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. Author manuscript; available in PMC: 2013 Jan 17.
Published in final edited form as: Circulation. 2011 Dec 5;125(2):354–363. doi: 10.1161/CIRCULATIONAHA.111.057596

The A2b Adenosine Receptor Regulates Hyperlipidemia and Atherosclerosis

Milka Koupenova 1,2, Hillary Johnston-Cox 2, Alexander Vezeridis 2, Haralambos Gavras 1,3, Dan Yang 2, Vassilis Zannis 1,3, Katya Ravid 1,2,3
PMCID: PMC3265935  NIHMSID: NIHMS347516  PMID: 22144568

Abstract

Background

The cAMP-elevating A2b adenosine receptor (A2bAR) controls inflammation via its expression in bone marrow cells.

Methods and Results

Atherosclerosis induced by high fat diet (HFD) in Apolipoprotein-E deficient mice was more pronounced in the absence of the A2bAR. Bone marrow transplantation experiments indicated that A2bAR bone marrow cell signals alone were not sufficient to elicit this effect. Intriguingly, liver expression of the A2bAR in wild type mice was vastly augmented by HFD, raising the possibility that this upregulation is of functional significance. A2bAR genetic ablation led to elevated levels of liver and plasma cholesterol and triglycerides, and to fatty-liver pathology typical of steatosis, assessed by enzymatic assays and analysis of liver sections. Western blotting and qPCR revealed elevated expression of the following molecules in the liver of A2bAR null mice: the transcription factor SREBP-1 and its downstream targets, and two regulators of lipogenesis, acetyl CoA carboxylase and fatty acid synthase. Pharmacological activation or inhibition of A2bAR in primary hepatocytes confirmed the regulation of SREBP-1 by this receptor. A2bAR-mediated changes in cAMP were found to regulate levels of the transcriptionally active form of SREBP-1. Finally, adenoviral-mediated restoration of the A2bAR in the liver of A2bAR-null mice reduced the lipid profile and atherosclerosis. Similarly, in vivo administration of the A2bAR ligand BAY 60-6853 in control mice on HFD reduced lipid profile and atherosclerosis.

Conclusions

This study provides the first evidence that the A2bAR regulates liver SREBP-1, hyperlipidemia and atherosclerosis, suggesting that this receptor may be an effective therapeutic target.

Keywords: atherosclerosis, lipids, adenosine

Introduction

Atherosclerosis is a major contributor to cardiovascular related mortality. Diets rich in cholesterol and saturated fats, lack of exercise, smoking, and high blood pressure, all contribute to the development of atherosclerosis. Numerous genes were identified in relationship to the development of this pathology, including those involved in synthesis and clearance of cholesterol1, inflammation2, and altered glucose metabolism3.

Extracellular adenosine is generated upon stress4 or inflammation5 and acts on four different G-protein coupled receptors, historically classified as adenylyl cyclase inhibiting (A1 and A3) or activating (A2a and A2b). Adenosine can regulate inflammation6–8, as well as metabolic processes involved in insulin homeostasis9–10, glucose metabolism11–13, lipolysis14–15 and cholesterol synthesis16. Of all four adenosine receptors, the A2bAR has the lowest affinity for adenosine17. It was not until the generation of the A2bAR knockout/β-galactosidase knock-in model that the importance of this receptor was clearly elucidated in vivo8. β-galactosidase expression in these mice indicated that in wild type mice the A2bAR exhibits widespread expression in the vasculature and inflammatory cells such as macrophages. Plasma analysis suggested a mildly enhanced inflammatory profile, with elevated IL-6 and TNF-α. These two cytokines were further augmented upon challenge with bacterial lipopolysaccharides, and this elevation in cytokine levels was dependent on A2bAR bone marrow cell signals. Further analysis of the vasculature of this knockout model showed elevated expression of vascular adhesion molecules, such as P-selectin, E-selectin, and ICAM-1, as well as an increased adhesion of leukocytes to the endothelial cells8. Additionally, in a model representative of human restenosis following angioplasty, A2bAR knockout mice showed increased lesion formation, an effect that was also dependent on A2bAR bone marrow cell signals18. Given these observations, we sought to elucidate the role of A2bAR in atherosclerosis induced by a high fat Western diet. For that purpose, we generated a double knockout model of A2bAR and apolipoprotein E (ApoE) genes and exposed the mice to a Western diet. Ablation of the A2bAR led to an elevation of plasma lipids and concomitant augmentation of the atherosclerotic profile. The pathophysiology of this hyperlipidemia and the consequent increase in atherosclerosis were due in part to altered SREBP-1 signaling in the liver.

Methods

Animals

C57BL/6J mice and ApoE KO mice were purchased from Jackson laboratory. A2bAR, ApoE double knockout mice (dKO) were generated by breeding the A2bAR KO, β-galactosidase knock-in8 and the ApoE KO mouse models. All experimental groups were on a C57BL/6J strain background, confirmed by PCR-based gene marker analysis using MAX-BAX (Charles River Laboratories). For this study, male mice were used unless specified otherwise. ApoE KO and dKO mice were matched for strain, sex, and age. All animals received humane care in accordance with their guidelines and approved by the Institutional Animal Care and Use Committee of the Boston University School of Medicine.

Diets

In all experiments, male mice at 12 weeks of age were subjected to either regular chow diet (Teklad, cat# TD2918) or Western diet (Teklad, cat# TD88137) for 8 or 16 weeks. Before collection, mice were starved for 16 hours.

Isolation and Treatment of Primary Hepatocytes

Hepatocytes from mice post HFD were isolated as previously described19, followed by treatments with agonists as indicated (details are provided in the online-only Data Supplement).

Cyclic AMP (cAMP) Measurements

Liver lobes were homogenized in 0.1N HCl and 100 μl of the homogenate was loaded on the plates and probed for cAMP according to the manufacturer’s protocol. Hepatocytes were also subjected to cAMP determination (details are provided in the online-only Data Supplement).

Aortic Lesion Analysis

Oil-red-O staining: Aortas were isolated after blood collection by heart puncture, and analyzed as described before20. Images were analyzed with NIH software-ImageJ (version 1.62; http://rsb.info.nih.gov/ij/).

Immunohistochemistry; Osmium Tetroxide Staining

Brachiocephalic arteries were cryo-embedded and sections were analyzed with F4/80 macrophage antibody (AbD Serotec) as described previously18. Pictures were taken with Olympus IX70 microscope. Lipid distribution in the liver was determined in cryo-embedded sections stained with OsO4 as described previously21. Briefly, cryo-sections of the embedded organ were stained with 0.1 % OsO4 in Palade buffer (0.03 M sodium barbital, 0.03 M sodium acetate buffer, 0.07 M potassium chloride, pH 7.4) for 10 min. The final concentration of 0.1% OsO4 was achieved by dissolving 1g of OsO4 in 100 mL of Barbital buffer. Pictures were taken with Nikon microscope Eclipse 50i.

β-Galactosidase Staining

Mice were anesthetized with isoflurane, blood was removed by heart puncture, and a small piece of the liver-lobe was excised and placed in 2% Paraformaldehyde (PFA) for 2 hours followed by incubation with 1X PBS supplemented with 2 mM MgCL2 for 1 hour. The piece was then stained overnight with x-galactosidase as described previously8.

Quantitative Polymerase Chain Reaction

RNA was isolated using RNeasy mini kit (Qiagen, cat# 74104) and cDNA was generated using the Superscript II kit (Invitrogen, cat# 18064-22) according to manufacturer’s instructions. mRNA levels of genes were quantified using Applied Biosystems (AB) TaqMan primers (details are provided in the online-only Data Supplement).

Western Blots

Liver or hepatocyte proteins were isolated with RIPA buffer8. Proteins were resolved on 8–10% SDS gels and probed with ACCα (Cell signaling); FAS (Novus Biologicals); SREBP-1 (Santa Cruz); β-Actin (Sigma) antibodies.

Cholesterol, Triglycerides Analysis

Plasma or liver was analyzed for triglycerides and cholesterol using Triglyceride Determination Kit (Sigma) and Infinity Cholesterol Reagent Kit (Thermo Scientific), according to manufacturers’ instructions. Liver cholesterol was measured as described previously22. Liver triglyceride isolation was done using ethanolic KOH as described earlier23. The concentration of TG was calculated by the following formula: [TG]= [Glycerol (mg/dL)×10 (μl loaded)×(415/200)×0.012 (dL)]/liver weight (g).

Fast Protein Liquid Chromatography (FPLC)

Plasma was resolved by fast pressure liquid chromatography in 25 fractions as described previously24–25. Each fraction was analyzed for TG and Cholesterol as described above24–25.

Thin Layer Chromatography (TLC)

Primary hepatocytes, plated at 1× 105/12 well-plate were starved overnight and then incubated for 24 hours with 5 μCi of Sodium-[1,2-14C]-acetate (Perkins Elmer) as described previously26 (details are provided in the online-only Data Supplement).

Adenoviral Injection

Human A2bAR cDNA (cDNA.org, cat# ADRA2B0000) was engineered in an adenovirus (AdV) system with GFP marker under CMV-promoter (CMV-A2bAR GFP-AdV vector). As a control, a vector containing only GFP was used (CMV-GFP-AdV vector). Both of the adenoviral constructs were cloned, replicated, and in vivo tittered by Vector Biolabs (Philadelphia, PA). Double knockout mice were tail-vein injected with 2×109 viral particles as described previously25. On the 4th day post injection mice were starved for 6 hours, and plasma and livers were collected for analysis.

BAY 60-6583 Injection

Mice were injected intraperitoneally for 12 weeks, every third day with BAY 60-6583 (2 μg/g mouse, dissolved in vehicle: 10% Ethanol, 50% PEG-400, 40% water27) or with equal volume of vehicle. At completion mice were starved for 16 hours, re-injected at 14.5 hours with BAY 60-6583 or vehicle, and collected for analysis.

Bone Marrow Transplantation

Bone marrow transplantation experiments were carried out essentially as described previously8 (details are provided in the online-only Data Supplement).

Statistical Analysis

The data from each experiment is expressed as means ± standard deviation (SD). Statistical comparison was done using two-tail exact (Mann Whitney) t-test considered significant when the medians were different with p≤0.05. When appropriate, and as indicated, we used one tail ANOVA followed by Bonferroni’s Multiple comparison test. All statistical analyses were performed using GraphPad Prism5 software.

Results

The A2bAR protects against atherosclerosis

Since wild-type mice on a C57BL/6J background do not develop atherosclerosis of all phases along the aortic tree1, a conventional approach to study this pathology is on an ApoE null background28–29. We monitored atherosclerosis in a mouse model in which the A2bAR was eliminated from ApoE knockout mice (A2bAR, ApoE double knockout (dKO)). Comparison of ApoE KO and A2bAR, ApoE dKO mice revealed that A2bAR was protective against lipid plaque formation induced by 8 weeks of Western high fat diet (HFD) in male mice (20 weeks old at collection time), as revealed by lipid staining with Oil-red-O of the entire aorta and the aortic arch (Figure 1A, 1B), and Hematoxylin and Eosin (H&E) staining of arterial tissue sections to determine lesion formation (Figure 1C, 1D). After 16 weeks of HFD diet, the amount of lipid plaques measured by Oil-red-O became saturated in the aortas of both mouse lines, however, the differences in aortic arch lesions were still statistically significant in the experimental groups (Supplemental Figures 1A,1B). An increase in lipid plaques was also noticed in older dKO mice (28 week old mice) on a chow diet (Supplemental Figures 1C, 1D). Of note, crude food consumption and body weights were similar in the two groups (data not shown).

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A2bAR role in plaque formation. The aortas of 20-week-old male mice following 8-weeks of high fat diet (HFD) were collected and subjected to various analyses. A. Representative composite images of the Oil-red-O staining of the arch and aorta of male ApoE knockout (KO) and A2bAR, ApoE double knockout (dKO) mice. Magnification is 8X for the aorta and 20X for the arch. The vessel was cut open in order to visualize and quantitate lipid deposition. The arch was open and the three branching arteries were dissected in a manner that would not disturb the plaques. B. Quantitation of the Oil-red-O staining of the aortas and arches was performed by using the NIH ImageJ software (version 1.41; http://rsb.info.nih.gov/ij/). The lesion area was calculated as a percent of the total aortic surface. Each rhomboid represents a reading from a different biological sample. Aorta p-value=0.0009; Arch p-value=0.001. C. Lesion formation in the brachiocephalic artery of ApoE KO and A2bAR, ApoE dKO mice was visualized by H&E staining of cryo-sections of the artery. Images were taken with Olympus IX70 microscope, with magnification of 200X. D. Quantitation of the lesion along the artery calculated by Image-Pro Plus analysis software. Lesion area of every 10th section of four mice per group was analyzed as a percent of intimal plus luminal area, as follows: % lesion area= (intimal area/intimal+luminal area)x100. The y-axis represents the average of % lesion area calculated for six slides per mouse and four different mice per group. p-value=0.0002.

The A2bAR controls the plasma lipid profile

Consistent with the pathogenesis of atherosclerosis, A2bAR, ApoE dKO mice harbor increased plasma levels of cholesterol and triglycerides (TG) at 8 weeks (Figure 2A) or 16 weeks post HFD (Supplemental Figure 2A). Fast protein liquid chromatography (FPLC) of the plasma showed that the increased amount of lipid is concentrated in the very low density lipoprotein (VLDL) particles (Figure 2B, 2C). In addition, A2bAR KO mice (expressing the ApoE gene) had augmented lipid levels post-HFD compared to wild type (Supplemental Figure 2B), but the absolute levels in both groups are much lower than in the atherosclerosis-inducing levels in ApoE null mice30–31 (compare to Figure 2A). Elevation of lipids was also observed in dKO mice on a regular chow diet compared to ApoE KO mice on a regular chow diet, again to lower extent than under HFD (Supplemental Table I compared to Figure 2A). Together, these data point to a role for the A2bAR in regulating lipid levels, regardless of ApoE expression or the type of diet.

Figure 2.

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Role of A2bAR in plasma lipid homeostasis. Plasma lipid levels were measured in 20-week-old male mice following 8 weeks of HFD and 16 hours post starvation. A. Cholesterol (n=6) and triglycerides (n=7) levels determined on ApoE null background (ApoE KO and A2bAR, ApoE dKO). Cholesterol p-value=0.0028; Triglyceride p-value=0.0126. FPLC of plasma was used to determine B. cholesterol and C. triglyceride distribution in the different lipoprotein particles. The FPLC profile was run for 3 different biological samples per group and the image here is representative of plasma drawn from one mouse per model.

Bone marrow derived signals from the A2bAR do not affect atherosclerosis or the lipid profile

Macrophages express high levels of the A2bAR, which controls the synthesis of inflammatory cytokines, such as TNF-α 8. TNF-α, in turn, was shown to affect lipid synthesis 32 and atherosclerosis 33. In order to determine the contribution of A2bAR bone marrow signals to atherosclerosis and to the lipid profile, bone marrow cells derived from A2bAR, ApoE dKO mice were transplanted into ApoE KO irradiated male mice. The results of this experiment indicate that A2bAR-bone marrow cell signals alone are insufficient to elicit effects on the progression of atherosclerosis or on plasma lipid accumulation post-HFD consumption (Figure 3). This is contrary to the protective effect of A2bAR bone marrow signals on vascular lesion development in a model reminiscent of restenosis 18. Effective transplantation was evaluated as described previously8 (Supplemental Figure 3).

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Role of A2bAR bone marrow cell signals in plaque formation. A. Oil-red-O staining of the aortas collected 8 weeks post HFD from irradiated ApoE KO male mice transplanted with ApoE KO or dKO bone marrow (the arrow denotes the bone marrow cell donor into the irradiated mouse). B. Quantification of the Oil-red-O staining of the aortas and arches was performed as in Figure 1. Aorta p-value=0.4418; Arch p-value=0.5054. C. Plasma lipid levels in the transplanted mice 8 weeks post HFD (n=6 for cholesterol, n=7 for TG levels). Cholesterol p-value=0.6742; Triglyceride p-value=0.5192.

The A2bAR is upregulated in the liver under high fat diet and affects liver cholesterol and triglycerides levels

The liver is the major organ responsible for endogenous cholesterol and triglyceride synthesis. Hence, we first sought to determine the level of expression of the A2bAR in this organ, and its lipogenic ability. A2bAR KO mice carry the β-galactosidase (β-gal) gene knocked-in under the control of the A2bAR gene promoter 8. Previously, we reported that β-gal staining of liver isolated from 12-week-old A2bAR KO mice on a regular diet showed no significant signal except in the vasculature 8. Here, we show that HFD robustly induces A2bAR gene expression in the entire organ, as illustrated by the blue color of the β-gal staining (Figure 4A). Furthermore, examination by qPCR of the endogenous levels of the A2bAR confirmed the increased expression of this receptor in the liver following HFD (Figure 4B). Additionally, elevation of lipid content post-HFD was significantly higher in the livers of mice lacking the A2bAR (Figure 4C). Lipid staining of liver sections showed that lipid accumulation increased in the A2bAR null mice post-HFD, resulting in a histopathology typical of steatosis (Figure 4D). Consistent with the observed liver steatosis, aspartate transaminase (AST) activity levels were increased in the plasma of mice lacking A2bAR after 8 weeks on HFD (Supplemental Figure 4A). Levels of alanine transaminase were not elevated in the dKO compared to ApoE KO after 8 weeks of HFD (Supplemental Figure 4B). Taken together, the data indicate that under HFD, the level of A2bAR expression has a central role in regulating lipid levels.

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Role of A2bAR in liver lipid homeostasis. A. HFD-induced upregulation of the A2bAR gene promoter in the liver, measured by β-galactosidase staining at 8-weeks post regular chow or HFD. Magnification is 8X. B. Liver A2bAR mRNA levels were measured by qPCR in ApoE KO mice 16 hours post-starvation. Twelve-week-old ApoE KO mice (with wild type A2bAR) (denoted as Control) were analyzed and compared to similar mice subjected to additional 8 weeks of regular chow diet (8W-RD) or HFD (8W-HFD). The expression of the receptor is relative to the baseline value, measured in 12 week-old-mice before the 8 weeks diet, and normalized to 18S rRNA. C. Liver cholesterol (n=6) and triglyceride (n=6) content in ApoE KO and dKO mice measured as described in Methods. Liver Triglyceride content p-value=0.0260; Liver Cholesterol content p-value=0.0152. D. Liver lipid content (n=3 different mice, and 10 slides per mouse) measured by osmium tetroxide at 8-weeks post HFD (denoted as 8W). Magnification of the sections is 400X.

A2bAR activation/deletion regulates SREBP-1, and triglycerides and cholesterol synthesis in liver and primary hepatocytes

To gain insight into the mechanisms by which A2bAR ablation affects liver lipid synthesis post-HFD, we used microarray analysis to measure gene expression in ApoE KO and dKO mice (limited repeats; data not shown). In our search for potential regulators of lipid synthesis, we noted that the mRNA level of sterol regulatory element binding protein one (SREBP-1) is upregulated in the dKO mice compared to ApoE KO mice. This was confirmed by qPCR, and Western blot analysis showing upregulation of the transcriptionally active form of SREBP-1 (68 kDa) (Figure 5A, Supplemental Figure 5). SREBP-1 is a transcription factor associated with regulation of genes, such as acetyl coenzyme-A carboxylase (ACC) and fatty acid synthase (FAS), that are involved in lipogenesis 34. As anticipated, the expression of ACC and FAS was elevated in livers from dKO mice (Figure 5A). qPCR also revealed that A2bAR elimination selectively affected SREBP-1c mRNA levels, as levels of SREBP-1a were unchanged (Figure 5B).

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A2bAR activation/deletion regulates triglyceride and cholesterol synthesis in the liver and in primary hepatocytes. Livers of mice 8-weeks post HFD and 16 hours post starvation were subjected to: A. Western blot analysis of proteins related to fatty acid and triglyceride (TG) synthesis, and their quantitation (mature SREBP-1 (68 kDa), p-value=0.0286; acetyl CoA caboxylase (ACC) (280 kDa), p-value=0.0286; fatty acid synthase (FAS) (180 kDa), p-value=0.0286), and B. qPCR analysis of the mRNA levels of the two forms of SREBP-1 normalized to 18S rRNA. SREBP-1a, p-value=0.8857; SREBP-1c, p-value=0.0286. Primary hepatocytes isolated from ApoE KO or dKO mice were subjected to the following analysis: C. Baseline synthesis of cholesterol and triglycerides determined by using 14C-acetate supplemented to serum depleted media for 24 hours, and resolved by thin layer chromatography (TLC), as detailed in Methods. Triglyceride p-value=0.0286; Cholesterol p-value=0.0294. D. cAMP levels and Western blot analysis of the processed form of SREBP-1 and ACC without treatment (Baseline, p-value=0.0286). E. cAMP and F. Western blot analysis post pharmacological treatment with the A2bAR specific agonist, BAY 60-6583 (denoted as BAY); A2bAR specific antagonist, CVT-6883 (CVT); or 8Br-cAMP (stable cAMP analog). In each case, data is an average of n=4 experiments, except in 5F where n=3. 5E was analyzed by ANOVA (p<0.0001) followed by Bonferroni’s comparison test, with the following p-values for ApoE KO-hepatocytes: DMSO vs. BAY p-value < 0.01; BAY vs. BAY+CVT p-value< 0.01; DMSO vs. BAY+CVT p-value >0.01; DMSO vs. CVT p-value = 0.92. P-values for cAMP in dKO samples were not significant. Quantitation of protein levels was done using NIH ImageJ software. Radioactive levels were quantitated by using Packard Instant Imager software.

To determine whether the effect of A2bAR deletion on SREBP-1 is systemic or dependent on signals originating from hepatocytes, the levels of triglycerides and cholesterol were measured in primary hepatocytes from ApoE KO and dKO mice. We first confirmed that hepatocytes isolated from the livers of the ApoE KO mice express the A2bAR, and found that they do, consistent with the β-galactosidase staining of the whole liver in dKO mice (Figure 4A and Supplemental Figure 6). Using 1,2-14C-labeled acetate and thin layer chromatography, it was shown that dKO cells have increased lipid synthesis (Figure 5C), similar to the observed liver profile (Figure 4C). Additionally, baseline cAMP levels in liver or hepatocytes isolated from mice lacking the A2bAR were lower than in the corresponding control cells (Figure 5D). Relevant to our findings, previous studies have shown that cAMP inhibits the processing and activation of SREBP-1 (68 kDa form) 35.

Hepatocytes derived from ApoE null mice (bearing normal A2bAR) under the above-described HFD regime were treated with an A2bAR-specific antagonist, CVT-6883 36. This resulted in attenuation of A2bAR agonist (BAY 60-6583 36)-mediated increases in cAMP (Figure 5E; left panel), and upregulation of the mature form of SREBP-1, and of its downstream target ACC (Figure 5F; left panel). CVT alone had no significant inhibitory effect on cAMP, as its influence is mainly manifested while blocking agonist activity, and when adenylyl cyclase activity is high enough (Figure 5E; left panel). Regulation of cAMP and SREBP-1, and consequently of ACC, by BAY 60-6583 was not observed in A2bAR, ApoE dKO hepatocytes used as control (Figures 5E and 5F; right panels). Of note, A2bAR-wild type (ApoE KO) or A2bAR, ApE dKO hepatocytes treated with a stable cAMP analog (8-Bromo-cAMP) that permeates cells (note cAMP levels in Figure 5E) had diminished levels of the transcriptionally active form of SREBP-1 (Figure 5F).

Since stimulation of the A2bAR with BAY 60-6583 or inhibition with CVT-6883 affected the levels of cAMP and processed SREBP-1 in a manner consistent with cAMP-mediated downregulation of active SREBP-1, we postulate that the mechanism by which A2bAR regulates SREBP-1 involves cAMP.

Restoration of the A2bAR in the liver and in vivo activation of this receptor reduced plasma lipids and atherosclerotic plaques

To examine the specific contribution of liver A2bAR to the observed hyperlipidemia in vivo, the receptor was restored in the livers of the dKO mice by adenoviral-mediated A2bAR expression (A2bAR-Ad). Adenoviruses are known to primarily infect the liver for the 1st week post injection, with marginal targeting of other tissues37 (also confirmed in our laboratories), due to the abundant expression of the coxsackie and adenoviral receptor, CAR. Preliminary studies demonstrated adenoviral-driven A2bAR expression in hepatocytes and in the liver of mice injected with adenovirus (Supplemental Figure 7). Restoration of the A2bAR in this organ, verified by qPCR expression studies and cAMP measurements, resulted in a decrease of plasma triglycerides and cholesterol levels as compared to dKO mice injected with vehicle (Figure 6A, 6B). The plasma lipid reduction was associated with downregulation of the mRNA and protein levels of ACC and FAS (Figure 6C-6F). Adenoviral restoration of the A2bAR in the liver resulted in a two-fold increase in cAMP level compared to wild type livers, signifying receptor overexpression rather than rescue to control levels (Figure 6G).

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Liver A2bAR restoration in vivo reduces the lipid profile. A2bAR was reinstated in the liver by tail vein injection of adenovirus carrying either control vector (denoted as Control AdV) or A2bAR-expressing vector (denoted as A2bAR AdV) as described in methods. The injection was carried out in male A2bAR, ApoE dKO mice 8-weeks post HFD. At five days post injection the following parameters were measured: A. plasma triglycerides (TG), p-value=0.0159; B. plasma cholesterol, p-value=0.0366; C. mRNA levels of liver fatty acid synthase (FAS), p-value=0.0286 and of D. Liver Acetyl-coenzyme A carboxylase (ACC), p-value=0.0286, determined by qPCR; and E. a representative profile of protein levels of liver FAS, ACC and SREBP-1 measured by Western blotting. To be able to assess early changes in SREBP-1 levels following adenoviral injection, in this case samples were collected two days post injection. F. Densitometry-quantitation of data from panel E, using ImageJ. In each case, data presented is the average of n=5 control AdV, and n=4 A2bAR AdV injected mice. SREBP-1c p-value=0.0286; FAS p-value=0.0286; ACC p-value=0.0286. G. cAMP levels in livers derived from ApoE KO mice compared to A2bAR, ApoE dKO mice (n=4, p-value =0.0286), as well as in livers derived from dKO mice described in panel A, i.e., following administration of adenoviral vector (V) or a vector carrying the A2bAR (n=4, p-value =0.0286).

To explore the potential therapeutic effect of the A2bAR on atherosclerosis, we injected ApoE null mice (with wild type A2bAR alleles) intraperitoneally with BAY 60-6583 or vehicle for twelve weeks and then examined plasma lipid levels and atherosclerotic plaque formation. Mice injected with BAY 60-6583 had reduced atherosclerotic plaque formation (Figures 7A, 7B) and circulating plasma lipids (Figures 7C, 7D) compared to mice injected with vehicle. Administration of BAY 60-6583 to the A2bAR, ApoE dKO mice had no effect on cholesterol and TG levels, supporting the conclusion that the lipid-lowering effect of BAY 60-6583 in the ApoE KO mice is due to a specific effect on the A2bAR (Figure 8A and Supplemental Figure 8B). These findings point to the therapeutic potential of this ligand, as well as to the need to develop additional A2bAR selective agonists. BAY 60-6583 injection lowered liver SREBP-1 levels and the levels of ACC and FAS (Figure 7E, 7F), suggesting this pathway may be involved in the mechanism by which the receptor regulates lipid levels. The effect on SREBP-1 was not observed in livers of agonist-injected dKO mice (Supplemental Figure 8C). The liver enzymes AST and ALT were not significantly affected by BAY 60-6583 injection (Figure 4A, Supplemental Figure 4B). These observations focus attention on the A2bAR as a therapeutic target for lowering cholesterol and triglycerides levels, and ameliorating atherosclerosis.

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Liver A2bAR activation in vivo reduces plasma lipids, liver SREBP-1 levels, and atherosclerosis. Twelve- week-old ApoE KO male mice were injected with A2bAR specific agonist BAY 60-6583 (denoted as BAY) for 12 weeks and A. cholesterol (n=8 per group, p-value =0.0148) and B. triglycerides (n=6 per group, p-value =0.0152) were measured. Atherosclerotic plaques were analyzed by C. Oil-red-O staining of the arch and aorta. D. Quantitation of the Oil-red-O images of the aorta is as described in Figure 1. Magnification is 8X for the aorta and 20X for the arch. BAY 60-6583 aorta p-value=0.0188; BAY 60-6583 arch p-value=0.0379. Liver SREBP-1 levels and its downstream targets ACC and FAS were analyzed by E. Western blot analysis and quantified by F. Densitometry-quantitation using ImageJ. At least n=4 were used for the quantification of each protein. SREBP-1 p-value=0.0286; ACC p-value=0.0286; FAS p-value=0.0286.

Discussion

Previous studies have described the A2bAR as anti-inflammatory 8, and protective against kidney ischemia 38, cardiac reperfusion injury 39, and restenosis 18, typically via bone marrow cell signals. Our results assign a novel function to the A2bAR with respect to atherosclerosis development as a result of Western diet. We have shown that the A2bAR is protective against the early stages of atherosclerosis that result from elevated consumption of dietary fat and cholesterol. Elevated cholesterol and triglyceride levels have been associated with an increase in cardiovascular pathology, such as atherosclerosis, due to plaque formation in the arteries 40. Macrophages play a key role in the process of atherosclerosis as they sequester oxidized LDL in the subendothelial space and give rise to the so-called fatty streaks that can progress to more complex plaques and form occlusions. A2bAR bone marrow (BM)-derived signals, which derive predominantly from the BM macrophages, did not have an effect on plaque formation. Rather, the role of A2bAR is in the reduction of liver lipid production and circulation of cholesterol and triglycerides which mediates the observed effect on plaque development. Interestingly, in similar settings, upon elimination of the other adenylyl cyclase activating receptor, A2aAR, aortic lesions were reduced, which the authors attributed to increased apoptosis of foam cells, resulting in a lower density of foam cells in atherosclerotic lesions 16. Bone marrow derived signals from A2aAR, ApoE dKO mice post-transplantation induced early apoptosis of macrophages and consequently reduced the size of atherosclerotic lesions 16. In contrast, our study shows that in the case of the A2bAR, its ablation promotes fatty liver formation, increase in liver and plasma triglycerides and cholesterol, and a consequent augmentation of atherosclerotic plaque formation. Consistent with this finding, elevation of lipid levels is also observed in A2bAR KO mice with normal ApoE gene expression, indicating that this phenomenon is not dependent on the absence of ApoE.

In an earlier study, we reported the inducibility of the A2bAR by oxidative stress or inflammation 41. Exposure to HFD vastly elevated the level of expression of the liver A2bAR, which could account for its protective effect against hyperlipidemia, hepatic steatosis and atherosclerosis; this upregulation of A2bAR in the liver is also seen in the presence of the ApoE gene following exposure to HFD and in older mice (28 week old and older) on a regular chow diet (data not shown). Since the pathogenesis of atherosclerosis and augmenteda lipid synthesis are associated with hypertension and impaired glucose clearance, we measured hemodynamic parameters post-HFD. There was no observable difference in heart rate or blood pressure between the ApoE KO and the A2bAR, ApoE dKO mice post-HFD (Supplemental Table II). Also, glucose clearance in response to glucose overload was similar in control and A2bAR KO mice under ApoE KO background (data not shown).

In the context of HFD-consumption, elimination of A2bAR resulted in an increase in liver cholesterol content, liver steatosis, as well as an increase in plasma cholesterol and triglyceride levels. On a molecular level, elimination of A2bAR in the liver in vivo caused upregulation of SREBP-1 and its downstream targets ACC and FAS. Various factors known to affect SREBP-1 might be altered upon total elimination of the A2bAR. However, in primary hepatocytes, there is still a direct influence of A2bAR signaling on SREBP-1 levels, as shown by direct pharmacological activation or inhibition of A2bAR.

While we found that the A2bAR regulated SREBP-1, there was no effect of A2bAR elimination on SREBP-2 or its downstream target, HMG CoA reductase (HMGCR), both of which are involved in the control of cholesterol synthesis. Acetyl-CoA is the primary substrate for both cholesterol and FA synthesis and its increase is associated with elevation in cholesterol production 42. It is possible, then, that the effect of A2bAR on cholesterol levels is not achieved by changing HMGCR levels but rather by affecting its activity or augmentation of the acetyl-CoA pool through the increase in circulating lipids. Interestingly, pharmacological treatments of hepatocyte cell lines did not yield an observable change in SREBP-1 level upon A2bAR stimulation or inhibition43. The new role we describe here for the A2bAR in vivo and in primary hepatocytes is associated with an effect on the SREBP-1c form, which is the predominant transcript in human and mouse livers, rather than SREBP-1a, the major isoform in cell lines 34. The role of liver A2bAR in the control of the lipid profile was examined by restoring this receptor in this organ in the context of total A2bAR knockout, and by studies in primary hepatocytes. Liver-specific restoration of the receptor led to downregulation of SREBP-1, and of its downstream targets ACC and FAS, and consequently lowered plasma lipid profile. This is consistent with other studies where deletion or knock down of SREBP-1 leads to downregulation of lipid synthesis44–46. Prolonged exposure to A2bAR specific agonist led not only to reduction of circulating plasma lipids but to decrease in plaque formation as well. This proposes a new role for the A2bAR in regulating SREBP-1 levels and in fighting hyperlipidemia and early stages of atherosclerosis.

In conclusion, this study reveals a novel protective role of the A2bAR with respect to hyperlipidemia, hepatic steatosis and atherosclerosis induced by elevated dietary fat and cholesterol consumption. Our study is the first to describe the vast upregulation of this liver receptor under HFD, and to highlight its importance in regulating triglyceride and cholesterol synthesis. Lipid regulation by A2bAR is associated with negative regulation of SREBP-1 and its downstream targets ACC and FAS, suggesting that this pathway is at least partially involved in the mechanism by which A2bAR regulates plasma lipid levels and atherosclerotic plaque formation.

Supplementary Material

1

CLINICAL PERSPECTIVE.

Western-high fat diet (HFD) has long been associated with obesity, elevation in circulating cholesterol and triglycerides, and altered metabolic disorders. The major problem or the ultimate outcome results is some form of cardiac occlusion or atherosclerosis. Although numerous studies have shown the negative effect of high fat, high cholesterol diets on the vasculature, including atherosclerosis and consequent mortality from occlusions of cardiac vessels, the general population would not change eating patterns to account for it. This has called for consideration of treatments that can potentially be used without a change in diet. Here, we employed a genetically modified mouse model lacking the A2b adenosine receptor, as well as a specific agonist for this receptor, BAY 60-6583 to identify a new link between receptor expression/activation, lipidemia and atherosclerosis. Our study showed augmented cholesterol and triglyceride levels, and atherosclerosis in the knockout mice, and a significant reduction in these parameters upon treatment of wild type mice with BAY 60-6583. Of importance, we observed a reduction in atherosclerosis across the aortic tree this treatment while the mice were on HFD. following We propose that activation of the A2b adenosine receptor can be a therapeutic target that can reduce levels of cholesterol and triglycerides and progression of atherosclerosis without significant change of western diet intake.

Acknowledgments

Funding Sources: This work was supported by the National Heart, Lung and Blood Institute Grant (HL93149) to K.R., an established Investigator with the American Heart Association. M.K. was supported by a Cardiovascular Training Grant from the NIH (HL007969).

Footnotes

Conflict of Interest Disclosures: None

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Supplementary Materials

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