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Atheroprotective effects of methotrexate on reverse cholesterol transport proteins and foam cell transformation in human THP-1 monocytemacrophages.

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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.
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