Atheroprotective effects of methotrexate on reverse cholesterol transport proteins and foam cell transformation in human THP-1 monocytemacrophages.код для вставкиСкачать
ARTHRITIS & RHEUMATISM Vol. 58, No. 12, December 2008, pp 3675–3683 DOI 10.1002/art.24040 © 2008, American College of Rheumatology Atheroprotective Effects of Methotrexate on Reverse Cholesterol Transport Proteins and Foam Cell Transformation in Human THP-1 Monocyte/Macrophages Allison B. Reiss,1 Steven E. Carsons,1 Kamran Anwar,1 Soumya Rao,1 Sari D. Edelman,1 Hongwei Zhang,1 Patricia Fernandez,2 Bruce N. Cronstein,2 and Edwin S. L. Chan2 Objective. To determine whether methotrexate (MTX) can overcome the atherogenic effects of cyclooxygenase 2 (COX-2) inhibitors and interferon-␥ (IFN␥), both of which suppress cholesterol efflux protein and promote foam cell transformation in human THP-1 monocyte/macrophages. Methods. Message and protein levels of the reverse cholesterol transport proteins cholesterol 27hydroxylase and ATP-binding cassette transporter A1 (ABCA1) in THP-1 cells were evaluated by real-time polymerase chain reaction and immunoblot, respectively. Expression was evaluated in cells incubated in the presence or absence of the COX-2 inhibitor NS398 or IFN␥, with and without MTX. Foam cell transformation of lipid-laden THP-1 macrophages was detected with oil red O staining and light microscopy. Results. MTX increased 27-hydroxylase message and completely blocked NS398-induced down-regulation of 27-hydroxylase (mean ⴞ SEM 112.8 ⴞ 13.1% for NS398 plus MTX versus 71.1 ⴞ 4.3% for NS398 alone; P < 0.01). MTX also negated COX-2 inhibitor–mediated down-regulation of ABCA1. The ability of MTX to reverse inhibitory effects on 27-hydroxylase and ABCA1 was blocked by the adenosine A2A receptor–specific antagonist ZM241385. MTX also prevented NS398 and IFN␥ from increasing transformation of lipid-laden THP-1 macrophages into foam cells. Conclusion. This study provides evidence supporting the notion of an atheroprotective effect of MTX. Through adenosine A2A receptor activation, MTX promotes reverse cholesterol transport and limits foam cell formation in THP-1 macrophages. This is the first reported evidence that any commonly used medication can increase expression of antiatherogenic reverse cholesterol transport proteins and can counteract the effects of COX-2 inhibition. Our results suggest that one mechanism by which MTX protects against cardiovascular disease in rheumatoid arthritis patients is through facilitation of cholesterol outflow from cells of the artery wall. Dr. Reiss’ work was supported by the NIH (National Heart, Lung, and Blood Institute grant HL-073814) and the Arthritis Foundation. Dr. Cronstein’s work was supported by the NIH (grants AA-13336, AR-41911, and GM-56268, and General Clinical Research Center grant M01-RR-00096), King Pharmaceuticals, and the Kaplan Cancer Center of New York University School of Medicine. Dr. Chan’s work was supported by the Arthritis Foundation, New York Chapter, and the Arthritis National Research Foundation. 1 Allison B. Reiss, MD, Steven E. Carsons, MD, Kamran Anwar, PhD, Soumya Rao, MD, Sari D. Edelman, DO, Hongwei Zhang, MD: Winthrop-University Hospital, Mineola, New York; 2 Patricia Fernandez, PhD, Bruce N. Cronstein, MD, Edwin S. L. Chan, MD: New York University School of Medicine, New York, New York. Dr. Cronstein has received consulting fees from King Pharmaceuticals, CanFite Biopharmaceuticals, Bristol-Myers Squibb, Cellzome, TAP Pharmaceuticals, Prometheus Laboratories, Regeneron (Westat, DSMB), Sepracor, Amgen, Endocyte, Protalex, Allos, Combinatorx, Kyowa Hakka, Hoffman-LaRoche, and Savient (less than $10,000 each) and has received speaking fees and/or honoraria from TAP Pharmaceuticals and Amgen (less than $10,000 each). He owns stock in CanFite Biopharmaceuticals, received for his membership in the Scientific Advisory Board. Dr. Cronstein also holds patents for the use of adenosine A1, A2A, and A2B receptor agonists for a variety of purposes, including promoting wound healing, inhibiting fibrosis, treating fatty liver, treating osteoporosis and other bone diseases, and preventing prosthesis loosening; King Pharmaceuticals is the licensee of these patents. Address correspondence and reprint requests to Allison B. Reiss, MD, Vascular Biology Institute, Winthrop-University Hospital, 222 Station Plaza North, Suite 502, Mineola, NY 11501. E-mail: AReiss@Winthrop.org. Submitted for publication September 25, 2007; accepted in revised form August 11, 2008. Methotrexate (MTX) has a long history of use in the treatment of various immunologic diseases, and has been used to treat rheumatoid arthritis (RA) and pso3675 3676 riasis since the 1960s (1–3). MTX was originally developed as a folate antagonist that inhibits dihydrofolate reductase activity and is used in high doses for the treatment of malignancies such as leukemia. Because of its antiinflammatory and immunosuppressive effects, MTX is also efficacious in the treatment of diseases such as asthma, systemic lupus erythematosus, Crohn’s disease, myositis, and vasculitis (4–8). A number of clinical studies show that MTX not only inhibits inflammatory cell proliferation through action on dihydrofolate reductase but also inhibits the conversion of 5-aminoimidazole-4-carboxamide ribonucleotide to 10-formyl-5-aminoimidazole-4-carboxamide ribonucleotide, thus increasing intracellular and extracellular levels of adenosine 5⬘-phosphate and adenosine (9,10). Adenosine is a nucleoside that acts as a signaling molecule by triggering activation of adenosine receptors. These receptors are expressed on the surface of a wide variety of cells and are implicated in cellular protection against ischemia-reperfusion injury (11) and anoxia (12). The risk of cardiovascular disease (CVD) is increased in patients with RA (13–15), and this elevated risk is not explained by traditional risk factors alone (16). Suggested explanations involve the inflammatory response that characterizes active RA, and adverse effects of glucocorticoid therapy or other medications. Findings of previous studies suggest that MTX has a beneficial effect on cardiovascular mortality that is not observed with other antirheumatic drugs (17,18). We previously reported that immune reactants interfere with cellular defense against cholesterol overload by diminishing expression of 2 proteins responsible for reverse transport of cholesterol out of the cell to the circulation for ultimate excretion: cholesterol 27hydroxylase and ATP-binding cassette transporter A1 (ABCA1) (19,20). The atherosclerosis-promoting cytokine interferon-␥ (IFN␥) reduced 27-hydroxylase and ABCA1 message and protein expression in human THP-1 monocyte/macrophages. Further, IFN␥-treated THP-1 macrophages exposed to acetylated low-density lipoprotein (LDL) formed foam cells more rapidly and in greater proportion than untreated control cells. Most recently, we have shown that inhibition of cyclooxygenase (COX) in THP-1 monocyte/macrophages acts in a proatherogenic manner by dose-dependently reducing 27-hydroxylase and ABCA1 levels (21). THP-1 macrophages show a significant increase in foam cell transformation in the presence of the selective COX-2 inhibitor NS398 compared with control (21). This work suggests that compromise of reverse cholesterol transport may REISS ET AL contribute to the known increase in cardiovascular risk in patients treated with COX-2 inhibitors (21,22). Our group also used a number of approaches to enhance expression of 27-hydroxylase and ABCA1. We discovered that these proteins can be up-regulated via activation of the adenosine A2A receptor with specific agonists including CGS21680 and MRE0094. Ligation of this receptor also inhibits macrophage foam cell transformation under cholesterol loading conditions (20). Since MTX is known to affect both adenosine release and cardiovascular risk, we investigated whether MTX modulates cholesterol metabolism and vulnerability to foam cell formation. We report here that MTX treatment counteracts propensity toward cholesterol overload in THP-1 monocyte/macrophages exposed to IFN␥ or selective COX-2 inhibition. This supports the hypothesis that MTX provides protection against atherosclerotic CVD by increasing expression of antiatherogenic molecules involved in cholesterol efflux, likely via a pathway involving adenosine release. MATERIALS AND METHODS Cells and reagents. THP-1 monocytes were obtained from American Type Culture Collection (Manassas, VA). Oil red O and OptiPrep Density Gradient Media were purchased from Sigma (St. Louis, MO). TRIzol reagent was purchased from Invitrogen (Grand Island, NY). All reverse transcriptase– polymerase chain reaction (RT-PCR) reagents were purchased from Applied Biosystems (Chicago, IL). Recombinant human IFN␥ was from R&D Systems (Minneapolis, MN). NS398 was purchased from RBI-Sigma (Natick, MA). MTX was purchased from Bedford Laboratories (Bedford, OH). Acetylated LDL was obtained from Intracel (Issaquah, WA). Cell culture. THP-1 monocytes were grown at 37°C in a 5% CO2 atmosphere, to a density of 106 cells/ml. Growth medium for THP-1 cells was RPMI 1640 (Gibco BRL, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS; Gibco BRL), 50 units/ml penicillin, and 50 units/ml streptomycin. To facilitate differentiation into macrophages, THP-1 monocytes (106 cells/ml) in 12-well plates were treated with 100 nM phorbol myristate acetate (Sigma) for 4 days at 37°C. Isolation of peripheral blood mononuclear cells (PBMCs). Blood from healthy donors was collected in EDTAtreated tubes, pooled, and kept at 4°C. The pooled blood was adjusted to a density of 1.120 with the addition of OptiPrep Density Gradient Media, according to the manufacturer’s instructions. The blood was then overlaid with a 1.074 density solution composed of complete RPMI containing 10% FBS and OptiPrep. A layer of complete RPMI containing 10% FBS was then overlaid on top to prevent monocytes from sticking to the plastic tube. The blood was centrifuged at 750g for 30 minutes at 4°C. After centrifugation, the monocyte interphase was collected from between the 1.074 and RPMI layers. The collected cells were diluted with 2 volumes of complete RPMI and harvested by centrifugation. The pellet was resuspended in complete RPMI. The monocytes were counted with a hemo- ATHEROPROTECTIVE EFFECTS OF MTX cytometer and plated at a density of 2 ⫻ 106 cells/well in a 6-well plate. Experimental conditions. When THP-1 cells had reached 106 cells/ml, medium was aspirated and cells were rinsed twice with Dulbecco’s phosphate buffered saline (PBS) without calcium or magnesium. The monocytes were then incubated for 24–48 hours in 6-well plates (37°C, 5% CO2), under the following conditions: 1) RPMI control, 2) RPMI containing MTX (5 M), 3) RPMI containing NS398 (50 M), 4) RPMI containing NS398 (50 M) and MTX (increasing doses of 0.1 M, 0.5 M, and 5 M), 5) RPMI containing IFN␥ (500 units/ml), and 6) RPMI containing IFN␥ (500 units/ml) and MTX (5 M). THP-1 macrophages were exposed to the following conditions: 1) RPMI control, 2) RPMI containing the adenosine A2A receptor–specific antagonist ZM241385 (10 M), 3) RPMI containing MTX (5 M), 4) RPMI containing IFN␥ (500 units/ml), 5) RPMI containing IFN␥ (500 units/ml) and MTX (5 M), 6) RPMI containing ZM241385 (10 M) and MTX (5 M), 7) RPMI containing IFN␥ (500 units/ml), ZM241385 (10 M), and MTX (5 M), and 8) RPMI containing NS398 (50 M), ZM241385 (10 M), and MTX (5 M). Immediately after the incubation period, the cells were collected and centrifuged at 1,500 revolutions per minute at room temperature, medium was aspirated, and cell protein and RNA were isolated. PBMCs were incubated for 18 hours in RPMI with 10% FBS with and without the addition of MTX at a concentration of 5 M. Cells were collected and RNA isolated. RNA isolation. RNA was isolated using 1 ml TRIzol per 106 cells and dissolved in nuclease-free water. The quantity of total RNA from each condition was measured by ultraviolet spectrophotometry (U2010 spectrophotometer; Hitachi, Brisbane, CA), by absorption at 260 and 280 wavelengths using quartz cuvettes. Analysis of 27-hydroxylase message by RT-PCR. Messenger RNA (mRNA) for 27-hydroxylase and ABCA1 was quantitated by real-time PCR. Complementary DNA (cDNA) was copied from 5 g total RNA using Moloney murine leukemia virus reverse transcriptase primed with oligo(dT). Equal amounts of cDNA were taken from each RT reaction mixture for PCR amplification, using cholesterol 27-hydroxylase– specific primers or ABCA1-specific primers as well as GAPDH control primers. The cholesterol 27-hydroxylase–specific primers span a 311-bp sequence encompassing nucleotides 491–802 of the human cholesterol 27-hydroxylase cDNA (23). ABCA1 primers yield a 234-bp amplified fragment (21). Real-time PCR analysis was performed using the SYBR Green PCR Reagents Kit (Applied Biosystems) with an MX3005P quantitative PCR System (Stratagene, La Jolla, CA). PCR was performed using techniques standardized in our laboratory. Each PCR employed 2.5 l of 10⫻ fluorescent green buffer, 3 l of 25 mM MgCl2, 2 l dNTP mix (2,500 M dCTP, 2,500 M dGTP, 2,500 M dATP, and 5,000 M dUTP), 0.15 l polymerase (5 units/l) (AmpliTaq Gold; Applied Biosystems), 0.25 l uracil N-glycosylase (1 unit/l) (AmpErase; Applied Biosystems), 0.5 l of the forward and reverse primers (10 M concentration), 4 l cDNA, and water to a final volume of 25 l. The thermal cycling parameters were as follows: 5 minutes at 95°C to activate the polymerase (AmpliTaq Gold), followed by 45 cycles of 30 seconds at 95°C and 45 seconds at 58°C, then a final step of 45 seconds at 72°C. 3677 Each reaction was performed in triplicate. The amounts of PCR products were estimated, using software provided by the manufacturer (Stratagene). After completion of PCR cycles, the reaction mixtures were heat denatured over a 35°C temperature gradient from 60°C to 95°C. To correct for differences in cDNA load among samples, the target PCRs were normalized to a reference PCR involving the endogenous housekeeping genes GAPDH and ␤-actin. Nontemplate controls were included for each primer pair to check for significant levels of any contaminants. Fluorescence emission spectra were monitored and analyzed. PCR products were measured by the threshold cycles (Ct) at which specific fluorescence became detectable. The Ct was used for kinetic analysis and was proportional to the initial number of target quantity copies in the sample. Melting curve analysis was performed to assess the specificity of the amplified PCR products. The quantity of the samples was calculated after the Ct levels of the serial dilutions were compared with a control. Quantitative RT-PCR standards were prepared by making 1:10 serial dilutions of a purified PCR product. Protein extraction and Western blot analysis. Total cell lysates were prepared for Western immunoblotting using radioimmunoprecipitation assay (RIPA) lysis buffer (98% PBS, 1% Igepal CA-630, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate). RIPA lysis buffer (100 l) and protease inhibitor cocktail (10 l) (Sigma) were added to the cell pellet from each condition and incubated on ice for 35 minutes with vortexing every 5 minutes. Supernatants were collected after centrifuging at 10,000g at 4°C for 10 minutes, using an Eppendorf 5415C centrifuge. The quantity of protein in each supernatant was measured by absorption at 560 nm using a Hitachi U2010 spectrophotometer. Total cell lysate was used for Western blot analysis. Protein samples (20 g/lane) were boiled for 5 minutes, loaded onto a 10% polyacrylamide gel, electrophoresed for 1.5 hours at 100V, and then transferred to a nitrocellulose membrane in a semidry transblot apparatus for 1 hour at 100V. The nitrocellulose membrane was blocked for 4 hours at 4°C in blocking solution (3% nonfat dry milk dissolved in 1⫻ Tris buffered saline–Tween 20 [TBST]), then immersed in a 1:300 dilution of primary antibody (18.7 g/ml) in blocking solution overnight at 4°C. The primary antibody was an affinity-purified rabbit polyclonal antipeptide antibody raised against residues 15–28 of the cholesterol 27-hydroxylase protein (24). The following day, the membrane was washed 5 times in TBST for 5 minutes per wash and then incubated at room temperature in a 1:3,000 dilution of enhanced chemiluminescence (ECL) donkey antirabbit IgG horseradish peroxidase (HRP)–linked speciesspecific whole antibody (no. NA934; Amersham Biosciences, Piscataway, NJ). The 5 washes in TBST were repeated, and then the immunoreactive protein was detected using ECL Western blotting detection reagent (no. RPN2106; Amersham Biosciences) and film development in SRX-101A (Konica Minolta, Tokyo, Japan). As a control, on the same transferred membrane, ␤-actin was detected using mouse anti–␤-actin (1:1,000) (no. ab6276; Abcam, Cambridge, MA) and ECL sheep anti-mouse IgG HRP-linked species-specific whole antibody (1:2,000) (no. NA931; Amersham Biosciences), with all other steps performed as described above. 3678 REISS ET AL For ABCA1 detection, macrophage cell lysates were electrophoresed for 1.5 hours at 100V (10% polyacrylamide gel), and then transferred to a nitrocellulose membrane. The membrane was blocked for 4 hours at 4°C in blocking solution and then incubated overnight at 4°C in a 1:200 dilution of rabbit anti-ABCA1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA). The following day, the membrane was washed 5 times in TBST for 5 minutes per wash and then incubated at room temperature in a 1:5,000 dilution of ECL donkey antirabbit IgG HRP-linked species-specific whole antibody. Development proceeded as described above for the 27-hydroxylase antibody. Foam cell formation and staining. THP-1–differentiated macrophages were washed 3 times with PBS and further incubated in RPMI (37°C, 5% CO2) for 48 hours under the following conditions: 1) acetylated LDL (50 g/ml), 2) acetylated LDL (50 g/ml) and IFN␥ (500 units/ml), 3) acetylated LDL (50 g/ml) and IFN␥-neutralizing antibody (1.2 g/ml), 4) acetylated LDL (50 g/ml), IFN␥ (500 units/ml), and IFN␥-neutralizing antibody (1.2 g/ml), 5) acetylated LDL (50 g/ml) and IFN␥ receptor antibody (125 ng/ml), and 6) acetylated LDL (50 g/ml), IFN␥ receptor antibody (125 ng/ ml), and IFN␥ (500 units/ml). Immediately following incubation, medium was aspirated and cells were fixed in the same 12-well plates used for incubation, with 4% paraformaldehyde in water, for 2–4 minutes. Cells were stained with 0.2% oil red O in methanol for 1–3 minutes. Cells were observed under a light microscope (Axiovert 25; Carl Zeiss, Gottingen, Germany) with 100⫻ magnification and then photographed using a DC 290 Zoom digital camera (Eastman Kodak, Rochester, NY). The number of foam cells formed under each condition were calculated manually and presented as percentage foam cell formation. Statistical analysis. Statistical analysis was performed using GraphPad, version 4.02 (GraphPad Software, San Diego, CA). All data were analyzed by one-way analysis of variance, and pairwise multiple comparisons were made between control and treatment conditions, using Bonferroni correction. P values less than 0.05 were considered significant. RESULTS MTX increases 27-hydroxylase message and blocks COX-2 inhibitor– and IFN␥-mediated 27hydroxylase down-regulation in THP-1 monocytes. MTX (5 M, 18 hours) increased 27-hydroxylase mRNA expression (mean ⫾ SEM 113.9 ⫾ 6.4% of control) and completely blocked NS398-induced down-regulation of 27-hydroxylase message (112.8 ⫾ 13.1% of control with NS398 plus MTX, versus 71.1 ⫾ 4.3% with NS398 alone; n ⫽ 3) (P ⬍ 0.01) (Figure 1A). MTX (5 M, 18 hours) also abrogated IFN␥-induced down-regulation of 27hydroxylase message (86 ⫾ 9.6% of control with IFN␥ plus MTX, versus 45 ⫾ 6.0% with IFN␥ alone; n ⫽ 3) (P ⫽ 0.02) (Figure 1B). This ability of MTX to overcome suppression of 27-hydroxylase expression by NS398 was observed with MTX in doses of 0.1 M, 0.5 M, and Figure 1. Effect of methotrexate (MTX) on 27-hydroxylase message in THP-1 monocytes and human peripheral blood mononuclear cells (PBMCs). After extraction of total RNA, expression of 27-hydroxylase mRNA was evaluated by quantitative reverse transcriptase–polymerase chain reaction. Signal obtained from the amplification of GAPDH message was used as an internal control. Values are the mean and SEM. A, Quantitation of 27-hydroxylase message in THP-1 cells treated with MTX (5 M, 18 hours), NS398 (NS) (a cyclooxygenase 2 [COX-2] inhibitor) (50 M, 18 hours), or both. The COX-2 inhibitor– mediated decrease in 27-hydroxylase mRNA was prevented by MTX. ⴱ ⫽ P ⬍ 0.05 versus control; # ⫽ P ⬍ 0.01 versus NS398 alone. B, Quantitation of 27-hydroxylase message in THP-1 cells treated with interferon-␥ (IFN␥) (500 units/ml, 18 hours), MTX (5 M, 18 hours), or both. The IFN␥-mediated decrease in 27-hydroxylase mRNA was prevented by MTX. ⴱⴱⴱ ⫽ P ⫽ 0.02 versus IFN␥ alone. C, Effect of MTX on 27-hydroxylase message in healthy donor PBMCs. Isolated PBMCs were incubated in RPMI 1640 with or without MTX (5 M, 18 hours). ⴱⴱⴱⴱ ⫽ P ⫽ 0.004 versus control. 5 M, at both the protein level (Figure 2A) and the message level (Figure 2B). Effect of MTX on 27-hydroxylase message in human donor PBMCs. To confirm that our THP-1 model accurately represented primary human monocyte ATHEROPROTECTIVE EFFECTS OF MTX 3679 down-regulation of ABCA1 message in THP-1 monocytes. Adenosine A2A receptor blockade with ZM241385 abolished the ability of MTX to counter the effects of COX-2 inhibitor on both 27-hydroxylase (Figure 3A) and ABCA1 (Figure 3B). Similarly, down-regulation of 27-hydroxylase and ABCA1 by IFN␥, which we demonstrated previously in THP-1 monocytes (19), was also demonstrated in THP-1 Figure 2. Detection and quantitation of cholesterol 27-hydroxylase (27-OHase) in NS398-treated THP-1 cells exposed to increasing doses of MTX. A, Cultured THP-1 monocytic cells were left untreated or exposed to NS398 (50 M, 18 hours) with or without exposure to increasing doses of MTX for 24 hours. Total cell protein was isolated, and 27-hydroxylase detected with a specific rabbit polyclonal antihuman 27-hydroxylase antibody. Western blotting was also performed with an anti–␤-actin antibody to confirm equal protein loading. The reduction in 27-hydroxylase protein in THP-1 monocytes treated with the COX-2 inhibitor NS398 was reversed with addition of MTX in increasing concentrations. B, Cultured THP-1 monocytic cells were incubated with NS398 (50 M, 48 hours) with or without exposure to increasing doses of MTX for 24 hours. Following isolation of total RNA, the RNA was reverse transcribed and cDNA amplified by reverse transcriptase–polymerase chain reaction. Signal obtained from the amplification of GAPDH message was used as an internal control. The COX-2 inhibitor–mediated suppression of 27-hydroxylase mRNA expression in THP-1 monocytes was reversed with addition of MTX in increasing concentrations. Values are the mean and SEM. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.01, versus control. # ⫽ P ⬍ 0.01 versus NS398 without MTX. See Figure 1 for other definitions. behavior, we isolated PBMCs from healthy human donors and incubated them for 18 hours in RPMI 1640 in the presence or absence of MTX (5 M). MTX exposure resulted in a nearly 4-fold increase in the level of 27-hydroxylase mRNA (mean ⫾ SEM 3.63 ⫾ 0.46–fold increase over the level in untreated cells; n ⫽ 3) (P ⫽ 0.004) as assessed by quantitative RT-PCR (Figure 1C). MTX reverses COX-2 inhibitor–induced and IFN␥-induced down-regulation of ABCA1 and 27hydroxylase via adenosine A2A receptor activation. MTX was also effective in blocking COX-2 inhibitor–mediated Figure 3. Detection and quantitation of cholesterol 27-hydroxylase and ATP-binding cassette transporter A1 (ABCA1) mRNA in NS398treated THP-1 cells exposed to MTX with or without the adenosine A2A receptor antagonist ZM241385 (ZM). THP-1 monocytes were treated with NS398 (50 M, 24 hours), NS398 (50 M, 24 hours) followed by addition of MTX (5 M, 24 hours), or NS398 (50 M) plus ZM241385 (10 M) (24 hours) followed by addition of MTX (5 M, 24 hours). After extraction of total RNA, expression of 27-hydroxylase mRNA was evaluated by quantitative reverse transcriptase–polymerase chain reaction. Signal obtained from the amplification of GAPDH message was used as an internal control. A, Quantitation of 27hydroxylase message. Suppression of 27-hydroxylase message in THP-1 cells by NS398 was reversed by MTX, and this reversal was blocked by ZM241385. B, Quantitation of ABCA1 message. Suppression of ABCA1 message in THP-1 cells by NS398 was reversed by MTX, and this reversal was blocked by ZM241385. Values are the mean and SEM. ⴱⴱ ⫽ P ⬍ 0.01 versus control; # ⫽ P ⬍ 0.01 versus NS398 plus MTX without ZM241385. See Figure 1 for other definitions. 3680 REISS ET AL macrophages and was prevented by MTX, and this effect of MTX was negated by ZM241385 (Figure 4). THP-1 macrophages exposed to MTX alone exhibited a substantial increase in expression of both 27-hydroxylase and ABCA1 relative to untreated THP-1 macrophages (Figure 4). Adenosine A2A receptor activation increases 27hydroxylase and blocks COX-2 inhibitor–mediated 27hydroxylase down-regulation. Addition of the adenosine A2A receptor agonist CGS21680 to THP-1 monocytes exposed to NS398 overcame the reduction in 27- Figure 4. Detection and quantitation of cholesterol 27-hydroxylase (27-OHase) mRNA and protein and ATP-binding cassette transporter A1 (ABCA1) mRNA in IFN␥-stimulated THP-1 macrophages exposed to MTX with or without the adenosine A2A receptor antagonist ZM241385 (ZM). A, THP-1 macrophages were treated with ZM241385 (10 M, 24 hours), MTX (5 M, 24 hours), IFN␥ (500 units/ml, 24 hours), IFN␥ (500 units/ml, 24 hours) followed by addition of MTX (5 M, 24 hours), ZM241385 (10 M, 24 hours) followed by addition of MTX (5 M, 24 hours), ZM241385 (10 M) plus IFN␥ (500 units/ml) (24 hours) followed by addition of MTX (5 M, 24 hours), or ZM241385 (10 M) plus NS398 (50 M) (24 hours) followed by addition of MTX (5 M, 24 hours). After extraction of total RNA, expression of 27-hydroxylase mRNA was evaluated by quantitative reverse transcriptase–polymerase chain reaction (RTPCR). Signal obtained from the amplification of GAPDH message was used as an internal control. Suppression of 27-hydroxylase message in THP-1 macrophages by IFN␥ was reversed by MTX, and this reversal was blocked by ZM241385. B, THP-1 macrophages were treated as described in A. Total cell protein was isolated, and 27-hydroxylase detected with a specific rabbit polyclonal anti-human 27-hydroxylase antibody. Western blotting was also performed with an anti–␤-actin antibody to confirm equal protein loading. C, THP-1 macrophages were treated as described in A. After extraction of total RNA, expression of 27-hydroxylase mRNA was evaluated by quantitative RT-PCR. Signal obtained from the amplification of GAPDH message was used as an internal control. Suppression of ABCA1 message in THP-1 macrophages by IFN␥ was reversed by MTX, and this reversal was blocked by ZM241385. Values in A and C are the mean and SEM. ⴱⴱ ⫽ P ⬍ 0.01 versus IFN␥ alone; # ⫽ P ⬍ 0.01 versus MTX alone. See Figure 1 for other definitions. Figure 5. Effect of the adenosine A2A receptor agonist CGS21680 (CGS) on NS398 (NS)–induced suppression of 27-hydroxylase (27OHase) expression in THP-1 monocytes. THP-1 monocytes were exposed to CGS21680 (10 M, 18 hours), the cyclooxygenase 2 inhibitor NS398 (50 M, 18 hours), or both. After extraction of total RNA, expression of 27-hydroxylase mRNA was evaluated by quantitative reverse transcriptase–polymerase chain reaction. Signal obtained from the amplification of GAPDH message was used as an internal control. A, Expression of 27-hydroxylase protein was decreased by NS398, and this decrease was reversed by addition of CGS21680. B, Levels of 27-hydroxylase message were decreased by NS398, and this decrease was reversed by addition of CGS21680. Values are the mean and SEM. ⴱ ⫽ P ⬍ 0.01 versus control; # ⫽ P ⬍ 0.001 versus NS398 alone. ATHEROPROTECTIVE EFFECTS OF MTX 3681 MTX attenuates foam cell transformation in lipid-laden THP-1 macrophages. Acetylated LDL– treated THP-1 macrophages showed a significant decrease in foam cell transformation in the presence of MTX (mean ⫾ SEM 29.7 ⫾ 2.0%, versus 39.3 ⫾ 5.0% in controls) (n ⫽ 3) (P ⬍ 0.001). With NS398 treatment, the percentage of foam cells was 72.7 ⫾ 4.9%, compared with only 36.3 ⫾ 3.2% with NS398 and MTX (n ⫽ 3) (P ⬍ 0.001). IFN␥ treatment prior to cholesterol loading with acetylated LDL resulted in 71.0 ⫾ 5.0% foam cells, while treatment with IFN␥ plus MTX resulted in only 46.0 ⫾ 7.2% foam cells (n ⫽ 3) (P ⬍ 0.001). Preincubation of THP-1 macrophages with the selective A2A receptor antagonist ZM241385 prior to MTX treatment ablated the antiatherogenic effect of MTX and resulted in a significant increase in foam cells (to 62.1 ⫾ 1.5%). Figure 6 shows photomicrographs of oil red O–stained lipid-laden THP-1 macrophages subjected to the various experimental conditions. DISCUSSION Figure 6. Effect of MTX on NS398- and IFN␥-induced foam cell transformation in lipid-laden THP-1 macrophages. A, Acetylated low-density lipoprotein–treated THP-1 macrophages show a significant decrease in foam cell transformation in the presence of MTX compared with control. B, MTX prevents an NS398-induced increase in foam cell formation. C, MTX prevents an IFN␥-induced increase in foam cell formation. D, The efficacy of MTX in decreasing foam cell formation is abolished by adenosine A2A receptor antagonism with ZM241385 (ZM). (Oil red O–stained; original magnification ⫻ 40.) See Figure 1 for other definitions. hydroxylase expression. This was demonstrated by immunoblot (Figure 5A) and quantitative RT-PCR (Figure 5B). The addition of CGS21680 to NS398-treated THP-1 cells resulted in a 184% increase in 27-hydroxylase mRNA (mean ⫾ SEM 167.2 ⫾ 8.57% of control in cells treated with CGS21680 plus NS398, versus 58.9 ⫾ 2.3% of control in cells treated with NS398 alone; n ⫽ 3) (P ⬍ 0.001) (Figure 5B). Methotrexate modulates the expression of numerous inflammatory cytokines. It has been used successfully in the treatment of many immune- or inflammatorymediated diseases (e.g., RA) based on its induction of generalized immunomodulation. Several studies have demonstrated that binding of adenosine to the A2 receptor inhibits lymphocyte proliferation and production of tumor necrosis factor, interleukin-8 (IL-8), and IL-12, as well as increasing secretion of IL-10 (25). Given the important role of inflammation mediators in the pathogenesis of atherosclerotic CVD, therapeutic modulation targeting these mediators might be a new and promising strategy for treating atherosclerotic CVD. Prior studies have demonstrated that adenosine A2A receptor ligation can help prevent atherosclerosis in an experimental intimal injury model (26). In this model, the recruitment of inflammatory cells to the intima following endothelial injury was markedly reduced. Our results suggest that MTX, which increases adenosine levels, may have a similar effect. Moreover, our results suggest that MTX and adenosine might reduce atherosclerosis not only by reducing inflammation in the vessel wall, but also by stimulating reverse cholesterol transport. The concentration of MTX used was in the range of those used in prior in vitro cell culture studies (27). In our experiments, the concentration of MTX used (5 M) falls within the span of serum concentrations described in the literature (28–31). Although previously 3682 measured serum MTX levels extend through a wide range (0.5–1,800 M), the relevance of serum levels to the effects of MTX is unclear since antiinflammatory activity has been attributed to multiple metabolites of MTX, some of which have vastly extended tissue halflives, rather than to MTX itself (32). Recent studies by Ghosh and colleagues (33) suggest that another mechanism by which COX-2 promotes atherosclerotic cardiovascular disease is by diminishing COX-2–mediated metabolism of endocannabinoids and downstream activation of peroxisome proliferator–activated receptor ␦, increasing endothelial tissue factor expression. Tissue factor promotes intravascular thrombosis, and the increased expression of tissue factor likely predisposes to the further development of atherosclerotic CVD. MTX may also reduce tissue factor expression by increasing adenosine levels and adenosine A2A receptor activation, since it has been known for some time that adenosine suppresses tissue factor production (34–36). In studies published recently, 27-hydroxycholesterol was shown to antagonize estrogen at its receptors, an effect that may cause loss of estrogen protection and therefore promote the development of atherosclerosis (37,38). Although this effect may be an important factor in preventing postmenopausal estrogen therapy from diminishing the risk of atherosclerosis, it may not be relevant to the development of atherosclerosis in men. Moreover, conjugated estrogens also reduce blood concentrations of such anticoagulants as plasminogen activator inhibitor 1 (39), and rising 27-hydroxycholesterol levels may reverse these estrogen-mediated effects. Furthermore, antiatherogenic, rather than proatherogenic, effects have been observed in vivo with selective estrogen receptor modulators such as raloxifene and tamoxifen. Potential explanations have included favorable alterations of antioxidant activity and cholesterol, fibrinogen, and homocysteine levels, as well as vascular tone (40). Thus, the overall effect of 27-hydroxycholesterol on possible estrogen receptor blockade–induced atherogenicity may be eclipsed by a multitude of other factors. This is the first reported evidence that any widely used pharmacotherapy can increase the expression of the antiatherogenic 27-hydroxylase or ABCA1 and can counteract the effects of COX-2 inhibition or IFN␥ exposure on gene expression. We have demonstrated that MTX inhibits foam cell formation under conditions of lipid overload. Our results suggest that the capacity of MTX to reduce the burden of atherosclerotic CVD in patients with RA may be ascribed, in part, to favorable alterations in cholesterol homeostasis mediated via ac- REISS ET AL tivation of the adenosine A2A receptor. Thus, adenosine receptor ligation may provide a suitable mechanism for development of a promising treatment paradigm with long term-benefit in atherosclerotic CVD. ACKNOWLEDGMENT We wish to thank Brian R. Malone, Director, Department of Pharmacy, Winthrop-University Hospital, for helpful discussions with regard to methotrexate. AUTHOR CONTRIBUTIONS Dr. Reiss had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study design. Reiss, Chan. Acquisition of data. Reiss, Anwar, Rao, Edelman, Zhang. Analysis and interpretation of data. Reiss, Carsons, Fernandez, Chan. Manuscript preparation. Reiss, Cronstein, Chan. Statistical analysis. Reiss, Anwar, Fernandez, Chan. REFERENCES 1. Ward JR. Historical perspective on the use of methotrexate for the treatment of rheumatoid arthritis. J Rheumatol 1985;12 Suppl 12:3–6. 2. Kremer JM. Historical overview of the treatment of rheumatoid arthritis with an emphasis on methotrexate. J Rheumatol Suppl 1996;44:34–7. 3. Alarcon GS. Methotrexate use in rheumatoid arthritis: a clinician’s perspective. Immunopharmacology 2000;47:259–71. 4. Moss RB. Alternative pharmacotherapies for steroid-dependent asthma. Chest 1995;107:817–25. 5. Wong JM, Esdaile JM. Methotrexate in systemic lupus erythematosus. Lupus 2005;14:101–5. 6. Feagan BG, Rochon J, Fedorak RN, Irvine EJ, Wild G, Sutherland L, et al, for the North American Crohn’s Study Group Investigators. Methotrexate for the treatment of Crohn’s disease. N Engl J Med 1995;332:292–7. 7. Carlson JA, Cavaliere LF, Grant-Kels JM. Cutaneous vasculitis: diagnosis and management. Clin Dermatol 2006;24:414–29. 8. Ytterberg SR. Treatment of refractory polymyositis and dermatomyositis. Curr Rheumatol Rep 2006;8:167–73. 9. Hornung N, Stengaard-Pedersen K, Ehrnrooth E, Ellingsen T, Poulsen JH. The effects of low-dose methotrexate on thymidylate synthase activity in human peripheral blood mononuclear cells. Clin Exp Rheumatol 2000;18:691–8. 10. Morabito L, Montesinos MC, Schreibman DM, Balter L, Thompson LF, Resta R, et al. Methotrexate and sulfasalazine promote adenosine release by a mechanism that requires ecto-5⬘nucleotidase-mediated conversion of adenine nucleotides. J Clin Invest 1998;101:295–300. 11. Xu Z, Mueller RA, Park SS, Boysen PG, Cohen MV, Downey JM. Cardioprotection with adenosine A2 receptor activation at reperfusion. J Cardiovasc Pharmacol 2005;46:794–802. 12. Sitkovsky MV, Lukashev D, Apasov S, Kojima H, Koshiba M, Caldwell C, et al. Physiological control of immune response and inflammatory tissue damage by hypoxia-inducible factors and adenosine A2A receptors. Annu Rev Immunol 2004;22:657–82. 13. Gonzalez A, Maradit Kremers H, Crowson CS, Ballman KV, Roger VL, Jacobsen SJ, et al. Do cardiovascular risk factors confer ATHEROPROTECTIVE EFFECTS OF MTX 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. the same risk for cardiovascular outcomes in rheumatoid arthritis patients as in non-rheumatoid arthritis patients? Ann Rheum Dis 2008;67:64–9. Gonzalez-Gay MA, Gonzalez-Juanatey C, Miranda-Filloy JA, Garcia-Porrua C, Llorca J, Martin J. Cardiovascular disease in rheumatoid arthritis. Biomed Pharmacother 2006;60:673–7. Kremers HM, Gabriel SE. Rheumatoid arthritis and the heart. Curr Heart Fail Rep 2006;3:57–63. Solomon DH, Curhan GC, Rimm EB, Cannuscio CC, Karlson EW. Cardiovascular risk factors in women with and without rheumatoid arthritis. Arthritis Rheum 2004;50:3444–9. Van Halm VP, Nurmohamed MT, Twisk JW, Dijkmans BA, Voskuyl AE. Disease-modifying antirheumatic drugs are associated with a reduced risk for cardiovascular disease in patients with rheumatoid arthritis: a case control study. Arthritis Res Ther 2006;8:R151. Choi HK, Hernan MA, Seeger JD, Robins JM, Wolfe F. Methotrexate and mortality in patients with rheumatoid arthritis: a prospective study. Lancet 2002;359:1173–7. Reiss AB, Awadallah NW, Malhotra S, Montesinos MC, Chan ES, Javitt NB, et al. Immune complexes and interferon-␥ decrease cholesterol 27-hydroxylase in human arterial endothelium and macrophages. J Lipid Res 2001;42:1913–22. Reiss AB, Rahman MM, Chan ES, Montesinos MC, Awadallah NW, Cronstein BN. Adenosine A2A receptor occupancy stimulates expression of proteins involved in reverse cholesterol transport and inhibits foam cell formation in macrophages. J Leukoc Biol 2004;76:727–34. Chan ES, Zhang H, Fernandez P, Edelman SD, Pillinger MH, Ragolia L, et al. Effect of COX inhibition on cholesterol efflux proteins and atheromatous foam cell transformation in THP-1 human macrophages: a possible mechanism for increased cardiovascular risk. Arthritis Res Ther 2007;9:R4. Iezzi A, Ferri C, Mezzetti A, Cipollone F. COX-2: friend or foe? Curr Pharm Des 2007;13:1715–21. Reiss AB, Martin KO, Rojer DE, Iyer S, Grossi EA, Galloway AC, et al. Sterol 27-hydroxylase: expression in human arterial endothelium. J Lipid Res 1997;38:1254–60. Cali JJ, Russell DW. Characterization of human sterol 27-hydroxylase. J Biol Chem 1991;266:7774–8. Cutolo M, Sulli A, Pizzorni C, Seriolo B, Straub RH. Antiinflammatory mechanisms of methotrexate in rheumatoid arthritis. Ann Rheum Dis 2001;60:729–35. McPherson JA, Barringhaus KG, Bishop GG, Sanders JM, Rieger JM, Hesselbacher SE, et al. Adenosine A2A receptor stimulation reduces inflammation and neointimal growth in a murine carotid ligation model. Arterioscler Thromb Vasc Biol 2001;21:791–6. Galivan J, Pupons A, Rhee MS. Hepatic parenchymal cell glu- 3683 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. tamylation of methotrexate studied in monolayer culture. Cancer Res 1986;46:670–5. Cheng KK. Association of plasma methotrexate, neutropenia, hepatic dysfunction, nausea/vomiting and oral mucositis in children with cancer. Eur J Cancer Care (Engl) 2008;17:306–11. Bacci G, Loro L, Longhi A, Bertoni F, Bacchini P, Versari M, et al. No correlation between methotrexate serum level and histologic response in the pre-operative treatment of extremity osteosarcoma. Anticancer Drugs 2006;17:411–5. Wallace CA, Bleyer WA, Sherry DD, Salmonson KL, Wedgwood RJ. Toxicity and serum levels of methotrexate in children with juvenile rheumatoid arthritis. Arthritis Rheum 1989;32:677–81. Ravelli A, Di Fuccia G, Molinaro M, Ramenghi B, Zonta L, Regazzi MB, et al. Plasma levels after oral methotrexate in children with juvenile rheumatoid arthritis. J Rheumatol 1993;20: 1573–7. Chan ES, Cronstein BN. Molecular action of methotrexate in inflammatory diseases. Arthritis Res 2002;4:266–73. Ghosh M, Wang H, Ai Y, Romeo E, Luyendyk JP, Peters JM, et al. COX-2 suppresses tissue factor expression via endocannabinoid-directed PPAR␦ activation. J Exp Med 2007;204:2053–61. Deguchi H, Takeya H, Wada H, Gabazza EC, Hayashi N, Urano H, et al. Dilazep, an antiplatelet agent, inhibits tissue factor expression in endothelial cells and monocytes. Blood 1997;90: 2345–56. Deguchi H, Takeya H, Urano H, Gabazza EC, Zhou H, Suzuki K. Adenosine regulates tissue factor expression on endothelial cells. Thromb Res 1998;91:57–64. Watanabe T, Tokuyama S, Yasuda M, Sasaki T, Yamamoto T. Involvement of adenosine A2 receptors in the changes of tissue factor-dependent coagulant activity induced by polymorphonuclear leukocytes in endothelial cells. Jpn J Pharmacol 2002;88: 407–13. Umetani M, Domoto H, Gormley AK, Yuhanna IS, Cummins CL, Javitt NB, et al. 27-hydroxycholesterol is an endogenous SERM that inhibits the cardiovascular effects of estrogen. Nat Med 2007; 13:1185–92. Dusell CD, Umetani M, Shaul PW, Mangelsdorf DJ, McDonnell DP. 27-hydroxycholesterol is an endogenous selective estrogen receptor modulator. Mol Endocrinol 2008;22:65–77. Skouby SO, Sidelmann JJ, Nilas L, Jespersen J. A comparative study of the effect of continuous combined conjugated equine estrogen plus medroxyprogesterone acetate and tibolone on blood coagulability. Hum Reprod 2007;22:1186–91. Leung FP, Tsang SY, Wong CM, Yung LM, Chan YC, Leung HS, et al. Raloxifene, tamoxifen and vascular tone. Clin Exp Pharmacol Physiol 2007;34:809–13.