Adenosine A2A receptor occupancy stimulates expression ... - CiteSeerX

Published on June 14, 2004 as DOI:10.1189/jlb.0204107

Adenosine A2A receptor occupancy stimulates expression of proteins involved in reverse cholesterol transport and inhibits foam cell formation in macrophages Allison B. Reiss,1 Mohammad M. Rahman, Edwin S. L. Chan, M. Carmen Montesinos, Nahel W. Awadallah, and Bruce N. Cronstein Department of Medicine, New York University Medical Center, New York

Abstract: Transport of cholesterol out of macrophages is critical for prevention of foam cell formation, the first step in the pathogenesis of atherosclerosis. Proteins involved in this process include cholesterol 27-hydroxylase and adenosine 5ⴕ-triphosphate-binding cassette transporter A1 (ABCA1). Proinflammatory cytokines and immune complexes (IC) down-regulate cholesterol 27-hydroxylase and impede cholesterol efflux from macrophages, leading to foam cell formation. Prior studies have suggested occupancy of the anti-inflammatory adenosine A2A receptor (A2AR) minimizes early atherosclerotic changes in arteries following injury. We therefore asked whether A2AR occupancy affects macrophage foam cell formation in response to IC and the cytokine interferon-␥. We found that the selective A2AR agonist 2-p-(2-carboxyethyl)phenethylamino-5ⴕ-N-ethylcarboxamido-adenosine (CGS21680) inhibited foam cell formation in stimulated THP-1 human macrophages, and the effects of CGS-21680 were reversed by the selective A2AR antagonist 4-(2-[7-amino-2-(2-furyl) [1, 2, 4]triazolo[2,3-a] [1, 3, 5]triazin-5-ylamino]ethyl)phenol. In confirmation of the role of A2AR in prevention of foam cell formation, CGS-21680 also inhibited foam cell formation in cultured murine peritoneal macrophages but did not affect foam cell formation in A2AR-deficient mice. Agents that increase foam cell formation also down-regulate cholesterol 27-hydroxylase and ABCA1 expression. Therefore, we determined the effect of A2AR occupancy on expression of these reverse cholesterol transport (RCT) proteins and found that A2AR occupancy stimulates expression of message for both proteins. These results indicate that one mechanism for the antiatherogenic effects of adenosine is stimulation of the expression of proteins involved in RCT. It is more important that these findings suggest a novel approach to the development of agents that prevent progression of atherosclerosis. J. Leukoc. Biol. 76: 000 – 000; 2004. Key Words: atherosclerosis 䡠 cholesterol 27-hydroxylase 䡠 ABCA1

INTRODUCTION There is increasing appreciation of the role of inflammation in the pathogenesis of atherosclerosis [1]. Beginning with recruitment of monocytes from the blood to the arterial wall at sites of vascular injury through accumulation of cholesterol-laden foam cells and infiltration by activated T cells and secretion of proinflammatory cytokines, inflammatory cells and molecules provide the basis for development of the earliest lesions (fatty streak) and the subsequent events that lead to the development of the complicated atherosclerotic plaques [2, 3]. There are mechanisms for control of these inflammatory processes, and reverse cholesterol transport (RCT) out of macrophages in the arterial wall is one important, protective mechanism against cholesterol overload and consequent development of foam cells and atherosclerotic plaques. Many of the enzymes and proteins that regulate RCT from the cells of the vessel wall to the liver for excretion are now known. It has recently been appreciated that the mitochondrial enzyme cholesterol 27-hydroxylase (EC1.14.13.15), expressed at high levels in liver and also present in most organs and tissues, is involved in extrahepatic cholesterol homeostasis and constitutes one of the first lines of defense against atherosclerosis [4 – 6]. Extrahepatic cholesterol is converted into 27hydroxycholesterol and 3␤-hydroxy-5-cholestenoic acid by the enzyme cholesterol 27-hydroxylase. These metabolites (oxysterols) are polar and therefore more readily excreted from cells than cholesterol. In the liver oxysterols are potent inhibitors of 3-hydroxy-3-methylglutaryl-Coenzyme A reductase, the ratelimiting enzyme in de novo cholesterol biosynthesis [7]. Patients with hereditary deficiency of cholesterol 27-hydroxylase suffer from cerebrotendinous xanthomatosis, a rare disease characterized by large extravascular cholesterol deposits, and many of these patients develop premature atherosclerosis despite normal serum lipids [8]. In our earlier studies, we demonstrated that the enzyme 27-hydroxylase is highly expressed in human arterial endothelium and circulating monocytes/ macrophages [4]. Another cellular protein that is critically

1 Correspondence: Department of Medicine, New Bellevue 16N1, New York University Medical Center, 550 First Ave., New York, NY 10016. E-mail: [email protected] Received February 23, 2004; revised April 14, 2004; accepted April 26, 2004; doi: 10.1189/jlb.0204107.

Journal of Leukocyte Biology Volume 76, September 2004 1

Copyright 2004 by The Society for Leukocyte Biology.

involved in RCT is adenosine 5⬘-triphosphate-binding cassette transporter A1 (ABCA1), which functions as a rate-controlling protein in the apolipoprotein A-I (apoA-I)-dependent active transport of cholesterol and phospholipids, facilitating the efflux of cellular cholesterol to extracellular apoA-I or highdensity lipoprotein (HDL) [9]. Evidence that ABCA1-mediated RCT is a critical homeostatic step in the prevention of atherosclerosis is provided by the observation that mutations in the ABCA1 gene result in Tangier disease, a heritable condition characterized by cholesteryl ester deposition in tissues, cholesterol ester accumulation in macrophages, and a high prevalence of atherosclerotic cardiovascular disease [10]. A number of immunologic reactants may be involved in the pathogenesis of atherosclerosis. Earlier studies by Palinski and Witztum [11] demonstrated that antigen-antibody complexes stimulate foam cell transformation of macrophages within the arterial wall. Immune complexes (IC), which have fixed the complement component C1q (IC-C1q), can stimulate the endothelial expression of adhesion molecules that are involved in the recruitment of macrophages to the arterial wall [12, 13] and may also stimulate macrophage secretion of other cytokines, such as interferon-␥ (IFN-␥), which are involved in the development of atherosclerosis [14 –16]. IC-C1q and IFN-␥ have also been found to suppress the expression of 27-hydroxylase [17]. Interest has recently been directed at developing agents that interact with adenosine A2A receptors (A2AR) to prevent or treat vascular and tissue injury. Ligation of adenosine A2AR prevents reperfusion injury and atherosclerosis in animal models, although the pharmacologic mechanism for prevention of atherosclerosis is unknown [18]. We report here that IFN-␥ and IC-C1q dramatically increase foam cell formation in vitro and in parallel, down-regulate the expression of mRNA for the proteins involved in RCT. We also find that adenosine A2AR ligation inhibits foam cell formation and prevents IC-C1q- and IFN-␥-mediated down-regulation of transcription for 27-hydroxylase and ABCA1 by a protein kinase A (PKA)-cyclic adenosine monophosphate (cAMP)-mediated mechanism. This is the first evidence that any physiologic regulator can increase expression of the enzyme cholesterol 27-hydroxylase. Suppression of RCT protein expression by immunologic reactants may represent a pathogenic mechanism of progression in atherosclerosis, which can potentially be prevented by stimulation of adenosine A2AR.

MATERIALS AND METHODS Cell culture reagents Standard tissue-culture media and reagents were obtained from Invitrogen (Carlsbad, CA) and Sigma Chemical Co. (St. Louis, MO) unless otherwise noted. Fetal bovine serum (FBS), bovine serum albumin (BSA), and L-glutamine were purchased from Invitrogen. The protein synthesis inhibitor cycloheximide and PKA inhibitor KT5720 were obtained from Sigma Chemical Co.

Murine model BALB/c and C57BL/6 mice were used for all experiments and were handled in compliance with the Guidelines for the Care and Use of Laboratory Animals approved by the Institutional Animal Care and Use Committee of New York

2

Journal of Leukocyte Biology Volume 76, September 2004

University Medical Center. BALB/c mice were purchased from Harlan Sprague Dawley (Indianapolis, IN). C57BL/6 mice deficient for the A2AR (A2A⫺/⫺) were a gift of Dr. J. F. Chen. C57BL/6 mice deficient for the A3R (A3⫺/⫺) were a gift of Dr. M. A. Jacobson [19, 20]. All mice were injected intraperitoneally with 1 ml thioglycollate media (10%) to induce inflammation. Macrophages were collected by peritoneal lavage after 3– 4 days.

Cell culture Mouse peritoneal macrophages were washed with sterile phosphate-buffered saline (PBS) and were incubated in six-well plates in RPMI-1640 media with 10% FBS (37°C, 5% CO2). The human monocyte-macrophage cell line (THP-1) was purchased from American Type Culture Collection (Manassas, VA) [21]. THP-1 cells were grown in the monocytic form in suspension in RPMI 1640 with 10% serum (37°C, 5% CO2).

Foam cell formation THP-1 cells (105–106 cells/ml) were treated with 100 nM phorbol 12-myristate 13-acetate (PMA) for 48 h (37°C, 5% CO2) to differentiate into macrophages, which were washed with low serum RPMI 1640 and further incubated with 50 ␮g/ml acetylated low-density lipoprotein (acLDL; Intracel, Frederick, MD) for 36 – 48 h under the following three conditions: untreated control, IFN-␥ (500 U/ml), and IC-C1q (0.48 mg/ml). An identical amount of acLDL was added to equal numbers of isolated murine peritoneal macrophages. Following incubation, lipid laden cells were stained with 2% oil red O. Stained cells were counted under a Zeiss-Axiovert 25 light microscope, and foam cells were determined by counting cells containing red-stained globules.

Preparation of BSA rabbit anti-BSA IC IC was prepared as described previously [17]. Briefly, 2 mg BSA was added to 5 ml rabbit immunoglobulin G fraction to BSA (ICN, Costa Mesa, CA). The mixture was incubated at 37°C for 2 h. The resulting IC were washed twice with PBS and then resuspended in PBS to a final concentration of ⬃5 mg/ml.

Total RNA isolation Total RNA from peritoneal macrophages and human THP-1 cells was extracted using Trizol (Invitrogen) according to the manufacturer’s instructions, and 1 ␮g total RNA was used for first-strand, complementary cDNA synthesis primed with oligo dT16 using an RNA polymerase chain reaction (PCR) core kit (Applied Biosystems, Foster City, CA).

Quantitative real-time PCR assay for message analysis For quantification of 27-hydroxylase and ABCA1 message by real-time PCR, the following primer pairs were used: (27-hydroxylase) 5⬘-tgcgccaggctctgaaccag-3⬘, 5⬘-tccacttggggaggaaggtg-3⬘, and (ABCA1) 5⬘-gttgctgctgtggaagaacctc3⬘, 5⬘-agataatcccctgaacccaagg-3⬘. Human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression was used as an internal standard (5⬘-tgaaggtcggagtcaacggatttggt-3⬘, 5⬘-catgtgggccatgaggtccaccac-3⬘) under parallel conditions. PCR amplifications were detected using the SYBR Green kit from Applied Biosystems.

Selective receptor agonists and antagonists The selective A2AR agonist 2-p-(2-carboxyethyl)phenethylamino-5⬘-N-ethylcarboxamido-adenosine (CGS-21680) was collected from Sigma Chemical Co. Another potent A2AR agonist 2-[2-(4-chlorophynyl)ethosy]adenosine (MRE0094) was a gift from King Pharmaceuticals (Bristol, TN), and the selective A2AR antagonist 4-(2-[7-amino-2-(2-furyl) [1, 2, 4]triazolo[2,3-a] [1, 3, 5]triazin-5-ylamino]ethyl)phenol (ZM-241385) was purchased from Tocris Cookson (Bristol, UK) [22].

Gene expression modulation experiments Mouse peritoneal macrophage and human THP-1 monocytoid cells were incubated with the selective A2AR agonists CGS-21680 and MRE-0094, with or without the selective A2AR antagonist ZM-241385, for 3 h prior to total RNA isolation for message analysis and overnight, prior to protein isolation and foam

http://www.jleukbio.org

cell formation experiments. Cells were preincubated with antagonist for 1–2 h before addition of agonists to the culture medium.

Statistical analysis of experimental data Reported as the mean ⫾ SEM, differences between treatment groups were analyzed by using one-way ANOVA followed by unpaired Student-NewmanKeuls test to evaluate two-tailed levels of significance. P ⬍ 0.05 was accepted as being statistically significant. All statistical analyses were performed with SigmaStat software v. 2.03 (SSPS).

RESULTS IFN-␥ and IC stimulate foam cell formation in PMA-differentiated THP-1 macrophages As macrophage-derived foam cells are important constituents of atheromatous lesions, we determined whether immune reactants initiate foam cell formation in differentiated macrophages. IFN-␥ increased the percentage of foam cell transformation by nearly threefold (from 12.8⫾0.5% to 36⫾2.4%, n⫽5, P⬍0.002, Fig. 1) in PMA-differentiated THP-1 macrophages. IC also stimulated a similar increase in foam cell formation (from 12.8⫾0.5% to 37⫾1.2%, n⫽5, P⬍0.002, Fig. 1).

Selective adenosine A2AR occupancy decreased foam cell formation in THP-1 human macrophages Occupation of adenosine A2AR has previously been reported to prevent development of atherosclerosis in animal models [18]; therefore, we determined whether A2AR occupancy modulates foam cell formation stimulated by IC or IFN-␥. Incubation of PMA-differentiated THP-1 macrophages with the A2AR agonist CGS-21680 (1 ␮M) diminishes foam cell transformation stimulated by IFN-␥ by nearly 50% (from 36⫾2.4% to 25⫾2.4%

of cells, n⫽5, P⬍0.001) and reduced IC-stimulated foam cell transformation by ⬃40% (from 37⫾1.1% to 27⫾3.1% of cells, n⫽5, P⬍0.03).

Adenosine A2AR occupancy protects against foam cell formation in murine macrophages To confirm that A2AR occupancy is responsible for prevention of foam cell formation, we studied the effect of CGS-21680 on this phenomenon in murine peritoneal macrophages taken from several strains of mice including mice rendered deficient in A2AR or A3R. As shown, the effect of IC on foam cell formation was much more marked in the murine peritoneal macrophages than the THP-1 cells, increasing by as much as tenfold (from 9⫾4% to 98⫾2% of cells from BALB/c mice that underwent foam cell transformation, n⫽3, P⬍0.001, Fig. 2). The addition of the selective A2AR agonist CGS-21680 completely abrogated the IC-induced foam cell transformation (6⫾2% of cells became foam cells, n⫽3, P⬍0.001) in BALB/c peritoneal macrophages, an effect reversed by the A2AR antagonist ZM241385 (87⫾11% of cells underwent foam cell transformation, P⫽NS vs. control, n⫽3). Similar results were observed in murine peritoneal macrophages from A3R knockout mice and their respective wild-type controls (Fig. 2). In contrast, CGS21680 did not affect IC-stimulated foam cell transformation in peritoneal macrophages from A2AR knockout mice (A2A⫺/⫺).

IFN-␥ and IC down-regulate expression of mRNA for 27-hydroxylase and ABCA1 in THP-1 monocytes/macrophages The immune reactants IFN-␥ and IC stimulate foam cell transformation, and the RCT proteins 27-hydroxylase and ABCA1 inhibit macrophage foam cell transformation [6, 9, 23]. We investigated the effects of IFN-␥ and IC on 27-hydroxylase and ABCA1 mRNA expression (Fig. 3). In THP-1 monocytes, IFN-␥ (500 U/ml) diminished the mRNA level for 27-hydroxylase (by 45⫾3.4%, n⫽5) and ABCA1 (by 29⫾6.8%, n⫽5). Similarly, treatment of PMA-differentiated THP-1 macrophages with IC also decreased the expression of the 27-hydroxylase and ABCA1 message level by 40 ⫾ 6% and 51 ⫾ 5%, respectively (Fig. 3).

Adenosine A2AR occupancy increases extrahepatic 27-hydroxylase and ABCA1 gene expression

Fig. 1. Effect of proinflammatory agents on foam cell transformation in THP-1 macrophages. PMA-treated THP-1 cells (104 cells/ml) were incubated for 24 h separately with IC in the presence of complement C1q (IC-C1q) and IFN-␥ (IFN-g; 500 U/ml), followed by another 36 – 48 h incubation with acLDL (50 ␮g/ml) for lipid loading. Cells were stained with oil Red O to visualize lipid-laden foam cells. The data were expressed as the percentage of foam cells out of the total number of macrophages plated.

The selective A2AR agonist CGS-21680 increased 27-hydroxylase mRNA expression in BALB/c murine macrophages by 47 ⫾ 6.2% (n⫽3, P⬍0.002). CGS-21680 increased the 27hydroxylase message in a dose-dependent manner by as much as 1.8-fold in THP-1 cells (Fig. 4A). A more selective A2AR agonist MRE-0094 affected 27-hydroxylase mRNA expression similarly (Fig. 4A), and the A2AR antagonist ZM-241385 reversed the effect of CGS-21680 and MRE-0094 on expression of mRNA for this protein. Similar results were observed when the effects of these agonists and antagonists were tested on expression of ABCA1 mRNA (Fig. 4B). Reiss et al. Adenosine increases reverse cholesterol transport

3

Fig. 2. Adenosine A2AR occupancy decreases foam cell formation in lipid-loaded murine macrophages. (A) Cultured peritoneal macrophages (thioglycolate method) from BALB/c, A2AR wild-type (A2A WT), A2AR knockout (A2A KO), A3AR WT, and A3AR KO mice were incubated with acLDL (50 ␮g/ml) for 36 – 48 h under the following conditions: untreated control, IC (0.48 mg/ml), selective A2AR agonist CGS21680 (1 ␮M) and IC (0.48 mg/ml), and selective A2AR agonist CGS21680 (1 ␮M) ⫹ selective A2AR antagonist ZM241385 (1 ␮M) ⫹ IC (0.48 mg/ml). Cells were treated with oil Red O to detect lipid-loaded foam cells. Foam cell transformation was expressed as the percentage of foam cells out of the total number of macrophages. (B) The IC-induced increase in foam cell formation in acLDL-treated murine macrophages is prevented by adenosine A2AR occupancy. Cultured peritoneal macrophages (thioglycolate) from BALB/c mice were incubated for 48 h (37°C, 5% CO2) with 50 ␮g/ml acLDL under the following four conditions: control (untreated), agonist (A2AR agonist CGS-21680, 10⫺6 M), IC (BSA–anti-BSA IC, 0.48 mg/ml), agonist ⫹ IC (A2AR agonist CGS-21680, 10⫺6 M⫹BSA–anti-BSA IC, 0.48 mg/ml). (C) Occupancy of the adenosine A2AR by a specific antagonist blocks the protective effect of the agonist against the IC-induced increase in foam cell formation in acLDL-treated murine macrophages. Cultured peritoneal macrophages (thioglycolate) from BALB/c mice were incubated for 48 h (37°C, 5% CO2) with 50 ␮g/ml acLDL under the following two conditions: antagonist ⫹ IC (A2AR antagonist ZM-241385, 10⫺5 M⫹BSA–anti-BSA IC, 0.48 mg/ml) and antagonist ⫹ agonist ⫹ IC (10⫺6 M⫹A2AR antagonist ZM-241385, A2AR agonist CGS-21680, 10⫺5ⴙBSA–anti-BSA IC, 0.48 mg/ml). All cells were stained with oil Red O to visualize lipid-loaded foam cells.

Up-regulation of ABCA1 following A2AR ligation does not depend on expression of 27-hydroxylase Oxysterols, such as 27-hydroxycholesterol, regulate the expression of ABCA1 mRNA by binding to the heterodimeric nuclear receptors, liver X receptor and retinoid X receptor, leading to translocation of the heterodimer to the nucleus, where it regulates gene expression [24 –26]. These observations suggested the hypothesis that up-regulation of the ABCA1 message is contingent on increased expression of 27-hydroxylase with increased production of 27-hydroxycholesterol. To test this hypothesis, we determined whether A2AR-stimulated activation of 27-hydroxylase is required for up-regulation of ABCA1 in monocyte/macrophages. We examined the effect of inhibition of protein synthesis by cycloheximide on 27-hydroxylase and ABCA1 mRNA expression. Treatment of THP-1 monocytes (106 cells/ml) with cycloheximide (50 ␮g/ml, 4 – 6 h) did 4


not affect the basal expression level of mRNA for 27-hydroxylase or ABCA1, as measured by quantitative real-time PCR. Moreover, inhibition of translation did not affect the CGS21680-stimulated increases in expression of mRNA for 27hydroxylase or ABCA1 (Fig. 5). Thus, the A2AR-mediated increase in ABCA1 mRNA expression does not depend only on increased synthesis of 27-hydroxycholesterol in THP-1 cells.

Adenosine A2AR occupancy stimulates expression of mRNA for RCT proteins by a cAMP–PKA-mediated mechanism Adenosine A2AR, which are G␣S-linked receptors, generally signal for changes in cellular function via increasing intracellular concentrations of cAMP, which, in turn, activates PKA to modulate cellular function. We therefore determined whether an inhibitor of PKA affected the A2AR-mediated increase in 27-hydroxylase or ABCA1. The PKA inhibitor KT5720 (0.2 http://www.jleukbio.org

activity, and A2AR and A2BR are stimulatory. At the sequence level, these receptors share varying degrees of homology with each other and in general, are highly conserved through evolution [32, 33]. The effects of adenosine on cardiac muscle, coronary and peripheral vessels, and immune cells have been well-docu-

Fig. 3. Down-regulation of RCT proteins 27-hydroxylase (27-OHase) and ABCA1 message levels in the presence of proinflammatory agents. THP-1 cells (106/ml) in suspension were incubated for 3 h (37°C, 5% CO2) in the presence or absence of the following agents: untreated, IFN-␥ (IFN-G; 500 U/ml), and IC (0.48 mg/ml). Following incubation, total RNA was analyzed for 27hydroxylase and ABCA1 mRNA level by real-time PCR and normalized to GAPDH levels. Gene expression levels were graphed as relative mRNA expression (% of control). Bars represent the mean and SE of multiple independent experiments.

␮M, 2– 4 h, 37°C, 5% CO2) blocked the adenosine A2ARstimulated increase in expression of mRNA for ABCA1 and 27-hydroxylase in THP-1 cells (Fig. 6).

DISCUSSION Our results demonstrate that immune reactants increase foam cell formation in PMA-stimulated human THP-1 monocytoid cells and murine peritoneal macrophages and diminish expression of mRNA for RCT proteins cholesterol 27-hydroxylase and ABCA1. We also observed that in keeping with the inverse relationship between expression of RCT proteins and foam cell formation, adenosine A2AR occupancy increased expression of RCT proteins and decreased foam cell transformation. To our knowledge, the increased expression of 27-hydroxylase that results from engagement of one of the four adenosine receptors (A1, A2A, A2B, and A3) [27] is the first demonstration that an endogenous agent can up-regulate expression of the antiatherogenic enzyme 27-hydroxylase. Attempts by Babiker et al. [28] to up-regulate 27-hydroxylase enzyme activity in macrophages by adding a variety of hormones were unsuccessful. Adenosine is a purine nucleoside produced by many cells and tissues in response to a variety of physical or metabolic stresses. It mediates a broad array of physiological responses, including central nervous system sedation, inhibition of platelet aggregation, and smooth muscle vasodilation [29]. The physiological effects of adenosine are mediated by G protein-coupled seven-transmembrane spanning receptors that exist on almost all cell types in humans [29 –31]. Adenosine receptors are subdivided into four classes based on their differential selectivity of adenosine analogs and molecular structure. The A1R and A3R inhibit stimulated adenylate cyclase

Fig. 4. (A) Selective A2AR agonist induced activation of 27-hydroxylase message in THP-1 cells (106/ml), which were incubated for 3 h (37°C, 5% CO2) alone or in the presence of CGS-21680 or MRE-0094. Cells pretreated with the selective A2AR antagonist ZM-241385 (1 ␮M, 90 min) were also incubated under the same conditions. ZM-241385-treated cells were unable to respond to CGS-21680 or MRE-0094 stimulation. Following incubation, total RNA was analyzed for 27-hydroxylase mRNA level by real-time PCR and was normalized to GAPDH levels. Gene expression levels were graphed as relative mRNA expression (% of control). The data represent the mean and SE of multiple independent experiments. (B) Selective A2AR agonist-induced activation of the ABCA1 message in THP-1 cells (106/ml), which were incubated for 3 h (37°C, 5% CO2) alone or in the presence of CGS-21680 or MRE-0094 at concentrations of 0, 0.01, 0.1, 1, and 10 ␮M. Cells pretreated with the selective A2AR antagonist ZM-241385 (1 ␮M, 90 min) were also incubated under the same conditions. ZM-241385-treated cells were unable to respond to CGS-21680 or MRE-0094 stimulation. Following incubation, total RNA was analyzed for ABCA1 mRNA level by real-time PCR and normalized to GAPDH levels. Gene expression levels were graphed as relative mRNA expression (% of control). The data represent the mean and SE of multiple independent experiments.

Reiss et al. Adenosine increases reverse cholesterol transport

5

Fig. 5. Independent regulation of RCT proteins 27-hydroxylase (27-OHase) and ABCA1 by a selective A2AR agonist. THP-1 monocytes were treated with the standard protein synthesis inhibitor cycloheximide (CHX; 50 ␮g/ml) for 4 – 6 h (n⫽4, 37°C, 5% CO2) with or without the addition of the selective A2AR agonist CGS-21680 (1 ␮M). Following incubation, total RNA was analyzed for 27-hydroxylase and ABCA1 mRNA level by real-time PCR and normalized to GAPDH expression levels. Gene expression levels were graphed as relative mRNA expression (% of control). Bars represent the mean and SE of multiple independent experiments.

mented over the past decade [34]. Adenosine plays an important role in the metabolic regulation of coronary blood flow. It is a powerful coronary vasodilator, the formation of which is sensitive to changes in tissue metabolic state. The vasodilatory effect of adenosine is mediated via extracellular A2R located on the smooth muscle and endothelium of the coronary artery. Further, adenosine has anti-inflammatory effects on leukocytes and endothelial cells mediated through its A2AR [35]. Molecular and pharmacological information suggests that the unique properties of specific adenosine receptors provide a fundamental basis for the design of useful cardiovascular, therapeutic ligands [36, 37]. Indeed, an animal model study by Linden and co-workers [18] demonstrated that A2AR occupancy prevents atherosclerosis, and studies in patients demonstrate that adenosine infusion may diminish myocardial injury following myocardial infarction [38]. The current study has raised the possibility that stimulation of specific adenosine receptors can effectively attenuate the early inflammatory responses to reduce macrophage cholesterol load and eventually prevent foam cell transformation. Prior in vitro studies demonstrated that increased expression of 27-hydroxylase can act as a protective mechanism against the accumulation of excess cholesterol in the arterial intima [7, 28]. Indeed, cycloheximide subjects deficient in 27-hydroxylase develop atherosclerosis prematurely [8]. This new role of adenosine, acting at the A2AR, is consistent with the well-described effects of adenosine on macrophage function [39]. In general, the A2AR modulates cellular function by activating adenylate cyclase leading to accumulation of cAMP and activation of PKA, which phosphorylates a variety of signaling proteins. Among these proteins is a cAMP response element-binding protein family member, which trans6


locates to the nucleus, where it interacts with a consensus cAMP-response element (CRE; 5⬘-TGACGTCA-3⬘) found in the promoter of target genes. Analysis of 1.1 kb upstream 27-hydroxylase gene (CYP27) sequence using the MatInspector program and the TRANSFAC database reveals three potential CREs upstream of the CYP27 antithymocyte globulin initiation site located at –1421, –2340, and –2342 positions [40]. Earlier reports have shown that ABCA1 function is cAMP-inducible, and inhibitors of cAMP can reduce ABCA1-mediated cholesterol efflux to apoA1 and HDL, although no analysis of the promoter region of this gene has been performed [41]. In the present study, we establish a possible link between the effects of IC and IFN-␥ on extrahepatic 27-hydroxylase and ABCA1 expression levels (in monocytes/macrophages) and the ability of these cells to handle high levels of cholesterol. Current and previous data suggested that IC- and IFN-␥treated macrophages exhibit increased percentage of foam cell transformation as compared with untreated cells upon exposure to acLDL. Physiologic changes in macrophages that increase their vulnerability to foam cell conversion may occur via IFN-␥ receptor, Fc receptor, or C1q receptor stimulation. The downregulatory effect of IC and IFN-␥ on 27-hydroxylase expression is highly correlated with the morphological transformation of lipid-laden macrophages into foam cells. Nonetheless, the macrophages of fatty streaks express little or no 27-hydroxylase, which is consistent with the hypothesis that RCT proteins are critical for maintenance of homeostasis in the arterial wall [42]. Patients with rheumatoid arthritis (RA) and systemic lupus erythematosus, diseases associated with elevated levels of IC and IFN-␥, are at greater risk for development of myocardial infarction [43– 45]. Recent studies have suggested that treatment of patients with low-dose methotrexate reduces this risk

Fig. 6. Mitogen-activated protein kinase pathway blockade prevents A2ARstimulated up-regulation of the RCT protein message. THP-1 monocytes were treated with the PKA inhibitor KT5720 (0.2 ␮M) with or without the addition of the selective A2AR agonist CGS-21680 (1 ␮M) for 2– 4 h at 37°C in 5% CO2. Following incubation, total RNA was analyzed for 27-hydroxylase (27-OHase) and ABCA1 mRNA level by real-time PCR and normalized to GAPDH expression levels. Gene expression levels were graphed as relative mRNA expression (% of control). Bars represent the mean and SE of multiple independent experiments.


in patients with RA [46]. Our lab previously demonstrated that many of the beneficial effects of methotrexate in the therapy of RA are mediated by adenosine acting on its receptors [47]. It is interesting to speculate that the reduction in atherosclerosis risk mediated by methotrexate therapy is also a result of the actions of adenosine acting at the A2AR to promote RCT and inhibit the transformation of macrophages in the wall of the coronary and carotid arteries to foam cells with a consequent reduction of atherosclerosis. In summary, treatment with potent and specific A2AR agonists such as CGS-21860 or MRE-0094 increased extrahepatic 27-hydroxylase and ABCA1 expression and significantly reduced early inflammatory events mediated by cytokines and IC and resulted in a marked and sustained attenuation in percentage of foam cell transformation in lipid-laden macrophages. These data support the hypothesis that adenosine A2AR activation provides a defense mechanism against atherosclerosis. Adenosine, released from endothelial cells, smooth muscle cells, platelets, and damaged cells in atherosclerosis, interacts with adenosine A2AR to promote RCT via up-regulation of proteins involved in this process, including cholesterol 27hydroxylase and ABCA1. Previous work in our laboratory [48] on adenosine A2AR in human monocytes has demonstrated that elevated levels of intracellular cAMP are involved in the diminution of the inflammatory response, a finding consistent with our observation that an effective blockade of early inflammatory events with novel A2AR agonists could potentially have therapeutic benefits in inflammation-mediated human cardiovascular diseases. Currently, we are investigating the possible signal transduction mechanism that governs 27-hydroxylase expression and related cholesterol reverse transport processes responsible for protecting against atherosclerosis.

ACKNOWLEDGMENTS This work was supported by grants from the Arthritis Foundation, New York Chapter; American Heart Association, Heritage Affiliate; National Institutes of Health (NIH; AR41911, GM56268); King Pharmaceuticals, Inc.; a general Clinical Research Center grant from NIH, National Center for Research Resources (M01RR00096, awarded to the New York University School of Medicine); and the Kaplan Cancer Center.

REFERENCES 1. Libby, P. (2002) Inflammation in atherosclerosis. Nature 420, 868 – 874. 2. Boisvert, W. A. (2001) The participation of inflammatory cells in atherosclerosis. Drugs Today 37, 173–179. 3. Takahashi, K., Takeya, M., Sakashita, N. (2002) Multifunctional roles of macrophages in the development and progression of atherosclerosis in humans and experimental animals. Med. Electron Microsc. 35, 179 –203. 4. Reiss, A. B., Martin, K. O., Rojer, D. E., Iyer, S., Grossi, E. A., Galloway, A. C., Javitt, N. B. (1997) Sterol 27-hydroxylase: expression in human arterial endothelium. J. Lipid Res. 38, 1254 –1260. 5. Shanahan, C. M., Carpenter, K. L. H., Cary, N. R. B. (2001) A potential role for sterol 27-hydroxylase in atherogenesis. Atherosclerosis 154, 269 – 276. 6. Escher, G., Krozowski, Z., Croft, K. D., Sviridov, D. (2003) Expression of sterol 27-hydroxylase (CYP27A1) enhances cholesterol efflux. J. Biol. Chem. 278, 11015–11019.

7. Bjorkhem, I., Andersson, O., Diczfalusy, U., Sevastik, B., Xiu, R. J., Duan, C., Lund, E. (1994) Atherosclerosis and sterol 27-hydroxylase: evidence for a role of this enzyme in elimination of cholesterol from human macrophages. Proc. Natl. Acad. Sci. USA 91, 8592– 8596. 8. Burnett, J. R., Moses, E. A., Croft, K. D., Brown, A. J., Grainger, K., Vasikaran, S. D., Leitersdorf, E., Watts, G. F. (2001) Clinical and biochemical features, molecular diagnosis and long-term management of a case of cerebrotendinous xanthomatosis. Clin. Chim. Acta 306, 63– 69. 9. Oram, J. F. (2003) HDL apolipoproteins and ABCA1: partners in the removal of excess cellular cholesterol. Arterioscler. Thromb. Vasc. Biol. 23, 720 –727. 10. Brooks-Wilson, A., Marcil, M., Clee, S. M., Zhang, L. H., Roomp, K., Dam, M. V., Yu, L., Brewer, C., Collins, J. A., Molhuizen, H. O., Loubser, O., Ouelette, B. F., Fichter, K., Ashbourne-Excoffon, K. J., Sensen, C. W., Scherer, S., Mott, S., Denis, M., Martindale, D., Frohlich, J., Morgan, K., Koop, B., Pimstone, S., Kastelein, J. J., Hayden, M. R., et al. (1999) Mutations in ABC1 in Tangier disease and familial high-density lipoprotein deficiency. Nat. Genet. 22, 336 –345. 11. Palinski, W., Witztum, J. L. (2000) Immune responses to oxidative neoepitopes of LDL and phospholipids modulate the development of atherosclerosis. J. Intern. Med. 247, 371–380. 12. Lozada, C., Levin, R., Huie, M., Hirchorn, R., Naime, D., Whitlow, M., Recht, P., Golden, B., Cronstein, B. N. (1995) Identification of C1q as the heat-labile serum cofactor required for immune complexes to stimulate endothelial expression of the adhesion molecules E-selectin and intercellular and vascular cell adhesion molecules 1. Proc. Natl. Acad. Sci. USA 92, 8378 – 8382. 13. Daha, M. R., Miltenburg, A. M., Hiemstra, P. S., Klar-Mohamad, N., Van Es, L. A., Van Hinsbergh, V. W. (1988) The complement subcomponent C1q mediates binding of immune complexes and aggregates to endothelial cells in vitro. Eur. J. Immunol. 18, 783–787. 14. Inagaki, Y., Yamagishi, S., Amano, S., Okamoto, T., Koga, K., Makita, Z. (2002) IFN-␥ induced apoptosis and activation of THP-1 macrophages. Life Sci. 71, 2499 –2508. 15. Jiang, K., Chen, Y., Xu, C. S., Jarvis, J. N. (2003) T cell activation by soluble C1q-bearing immune complexes: implications for the pathogenesis of rheumatoid arthritis. Clin. Exp. Immunol. 131, 61– 67. 16. Buono, C., Come, C. E., Stavrakis, G., Maguire, G. F., Connelly, P. W., Lichtman, A. H. (2003) Influence of IFN-␥ on the extent and phenotype of diet-induced atherosclerosis in the LDLR-deficient mice. Arterioscler. Thromb. Vasc. Biol. 23, 454 – 460. 17. Reiss, A. B., Awadallah, N. W., Malhotra, S., Montesinos, M. C., Chan, E. S., Javitt, N. B., Cronstein, B. N. (2001) Immune complexes and IFN-␥ decrease cholesterol 27-hydroxylase in human arterial endothelium and macrophages. J. Lipid Res. 42, 1913–1922. 18. McPherson, J. A., Barringhaus, K. G., Bishop, G. G., Sanders, J. M., Rieger, J. M., Hesselbacher, S. E., Gimple, L. W., Powers, E. R., Macdonald, T., Sullivan, G., Linden, J., Sarembock, I. J. (2001) Adenosine A(2A) receptor stimulation reduces inflammation and neointimal growth in a murine carotid ligation model. Arterioscler. Thromb. Vasc. Biol. 21, 791–796. 19. Chen, J. F., Huang, Z. M. J., Zhu, J., Moratalla, R., Standaert, D., Moskowitz, M. A., Fink, J. S., Schwarzchild, M. A. (1999) A2A adenosine receptor deficiency attenuates brain injury induced by transient focal ischemia in mice. J. Neurosci. 19, 9192–9200. 20. Salvatore, C. A., Tilley, S. L., Latour, A. M., Fletcher, D. S., Koller, B. H., Jacobson, M. A. (2000) Disruption of the A(3) adenosine receptor gene in mice and its effect on stimulated inflammatory cells. J. Biol. Chem. 275, 4429 – 4434. 21. Tsuchiya, S., Yamabe, M., Yamaguchi, Y., Kobayashi, Y., Konno, T., Tada, K. (1980) Establishment and characterization of a human acute monocytic leukemia cell line (THP-1). Int. J. Cancer 26, 171–176. 22. Victor, V. C., Desai, A., Montesinos, M. C., Cronstein, B. N. (2002) Adenosine A2A receptor agonists promote more rapid wound healing than recombinant human platelet-derived growth factor. Inflammation 26, 19 –24. 23. Singaraja, R. R., Brunham, L. R., Visscher, H., Kastelein, J. J., Hayden, M. R. (2003) Efflux and atherosclerosis: the clinical and biochemical impact of variations in the ABCA1 gene. Arterioscler. Thromb. Vasc. Biol. 23, 1322–1332. 24. Fu, X., Menke, J. G., Chen, Y., Zhou, G., MacNaul, K. L., Wright, S. D., Sparrow, C. P., Lund, E. G. (2001) 27-Hydroxycholesterol is an endogenous ligand for liver X receptor in cholesterol-loaded cells. J. Biol. Chem. 276, 38378 –38387. 25. Sparrow, C. P., Baffic, J., Lam, M. H., Lund, E. G., Adams, A. D., Fu, X., Hayes, N., Jones, A. B., Macnaul, K. L., Ondeyka, J., Singh, S., Wang, J., Zhou, G., Moller, D. E., Wright, S. D., Menke, J. G. (2002) A potent synthetic LXR agonist is more effective than cholesterol loading at induc-

Reiss et al. Adenosine increases reverse cholesterol transport

7

26. 27. 28.

29. 30. 31. 32. 33. 34.

35. 36. 37. 38.

8

ing ABCA1 mRNA and stimulating cholesterol efflux. J. Biol. Chem. 277, 10021–10027. Wang, N., Tall, A. R. (2003) Regulation and mechanisms of ATP-binding cassette transporter A1-mediated cellular cholesterol efflux. Arterioscler. Thromb. Vasc. Biol. 23, 1178 –1184. Cronstein, B. N. (1995) A novel approach to the development of antiinflammatory agents: adenosine release at inflamed sites. J. Investig. Med. 43, 50 –57. Babiker, A., Andersson, O., Lund, E., Xiu, R. J., Deeb, S., Reshef, A., Leitersdorf, E., Bjorkhem, I. (1997) Elimination of cholesterol in macrophages and endothelial cells by the sterol 27-hydroxylase mechanism. J. Biol. Chem. 272, 26253–26261. Stiles, G. L. (1992) Adenosine receptors. J. Biol. Chem. 267, 6451– 6467. Palmer, T. M., Stiles, G. L. (1995) Adenosine receptors. Neuropharmacology 34, 683–701. Ramkumar, V., Pierson, G., Stiles, G. L. (1988) Adenosine receptors: clinical implications and biochemical mechanisms. Prog. Drug Res. 32, 195–204. Ralevic, V., Burnstock, G. (1998) Receptors for purines and pyrimidines. Pharmacol. Rev. 50, 413– 418. Londos, C., Cooper, D. M., Wolff, J. (1980) Subclasses of external adenosine receptors. Proc. Natl. Acad. Sci. USA 77, 2551–2558. Vinten-Johansen, J., Zhao, Z. Q., Corvera, J. S., Morris, C. D., Budde, J. M., Thourani, V. H., Guyton, R. A. (2003) Adenosine in myocardial protection in on-pump and off-pump cardiac surgery. Ann. Thorac. Surg. 75, s691–s699. Shryock, J. C., Belardinelli, L. (1997) Adenosine and adenosine receptors in the cardiovascular system: biochemistry, physiology, and pharmacology. Am. J. Cardiol. 79, 2–10. Burnstock, G. (2002) Purinergic signaling and vascular cell proliferation and death. Arterioscler. Thromb. Vasc. Biol. 22, 364 –373. Hayes, E. S. (2003) Adenosine receptors and cardiovascular disease: the adenosine-1 receptor (A1) and A1 selective ligands. Cardiovasc. Toxicol. 3, 71– 88. Hodge, K. R., Bailey, J. K., Lobl, D. A., Laudon, A. B., Gibbons, R. J. (1998) Intravenous adenosine and lidocaine in patients with acute mycocardial infarction. Am. Heart J. 136, 196 –204.


39. Montesinos, M. C., Cronstein, B. N. (2001) Role of P1 receptors in inflammation. In Handbook of Experimental Pharmacology Vol. 151/II: Purinergic and Pyrmidinergic Signalling II Cardiovascular, Respiratory, Immune, Metabolic and Gastrointestinal Tract Function (M. P. Abbrachio, M. Williams, eds.), Berlin, Germany, Springer-Verlag, 303–321. 40. Jegga, A. G., Sherwood, S. P., Carman, J. W., Pinski, A. T., Phillips, J. L., Pestian, J. P., Aronow, B. J. (2002) Detection and visualization of compositionally similar cis-regulatory element clusters in orthologous and coordinately controlled genes. Genome Res. 12, 1408 –1417. 41. Zheng, P., Horwitz, A., Waelde, C. A., Smith, J. D. (2001) Stably transfected ABCA1 antisense cell line has decreased ABCA1 mRNA and cAMP-induced cholesterol efflux to apolipoprotein A1 and HDL. Biochim. Biophys. Acta 1534, 121–128. 42. Wang, X. Q., Panousis, C. G., Alfaro, M. L., Evans, G. F., Zuckerman, S. H. (2002) IFN-␥-mediated downregulation of cholesterol efflux and ABCA1 expression is by the Stat1 pathway. Arterioscler. Thromb. Vasc. Biol. 22, e5– e9. 43. Wick, G., Xu, Q. (1999) Atherosclerosis–an autoimmune disease. Exp. Gerontol. 34, 559 –566. 44. Esdaile, J. M., Abrahamowicz, M., Grodzicky, T., Li, Y., Panaritis, C., du Berger, R., Cote, R., Grover, S. A., Fortin, P. R., Clarke, A. E., Senecal, J. L. (2001) Traditional Framingham risk factors fail to fully account for accelerated atherosclerosis in systemic lupus erythematosus. Arthritis Rheum. 44, 2331–2337. 45. Goodson, N. (2002) Coronary artery disease and rheumatoid arthritis. Curr. Opin. Rheumatol. 14, 115–120. 46. Choi, H. K., Hernan, M. A., Seeger, J. D., Robins, J. M., Wolfe, F. (2002) Methotrexate and mortality in patients with rheumatoid arthritis: a prospective study. Lancet 359, 1173–1177. 47. Chan, E. S., Cronstein, B. N. (2002) Molecular action of methotrexate in inflammatory disease. Arthritis Res. 4, 266 –273. 48. Khoa, N. D., Montesinos, M. C., Reiss, A. B., Delano, D. L., Awadallah, N. W., Cronstein, B. N. (2001) Inflammatory cytokines regulate the expression of adenosine receptors in human monocytic THP-1 cells and human microvascular endothelial cells. J. Immunol. 167, 4026 – 4032.
