosis of AIM−/− macrophages in the lesions.7 In other studies, Tabas et al have shown that macrophages undergo apoptosis when they are treated in vitro with ox-LDL or acetylated-LDL together with an inhibitor of acyl-coenzyme A (CoA):cholesterol acyltransferase (ACAT).26,27 The ACAT inhibitor is critical as it prevents cholesterol esterification and thus results in an increase in intracellular levels of unesterified cholesterol which in turns activates the unfolded protein response in the endoplasmic reticulum, p38 and JNK, resulting in increased apoptosis.27 Tabas has proposed that this process may account for the presence of apoptotic cells in atherosclerotic lesions.27 The current study provides in vivo support for this model. Importantly, we have shown that increased apoptosis (Figure 4) and deposition of unesterified cholesterol crystals in Abcg1−/− macrophages15 does not require ACAT inhibitors, but is dependent on loss of ABCG1 function. We hypothesize that macrophages in the aortic root/aorta also accumulate unesterified cholesterol and that this ultimately results in apoptosis and decreased lesion development. However, several pro-apoptotic factors, that include oxysterols, tumor necrosis factor (TNF)-α and nitric oxide, have been shown to be generated after uptake of ox-LDL by macrophages.27 In addition, scavenger receptors have been linked to macrophage apoptosis.26 Consequently, additional studies will be needed to determine how loss of ABCG1 affects all these pro- and anti-apoptotic pathways and whether such changes are restricted to lesions or are also present in other tissues.
It is formally possible that the presence of LDL receptor, or ApoE, in the transplanted Abcg1−/− bone marrow cells influences lesion development. However, based on previous transplantation studies into Ldlr−/− mice19,28–30 and the finding that lesions in the aortic roots of hyperlipidemic Ldlr−/− mice fed a high-fat diet were similar after transplantation with either Ldlr+/+ or Ldlr−/− bone marrow,31,32 we suggest that this is unlikely. Nonetheless, in a different model system, Linton et al32 and Herijgers et al33 reported that lesion size was affected by the presence or absence of Ldlr in the donor bone marrow cells. However, the hyperlipidemia was mild, the lesions small, the recipients were C57BL/6 mice, and the diet contained cholic acid, which is known to promote inflammation and to drastically affect bile acid and cholesterol metabolism.34 Thus, the relevance of the latter studies to those presented here and elsewhere19,28–30 is unclear. Nonetheless, additional studies that use transplantation of either Ldlr−/−/Abcg1−/−, or ApoE−/−/Abcg1−/− donor cells into either Ldlr−/− or ApoE−/− mice, respectively, should provide additional insights into the role of ABCG1 in mice that lack LDLR or ApoE in macrophages.
We have previously demonstrated that liver and lungs of transgenic mice, that express both human and mouse ABCG1, are protected from lipid deposition that follows administration of a HF/HC diet.15 Consequently, we hypothesize that transplantation of bone marrow from ABCG1 transgenic mice into atherosclerosis-prone animals will be atheroprotective. Additional studies will be required to test this hypothesis and to also determine whether the anti-atherogenic effects of LXR agonists19,35 result from altered expression of ABCG1.
We thank Tony Mottino from the Department of Medicine at UCLA for histological analysis and lesion measurement technical assistance. We also thank the members of the Edwards laboratory for critical reading of the manuscript.
Sources of Funding
Á.B. and P. Tarr are recipients of an American Heart Association (Western Affiliate) Postdoctoral Fellowship (0525010y) and a NIH Predoctoral Fellowship (NHLBI T32 69766), respectively. This work was supported by National Institutes of Health Grants NIH30568 and NIH68445 (to P.A.E.) and HL66088 (to P. Tontonoz), a grant from the Laubisch Fund (to P.A.E.), a grant from Pfizer, Inc (to P.A.E.), and an AHA Western Affiliated State Beginning grant-in-aid (WS-046508) (to A.C.L.).
Original received April 24, 2006; final version accepted July 26, 2006.
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