Published online before print July 3, 2008, doi: 10.1161/ATVBAHA.108.168542
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© 2008 American Heart Association, Inc.
Integrative Physiology/Experimental Medicine |
Overexpression of Human ABCG1 Does Not Affect Atherosclerosis in Fat-Fed ApoE-Deficient Mice
From the Department of Pathology and Laboratory Medicine (B.B., K.N., J.C., V.H.-R., G.T., A.W., J.F., J.D., D.B., T.T., G.O., C.W.), Child and Family Research Institute, University of British Columbia, Vancouver, Canada; ICapture Centre (L.M., B.M.), University of British Columbia, Vancouver, Canada; the Department of Pediatrics (B.C., R.D., S.I.), Child and Family Research Institute, University of British Columbia, Vancouver, Canada; and the Department of Clinical Pharmacology (D.L.), University of Bonn, Germany.
Correspondence to Dr Cheryl L. Wellington, Department of Pathology and Laboratory Medicine, University of British Columbia, 980 West 28th Avenue, Vancouver, British Columbia, Canada. E-mail Cheryl{at}cmmt.ubc.ca
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Abstract |
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Objective— The purpose of this study was to evaluate the effects of whole body overexpression of human ABCG1 on atherosclerosis in apoE–/– mice.
Methods and Results— We generated BAC transgenic mice in which human ABCG1 is expressed from endogenous regulatory signals, leading to a 3- to 7-fold increase in ABCG1 protein across various tissues. Although the ABCG1 BAC transgene rescued lung lipid accumulation in ABCG1–/– mice, it did not affect plasma lipid levels, macrophage cholesterol efflux to HDL, atherosclerotic lesion area in apoE–/– mice, or levels of tissue cholesterol, cholesterol ester, phospholipids, or triglycerides. Subtle changes in sterol biosynthetic intermediate levels were observed in liver, with chow-fed ABCG1 BAC Tg mice showing a nonsignificant trend toward decreased levels of lathosterol, lanosterol, and desmosterol, and fat-fed mice exhibiting significantly elevated levels of each intermediate. These changes were insufficient to alter ABCA1 expression in liver.
Conclusions— Transgenic human ABCG1 does not influence atherosclerosis in apoE–/– mice but may participate in the regulation of tissue cholesterol biosynthesis.
We developed transgenic mice expressing functional human ABCG1. Elevated ABCG1 levels did not affect plasma lipids, macrophage cholesterol efflux, atherosclerotic lesion area in apoE–/– mice, or levels of tissue cholesterol, cholesterol ester, phospholipids, or triglycerides. Transgenic ABCG1 was, however, associated with altered sterol intermediate levels in liver.
Key Words: ABCG1 • cholesterol • atherosclerosis • cholesterol intermediate
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Introduction |
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The ABCA and ABCG classes of ATP-binding cassette transporters form a network of proteins that regulate high-density lipoprotein (HDL) metabolism, reverse cholesterol transport, atherosclerosis, and cell and body sterol homeostasis.1–3 ABCA1 catalyzes cholesterol and phospholipid efflux to lipid-free apolipoprotein A-I (apoA-I) to form pre-β HDL.4–7 Deficiency of human ABCA1 results in Tangier Disease, characterized by virtually undetectable plasma HDL, tissue deposition of cholesterol esters (CE), and increased atherosclerosis risk.6–8 Conversely, selective overexpression of ABCA1 in mice increases plasma HDL levels9,10 and reduces atherosclerotic lesion progression.11,12 Plasma HDL levels are regulated largely by liver and intestinal ABCA1.13,14 Macrophage ABCA1 makes only a minor contribution to plasma HDL but plays a key role in atherosclerosis.12,15–17
ABCG1 is the founding member of the ABCG subclass of ABC transporters.18 In vitro and in vivo studies have shown that ABCG1 mediates cholesterol efflux to HDL and other phospholipid-enriched but not lipid-free apolipoproteins.19–27 In cells, ABCG1 also redistributes cellular cholesterol to cholesterol oxidase-accessible membrane domains.28 Both the cholesterol efflux and redistribution activities are present when ABCG1 is selectively overexpressed in cells, showing that ABCG1 functions as a homodimer.19,26–28 In vivo, ABCG1 is broadly expressed20,20,26,29 and is induced by lipid loading as well as by Liver X Receptor/Retinoic Acid Receptor (LXR/RXR) agonists.19,30–33 Under basal conditions, liver ABCG1 is 76-fold more abundant in Kupffer cells and 27-fold more abundant in endothelial cells compared to hepatocytes.32 A high-cholesterol diet specifically increases hepatocyte ABCG1 expression,32 suggesting that hepatocyte ABCG1 is sensitive to regulatory pathways important for tissue lipid homeostasis. Macrophage ABCG1 is also induced by lipid loading34 and is upregulated in macrophages isolated from individuals with Tangier Disease.35 Conversely, cholesterol efflux from lipid-laden macrophages suppresses ABCG1 expression,34 and antisense inhibition of ABCG1 reduces macrophage cholesterol efflux in a dose-dependent manner.19,34 Phospholipid-enriched apoA-I particles generated by selective ABCA1 activity are efficient lipid acceptors for ABCG1,27,36 suggesting that sequential ABCA1 and ABCG1 activity may provide functional synergy for cholesterol efflux in cells such as macrophages where they are coexpressed.
In vivo, ABCG1 deficiency causes accumulation of neutral lipids within tissues, particularly in response to a high-fat high-cholesterol diet.20,20,24 Conversely, transgenic ABCG1 has been reported to protect tissues from dietary-induced lipid deposition.20 Although adenoviral-mediated overexpression of ABCG1 in the liver was reported to reduce plasma HDL-C levels,37 it has subsequently been demonstrated that plasma lipid levels are unaffected by loss or gain of ABCG1, on otherwise wild-type as well as on apoE–/– or LDLR–/– genetic backgrounds.20,21,23,24,38
Five studies have addressed the impact of ABCG1 deficiency on atherosclerosis. Out et al reported a 33% to 36% increase in aortic root lesion area when ABCG1–/– bone marrow was transplanted into LDLR–/– recipients and fed a HFD of 15% fat, 0.25% cholesterol.23 This group also reported that complete ABCG1 deficiency led to a 1.9-fold increase in aortic lesion area in animals fed a HFD of 15% fat, 1% cholesterol, and 0.5% cholate from 12 to 24 weeks of age.24 In contrast, Baldán and colleagues reported a significant 40% or 35% decrease in lesion area in aortic root and en face preparations, respectively, in LDLR–/– recipients transplanted with ABCG1–/– donors after 16 weeks on a HFD of 21% fat, 1.25% cholesterol.20 Ranalletta et al also observed decreased lesion area on transplanting ABCG1–/– bone marrow into LDLR–/– recipients after feeding a HFD containing 21.2% fat, 0.2% cholesterol for 11 weeks.21 Finally, LDLR–/– mice transplanted with ABCA1/ABCG1 doubly-deficient macrophages exhibit increased aortic root lesions, but little effect was observed with singly ABCG1-deficient animals.25 These studies show that ABCG1 deficiency can impact atherosclerosis and indicate that the experimental conditions used may dictate whether loss of ABCG1 is functionally neutral, pro-, or antiatherogenic.
Recently, Basso et al developed a BAC Tg model of murine ABCG1 overexpression and observed a 39% and 52% increase in lesion area in en face and cross sectional analyses, respectively, when crossed to LDLR–/– mice and fed a HFD of 21.2% fat and 0.2% cholesterol for 12 weeks.22 Increased lesions were associated with increased levels of the proinflammatory markers tumor necrosis factor (TNF)-
and MCP-1, arguing for a proatherogenic and proinflammatory role of ABCG1, which presumably overrides the enhanced efflux to HDL also observed in macrophages from this model.
We generated a BAC Tg animal model in which human ABCG1 is expressed under the control of its endogenous regulatory signals, similar to the model previously reported by Kennedy et al.20 Although our mice have robust expression of functional human ABCG1 in appropriate tissues, they exhibit no change in plasma lipid levels, macrophage cholesterol efflux to HDL, or atherosclerotic lesion area when crossed to apoE–/– mice and fed a HFD of 21.2% fat and 0.2% cholesterol for 14 weeks. The only changes observed in these mice were alterations in the levels of the sterol intermediates lanosterol, lathosterol, and desmosterol in liver, which were significantly increased in fat-fed animals. These observations suggest that, in vivo, selective overexpression of ABCG1 is insufficient to influence atherogenesis in apoE–/– mice, but may rather play a role in tissue cholesterol homeostasis.
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Methods |
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For detailed methodology, please see the supplemental materials (available online at http://atvb.ahaournals.org). Briefly, ABCG1 BAC Tg mice were generated using a 141-kb BAC containing the complete human ABCG1 sequence with no other genes and maintained on a C57Bl/6 background. ABCG1 expression was evaluated by species-specific quantitative real-time polymerase chain reaction (QRT-PCR) and Western blotting from crude membrane fractions prepared from tissues or thioglycollate-elicited primary peritoneal macrophages. Transgene function was assessed by breeding to ABCG1–/– mice and evaluating lung phenotypes by oil red O staining and lipid quantification. To assess atherosclerosis, ABCG1 BAC Tg mice were bred to apoE–/– animals and fed a HFD of 21.2% fat, and 0.2% cholesterol for 14 weeks, followed by aortic root lesion quantification using 4 sections per mouse. Isotopic 3H-cholesterol efflux assays were performed on thioglycollate-elicited primary peritoneal macrophages. Plasma lipids were analyzed by enzymatic colorimetric assays, and plasma apoE levels were determined by Western blot. Tissue lipids were quantified either by high-performance liquid chromatography (HPLC) or by GC-MS.
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Results |
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Human ABCG1 Is Appropriately Expressed in ABCG1 BAC Tg Mice
To develop a physiologically accurate mouse model of human ABCG1 overexpression, we microinjected a 141-kb BAC containing the complete human ABCG1 gene into murine oocytes. Three independent congenic C57Bl/6 lines were developed, and the line with highest human ABCG1 expression, estimated at approximately 9 BAC copies, was used in this study. QRT-PCR analysis confirmed expression of human ABCG1 mRNA across multiple tissues (supplemental Figure I). Western blot analysis of crude membrane fractions revealed that ABCG1 protein levels in ABCG1 BAC Tg mice are elevated in liver, lung, and spleen, and are increased further after 14 weeks on a HFD (Figure 1). In peritoneal macrophages, human ABCG1 mRNA is responsive to LXR stimulation or cholesterol loading, similar to murine Abcg1 mRNA (Figure 2A). We observed approximately 7-fold more ABCG1 protein in macrophages from ABCG1 BAC Tg mice 2 hours after harvesting (Figure 2B), but after 48 hours ABCG1 protein levels decreased to approximately 2-fold over that observed in control macrophages (Figure 2B). These increases in ABCG1 expression were maintained in apoE–/– mice (not shown). Because the ABCG1 antibody used in these experiments may not recognize murine and human ABCG1 equally well, it is not possible to determine the contribution of human ABCG1 to total ABCG1 protein levels in our mice.
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Human ABCG1 Functionally Compensates for Murine ABCG1 in Tissue Lipid Homeostasis
To confirm functionality of the human ABCG1 transgene, we bred ABCG1 BAC Tg mice onto an ABCG1–/– backgound (Figure 3A). Humanized ABCG1 BAC Tg mice and ABCG1–/– and ABCG1+/– controls were fed a high-fat high-cholesterol diet (21% fat, 1.25% cholesterol) for 6 weeks, followed by histological and quantitative assessments of lipid accumulation in lung. Expression of human ABCG1 completely prevented neutral lipid accumulation in ABCG1–/– lung (Figure 3B). HPLC was then used to quantify lung lipids from wild-type, ABCG1 BAC Tg, ABCG1–/–, and humanized ABCG1 mice after an 8-week exposure to the high-fat high-cholesterol diet (Figure 3C and 3D). Compared to wild-type controls, selective overexpression of ABCG1 did not alter the levels of any lipids, unlike the results reported by Kennedy et al20 who found that excess ABCG1 protected from diet-induced tissue lipid accumulation. Compared to wild-type controls, ABCG1–/– mice exhibited significantly elevated CE (Figure 3C) and significantly decreased TG levels (Figure 3D), and both of these phenotypes were completely rescued by expression of human ABCG1 in lieu of endogenous murine ABCG1. Lung lipid levels in rescued mice were not statistically different than those in WT or Tg animals. These data confirm that the human ABCG1 transgene functionally compensates for murine ABCG1 in vivo.
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Notably, ABCG1 BAC Tg mice fed a HFD (21.2% fat, and 0.2% cholesterol) for 14 weeks also showed no protection from lipid accumulation in lung and liver compared to wild-type controls, whether assessed by oil red O histological analysis or by HPLC quantitation of lipids (supplemental Figure II). Our data show that transgenic ABCG1 is not sufficient to protect from diet-induced lipid accumulation in tissues but can restore tissue lipid homeostasis to baseline levels in the absence of endogenous ABCG1.
Overexpression of Human ABCG1 Does Not Influence Plasma Lipid or ApoE Levels
ABCG1 BAC transgenic mice were then crossed to apoE–/– mice and fed a HFD (21.2% fat, and 0.2% cholesterol) for 14 weeks. No differences in plasma TC, HDL-cholesterol, or TG were observed between control and ABCG1 BAC Tg mice irrespective of apoE genotype (supplemental Figure III). Because plasma apoE levels have been reported to be increased in ABCG1–/– mice,21 we tested the converse possibility that overexpression of ABCG1 may lead to decreased apoE levels. On a chow diet, there were no differences in plasma apoE levels in ABCG1 BAC Tg mice relative to wild-type controls. On the HFD, we observed a nonsignificant trend toward reduced apoE levels in ABCG1 BAC Tg mice (supplemental Figure IV).
ABCG1 BAC Tg Mice Exhibit no Change in Atherosclerosis, Macrophage Cholesterol Efflux, or ABCA1 Expression
Examination of aortic roots in apoE–/– mice with and without transgenic ABCG1 after 14 weeks on the HFD revealed no differences in lesion area (P<0.05, n=9 to 11 mice; Figure 4A and 4B) or complexity (not shown). Thioglycollate-elicited peritoneal macrophages were then isolated from animals of all 4 genotypes and used for cholesterol efflux assays to HDL. Despite clear overexpression of ABCG1 in macrophages (Figure 2), we found that transgenic ABCG1 had no effect on cholesterol efflux to apoA-I or HDL2/3 under our experimental conditions (P<0.05, n=triplicate measurements from at least 9 mice/genotype; Figure 4C). ABCA1 mRNA levels were unchanged in macrophages cultured from wild-type and ABCG1 BAC Tg mice (1.00±0.295 versus 0.930±0.352, n=3 mice measured in triplicate). This lack of elevated cholesterol efflux activity may explain the lack of effect on atherosclerosis in ABCG1 BAC Tg mice.
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Overexpression of ABCG1 Is Associated With Altered Levels of Sterol Biosynthetic Intermediates in Liver
GC:MS was used to sensitively quantify the levels of cholesterol, sterol intermediates, and metabolites in liver and lung of chow- and fat-fed wild-type and ABCG1 BAC Tg mice. Compared to controls, cholesterol levels were unchanged in ABCG1 BAC Tg liver on chow, but significantly increased after 14 weeks on a HFD (Figure 5A). On chow, transgenic ABCG1 was associated nonsignificantly decreased levels of the cholesterol intermediates lathosterol, lanosterol, and desmosterol in liver (Figure 5B). On a HFD, however, the levels of these intermediates were significantly elevated in ABCG1 BAC Tg liver (Figure 5C). No significant changes in sterol intermediate levels were found in lung (not shown). ABCG1 overexpression did not affect the levels of cholesterol metabolites 7-hydroxycholesterol, 24(S)-hydroxycholesterol, 27-hydroxycholesterol, nor cholestanol (Figure 5B and 5C).
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Altered Desmosterol Levels in ABCG1 BAC Tg Mice Do Not Influence ABCA1 Expression
Desmosterol, an immediate precursor to cholesterol, has been reported to act as an endogenous LXR ligand.39 We investigated whether altered desmosterol levels affected ABCA1 expression, as a known LXR target. In both chow- and fat-fed mice, ABCA1 mRNA levels were slightly reduced in ABCG1 BAC Tg livers compared to WT controls, but these trends were not significant (WT chow: 1.025±0.132 versus ABCG1 chow: 0.917±0.125, P=0.5731; WT fat: 1.270±0.141 versus ABCG1 chow: 0.985±0.081, P=0.130, n=4). ABCA1 protein levels were indistinguishable in wild-type and ABCG1 BAC Tg mice, regardless of diet (not shown), indicating that the changes in desmosterol levels observed in our ABCG1 model are insufficient to alter ABCA1 expression.
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Discussion |
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This study was designed to determine the impact of whole-body overexpression of human ABCG1 on atherosclerosis. ABCG1–/– mice clearly exhibit impaired cholesterol efflux to HDL and accumulate tissue lipids when challenged with a high-fat high-cholesterol diet.20,21,24 However, the question of whether loss or gain of ABCG1 is pro- or antiatherogenic remains to be fully resolved. Our ABCG1 BAC Tg mice exhibit elevated levels of functional ABCG1 protein, but this does not affect atherosclerotic lesion area in fat-fed apoE–/– mice. Our results differ from those of Basso et al, who reported that selective overexpression of murine ABCG1 led to increased macrophage efflux to HDL and increased lesion area in LDLR–/– mice fed the identical HFD diet used here.22 It is possible that differences in BAC copy number, the model of atherosclerosis, or other variations in experimental methodology may explain these discrepancies. The differences in our findings are unlikely to be attributable to species differences given that ABCG1 exhibits 96.8% amino acid identity between mice and humans, which is significantly higher than the average of approximately 80% identify for proteins across these species.
It is possible that the robust pathogenesis in the apoE–/– model may overwhelm the impact of selective ABCG1 overexpression on atherosclerosis. It is also possible that effects of ABCG1 on lesion area may require functional apoE. Additional experiments using bone marrow transplants may further clarify the role of selective ABCG1 overepression in atherosclerosis. Notably, Out et al observed that TC levels correlate with the impact of ABCG1 deficiency on lesion area.24 In our study, TC levels were approximately 700 mg/dL, perhaps a range where ABCG1 has little effect on atherosclerosis.
To further investigate why no change in lesion area was observed in our study, we performed several experiments to reveal the impact of elevated ABCG1 levels in our mice. Although an established assay for ABCG1 activity is cholesterol efflux to HDL,19,26,27 and primary macrophages from ABCG1–/– mice are clearly impaired in this activity, primary peritoneal macrophages cultured from our ABCG1 BAC Tg mice exhibited no increase in cholesterol efflux to HDL2/3 compared to wild-type macrophages, regardless of apoE genotype, despite abundant expression of functional human ABCG1. Although there is no question that ABCG1 deficiency compromises cholesterol efflux to HDL, whether excess ABCG1 facilitates efflux is more challenging. Many studies that assay excess ABCG1 have used transfected cells that permit expression of supraphysiological levels of ABCG1, and the impact on cholesterol efflux to HDL in these studies is subtle if expressed as fold increase in ABCG1-expressing versus control cells, rather than raw increases in % efflux. Furthermore, despite containing approximately 30 copies of a murine ABCG1 BAC in the mice developed by Basso et al, ABCG1 mRNA and protein levels were modestly increased by at most 2.7- and 1.5-fold, respectively, and the maximum increase in cholesterol efflux observed was only 1.4-fold.22 Observations in both cellular and animal models therefore suggest that cholesterol efflux to HDL may be a relatively insensitive assay of excess ABCG1 activity. BecauseABCA1 and ABCG1 have been reported to act sequentially to remove cellular cholesterol,27,36 it is also possible that the impact of excess ABCG1, especially in vivo, may be best observed when ABCA1 is also overexpressed, to provide the substrates for ABCG1-mediated efflux. Finally, we noted that ABCG1 protein levels decline rapidly after primary peritoneal macrophages are plated (Figure 2), so it is possible that little ABCG1 overexpression remained by the time our efflux assays were completed.
ABCG1 BAC Tg mice exhibited a nonsignificant trend toward decreased levels of cholesterol biosynthetic intermediates in the livers of chow-fed animals, and a significant increase in cholesterol, lathosterol, lanosterol, and desmosterol in fat-fed liver when quantified by mass spectrometry as a function of tissue dry weight. Although these findings are of potential interest because of the recent demonstration that desmosterol can act as an endogenous LXR ligand,39 we found that these changes were insufficient to alter liver ABCA1 levels. Understanding whether cholesterol intermediate levels are altered in ABCG1-deficicient mice may shed further light on the potential role for ABCG1 in sterol biosynthesis. Notably, lung did not show these trends despite our demonstration of the functionality of the human ABCG1 transgene in this tissue. This suggests that either the role of ABCG1 in lipid homeostasis may be highly tissue-specific, or that it may only be revealed under conditions of high dietary cholesterol.
Despite confirmation of abundant overexpression of functional ABCG1 in our transgenic model, the in vivo effect of transgenic ABCG1 can be subtle. In our study, selective overexpression of ABCG1 had no effect on atherosclerosis in the apoE–/– model or macrophage cholesterol efflux to HDL, but may be associated with changes in hepatic sterol biosynthesis in response to dietary fat. Additional studies will be required to understand the mechanisms by which ABCG1 participates in tissue lipid homeostasis.
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Acknowledgments |
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We thank Anja Kerksiek and Silvia Friedrichs for technical assistance in GC and GC-MS analysis.
Sources of Funding
B.L.B. is supported by the British Columbia Child and Family Research Institute; VHR by a postdoctoral fellowship from the Canadian Institutes of Health Research (CIHR); CLW by a CIHR New Investigator Salary Award and operating funding from the Heart and Stroke Foundation of Canada.
Disclosures
None.
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Footnotes |
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Original received January 3, 2008; final version accepted June 23, 2008.
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