Integrative Physiology/Experimental Medicine

Impaired Development of Atherosclerosis in Abcg1−/− Apoe−/− Mice

Identification of Specific Oxysterols That Both Accumulate in Abcg1−/− Apoe−/− Tissues and Induce Apoptosis

Elizabeth J. Tarling, Dragana D. Bojanic, Rajendra K. Tangirala, Xuping Wang, Anita Lovgren-Sandblom, Aldons J. Lusis, Ingemar Bjorkhem, Peter A. Edwards
https://doi.org/10.1161/ATVBAHA.110.205617
Arteriosclerosis, Thrombosis, and Vascular Biology. 2010;30:1174-1180
Originally published May 19, 2010

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Abstract

Objective— To generate Abcg1−/− Apoe−/− mice to understand the mechanism and cell types involved in changes in atherosclerosis after loss of ABCG1.

Methods and Results— ABCG1 is highly expressed in macrophages and endothelial cells, 2 cell types that play important roles in the development of atherosclerosis. Abcg1−/− Apoe−/− and Apoe−/− mice and recipient Apoe−/− mice that had undergone transplantation with bone marrow from Apoe−/− or Abcg1−/− Apoe−/− mice were fed a Western diet for 12 or 16 weeks before quantification of atherosclerotic lesions. These studies demonstrated that loss of ABCG1 from all tissues, or from only hematopoietic cells, was associated with significantly smaller lesions that contained increased numbers of TUNEL- and cleaved caspase 3–positive apoptotic Abcg1−/− macrophages. We also identified specific oxysterols that accumulate in the brains and macrophages of the Abcg1−/− Apoe−/− mice. These oxysterols promoted apoptosis and altered the expression of proapoptotic genes when added to macrophages in vitro.

Conclusion— Loss of ABCG1 from all tissues or from only hematopoietic cells results in smaller atherosclerotic lesions populated with increased apoptotic macrophages, by processes independent of ApoE. Specific oxysterols identified in tissues of Abcg1−/− Apoe−/− mice may be critical because they induce macrophage apoptosis and the expression of proapoptotic genes.

The ATP binding cassette transporter, subfamily G, member 1 (ABCG1) is 1 member of a large superfamily of membrane proteins that function to transport substrates across specific membranes.1,2 Studies3–7 with Abcg1−/− β-galactosidase (LacZ) knock-in mice have demonstrated that ABCG1 is expressed in numerous organs and cell types, with particularly high expression in macrophages, endothelial and epithelial cells, and neurons.

Numerous studies have shown that ABCG1 can function to efflux cholesterol and/or other sterols from cells to various exogenous acceptors, including high-density lipoprotein.8 Studies3,6,9,10 with Abcg1−/− mice demonstrated that pulmonary macrophages accumulate massive levels of cholesterol and sterol esters, consistent with these cells being particularly sensitive to loss of function of this transporter. These lipid-loaded Abcg1−/− pulmonary macrophages also undergo increased apoptosis.11 These data are consistent with the normal role of pulmonary macrophages in clearing cholesterol-containing surfactant from the extracellular space,12 with ABCG1 then functioning to eliminate the sterols from the cells to maintain cellular sterol homeostasis.

Loss of ABCG1 from macrophages results in increased expression of multiple inflammatory genes, consistent with a stimulatory effect of the accumulating cellular sterols on inflammation.9,11,13 Earlier studies demonstrated that endothelial cells, like macrophages, express particularly high levels of ABCG1.14 Interestingly, the administration of a Western diet to Abcg1−/− mice was recently shown to increase the inflammatory status and sterol levels of endothelial cells.13 Taken together, these data suggest that loss of ABCG1 results in subtle or gross changes in cellular sterols that may result in induction of inflammatory genes and/or increased apoptosis in 2 cell types (endothelial cells and macrophages) that are known to play critical roles in the development of atherosclerosis.

Atherosclerosis is a complex disease that is characterized in the early stages by the accumulation of lipid-loaded macrophages (foam cells) in the intima.15 The initial findings that loss of ABCG1 led to the accumulation of sterol-loaded macrophages in the lungs of Abcg1−/− mice3,6,10,11 suggested that hyperlipidemic mice lacking functional ABCG1 would exhibit accelerated atherosclerosis. However, Abcg1−/− mice have a normal plasma lipoprotein profile and, thus, do not develop significant atherosclerotic lesions even when fed a Western diet.16 To assess the role of ABCG1 in the development of atherosclerosis, 3 groups independently performed bone marrow transplantation studies using donor cells from wild-type or Abcg1−/− C57BL/6 mice and recipient hyperlipidemic low-density lipoprotein receptor null (Ldlr−/−) mice. Although the protocols used in these studies were similar, the conclusions were not; one group reported that transplantation of Abcg1−/− bone marrow led either to a modest but significant increase in atherosclerotic lesions in the Ldlr−/− recipient mice6,17 or to no change in lesion size.17,18 In contrast, Baldan et al16 and Ranalletta et al19 observed a significant decrease in lesion size in Ldlr−/− mice receiving Abcg1−/− cells. It remains to be determined whether these inconsistencies result from differences in the genetic backgrounds of the mice, from different concentrations of cholesterol in the diet, or from different treatment times that affect lesion progression.

Alternative mechanisms, which are not necessarily exclusive, were invoked to explain the unexpected decrease in lesion size noted in 2 of the transplantation studies.16,19 Ranalletta et al19 proposed that the Abcg1−/− macrophages secreted increased amounts of ApoE protein, a known antiatherogenic protein. It was also suggested that increased expression of a second sterol transporter, ABCA1, in the Abcg1−/− macrophages might reduce sterol accumulation in the foam cells and, thus, impair lesion development.19 In contrast, Baldan et al16 proposed that the smaller lesions were a result of increased apoptosis of the Abcg1−/− macrophages that populated the atherosclerotic lesions of Ldlr−/− mice. A role for ABCG1 in protection against apoptosis is consistent with studies showing that the lungs of Abcg1−/− mice contain increased TUNEL-positive apoptotic cells11 and that overexpression of ABCG1 in cultured cells attenuates oxysterol-induced cell death, possibly by stimulating the efflux of either 7β-hydroxycholesterol20 or 7-ketocholesterol21 to exogenous high-density lipoprotein.

Apoptosis plays an important role in the development of atherosclerotic lesions.22–24 An increase in macrophage apoptosis in early lesions has been associated with decreased lesion progression.22 In contrast, an increase in macrophage apoptosis in advanced lesions is thought to promote the development of the necrotic core, a key factor in vulnerable plaque formation and acute thrombosis.24 The increase in apoptotic cells in lesions may result from the accumulation of unesterified cholesterol and/or oxysterols because these lipids are known to stimulate proapoptotic processes.25,26 Support for a role for ABCG1 in preventing apoptosis16 came from studies16,27 showing that Abcg1−/− or Abcg1−/− Abca1−/− macrophages exhibit increased apoptosis in vitro compared with wild-type cells after a challenge with oxidized low-density lipoprotein (oxLDL).

We report that hyperlipidemic apolipoprotein E null (Apoe−/−) mice lacking ABCG1 in all tissues or in hematopoietic cells only exhibit decreased lesions, decreased aortic lesion calcification, and increased macrophage apoptosis as a result of the accumulation of specific proapoptotic oxysterols.

Methods

We fed a Western diet for 12 or 16 weeks to Abcg1−/− Apoe−/− and Apoe−/− mice and to recipient Apoe−/− mice that had undergone transplantation with bone marrow from Apoe−/− or Abcg1−/− Apoe−/− mice. Atherosclerotic lesion size, apoptosis, and oxidized sterol concentrations in macrophages in these mice were determined. For details and further methods, please see the Supplemental Material (available online at http://atvb.ahajournals.org).

Results

Characterization of Abcg1−/−Apoe−/− Mice

Endothelial cells of Abcg1−/− mice fed a Western diet accumulate 7-ketocholesterol, a nonenzymatic product of cholesterol autoxidation.13 Abcg1−/− mice also exhibit decreased endothelium-dependent vasodilatation and decreased endothelial nitric oxide synthase activity.13 Macrophages from Abcg1−/− mice also exhibit increased expression of inflammatory genes and accumulate intracellular 7-ketocholesterol.9,11 In addition, loss of ABCG1 from macrophages has been reported to result in increased secretion of ApoE protein,19 an antiatherosclerotic protein.28

Consequently, to better understand the effect of loss of function of ABCG1 from all cell types, including macrophages and endothelial cells, and to remove any confounding effects that could arise from altered secretion of ApoE from macrophages, we generated Abcg1−/− Apoe−/− double-knockout (DKO) mice. Analysis of the plasma showed that, compared with Apoe−/− mice, DKO mice had increased hemoglobin (14.00±0.67 versus 12.68±0.96; P<0.04; mean±SEM n=10) and hematocrit (40.42±1.94 versus 36.80±2.14; mean±SEM, n=10; P<0.03) values; however, lipid and lipoprotein levels and a broad array of hematology values did not significantly differ between the 2 genotypes (data not shown).

To accelerate the development of atherosclerosis, DKO and Apoe−/− mice were challenged with a Western diet (21% fat and 0.2% cholesterol). After 16 weeks, analysis of plasma from DKO and Apoe−/− mice indicated that there was no significant difference in lipid levels (Supplemental Table I) or the lipoprotein profile (Supplemental Figure IA). Lungs of the DKO, but not the Apoe−/− mice, appeared white and stained positive with oil red O, especially in areas enriched in LacZ-expressing macrophages (Supplemental Figure II; data not shown). In addition, the red and white pulp of spleens of the DKO, but not Apoe−/− mice, contained cells that were positive for both LacZ and oil red O (Supplemental Figure II). Thus, multiple tissues of the DKO mice exhibited evidence of neutral lipid accumulation.

Abcg1−/−Apoe−/− Mice Have Decreased Atherosclerotic Lesions Containing Increased Numbers of Apoptotic Macrophages

After 16 weeks on the Western diet, atherosclerotic lesions were determined both by en face analysis of the Sudan IV–stained descending aorta or after analysis of stained frozen sections (25 to 30 sections per mouse) of the aortic root.

The data show that Abcg1−/− Apoe−/− mice had significantly smaller lesions than Apoe−/− mice in both the aortic root (Figure 1A and B, panel b versus a) and in the descending aorta (Figure 1D and E). The lesions of the DKO mice contained numerous LacZ-positive macrophages (Supplemental Figure III), thus excluding the possibility that DKO macrophages do not enter the subendothelial space. Calcified deposits in the lesions of the aortic root, identified after staining of sections with either von Kossa or oil red O, hematoxylin, and fast green, were significantly reduced in the Abcg1−/− Apoe−/− mice (Figure 1C; and Figure 1B, panel d versus c and b versus a), consistent with the smaller lesions in the DKO mice. Photomicrographs taken at a higher magnification illustrate that the calcium deposition occurs within the aortic lesions, adjacent to the medial layer (Supplemental Figure IV).

Figure1

Figure 1. Atherosclerosis and lesion calcification are reduced in Abcg1−/− Apoe−/− compared with Apoe−/− mice. DKO and Apoe−/− mice (6 to 8 mice per group) were fed a Western diet for 16 weeks. A, Frozen sections (25 to 30 sections per mouse; n=6 mice per group) from the aortic root were stained with oil red O and counterstained with hematoxylin and fast green before lesion areas were determined, as described in the “Methods” section. Each point represents an individual mouse. **P<0.01. B, Representative oil red O–stained sections counterstained with hematoxylin and fast green (a and b) and adjacent sections stained with von Kossa (c and d). Calcification/calcium phosphate deposits are indicated by arrows in panels a and c. C, Quantification of lesion calcification. *P<0.05. D, Lesions in the descending aorta were identified by en face analysis and quantified as described in the “Methods” section (n=8 mice per group). Data represent mean±SEM. **P<0.01. E, Representative Sudan IV–stained aortas are shown from the 2 genotypes.

Loss of ABCG1 From Hematopoietic Cells Delays the Development of Atherosclerosis and Increases Apoptotic Macrophages in the Lesions, Independent of ApoE

To determine the relative importance of hematopoietic and nonhematopoietic Abcg1−/− cells on the observed changes in atherosclerosis noted in the Abcg1−/− Apoe−/− mice (Figure 1), we performed bone marrow transplantation studies in which bone marrow from either Apoe−/− or DKO mice was transplanted into recipient Apoe−/− animals. After a 4-week recovery period, the mice were fed a Western diet for 12 weeks. An analysis of the lungs of the Apoe−/− recipients indicated that mice that had been transplanted with DKO donor cells, but not those receiving Apoe−/− cells, contained LacZ-positive cells and white patches consistent with lipid deposition in macrophages (data not shown).

Neither plasma lipid levels (Supplemental Table II) nor plasma lipoprotein profiles (Supplemental Figure IB) were significantly different between the 2 groups of recipient Apoe−/− mice. Compared with wild-type mice, all transplanted mice contained elevated levels of very low-density lipoprotein and LDL and decreased levels of high-density lipoprotein independent of the genotype of the donor cells (Supplemental Figure IB; data not shown).

Quantification of the atherosclerotic lesions showed that they were significantly smaller in mice transplanted with DKO compared with Apoe−/− donor bone marrow (Figure 2A; and Figure 2B, panel b versus a). Interestingly, and in agreement with the studies using whole-body DKO mice, calcification in the lesions of the aortic root was also significantly decreased in mice transplanted with Abcg1−/− Apoe−/− donor bone marrow (Figure 2C; and Figure 2B, panel d versus c and b versus a), consistent with smaller lesions in the latter mice.

Figure2

Figure 2. Apoe−/− mice lacking ABCG1 in hematopoietic cells have reduced atherosclerotic lesions. Apoe−/− mice were transplanted with bone marrow from Apoe−/− or DKO mice before being fed a Western diet for 12 weeks. All analyses were performed as described in Figure 1. A, C, and D, Lesion size (A) and calcification (C) in the aortic root sections (10 to 13 mice per group) and lesion size in the descending aorta (D) (13 to 16 mice per group) are shown, with each point representing 1 mouse. B, Representative sections from the aortic root after staining with oil red O and counterstaining with hematoxylin and fast green (a and b) or adjacent sections stained with von Kossa (c and d). Arrows identify calcium deposits. E, Representative Sudan IV–stained sections of the descending aorta. Data represent mean±SEM. **P<0.01 and ***P<0.001.

En face analysis of the descending aorta indicated a trend toward lower lesions in those mice receiving bone marrow from Abcg1−/− Apoe−/− mice, although the difference failed to reach statistical significance (Figure 2D and E). However, lesion coverage in the thoracic and abdominal sections, but not the proximal sections, was significantly smaller in mice transplanted with DKO cells (Supplemental Figure V), consistent with slower lesion progression in mice receiving DKO donor cells.

Increased Macrophage Apoptosis in Lesions of DKO Mice

After 16 weeks on the Western diet, the aortic root lesions of DKO mice contained significantly more TUNEL-positive cells, often present as multicell aggregates (Figure 3A). A similar difference was seen in the bone marrow transplantation studies in which we observed a 22-fold increase in TUNEL-positive cells in lesions of Apoe−/− mice transplanted with DKO compared with Apoe−/− donor cells (Figure 3C).

Figure3

Figure 3. ABCG1 deficiency results in increased numbers of apoptotic macrophages in atherosclerotic lesions. A through D, The indicated whole-body knockout mice (A and B) or bone marrow–transplanted mice (C and D) were fed a Western diet, as described in the legends to Figure 1 and Figure 2. TUNEL- and 4′,6-diamidino-2-phenylindole–positive cells (green and blue, respectively) were determined in adjacent sections of the aortic roots (A and C) of the indicated mice. Aggregated TUNEL-positive cells are indicated by arrows. Graphs show the percentage of TUNEL-positive cells in the lesions. B and D, Adjacent frozen sections were also stained with either antibody to cleaved caspase 3 (green foci marked by arrows) or macrophages (red). The merged images are also shown (B and D). Data are representative of multiple stained sections (n=15 per section per mouse; 3 mice per genotype). Data are expressed as mean±SEM. ***P<0.001.

One of the late events in apoptosis involves cleavage of the precursor form of caspase 3 to form an active protease.29 To identify cells undergoing apoptosis within the lesions of the aortic root, frozen sections from the aortic roots of mice were immunostained with antibodies to macrophages and to the cleaved form of caspase 3. An analysis of multiple stained sections indicated that lesions of Apoe−/− mice had few active caspase 3–positive cells, whereas numerous active caspase 3–positive cells, often present as aggregates, were present in the lesions of DKO mice (Figure 3B) and in Apoe−/− mice that were the recipients of the DKO bone marrow (Figure 3D). These tissue sections also stained positive for macrophages when costained with anti-Mac3 (Figure 3B, D). An analysis of the merged Figure showed that cleaved caspase 3–positive and anti-Mac3–positive cells colocalized in the lesions of mice lacking ABCG1 (Figure 3B and D), thus identifying the apoptotic cells as macrophages. Interestingly, cleaved caspase 3– or TUNEL-positive endothelial cells were never observed in any section, suggesting that loss of ABCG1 from endothelial cells did not result in accelerated apoptosis in vivo (data not shown).

Taken together, the data from studies with whole-body DKO mice and after bone marrow transplantation demonstrate that loss of ABCG1 from hematopoietic cells alone is sufficient to slow the progression of atherosclerotic lesions. This is associated with an increase in the number of apoptotic cells in the lesions and decreased calcification within the lesion. All these changes occur by mechanisms that are independent of ApoE.

Identification of Specific Oxysterols Accumulating in Abcg1−/−Apoe−/− Macrophages

The identification of specific sterols that accumulate within macrophages in atherosclerotic lesions is complicated by the inability to obtain sufficient numbers of cells. Consequently, we performed bronchoalveolar lavage on Apoe−/− and Abcg1−/− Apoe−/− mice and recovered alveolar macrophages. Analyses of these samples using isotope dilution mass spectrometry identified a number of oxysterols, including 24-, 25-, and 27-hydroxycholesterols that accumulate in the Abcg1−/− Apoe−/− and Abcg1−/− cells compared with wild-type or Apoe−/− cells (Table). Also, compared with Apoe−/− mice, 25- and 27-hydroxycholesterol levels are significantly increased in the brain specimens of the DKO mice (Table). Therefore, the increase in the levels of these enzymatically synthesized oxysterols is not limited to macrophages.

View this table:

Table. Accumulation of Specific Oxysterols in Macrophages and Brains of Mice Lacking ABCG1

Abcg1−/− Bone Marrow–Derived Macrophages Display a Proapoptotic Phenotype and an Altered Sensitivity to Oxysterols

The data of Figure 4A show that after 7 days in culture, Abcg1−/− and Abcg1−/− Apoe−/− bone marrow–derived macrophages (BMDMs) exhibited a 3- to 6-fold increase in TUNEL-positive cells, compared with wild-type or Apoe−/− cells. Although exposure of all these cells to oxLDL for 8 hours increased TUNEL staining, the highest levels of apoptosis/TUNEL staining were seen when cells lacked ABCG1 (Figure 4A), consistent with the proposal that ABCG1 is critical for limiting apoptosis in response to lipid loading. As expected, oxLDL treatment of wild-type, Apoe−/−, Abcg1−/−, or Abcg1−/− Apoe−/− BMDMs increased the expression of the Liver X Receptor (LXR) target genes Abca1 and Srebp1c and the antiapoptotic gene Aim (Supplemental Figure VIA-C).

Figure4

Figure 4. Macrophages lacking ABCG1 exhibit increased apoptosis in response to oxLDL or oxysterols and enhanced induction of proapoptotic genes. BMDMs in quadruplicate were differentiated in L929-conditioned media containing 10% fetal bovine serum. After 7 days, the media were replaced with media containing 0.2% BSA with or without oxLDL, 50 μg/mL (A), or the indicated oxysterol, 10 μmol/L (B–D). After 8 hours, the number of TUNEL-positive apoptotic cells (total, >1000 cells per field; 6 fields per genotype) (A and B) or the mRNA levels of Bid and Bok (C and D) were determined. Data are expressed as mean±SEM and are representative of 2 experiments. In A, * indicates significantly different from controls that were incubated with buffer (P<0.001); # indicates significantly different from buffer-treated WT and Apoe−/− cells (P<0.001). In B, # indicates significantly different from dimethylsulfoxide-(DMSO) treated Apoe−/− cells (P<0.001) and $ indicates significantly different from DMSO-treated DKO cells (P<0.001). In C and D, bars with different letters (a, b, c, d) are significantly different from one another at P<0.001.

Based on the finding that specific oxysterols accumulate in alveolar macrophages and the brain specimens of Abcg1−/− Apoe−/− mice (Table), we next investigated whether cells lacking ABCG1 and/or ApoE are particularly sensitive to these same oxysterols. In the absence of added oxysterols, the number of BMDMs undergoing apoptosis was 4-fold greater in Abcg1−/− Apoe−/− compared with Apoe−/− cells (Figure 4B). The addition of 10 μmol/L 7-ketocholesterol, 25-hydroxycholesterol, or 27-hydroxycholesterol increased the number of TUNEL-positive cells (Figure 4B). More important, the percentage of apoptotic cells was greatest after the addition of oxysterols to the DKO BMDMs (Figure 4B).

The increased sensitivity of cells lacking ABCG1 to oxysterol-induced apoptosis suggested that these cells might also exhibit altered expression of genes involved in apoptosis. Consequently, we performed a PCR-based screen to identify apoptotic genes that were altered after exposure of cells to 50 μg/mL oxLDL (data not shown). Confirmation of altered gene expression came from subsequent quantitative RT-PCR analysis that showed that incubation of cells with specific oxysterols increased the expression of Bid and Bok, 2 members of the Bcl-2 proapoptotic gene family (Figure 4C and D). More important, the expression of Bid and Bok was higher in DKO or Abcg1−/− BMDMs compared with wild-type or Apoe−/− cells (Figure 4C and D).

Discussion

Herein, we report on the generation and initial characterization of Abcg1−/− Apoe−/− mice. Studies with both whole-body Abcg1−/− Apoe−/− mice and after bone marrow transplantation into Apoe−/− mice demonstrate that atherosclerotic lesion progression is reduced when mice lack ABCG1 in either all tissues or macrophages and other hematopoietic cells (Figure 1 and Figure 2). In preliminary studies, we also noted that neutrophils accumulated in the adventitia, adjacent to lesions of Apoe−/− and Abcg1−/− Apoe−/− transplanted mice (data not shown), consistent with increased inflammation. Despite this latter finding, and the observation that endothelial cells lacking ABCG1 exhibit increased inflammatory properties,13 the current data suggest that the decrease in lesion size is dependent on loss of ABCG1 from hematopoietic cells and occurs by processes independent of ApoE. Whether the profound decrease in calcium deposition in the atherosclerotic lesions (Figure 1 and Figure 2 and Supplemental Figure IV) is simply a consequence of the smaller lesions or of the increase in apoptotic macrophages in the lesions will require additional studies.

The importance of macrophage apoptosis in affecting early lesion development was initially reported by Arai et al22 as a result of studies with mice lacking the antiapoptotic gene Aim. More important, the expression of Aim is largely restricted to macrophages.22 Arai et al demonstrated a remarkable (>90%) attenuation of atherosclerotic lesions in hyperlipidemic Aim−/− Ldlr−/− mice, compared with Aim+/+ Ldlr−/− mice.22 These data suggested that increased apoptosis of macrophages limited early development and progression of atherosclerotic lesions.22 Strikingly, in the current study, we show that cells lacking ABCG1 are more susceptible to oxysterols/induced apoptosis despite increased expression of Aim mRNA.

The current data extend a previous report in which it was shown that atherosclerotic lesions were decreased in Apoe−/− recipient mice after repopulation with Abcg1−/− Apoe+/+ cells, compared with Abcg1+/+Apoe+/+ (wild-type) bone marrow.16 However, the interpretation of the latter result was complicated by the fact that the expression of ApoE in the donor marrow cells is sufficient to attenuate atherosclerosis in recipient Apoe−/− mice.30 It was further complicated by the report18 that loss of ABCG1 from macrophages resulted in increased secretion of ApoE. Although we have not been able to confirm the latter finding, the current data demonstrate that the decrease in lesion progression after deletion of ABCG1 can occur independent of ApoE.

Lammers et al17 recently reported that atherosclerotic lesions of Ldlr−/− Apoe+/+ mice were increased after transplantation with Abcg1−/− Apoe−/− bone marrow.17 However, the finding that the transplanted mice expressed ApoE in many nonhematopoietic cells makes comparison with the current studies difficult. This same group previously reported either no change or an increase6,18 in lesion size after transplantation of Abcg1−/− bone marrow into hyperlipidemic Ldlr−/− mice. Whether these differences in lesion progression relate to differences in serum cholesterol levels31 or to differences in genetic background of the mice, length of time on different diets, and the extent of the disease remains unclear.

The results of the present, and previous, work are consistent with a more important role of the ABCG1 transporter for efflux of a number of oxysterols than for efflux of cholesterol.5,20,21,32,33 Thus, loss of this transporter leads to accumulation of 7-β-hydroxycholesterol, 7-ketocholesterol, and 24-, 25-, and 27-hydroxycholesterol as well as cholesterol. The accumulation of the side chain–oxidized oxysterols is surprising in view of their physiochemical properties, which allow them to pass biomembranes at a much higher rate than cholesterol.34 In addition to oxysterols, desmosterol (an intermediate in the cholesterol biosynthetic pathway) has been shown to accumulate in ABCG1-deficient cells.33 The cytotoxic and apoptotic properties of the side chain–oxidized oxysterols are well documented.34,35 We show here that exposure of macrophages lacking ABCG1 to 7-ketocholesterol, 25-hydroxycholesterol, and 27-hydroxycholesterol leads to increased apoptosis.

In summary, our results are consistent with the possibility that at least part of the apoptotic effects of a loss of the ABCG1 transporter is due to the accumulation of oxysterols. We also demonstrate that loss of ABCG1 from macrophages results in increased expression of the 2 proapoptotic genes Bid and Bok. Whether such changes in proapoptotic genes are sufficient to contribute to the increased apoptosis, despite an increase in the expression of the antiapoptotic gene Aim, is unknown.

Acknowledgments

We thank A. Fogelman, MD and M. Navab, PhD for providing the Apoe−/− mice. Drs Tarling and Bojanic contributed equally to this study.

Sources of Funding

This study was supported by grants HL086566 and DK063491 (Dr Tangirala), NIH30568 (Drs Lusis and Edwards), HL094322 (Dr Lusis), and NIH68445 (Dr Edwards) from the National Institutes of Health; grants from the Laubisch Fund (Drs Lusis and Edwards); and grants from the Swedish Council and Swedish Brain Power (Dr Bjorkhem).

Disclosures

None.

Footnotes

  • Received on: August 27, 2009; final version accepted on: March 5, 2010.

References

  1. ↵
    Dean M, Hamon Y, Chimini G. The human ATP-binding cassette (ABC) transporter superfamily. J Lipid Res. 2001; 42: 1007–1017.
    OpenUrlAbstract/FREE Full Text
  2. ↵
    Higgins CF, Linton KJ. The ATP switch model for ABC transporters. Nat Struct Mol Biol. 2004; 11: 918–926.
    OpenUrlCrossRefPubMed
  3. ↵
    Kennedy MA, Barrera GC, Nakamura K, Baldan A, Tarr P, Fishbein MC, Frank J, Francone OL, Edwards PA. ABCG1 has a critical role in mediating cholesterol efflux to HDL and preventing cellular lipid accumulation. Cell Metab. 2005; 1: 121–131.
    OpenUrlCrossRefPubMed
  4. ↵
    Tarr PT, Edwards PA. ABCG1 and ABCG4 are coexpressed in neurons and astrocytes of the CNS and regulate cholesterol homeostasis through SREBP-2. J Lipid Res. 2008; 49: 169–182.
    OpenUrlAbstract/FREE Full Text
  5. ↵
    Bojanic DD, Tarr PT, Gale GD, Smith DJ, Bok D, Chen B, Nusinowitz S, Lovgren-Sandblom A, Bjorkhem I, Edwards PA. Differential expression and function of ABCG1 and ABCG4 during development and ageing. J Lipid Res. 2010; 51: 161–181.
    OpenUrl
  6. ↵
    Out R, Hoekstra M, Hildebrand RB, Kruit JK, Meurs I, Li Z, Kuipers F, Van Berkel TJ, Van Eck M. Macrophage ABCG1 deletion disrupts lipid homeostasis in alveolar macrophages and moderately influences atherosclerotic lesion development in LDL receptor-deficient mice. Arterioscler Thromb Vasc Biol. 2006; 26: 2295–2300.
    OpenUrlAbstract/FREE Full Text
  7. ↵
    Wang N, Yvan-Charvet L, Lutjohann D, Mulder M, Vanmierlo T, Kim TW, Tall AR. ATP-binding cassette transporters G1 and G4 mediate cholesterol and desmosterol efflux to HDL and regulate sterol accumulation in the brain. FASEB J. 2008; 22: 1073–1082.
    OpenUrlAbstract/FREE Full Text
  8. ↵
    Tarr PT, Tarling EJ, Bojanic DD, Edwards PA, Baldan A. Emerging new paradigms for ABCG transporters. Biochim Biophys Acta. 2009; 1791: 584–593.
    OpenUrlPubMed
  9. ↵
    Baldan A, Gomes AV, Ping P, Edwards PA. Loss of ABCG1 results in chronic pulmonary inflammation. J Immunol. 2008; 180: 3560–3568.
    OpenUrlAbstract/FREE Full Text
  10. ↵
    Baldan A, Tarr P, Vales CS, Frank J, Shimotake TK, Hawgood S, Edwards PA. Deletion of the transmembrane transporter ABCG1 results in progressive pulmonary lipidosis. J Biol Chem. 2006; 281: 29401–29410.
    OpenUrlAbstract/FREE Full Text
  11. ↵
    Wojcik AJ, Skaflen MD, Srinivasan S, Hedrick CC. A critical role for ABCG1 in macrophage inflammation and lung homeostasis. J Immunol. 2008; 180: 4273–4282.
    OpenUrlAbstract/FREE Full Text
  12. ↵
    Wright JR. Immunoregulatory functions of surfactant proteins. Nat Rev Immunol. 2005; 5: 58–68.
    OpenUrlCrossRefPubMed
  13. ↵
    Terasaka N, Yu S, Yvan-Charvet L, Wang N, Mzhavia N, Langlois R, Pagler T, Li R, Welch CL, Goldberg IJ, Tall AR. ABCG1 and HDL protect against endothelial dysfunction in mice fed a high-cholesterol diet. J Clin Invest. 2008; 118: 3701–3713.
    OpenUrlCrossRefPubMed
  14. ↵
    O'Connell BJ, Denis M, Genest J. Cellular physiology of cholesterol efflux in vascular endothelial cells. Circulation. 2004; 110: 2881–2888.
    OpenUrlAbstract/FREE Full Text
  15. ↵
    Lusis AJ. Atherosclerosis. Nature. 2000; 407: 233–241.
    OpenUrlCrossRefPubMed
  16. ↵
    Baldan A, Pei L, Lee R, Tarr P, Tangirala RK, Weinstein MM, Frank J, Li AC, Tontonoz P, Edwards PA. Impaired development of atherosclerosis in hyperlipidemic Ldlr−/− and Apoe−/− mice transplanted with Abcg1−/− bone marrow. Arterioscler Thromb Vasc Biol. 2006; 26: 2301–2307.
    OpenUrlAbstract/FREE Full Text
  17. ↵
    Lammers B, Out R, Hildebrand RB, Quinn CM, Williamson D, Hoekstra M, Meurs I, Van Berkel TJ, Jessup W, Van Eck M. Independent protective roles for macrophage Abcg1 and Apoe in the atherosclerotic lesion development. Atherosclerosis. 2009; 205: 420–426.
    OpenUrlCrossRefPubMed
  18. ↵
    Out R, Hoekstra M, Habets K, Meurs I, de Waard V, Hildebrand RB, Wang Y, Chimini G, Kuiper J, Van Berkel TJ, Van Eck M. Combined deletion of macrophage ABCA1 and ABCG1 leads to massive lipid accumulation in tissue macrophages and distinct atherosclerosis at relatively low plasma cholesterol levels. Arterioscler Thromb Vasc Biol. 2008; 28: 258–264.
    OpenUrlAbstract/FREE Full Text
  19. ↵
    Ranalletta M, Wang N, Han S, Yvan-Charvet L, Welch C, Tall AR. Decreased atherosclerosis in low-density lipoprotein receptor knockout mice transplanted with Abcg1−/− bone marrow. Arterioscler Thromb Vasc Biol. 2006; 26: 2308–2315.
    OpenUrlAbstract/FREE Full Text
  20. ↵
    Engel T, Kannenberg F, Fobker M, Nofer JR, Bode G, Lueken A, Assmann G, Seedorf U. Expression of ATP binding cassette-transporter ABCG1 prevents cell death by transporting cytotoxic 7beta-hydroxycholesterol. FEBS Lett. 2007; 581: 1673–1680.
    OpenUrlCrossRefPubMed
  21. ↵
    Terasaka N, Wang N, Yvan-Charvet L, Tall AR. High-density lipoprotein protects macrophages from oxidized low-density lipoprotein-induced apoptosis by promoting efflux of 7-ketocholesterol via ABCG1. Proc Natl Acad Sci U S A. 2007; 104: 15093–15098.
    OpenUrlAbstract/FREE Full Text
  22. ↵
    Arai S, Shelton JM, Chen M, Bradley MN, Castrillo A, Bookout AL, Mak PA, Edwards PA, Mangelsdorf DJ, Tontonoz P, Miyazaki T. A role for the apoptosis inhibitory factor AIM/Spalpha/Api6 in atherosclerosis development. Cell Metab. 2005; 1: 201–213.
    OpenUrlCrossRefPubMed
  23. ↵
    Tabas I. Consequences and therapeutic implications of macrophage apoptosis in atherosclerosis: the importance of lesion stage and phagocytic efficiency. Arterioscler Thromb Vasc Biol. 2005; 25: 2255–2264.
    OpenUrlAbstract/FREE Full Text
  24. ↵
    Gautier EL, Huby T, Witztum JL, Ouzilleau B, Miller ER, Saint-Charles F, Aucouturier P, Chapman MJ, Lesnik P. Macrophage apoptosis exerts divergent effects on atherogenesis as a function of lesion stage. Circulation. 2009; 119: 1795–1804.
    OpenUrlAbstract/FREE Full Text
  25. ↵
    Feng B, Yao PM, Li Y, Devlin CM, Zhang D, Harding HP, Sweeney M, Rong JX, Kuriakose G, Fisher EA, Marks AR, Ron D, Tabas I. The endoplasmic reticulum is the site of cholesterol-induced cytotoxicity in macrophages. Nat Cell Biol. 2003; 5: 781–792.
    OpenUrlCrossRefPubMed
  26. ↵
    Myoishi M, Hao H, Minamino T, Watanabe K, Nishihira K, Hatakeyama K, Asada Y, Okada K, Ishibashi-Ueda H, Gabbiani G, Bochaton-Piallat ML, Mochizuki N, Kitakaze M. Increased endoplasmic reticulum stress in atherosclerotic plaques associated with acute coronary syndrome. Circulation. 2007; 116: 1226–1233.
    OpenUrlAbstract/FREE Full Text
  27. ↵
    Yvan-Charvet L, Ranalletta M, Wang N, Han S, Terasaka N, Li R, Welch C, Tall AR. Combined deficiency of ABCA1 and ABCG1 promotes foam cell accumulation and accelerates atherosclerosis in mice. J Clin Invest. 2007; 117: 3900–3908.
    OpenUrlCrossRefPubMed
  28. ↵
    Curtiss LK, Boisvert WA. Apolipoprotein E and atherosclerosis. Curr Opin Lipidol. 2000; 11: 243–251.
    OpenUrlCrossRefPubMed
  29. ↵
    Salvesen GS, Abrams JM. Caspase activation: stepping on the gas or releasing the brakes? lessons from humans and flies. Oncogene. 2004; 23: 2774–2784.
    OpenUrlCrossRefPubMed
  30. ↵
    Boisvert WA, Spangenberg J, Curtiss LK. Treatment of severe hypercholesterolemia in apolipoprotein E-deficient mice by bone marrow transplantation. J Clin Invest. 1995; 96: 1118–1124.
    OpenUrlCrossRefPubMed
  31. ↵
    Out R, Hoekstra M, Meurs I, de Vos P, Kuiper J, Van Eck M, Van Berkel TJ. Total body ABCG1 expression protects against early atherosclerotic lesion development in mice. Arterioscler Thromb Vasc Biol. 2007; 27: 594–599.
    OpenUrlAbstract/FREE Full Text
  32. ↵
    Burgess BL, Parkinson PF, Racke MM, Hirsch-Reinshagen V, Fan J, Wong C, Stukas S, Theroux L, Chan JY, Donkin J, Wilkinson A, Balik D, Christie B, Poirier J, Lutjohann D, Demattos RB, Wellington CL. ABCG1 influences the brain cholesterol biosynthetic pathway but does not affect amyloid precursor protein or apolipoprotein E metabolism in vivo. J Lipid Res. 2008; 49: 1254–1267.
    OpenUrlAbstract/FREE Full Text
  33. ↵
    Yvan-Charvet L, Welch C, Pagler TA, Ranalletta M, Lamkanfi M, Han S, Ishibashi M, Li R, Wang N, Tall AR. Increased inflammatory gene expression in ABC transporter-deficient macrophages: free cholesterol accumulation, increased signaling via toll-like receptors, and neutrophil infiltration of atherosclerotic lesions. Circulation. 2008; 118: 1837–1847.
    OpenUrlAbstract/FREE Full Text
  34. ↵
    Bjorkhem I, Diczfalusy U. Oxysterols: friends, foes, or just fellow passengers? Arterioscler Thromb Vasc Biol. 2002; 22: 734–742.
    OpenUrlAbstract/FREE Full Text
  35. ↵
    Nishio E, Watanabe Y. Oxysterols induced apoptosis in cultured smooth muscle cells through CPP32 protease activation and bcl-2 protein downregulation. Biochem Biophys Res Commun. 1996; 226: 928–934.
    OpenUrlCrossRefPubMed
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  • Impaired Development of Atherosclerosis in Abcg1−/− Apoe−/− Mice
    Elizabeth J. Tarling, Dragana D. Bojanic, Rajendra K. Tangirala, Xuping Wang, Anita Lovgren-Sandblom, Aldons J. Lusis, Ingemar Bjorkhem and Peter A. Edwards
    Arteriosclerosis, Thrombosis, and Vascular Biology. 2010;30:1174-1180, originally published May 19, 2010
    https://doi.org/10.1161/ATVBAHA.110.205617
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