Published online before print November 15, 2007, doi: 10.1161/ATVBAHA.107.156935
© 2008 American Heart Association, Inc.
Integrative Physiology/Experimental Medicine |
Combined Deletion of Macrophage ABCA1 and ABCG1 Leads to Massive Lipid Accumulation in Tissue Macrophages and Distinct Atherosclerosis at Relatively Low Plasma Cholesterol Levels
From the Division of Biopharmaceutics (R.O., M.H., K.H., I.M., V.d.W., R.B.H., Y.W., J.K., T.J.C.V.B., and M.V.E.), Leiden/Amsterdam Center for Drug Research, Gorlaeus Laboratories, Leiden University, The Netherlands; and the Centre d’Immunologie de Marseille Luminy (G.C.), Institut National de la Santé et de la Recherche Médicale, Centre National de la Recherche Scientifique, Université de la Méditerranée, Marseille, France.
Correspondence to R. Out, Division of Biopharmaceutics, Gorlaeus Laboratories, Einsteinweg 55, 2333CC Leiden, The Netherlands. E-mail r.out{at}lacdr.leidenuniv.nl
Abstract
Objective— The purpose of this study was to evaluate the effect of the combined deletion of ABCA1 and ABCG1 expression in macrophages on foam cell formation and atherosclerosis.
Methods and Results— LDL receptor knockout (KO) mice were transplanted with bone marrow from ABCA1/ABCG1 double KO (dKO) mice. Plasma cholesterol levels after 6 weeks of Western-type diet (WTD) feeding were significantly lower in dKO transplanted mice than ABCA1 KO, ABCG1 KO, and control transplanted animals. Extreme foam cell formation was present in macrophages of various tissues and the peritoneal cavity of dKO transplanted animals. Furthermore, severe hypoplasia of the thymus and a significant decrease in CD4-positive T cells in blood was observed. Despite relatively low plasma cholesterol levels dKO transplanted animals developed lesion sizes of 156±19x103 µm2 after only 6 weeks of WTD feeding. Lesions, however, were smaller than single ABCA1 KO transplanted animals (226±30x103 µm2; P<0.05) and not significantly different from single ABCG1 KO (117±22x103 µm2) and WT transplanted mice (112±15x103 µm2).
Conclusions— Macrophage ABCA1 and ABCG1 play a crucial role in the prevention of macrophage foam cell formation, whereas combined deletion only modestly influences atherosclerosis which is associated with an attenuated increase in WTD-induced plasma cholesterol and decreased proinflammatory CD4-positive T cell counts.
To assess the combined role of macrophage ABCA1 and ABCG1 in atherosclerosis, we generated LDL receptor knockout mice that are selectively deficient in ABCA1 and ABCG1 by using bone marrow transfer. Despite relatively low plasma cholesterol levels these mice show distinct atherosclerosis and massive accumulation of lipids in tissue macrophages.
Key Words: ABCA1 • ABCG1 • atherosclerosis • macrophage • transplantation • cholesterol
The transport of excess cholesterol by HDL from macrophages in the periphery back to the liver for catabolism and excretion in bile and feces, called reverse cholesterol transport (RCT), plays an important protective role in the development of atherosclerosis.1 Several ATP-binding cassette (ABC) transporters have been implicated in macrophage lipid metabolism, RCT, and atherosclerosis.2,3 The full-transporter ABCA1 is highly induced in lipid-laden macrophages where it facilitates cellular cholesterol and phospholipid efflux to lipid-poor apoproteins like apoAI or ApoE.4 As cholesterol efflux from macrophages results in decreased cellular lipid accumulation, macrophage ABCA1 expression has been suggested to protect against atherosclerosis. Information on the critical role of macrophage ABCA1 in lesion formation and progression was provided by bone marrow transplantation studies, using mostly LDL receptor (LDLr) KO mice as recipients with ABCA1 KO mice or ABCA1 overexpressor mice as donors leading to the anticipated induction and inhibition of lesion formation, respectively.5–7
More recently, in macrophages a second ABC-transporter, ABCG1, was identified as a transporter for cholesterol from cells to HDL.8–10 Because, similarly to ABCA1, ABCG1 is highly induced in lipid-laden macrophages and able to facilitate efflux of cholesterol from macrophages,8,9 it was anticipated that ABCG1 would add to the protective function of ABCA1 in lesion formation. This hypothesis was strengthened by data from Kennedy et al who showed that administration of a high-fat/high-cholesterol diet to ABCG1 KO mice resulted in the massive accumulation of lipids in tissue macrophages whereas overexpression of human ABCG1 protected the murine tissues from dietary fat-induced lipid accumulation.8 In addition, total-body deficiency of ABCG1 led to increased susceptibility to atherosclerosis.11 However, transplantation studies of ABCG1-deficient bone marrow into LDLr KO mice led to contrasting effects on atherosclerotic lesion formation.12–14 We found a moderate protection of macrophage ABCG1 against atherosclerotic lesion formation (33% to 36%) in male LDLr KO mice fed a Western-type diet containing 15% fat and 0.25% cholesterol for 6 and 12 weeks.13 In contrast, Baldán et al12 observed a decrease in lesion formation of 35% to 40% with ABCG1 KO transplanted female LDLr KO mice fed a diet with 21% fat and 1.25% cholesterol for 12 weeks, whereas Ranalletta et al found in ABCG1 KO transplanted female LDLr KO mice on a 42% fat diet no effect on lesion formation at 7 weeks and a 32% decrease in lesion area in the absence of macrophage ABCG1 at 11 weeks,14 indicating that ABCG1 expression is not protective under their conditions. Furthermore, enhanced ABCG1 expression increases atherosclerosis in LDLr KO mice on a Western diet.15 These contrasting effects of macrophage ABCG1 deficiency on atherosclerotic lesion development can be explained by compensatory upregulation of alternative cholesterol transporters like ABCA1 on high cholesterol loading, thereby masking the primary protective function of ABCG1 in preventing foam cell formation and thus atherosclerotic lesion development. To appreciate the function of ABCG1 it is important to study the effect of ABCG1 deficiency in the absence of compensatory proteins, like ABCA1.
Very recent in vitro evidence suggests that ABCA1 and ABCG1 may act sequentially to remove cellular cholesterol.16,17 In the present study, we compared the effects of combined deletion of ABCA1 and ABCG1 in bone marrow–derived cells with single ABCA1 and ABCG1 deficiency in bone marrow–derived cells to study putative sequential actions of these transporters on lipoprotein metabolism and atherosclerosis in vivo.
Materials and Methods
For detailed methodology, please see the data supplement, available online at http://atvb.ahajournals.org. Briefly, bone marrow transplantations were performed with ABCA1xABCG1 dKO mice as donors and LDLr KO mice as recipients. Plasma lipids were determined by enzymatic colorimetric assays. For histological analysis cryostat sections were routinely stained with Oil red O. Peritoneal leukocytes and white blood cells were analyzed with a hematology cell analyzer. CD4- and CD8-positive lymphocytes were analyzed by flow cytometric analysis. Atherosclerotic lesion areas in Oil red O stained cryostat sections of the aortic root were quantified using the Leica image analysis system.
Results
Effect of Combined Macrophage ABCA1 and ABCG1 Disruption on Plasma Lipid Levels
To investigate the effect of the combined deletion of macrophage ABCA1 and ABCG1 on lipoprotein metabolism and atherogenesis, we used the technique of bone marrow transplantation (BMT) to selectively disrupt ABCA1 and ABCG1 in hematopoietic cells. Bone marrow from WT, ABCA1 KO, ABCG1 KO, and dKO mice was transplanted into LDLr KO mice, which represent an established model for the development of atherosclerosis. Successful reconstitution of recipient mice with donor bone marrow was confirmed by analysis of ABCA1 and ABCG1 transcripts in genomic DNA from bone marrow of transplanted mice as described previously6,13 (data not shown). Deletion of ABCA1, ABCG1, or both ABCA1 and ABCG1 in bone marrow–derived cells did not affect plasma total cholesterol levels when fed regular chow diet, as is shown in Figure 1A. At 8 weeks after bone marrow transplantation, the diet was switched from regular chow diet to WTD (containing 0.25% [w/w] cholesterol and 15% [w/w] fat) to induce atherosclerotic lesion formation. As a result, the total plasma cholesterol levels in both the WT and ABCG1 KO bone marrow transplanted mice increased approximately 3.5-fold (Figure 1B). In agreement with previous studies,6 deletion of ABCA1 in bone marrow cells resulted in lower plasma cholesterol levels on a challenge with the WTD (Figure 1B). Interestingly, more dramatically lower plasma cholesterol levels were found in the dKO transplanted animals fed the WTD (Figure 1B). In the WT and ABCG1 KO transplanted groups the increase in plasma cholesterol levels was mainly attributable to increased VLDL and LDL levels, as is shown in Figure 1C. Also in the mice reconstituted with ABCA1 KO marrow a clear increase in VLDL and LDL levels was observed, although less extensive as compared with WT and ABCG1 KO transplanted animals (Figure 1C). The increase in VLDL and LDL in the dKO transplanted animals, however, was largely attenuated as compared with the other 3 groups (Figure 1C).
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Macrophage ABCA1 and ABCG1 Disruption and Lipid Homeostasis in Tissues
Necropsy of the WTD-fed transplanted LDLr KO mice revealed profound changes in tissues rich in macrophages, including the liver, spleen, lymph nodes, and the Peyer patches. Severe splenomegaly (supplemental Figure IA) and enlargement of lymph nodes and Peyer patches was observed in dKO transplanted mice, whereas no such phenotype was present in WT, ABCA1 KO, and ABCG1 KO transplanted mice (except for the spleen of ABCA1 KO transplanted animals which was also enlarged). Furthermore, compared with the WT, ABCA1 KO, and ABCG1 KO bone marrow transplanted groups, severe hypoplasia of the thymus was observed in the dKO transplanted animals leading to the absence of the thymus. In contrast to WT, ABCA1 KO, or ABCG1 KO transplanted mice, dKO transplanted mice did not gain weight on the WTD (supplemental Figure IB). No obvious difference in food intake was, however, observed between the 4 groups. The relative liver weight was slightly increased when expressed as % of body weight, whereas the spleen weight was also absolutely increased (supplemental Figure IA). The liver (supplemental Figure IC) and spleen (supplemental Figure ID) of dKO transplanted mice contained many pale white foci, whereas the Peyer patches were present as "white marbles" on top of the intestine (supplemental Figure IE).
These morphological changes observed in dKO transplanted mice are consistent with lipid accumulation. In agreement, cryostat sections of the Peyer patches showed massive neutral lipid accumulation, as indicated by Oil red O staining (Figure 2). Furthermore, Oil red O staining was also consistently stronger in the spleen, lymph nodes (not shown), and ileum of the dKO transplanted mice as compared with WT and the single KO transplanted animals (Figure 2). Within the different tissues of the dKO mice fed the WTD, Oil red O staining was mainly observed in macrophage-rich areas like the red pulp of the spleen and Peyer patches. Colocalization of Oil red O staining with MOMA-2 macrophage staining (data not shown) confirmed that in dKO transplanted animals lipid accumulation primarily occurred in macrophages. Furthermore, virtually no Oil red O staining was observed in the spleen, Peyer patches, and ileum of WT, ABCA1 KO, and ABCG1 KO transplanted mice (Figure 2). In ABCA1 KO and ABCG1 KO transplanted mice lipid accumulation was observed in the lungs (Figure 2), which is in agreement with earlier observations.8,18 Furthermore, on WTD feeding the liver of all transplanted groups showed lipid accumulation in hepatocytes, whereas only in the liver of the dKO transplanted animals heavy accumulation of lipids was observed in Kupffer cells (Figure 2).
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Macrophage ABCA1 and ABCG1 Disruption and Peripheral Blood Leukocyte Counts
Because of the proposed role of the thymus in T lymphocyte maturation and the hypoplasia of the thymus observed in dKO transplanted animals, we characterized circulating immune cells in dKO transplanted mice using a hematology analyzer with 5 differential leukocyte population counting (Figure 3A). A trend toward a larger amount of white blood cells was observed in dKO transplanted animals compared with WT, ABCA1 KO, and ABCG1 KO transplanted mice (P=0.065) which was mainly caused by a significant increase in the circulating numbers of neutrophils (P<0.001) and monocytes (P<0.01), which is most likely a protective response to the accumulation of severely lipid-laden macrophages in the different tissues of the dKO transplanted animals. No accumulation of neutrophils was observed in the liver and spleen of dKO transplanted animals (not shown). Interestingly, also a trend to a decrease in the amount of lymphocytes was observed in ABCA1 KO and dKO transplanted mice. To investigate in more detail whether T cell numbers were affected, lymphocytes were harvested from blood and the percentages of CD4+ and CD8+ T cells were determined by flow cytometric analysis (Figure 3B). A significant decrease in the percentage of CD4+ T cells was observed in ABCA1 KO and ABCG1 KO transplanted mice compared with WT transplanted animals (1.3-fold [P<0.05] and 1.5-fold [P<0.05], respectively). Compared with WT, ABCA1 KO, and ABCG1 KO transplanted mice, a more pronounced decrease in the percentage of CD4+ T cells in peripheral blood was detected in dKO transplanted animals (2.6-fold [P<0.001]). Also the absolute number of CD4+ T cells in peripheral blood of dKO transplanted mice was significantly reduced when compared with WT transplanted mice (0.46±0.06 x109/L versus 0.80±0.02 x109/L, respectively; P<0.05). In addition, a small decrease was observed in the CD8+ T cell population in dKO transplanted animals, although this decrease did not reach statistical significance.
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Foam Cell Formation in the Peritoneal Cavity
Lipid loading of macrophages and their transformation into foam cells is considered a decisive process in the formation of atherosclerotic lesions.19 In this context, it is relevant that foam cell formation within the peritoneal cavity parallels their effects on the development of atherosclerotic lesions.20 Therefore, we subsequently examined the effect of combined deletion of macrophage ABCA1 and ABCG1 on the presence of foam cells within the peritoneal cavity by isolating resident peritoneal leukocytes. The collected cells were analyzed using an hematology analyzer. In dKO transplanted animals, lipid-laden peritoneal cells were more numerous as compared with the corresponding populations in WT, ABCA1 KO, and ABCG1 KO transplanted mice (24.1±3.9% (P<0.001), 0.58±0.3%, 4.6±0.6%, and 0.70±0.2%, respectively; Figure 4B). The collected cells were cytospun and stained for lipids with Oil red O to visualize foam cell formation (See Figure 4A for representative photomicrographs). These data confirm the essential role for the combined action of ABCA1 and ABCG1 in maintaining macrophage lipid homeostasis.
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Effect of Macrophage ABCA1 and ABCG1 Disruption on Cellular Cholesterol Efflux
To test whether the lipid accumulation in the cells of animals transplanted with dKO bone marrow was attributable to a combined defect in the transport of cholesterol out of the cells to exogenous lipid acceptors, cholesterol efflux to apoAI (supplemental Figure IIA) and HDL (supplemental Figure IIB) was determined in bone marrow–derived macrophages. Compared with control cells expressing ABCA1 and ABCG1, the percentage of efflux toward apoAI from cells lacking ABCG1 was not different, whereas cells lacking ABCA1 or the combination of ABCA1 and ABCG1 led to a highly diminished efflux to apoAI (both 
80% decrease, P<0.01 and P<0.001, respectively; supplemental Figure IIA). The efflux toward HDL from cells lacking ABCG1 was not significantly different compared with control cells, whereas the absence of ABCA1 showed a slight but significant 10% decrease (P<0.05; supplemental Figure IIB). Compared with control cells and cells lacking either ABCA1 or ABCG1, a highly significant 34% reduction in efflux toward HDL in cells lacking both ABCA1 and ABCG1 was observed (P<0.001; supplemental Figure IIB).
Effect of Macrophage ABCA1 and ABCG1 Disruption on Atherosclerotic Lesion Formation
To investigate the importance of macrophage ABCA1 and ABCG1 for atherosclerotic lesion development, we analyzed the aortic root of the 4 transplanted groups after 6 weeks of WTD feeding (Figure 5). Representative photomicrographs of the aortic root of the 4 differentially transplanted groups are shown in Figure 5A. After 6 weeks on the WTD atherosclerotic lesion size in the aortic root was 1.13±0.15x105 µm2 in WT (n=18), 2.28±0.31x105 µm2 (n=17) in ABCA1 KO, 1.18±0.22x105 µm2 (n=15) in ABCG1 KO, and 1.58±0.19x105 µm2 (n=16) in dKO transplanted LDLr KO mice (Figure 5B). It is striking that, despite the relatively low plasma cholesterol levels observed in the LDLr KO mice transplanted with double deficient ABCA1/ABCG1 macrophages, still lesions develop in these mice (Figure 5C). Lesions in the dKO transplanted animals, however, were 1.4-fold smaller (P<0.05) as compared with single ABCA1 deletion in macrophages. No differences in lesion composition were observed. Lesions are mainly composed of macrophages (>90%) as determined by MOMA-2 macrophage staining (data not shown).
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Discussion
The induction of persistent hypercholesterolemia to levels higher than approximately 300 mg/dL is required for the development of experimental atherosclerosis in LDLr KO mice.21 In the current study we show that the combined specific deletion of both ABCA1 and ABCG1 in bone marrow–derived cells of LDLr KO mice led to the development of atherosclerotic lesions, despite cholesterol levels of 
250 mg/dL. In addition, massive lipid accumulation was found in peritoneal macrophages as well as macrophage-rich tissues like the liver and spleen, leading to severe splenomegaly and decreased body weight, which is often associated with splenomegaly.22,23 Furthermore, lymph nodes and Peyer patches were larger in size. No such phenotype was evident in the ABCA1 and ABCG1 single knockout or wild-type transplanted animals.
The dramatic lipid accumulation in macrophages lacking both ABCA1 and ABCG1 confirm the important role of these proteins in lipid metabolism in macrophages.16,17,24 In vitro studies showed that the transfer of lipids to apoAI mediated by ABCA1 activity is sufficient to generate an efficient acceptor for ABCG1-mediated cholesterol efflux, which implies that ABCA1 cooperatively works with ABCG1 in cholesterol transport.16,17 Furthermore, during the preparation of our manuscript, Wang et al24 described the role of ABCA1, ABCG1, and SR-BI in macrophage-specific in vivo reverse cholesterol transport. By using partial combined knockdown of ABCA1 and ABCG1 in J774 cells it was concluded that ABCA1 and ABCG1 cooperatively contribute to macrophage RCT in vivo, although there was a substantial level of RCT remaining in these C57BL/6J mice. The observed dramatic enhancement of macrophage foam cell formation as a result of combined total deletion of ABCA1 and ABCG1 and the highly reduced efflux capacity toward HDL as compared with single deletion of ABCA1 and ABCG1 in this study would indeed favor such a model.
Several recent studies have provided conflicting results on the role of macrophage ABCG1 in the development of atherosclerosis in mice. We have shown that macrophage deletion of ABCG1 led to a modest, but significant increase in atherosclerotic lesion development in LDLr KO mice,13 whereas 2 other studies observed a decrease in atherosclerotic lesion development in LDLr KO mice transplanted with ABCG1 KO bone marrow.12,14 In the latter studies, lipid loading of the macrophages, especially at high plasma cholesterol levels, might have led to compensatory upregulation of ie, ABCA1 thereby masking the primary protective effect of ABCG1 in cholesterol efflux. More recently, we proposed a model in which the in vivo effects of macrophage ABCG1 deficiency on atherosclerosis are a function of the serum cholesterol levels whereby above 900 mg/dL compensatory mechanisms mask the primary effect of ABCG1 deletion on atherosclerosis.11 In agreement, in this study at plasma cholesterol levels close to the switch point value of 900 mg/dL, no effect of macrophage ABCG1 deficiency on atherosclerotic lesion development was observed. Also in agreement with earlier studies, transplantation of ABCA1 KO bone marrow into LDLr KO mice resulted in enhanced atherosclerotic lesion development.5,6 Although combined deletion of ABCA1 and ABCG1 leads to a dramatic enhancement of macrophage foam cell formation, in the dKO transplanted animals we could not demonstrate an additive effect of ABCA1 and ABCG1 deletion in macrophages on atherosclerotic lesion development, which might be caused by the lower plasma cholesterol levels observed in the dKO mice as compared with WT, ABCA1 KO, and ABCG1 KO transplanted animals. Getz and Reardon already concluded that persistent hypercholesterolemia to levels higher than approximately 300 mg/dL is required for the development of experimental atherosclerosis in mice.21 So it is remarkable that at plasma cholesterol levels of 250 mg/dL combined deletion of both ABCA1 and ABCG1 in bone marrow–derived cells of LDLr KO mice does lead to the development of highly significant atherosclerotic lesions, suggesting that the combined presence of ABCA1 and ABCG1 does protect against atherogenesis.
The attenuated increase in plasma cholesterol levels of ABCA1 KO transplanted and more extremely in the dKO transplanted mice fed a WTD maybe the consequence of an altered immune system. We observed severe hypoplasia of the thymus in the dKO transplanted animals, likely resulting in the decrease in CD4-positive T cells in the circulation. In line with our present data, T- and B-cell deficiency is associated with an attenuated increase in WTD-induced plasma cholesterol levels in LDLr KO mice.25 Furthermore, reconstitution of LDLr KO mice with bone marrow from mice lacking LIGHT or lymphotoxin, potent proinflammatory ligands expressed on T cells, leads to a reduction in CD4-positive and CD8-positive T cells and a corresponding reduction in plasma cholesterol levels.26 Thus, the reduced plasma cholesterol levels observed in the dKO transplanted mice are likely a direct effect of the decrease in CD4-positive T cells in these mice. Adaptive immunity, in particular T cells, is also highly involved in atherogenesis (reviewed by Hansson27). CD4-positive T cells are the predominant T cell subset in atherosclerotic lesions in ApoE- and LDLr KO mice.28 Furthermore, CD4-positive T cell deficiency results in reduced atherosclerosis in these mice,29–31 whereas transfer of CD4-positive T cells aggravated atherosclerosis.32 In addition to the lower plasma cholesterol levels, the decrease in CD4-positive T cells might thus also have contributed to the reduced atherosclerosis in the ABCA1/ABCG1 dKO transplanted mice as compared with single ABCA1 KO transplanted animals.
In conclusion, our results indicate that the combined deletion of macrophage ABCA1 and ABCG1 leads to only modest atherosclerosis which is associated with relatively low plasma cholesterol levels and decreased proinflammatory CD4-positive T cell counts. Despite the low plasma cholesterol levels ABCA1 and ABCG1 deficiency resulted in heavy lipid accumulation in tissue macrophages, providing clear evidence for the combined protective function of macrophage ABA1 and ABCG1 in the prevention of macrophage foam cell formation.
Acknowledgments
The authors belong to the European Vascular Genomics Network (http://www.evgn.org), a Netwerk of Excellence supported by the European Community’s Sixth Framework Program for research Priority I (Life Sciences, Genomics, and Biotechnology for Health; contract LSHM-CT-2003-503254).
Sources of Funding
This work was supported by The Netherlands Heart Foundation (Grants 2003B134 (R.O.), 2001T041 (M.V.E.), and 2001B043 (J.K.), and The Netherlands Organization for Scientific Research (VIDI Grant 917.66.301 [M.V.E.]).
Disclosures
None.
Footnotes
Original received August 23, 2007; final version accepted November 7, 2007.
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