Erythropoietin Suppresses the Formation of Macrophage Foam Cells
Role of Liver X Receptor α
Background— In addition to the hematopoietic effect of erythropoietin, increasing evidence suggests that erythropoietin also exerts protective effects for cardiovascular diseases. However, the role of erythropoietin and its underlying mechanism in macrophage foam cell formation are poorly understood.
Methods and Results— Compared with wild-type specimens, erythropoietin was increased in atherosclerotic aortas of apolipoprotein E–deficient (apoE−/−) mice, mainly in the macrophage foam cells of the lesions. Erythropoietin levels in culture medium and macrophages were significantly elevated in response to oxidized low-density lipoprotein in a dose-dependent manner. Furthermore, erythropoietin markedly attenuated lipid accumulation in oxidized low-density lipoprotein–treated macrophages, a result that was due to an increase in cholesterol efflux. Erythropoietin treatment significantly increased ATP-binding cassette transporters (ABC) A1 and ABCG1 mRNA and protein levels without affecting protein expression of scavenger receptors, including scavenger receptor-A, CD36, and scavenger receptor-BI. The upregulation of ABCA1 and ABCG1 by erythropoietin resulted from liver X receptor α activation, which was confirmed by its prevention on expression of ABCA1 and ABCG1 after pharmacological or small interfering RNA inhibition of liver X receptor α. Moreover, the erythropoietin-mediated attenuation on lipid accumulation was abolished by such inhibition. Finally, reduced lipid accumulation and marked increase in ABCA1 and ABCG1 were demonstrated in erythropoietin-overexpressed macrophages.
Conclusion— Our data suggest that erythropoietin suppresses foam cell formation via the liver X receptor α–dependent upregulation of ABCA1 and ABCG1.
Received April 30, 2009; accepted February 25, 2010.
Complications of atherosclerosis are the leading causes of death in Western societies. Accumulation of macrophage foam cells in atherosclerotic lesions is the hallmark of early-stage atherosclerosis.1,2 The formation of foam cells is due mainly to uncontrolled uptake of modified low-density lipoprotein (LDL by macrophages, resulting in excessive lipoprotein-derived cholesterol accumulation inside the cells.2,3 It has been established that several types of macrophage scavenger receptors (SRs) such as class A SRs (SR-AI, SR-AII, SR-AIII) and class B SRs (SR-BI, SR-BII, CD36) are responsible for the internalization of oxidized LDL (oxLDL) that promotes the cellular accumulation of cholesterol.1–3 Among them, SR-A and CD36 account for >75% of oxLDL uptake.4–6 In contrast, the cholesterol efflux of macrophages is mediated through SR-BI and ATP-binding cassette (ABC) transporters, including ABCA1 and ABCG1.7–9 Thus, the cellular lipid level and foam cell formation are dynamically regulated by these SRs and cholesterol efflux transporters. In fact, several groups have reported that cytokines secreted from macrophage-derived foam cells such as tumor necrosis factor-α, interleukin-4, or interleukin-10 regulate the expression of SRs or cholesterol efflux transporters in macrophages and result in the progression or regression of atherosclerosis.10–13
Clinical Perspective on p 1837
Erythropoietin, a 165–amino acid glycoprotein hematopoietic hormone, is required for proliferation, differentiation, and survival of erythroid precursors.14,15 Erythropoietin and its receptor, erythropoietin receptor (EPOR), are expressed in a variety of tissues, including the vascular system.16–18 Beside its role in erythropoiesis, increasing evidence suggests that erythropoietin exerts several extrahematopoietic protective effects for cardiovascular diseases. For example, erythropoietin protects cardiomyocytes from apoptosis, reduces infarction size, and preserves cardiac function during myocardial ischemia/reperfusion.19,20 Erythropoietin also enhances reendothelialization and inhibits neointimal formation after vascular injury.21 Additionally, erythropoietin suppresses the progression of atherosclerosis and significantly decreases the ratio of plasma LDL cholesterol to high-density lipoprotein cholesterol in chronic kidney disease patients.22–24 However, little is known about the interaction between erythropoietin and foam cells. To this end, further investigation delineating the expression and the mechanisms of erythropoietin on foam cell formation is warranted.
The purpose of this study was to investigate the expression of erythropoietin in atherosclerotic lesions of apolipoprotein E–deficient (apoE−/−) mice and the effect of oxLDL on erythropoietin expression in macrophages. We also studied the influence of erythropoietin on macrophage foam cell formation and its molecular mechanisms.
Reagents and Assay Kits
Information on the reagents and assay kits is given in the online-only Data Supplement.
All animal experiments were approved by the Animal Care and Utilization Committee of National Yang-Ming University. C57BL/6 and SJL mice were purchased from the National Laboratory Animal Center, National Science Council (Taipei, Taiwan); apoE−/− mice and erythropoietin transgenic (EPO-Tg) mice were obtained from the Jackson Laboratory (Bar Harbor, ME).
Formalin-fixed, paraffin-embedded tissue blocks were cut into 8-μm sections. Sections were deparaffinized, rehydrated, and then covered with 3% H2O2 for 10 minutes. After blocking with BSA, slides were incubated with primary antibodies for 1 hour at 37°C and then with correspondingly secondary antibodies for an additional hour. Antigenic sites were visualized by the addition of DAB. Slides were counterstained with hematoxylin.
Mouse bone marrow–derived macrophages were prepared as previously described.25 Briefly, mice were killed by CO2 exposure, and mononuclear cells from the femurs were harvested by Percoll (1.073 g/cm3) density gradient centrifugation. The cells were then seeded in minimum essential medium α supplemented with 50 ng/mL macrophage colony-stimulating factor, 10% FBS, and penicillin (100 U/mL)/streptomycin (100 μg/mL) at 37°C for 5 days. Murine macrophage J774.A1 cells (ATCC, TIB-67) were cultured in RPMI 1640 medium supplemented with 10% FBS, penicillin, and streptomycin.
The oxLDL was prepared as described previously.26 LDL was exposed to 5 μmol/L CuSO4 for 24 hours at 37°C; then, Cu2+ was removed by extensive dialysis. The extent of modification was determined by measurement of thiobarbituric acid–reactive substances. OxLDL containing ≈30 to 60 nmol thiobarbituric acid–reactive substances defined as malondialdehyde equivalents per milligrams of LDL protein was used for experiments.
Oil Red O Staining
Cells were fixed with 4% paraformaldehyde and then stained by 0.5% Oil Red O. Hematoxylin was used as a counterstain. The density of lipid content was evaluated by alcohol extraction after Oil Red O staining. The absorbance at 540 nm was measured with a microplate reader (BioTek Instruments Inc, Winooski, Vermont).
Cholesterol Efflux Assay
Macrophages were treated with various concentrations of erythropoietin for 12 hours, followed by the equilibration of NBD-cholesterol (1 μg/mL) for an additional 6 hours in the presence of erythropoietin. NBD-cholesterol–labeled cells were washed with PBS and incubated in RPMI 1640 medium for 6 hours. The fluorescence-labeled cholesterol released from the cells into the medium was measured with a multilabel counter (PerkinElmer, Waltham, Mass). Cholesterol efflux was expressed as a percentage of fluorescence in the medium relative to the total amount of fluorescence (cells and medium).
Cholesterol and Triglyceride Measurement
Cellular cholesterol and triglycerides were extracted by hexane/isopropanol (3/2, vol/vol). After cellular debris was removed, the supernatant was dried under nitrogen flush. The level of cholesterol and triglyceride was measured with cholesterol and triglyceride assay kits.
Quantitative Real-Time Polymerase Chain Reaction
Total RNA was isolated from cells by Tri reagent and was converted into complementary DNA by reverse transcriptase (Biolabs, Ipswich, Mass) with oligo-dT primer. The obtained complementary DNAs were then used as the templates for quantitative real-time polymerase chain reaction (PCR). The reaction of quantitative real-time PCR was performed by the TaqMan probe–based real-time quantification system (Applied Biosystems, Foster, Calif). The relative amount of messenger RNAs (mRNAs) was calculated with GADPH mRNA as the invariant control.
Preparation of Nuclear Extracts
The nuclear extracts were prepared as described previously.27 Cells were lysed in 10 mmol/L HEPES (pH 7.9), 10 mmol/L KCl, 1.5 mmol/L MgCl2, 0.5% Nonidet P-40, 1 μg/mL leupeptin, 10 μg/mL aprotinin, and 1 mmol/L phenylmethylsulfonyl fluoride. Nuclei were pelleted at 5000g for 5 minutes at 4°C, and the resulting supernatants were used as cytosolic fraction. Nuclei were resuspended in lysis buffer and sheared for 15 seconds with a microprobe sonicator and incubated on ice for 5 minutes. After centrifugation at 12 000g for 5 minutes at 4°C, the supernatants were collected as nuclear extracts.
Aliquots (50 μg) of cell lysates or nuclear extracts were separated on 8% or 12% SDS-PAGE and then transblotted onto the Immobilon-P membrane (Millipore, Bedford, Mass). After being blocked with 5% skim milk, blots were incubated with various primary antibodies and followed by secondary antibodies. The protein bands were detected with an enhanced chemiluminescence kit (PerkinElmer) and quantified by ImageQuant 5.2 software (Healthcare Bio-Sciences, Philadelphia, Pa).
Chromatin Immunoprecipitation Assay
Chromatin immunoprecipitation assays were performed according to the manufacturer’s instructions (Upstate Biotechnology, Lake Placid, NY) with minor modifications. J774.A1 cells were cultured in RPMI 1640 medium with or without preceding treatment of erythropoietin (5 U/mL) for 6 hours and fixed by formaldehyde for 15 minutes at room temperature. After lysing and sonicating cells, chromatin solution was diluted and incubated overnight with rabbit anti–liver X receptor α (LXRα) antibody or rabbit immunoglobulin G at 4°C. Immunocomplexes were precipitated with salmon sperm DNA/protein A agarose and collected by centrifugation. After being washed, chromatin DNA was eluted, purified, and subjected to PCR analysis. We used 1% chromatin solution as an input control. The mouse ABCA1 gene promoter containing LXR binding element was amplified by PCR with the following primers: 5′-CCA CGT GCT TTC TGC TGA GT-3′ and 5′-TGC CGC GAC TAG TTC CTT TT-3′. PCR products were resolved on a 2% agarose gel and visualized with ethidium bromide staining.
Transient Transfection and Luciferase Reporter Assay
In the promoter activation assay, cells were transfected with one of the following plasmids with the TurboFect reagent: 3xLXRE-Luc, a reporter construct containing 3 copies of LXRE; phABCA1(-928)-Luc, a reporter plasmid of human ABCA1 promoter; or the mutant of LXRE, phABCA1-DR4m-Luc, which were kindly provided by Dr Song-Kun Shyue (Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan). The pGL3-renilla was cotransfected as a transfection control. Twenty-four hours after transfection, the cells were treated with erythropoietin for another 12 hours. The cells were then lysed for Luc and renilla activity assays.
Results are reported as medians and 25% and 75% percentiles, and whiskers in plots represent ranges from 5 independent experiments. The Mann-Whitney test was used to compare 2 independent groups. The Kruskal-Wallis test followed by Bonferroni posthoc analysis was performed for testing multiple groups. SPSS version 8.0 (SPSS Inc, Chicago, Ill) was used for analysis. Difference was considered statistically significant at values of P<0.05.
Expression of Erythropoietin and EROR in Atherosclerotic Lesions
We investigated first the expression of erythropoietin in atherosclerotic lesions. As shown in Figure 1A, aortas were collected from apoE−/− and wild-type mice at 5 months of age and subjected to Western blotting. Erythropoietin was markedly upregulated in apoE−/− mice; furthermore, immunohistochemical staining for erythropoietin and EPOR in atherosclerotic lesions of apoE−/− mice demonstrated that positive signals were restricted mainly in macrophages (Figure 1B). These results imply that erythropoietin may play a role in the regulation of macrophage foam cell formation via autocrine action.
Effect of OxLDL on Expression of Erythropoietin and EPOR in Macrophages
It has been demonstrated that modified LDL is proatherogenic in the pathogenesis of atherosclerosis in both animals and humans, in particular the critical role of oxLDL in cytokine release. We therefore examined the effect of oxLDL on the expression of erythropoietin in macrophages. Macrophages stimulated with oxLDL (25, 50, or 100 μg/mL) for 24 hours significantly induced erythropoietin secretion from cells into culture medium in a dose-dependent manner (Figure 2A). Moreover, oxLDL also increased erythropoietin protein expression in macrophages (Figure 2B).
Erythropoietin Confers Protection From Foam Cell Formation
Abundant lipid-laden macrophage foam cells in the fatty streak and atherosclerotic plaques imply their importance in the atherosclerosis. Because erythropoietin was upregulated by oxLDL, we speculated that it was also involved in oxLDL-induced foam cell formation. Combined treatment with recombinant erythropoietin and oxLDL significantly ameliorated intracellular lipid accumulation in macrophages compared with the oxLDL-treated group (Figure 3A and 3B). This reduction in intracellular lipid accumulation was also evidenced by a decrease in cellular level of cholesterol and triglycerides (Figure 3C and 3D). These data suggest that erythropoietin confers a protective role in the formation of macrophage foam cells.
Erythropoietin Enhances Cholesterol Efflux Without Altering the Protein Expression of Lipid Synthesis–Related Genes
We then investigated the effect of erythropoietin on cholesterol efflux and endogenous lipid synthesis–related gene expression. Our data showed that treatment with erythropoietin increased the cholesterol efflux in a dose-dependent manner (Figure 4A). However, erythropoietin exposure did not alter the cleavage of sterol regulatory element binding protein (SREBP) 1 and SREBP2, 2 key transcription factors regulating multiple genes involved in the synthesis and metabolism of cholesterol and fatty acids. Additionally, neither the protein expression of SREBP1-targeted genes (acetyl-CoA carboxylase and fatty acid synthase) nor that of SREBP2-targeted genes (3-hydroxy-3-methylglutaryl coenzyme A reductase [HMGCR] and LDL receptor) was influenced by erythropoietin treatment (Figure 4B through 4E). Collectively, these data suggest that the erythropoietin-induced suppression of intracellular lipid accumulation is likely due to the increase in cholesterol efflux, not the inhibition of endogenous lipid synthesis.
Erythropoietin Promotes Expression of ABCA1 and ABCG1 Rather Than SR-A, CD36, and SR-BI
SR-A, CD36, SR-BI, ABCA1, and ABCG1 have shown their crucial role in cholesterol homeostasis during foam cell formation.4–9 We therefore delineated the mechanisms of erythropoietin to attenuate lipid accumulation by examining the alterations of these receptors and transporters. As shown in Figure 5A through 5F, macrophages treated with erythropoietin (1.25, 2.5, or 5 U/mL) for 24 hours dose dependently increased protein levels of ABCA1 and ABCG1, whereas the protein expression of SR-A, CD36, and SR-BI was not affected. Moreover, treatment with erythropoietin at various time points also significantly enhanced the mRNA expression of ABCA1 and ABCG1 (Figure 5G and 5H).
LXRα Activation Mediates the Antiatherogenic Effect of Erythropoietin
To address whether the LXRα/retinoid X receptor (RXR) system is involved in erythropoietin-induced expression of ABCA1 and ABCG1, we determined the nuclear protein level of LXRα and RXR in erythropoietin-treated macrophages. Erythropoietin treatment increased the nuclear level of LXRα and RXR in a time-dependent manner (Figure 6A), and enhanced binding of LXRα to LXRE in the ABCA1 promoter was observed via chromatin immunoprecipitation assay (Figure 6B). Furthermore, to explore the transcriptional regulation of LXRα in erythropoietin-treated macrophages, LXR activation assays were performed by transfecting with 3xLXRE-Luc, phABCA1(-928)-Luc, or phABCA1-DR4m-Luc (the reporter plasmid with a mutation in the LXRE), followed by erythropoietin treatment. Compared with the control group, erythropoietin markedly increased LXRE-mediated luciferase activity by 3.7-fold, which was prevented by geranylgeranyl pyrophosphate (GGPP), a pharmacological LXRα antagonist (Figure 6C). In contrast to phABCA1-Luc, induction was blunted in phABCA1-DR4m-Luc by either erythropoietin or TO901317, an LXRα agonist, as positive control (Figure 6D). Moreover, coincubation with GGPP or LXRα small interfering RNA (siRNA) abolished the induction of ABCA1 and ABCG1 by erythropoietin (Figure 7A and 7C), and inhibition of LXR activation by GGPP or siRNA further abrogated the inhibitory effect of erythropoietin on lipid accumulation (Figure 7B and 7D). Additionally, erythropoietin did not affect the protein level of nuclear LXRβ, and gene knockdown of LXRβ by siRNA transfection did not alter the erythropoietin-induced increase in protein expression of ABCA1 and ABCG1 (Figure I in the online-only Data Supplement). These results imply the essential role of LXRα activation in erythropoietin-regulated gene expression of ABCA1 and ABCG1 and thus may contribute to the suppressive effect of erythropoietin in macrophage foam cell transformation in vitro.
Overexpression of Erythropoietin in Macrophages Restricts OxLDL-Mediated Lipid Accumulation
Finally, we elucidated the impact of erythropoietin overexpression on ABCA1, ABCG1, and foam cell formation by using bone marrow–derived macrophages isolated from wild-type and EPO-Tg mice. As shown in Figure 8A, the protein expression of ABCA1 and ABCG1 was significantly higher in EPO-Tg macrophages compared with wild-type macrophages. Furthermore, oxLDL-induced lipid accumulation was markedly attenuated in EPO-Tg macrophages, and this suppressive effect was abolished by pretreatment with erythropoietin-neutralizing antibody or GGPP (Figure 8B and 8C).
Erythropoietin was first identified in kidney to function as a key regulator for red blood cell differentiation.14,15 Over the past decade, several lines of evidence have indicated that erythropoietin and EPOR exhibit a widespread distribution among a variety of organs in addition to kidney.15–18 A large body of additional studies has shown that erythropoietin protects cardiac function by increasing blood flow and preventing apoptosis in ischemia models.19,20 Moreover, erythropoietin plays a critical role in endothelial cell integrity and promoting angiogenesis in the vascular system.17,21,28 Although Buemi et al22 reported that erythropoietin administration significantly decreased the cholesterol ester content of atherosclerotic aortas in Watanabe heritable hyperlipidemic rabbits, the effect of erythropoietin in the transformation of macrophage foam cells, a crucial step for the initiation and progression of atherosclerosis, has never been established.
Here, we demonstrated a novel effect of erythropoietin and its underlying molecular mechanism in suppressing macrophage foam cell formation. We first validated that erythropoietin was significantly increased in atherosclerotic aortas and in particular the intralesional macrophage foam cells (Figure 1). The cellular localization implies the possible role of erythropoietin during the transformation of such cells. Modified LDL such as oxLDL is the most critical modulator in the initial stage of atherogenesis. It has been reported to induce lipid accumulation by regulating expression of SRs and transporters of reverse cholesterol transport or modulating inflammatory responses by cytokine induction.1,2,29 In this study, we found that oxLDL treatment induced an increase in erythropoietin both in macrophage culture medium and within the macrophages (Figure 2). This finding is consistent with previous results that oxLDL triggers hypoxia-inducible factor-1α–mediated induction in erythropoietin promoter activity via a reactive oxygen species–dependent mechanism.30 On the basis of these observations, it is pertinent to ask whether erythropoietin affects the lipid metabolism in macrophages. Interestingly, our data showed that erythropoietin indeed ameliorated the oxLDL-induced lipid accumulation in mouse macrophages (Figures 3 and 8⇑). Using this experimental cell culture model, we then investigated the molecular mechanisms underlying the beneficial function of erythropoietin during foam cell formation.
The intracellular lipid homeostasis of foam cells is dynamically regulated by oxLDL internalization and cholesterol efflux. The importance of SR-A and CD36 in foam cell formation and atherogenesis is well established. Macrophages from mice lacking SR-A and/or CD36 have a reduction in their internalization of oxLDL and are less prone to foam cell formation in vitro.4–6 On the other hand, ABCA1, ABCG1, and SR-BI, the 3 major transporters for cholesterol efflux of foam cells,7–9 are pivotal in maintaining the cholesterol homeostasis in macrophages. Studies using gene-manipulated mice demonstrated that foam cell accumulation and atherosclerotic lesions are significantly promoted in individual transporter-deficient animals.31–33 Expression of corresponding SRs or transporters is known to be regulated by several mediators, including tumor necrosis factor-α, interferon-γ, interleukin-4, reactive oxygen species, interleukin-10, and interleukin-20, which are all present within the atherosclerotic lesions.10,11,34–37 Therefore, we examined the regulatory role of erythropoietin and showed that erythropoietin augmented both mRNA and protein expression of ABCA1 and ABCG1 but not SR-A, CD36, and SR-BI (Figure 5). This finding indicates that erythropoietin may affect cholesterol efflux but not oxLDL uptake during the transformation of foam cells, and it is also consistent with previous studies showing that cytokine-induced expression of ABCA1 or ABCG1 would mitigate the lipid accumulation in foam cells.34,38
More important, we also showed that the upregulation of ABCA1 and ABCG1 by erythropoietin was accompanied by increased nuclear LXRα levels and enhanced binding of LXRα to ABCA1 promoter (Figure 6A and 6B). This finding is further supported in promoter activation assays in which erythropoietin-induced promoter activity was abrogated in mutant of LXR binding element (phABCA1-mDR4-Luc; Figure 6C and 6D). Moreover, inhibition of LXRα activation by GGPP or siRNA abolished the erythropoietin-mediated upregulation of ABCA1 and ABCG1 (Figure 7A and 7C). These results suggest that LXRα-mediated transcriptional regulation is required for the induction of ABCA1 and ABCG1 by erythropoietin. Our findings are in agreement with those of Beyea et al39 and Joseph et al,40 who found that the endogenous LXR ligand 24 (S) 25-epoxycholesterol upregulated ABCA1 and ABCG1 and that synthetic LXR ligands inhibited the development of atherosclerosis in mice, respectively. Despite the unique pathway discovered in this study, the detailed mechanisms of how erythropoietin affects cholesterol efflux merit further study. In functional analysis, inhibition of LXRα activation resulted in intracellular lipid accumulation that was even higher than that of the group treated with oxLDL only (Figure 7B and 7D). One possible explanation is that oxLDL itself is known to upregulate ABCA1 and ABCG1 via an LXRα-dependent mechanism.40 Therefore, extra lipid accumulation may occur in response to oxLDL when the LXRα pathway is inhibited. Finally, congruous data are observed in the experiment using EPO-Tg bone marrow–derived macrophages, which again demonstrated the protective effects of erythropoietin on ABCA1 and ABCG1 expression and foam cell formation (Figure 8).
This study demonstrates a unique protective effect of erythropoietin that could reduce lipid accumulation in foam cells via the upregulation of ABCA1 and ABCG1 and could be transcriptionally modulated mediated by LXRα activation. Our findings suggest that erythropoietin may potentially be of therapeutic value in inhibiting the process of atherosclerosis.
We thank Dr Shiao-Chi Wu for helpful suggestions for statistical analyses and Dr Song-Kun Shyue for technical assistance.
Sources of Funding
This study was supported by grants from National Science Council (NSC-96-2320-B-010-031-MY3), National Health Research Institutes (NHRI-EX97-9608SC), VGHUST Joint Research Program, Tsou’s Foundation (97-P6-27), Department of Health of Taipei City Government (97001-62-016), Chang-Gung Institute of Technology (09511N024), and the Aim for the Top University Plan supported by the Ministry of Education (97A-C-T113), Taiwan.
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Atherosclerosis is a major cause of death resulting from cardiovascular diseases. Atherogenesis starts with an increase in circulating cholesterol levels and subsequently involves a complex cascade of events, including lipid modification, foam cell formation, and recurrent inflammation within the artery wall. Currently, the most widely prescribed medications for treating atherosclerosis are statins, a group of drugs that mainly aim to lower circulating cholesterol levels. Although clinical trials have favorably shown a reduction in atherogenic events by statins, this therapeutic strategy is not optimal. Foam cells derived from macrophages play a critical role in the initiation and progression of atherosclerosis. They not only accumulate lipids but also release inflammatory and chemotactic cytokines as atherogenic factors. Thus, therapeutic approaches for reducing form cell formation may also represent an important strategy for the prevention and treatment of atherosclerosis. Erythropoietin is known as a glycoprotein hormone that controls erythropoiesis. Erythropoietin has been used as a therapeutic agent to treat anemia in patients with chronic kidney disease or cancer. Although interest in other beneficial functions of erythropoietin has recently emerged, its role in atherogenesis is unknown. This study demonstrates that erythropoietin is increased mainly in the macrophage foam cells present in atherosclerotic lesions. Either treatment with exogenous erythropoietin or overexpression of erythropoietin markedly reduces foam cell formation by increasing the cholesterol efflux from macrophages, suggesting a novel antiatherogenic function of erythropoietin. Our findings may shed new light on the potential therapeutic application of erythropoietin to treat atherosclerosis.
The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.109.876839/DC1.