Dr. Cronstein has received consulting fees (less than $10,000) from King, TAP, Canfite, Bristol-Myers-Squibb, Regeneron, and Centocor, owns stock in Canfite, and has provided expert testimony for Merck and Amgen.
Drs. Cronstein and Chan hold patents for adenosine receptor antagonists for fibrosis, and have received royalties from King Pharmaceuticals.
Adenosine regulates inflammation and tissue repair, and adenosine A2A receptors promote wound healing by stimulating collagen matrix production. We therefore examined whether adenosine A2A receptors contribute to the pathogenesis of dermal fibrosis.
Collagen production by primary human dermal fibroblasts was analyzed by real-time polymerase chain reaction, 14C-proline incorporation, and Sircol assay. Intracellular signaling for dermal collagen production was investigated using inhibitors of MEK-1 and by demonstration of ERK phosphorylation. In vivo effects were studied in a bleomycin-induced dermal fibrosis model using adenosine A2A receptor–deficient wild-type littermate mice, C57BL/6 mice, and mice treated with adenosine A2A receptor antagonist. Morphometric features and levels of hydroxyproline were determined as measures of dermal fibrosis.
Adenosine A2A receptor occupancy promoted collagen production by primary human dermal fibroblasts, which was blocked by adenosine A2A, but not A1 or A2B, receptor antagonism. Adenosine A2A receptor ligation stimulated ERK phosphorylation, and A2A receptor–mediated collagen production by dermal fibroblasts was blocked by MEK-1 inhibitors. Adenosine A2A receptor–deficient and A2A receptor antagonist–treated mice were protected from developing bleomycin-induced dermal fibrosis.
These results demonstrate that adenosine A2A receptors play an active role in the pathogenesis of dermal fibrosis and suggest a novel therapeutic target in the treatment and prevention of dermal fibrosis in diseases such as scleroderma.
Adenosine, a product of ATP catabolism, is released from cells and tissues under conditions of stress or hypoxia and is a potent endogenous physiologic and pharmacologic mediator. Adenosine regulates cellular and organ function via interaction with a family of 4 G protein–coupled receptors, A1, A2A, A2B, and A3. Among other pharmacologic effects, the stimulation of adenosine A2A receptors promotes excisional wound closure in both normal mice and diabetic rats, and the enhancement in dermal wound healing is accompanied by an increase in matrix (collagen) in the wounds (1, 2). Indeed, adenosine A2A receptor–mediated stimulation of wound healing by the novel, potent, and selective A2A receptor agonist MRE0094 (2-[2-(4-chlorophenyl)-ethoxy]-adenosine) promotes wound closure at a rate substantially greater than that by recombinant human platelet-derived growth factor, an agent currently marketed for the promotion of wound healing in patients with diabetic ulcers (3). Further confirmation of the role of A2A receptors in promoting wound healing is provided by the observation that adenosine A2A agonists do not promote wound healing in adenosine A2A receptor–deficient mice (2).
Since the adenosine A2A receptor mediates dermal wound closure and increases dermal matrix deposition, we hypothesized that adenosine, acting at the adenosine A2A receptor, may also play a role in fibrosis in pathologic conditions such as scleroderma or hypertrophic scar formation. We therefore determined the effect of A2A receptors on collagen production by primary human dermal fibroblasts, and examined the role of A2A receptors in bleomycin-induced dermal fibrosis, a murine model of scleroderma.
We provide evidence that endogenously released adenosine plays an important role in the pathogenesis of dermal fibrosis. Occupancy by the adenosine A2A receptor stimulates collagen production by primary human dermal fibroblasts via an ERK-dependent pathway. Unlike wild-type (WT) mice that were otherwise genetically identical, adenosine A2A receptor–knockout mice were protected from developing bleomycin-induced dermal fibrosis as were mice treated with an adenosine A2A receptor antagonist. These data suggest that the adenosine A2A receptor is a key player in dermal fibrogenesis, and adenosine A2A receptor antagonism may be a novel therapeutic target for the treatment of dermal fibrosis in scleroderma.
MATERIALS AND METHODS
Role of the funding source.
King Pharmaceuticals had a role in study design, data collection, data analysis, and interpretation of the data. All authors contributed to the manuscript and approved the content prior to submission.
Cell cultures and reagents.
Primary human dermal fibroblasts were isolated from normal human foreskin by investigators at the Weill Medical College laboratory, and primary adult human dermal fibroblasts were purchased from Cambrex BioWhittaker (Walkersville, MD) and used within the first 5 passages. CGS-21680, collagenase, 8-cyclopentyldipropylxanthine (DPCPX), 3-propylxanthine (enprofylline), ascorbic acid, and β-aminopropionitrile were purchased from Sigma (St. Louis, MO). Bleomycin sulfate was from Bedford Laboratories (Bedford, OH). ZM241385 and MRS1706 were from Tocris (Ballwin, MO), and 14C-labeled proline was from Moravek Biochemicals (Brea, CA). SB203580, PD098059, and UO126 were obtained from Biomol Research Laboratory (Plymouth, MA). Antibodies to type I collagen were from Southern Biotechnology Associates (Birmingham, AL). Antibodies to adenosine receptor A2A were from Upstate Cell Signaling (Charlottesville, VA). Antibodies to matrix metalloproteinase 14 (MMP-14) were from Chemicon (Temecula, CA). Anti–phosphorylated ERK antiserum (anti–active MAPK pAB; sc-7383-pERK) mouse monoclonal IgG2a was from Promega (Madison, WI). Antisera specific for p44erk1 (sc-93-ERK-1) rabbit polyclonal IgG and p42erk2 (sc-154-ERK-2) rabbit polyclonal IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Isolation of messenger RNA (mRNA) and semiquantitative reverse transcription–polymerase chain reaction (RT-PCR).
Messenger RNA was isolated from primary human dermal fibroblasts using a MicroFastTrack kit (Invitrogen, Carlsbad, CA) following the manufacturer's protocol. First-strand complementary DNA (cDNA) was synthesized and subsequent PCR was performed using the GeneAmp RNA-PCR Core kit (PerkinElmer, Branchburg, NJ). The PCRs were performed using primers specific for human adenosine receptors and GAPDH, a housekeeping gene. The cycle number was adjusted to allow the PCR to proceed in a linear range. The primer sequences were 5′-TCCATCTCAGCTTTCCAGGC-3′ (forward) and 5′-CTCGAACTCGCACTTGATCAC-3′ (reverse) for the A1 receptor, 5′-ACCTGCAGAACGTCACCAAC-3′ (forward) and 5′-TCTGCTTCAGCTGTCGTCGC-3′ (reverse) for the A2A receptor, 5′-CACAGGACGCGCTGTACGTG-3′ (forward) and 5′-TTCTGTGCAGTTGTTGGTGG-3′ (reverse) for the A2B receptor, 5′-AACGTGCTGGTCATCTGCGTGGTC-3′ (forward) and 5′-GTAGTCCATTCTCATGACGGAAAC-3′ (reverse) for the A3 receptor, and 5′-ACCATCATCCCTGCCTCTAC-3′ (forward) and 5′-CCTGTTGCTGTAGCCAAAT-3′ (reverse) for GAPDH. All primers were designed to amplify cDNA that crossed an intron in the genomic DNA, and the amplicon was sequenced to confirm the identity of the cDNA amplified. Aliquots of PCR product were loaded onto ethidium bromide–stained agarose gel, visualized with an ultraviolet transilluminator, and digitally photographed. The amplicon was quantitated densitometrically using Kodak Digital Science software (Stuttgart, Germany), and all values were normalized to the GAPDH amplicon.
RT and real-time PCR for collagen.
Total RNA from treated primary human dermal fibroblasts was isolated using TRIzol (Invitrogen, Carlsbad, CA) following stimulation with CGS-21680 (10 μM) or transforming growth factor β (TGFβ) (10 ng/ml) according to the manufacturer's protocol. RT was performed using the GeneAmp RNA Core kit (Applied Biosystems, Branchburg, NJ) in a volume of 50 μl using oligo(dT) primers and murine leukemia virus reverse transcriptase according to the manufacturer's protocol. Real-time PCRs were performed using the SYBR Green PCR kit (Applied Biosystems, Foster City, CA) following the manufacturer's instructions, and carried out on the Mx3005P Q-PCR system (Stratagene, La Jolla, CA). Aliquots of RT reactions were subjected to PCR in 25-μl reactions with SYBR Green (PerkinElmer) using primers for collagen α1(I) (Col1a1), 5′-TGTTCAGCTTTGTGGACCTCCG-3′ (forward) and 5′-CCGTTCTGTACGCAGGTGATTG-3′ (reverse), Col3a1, 5′-GAAGATGTCCTTGATGTGC-3′ (forward) and 5′-AGCCTTGCGTGTTCGATAT-3′ (reverse), and GAPDH, 5′-ACCATCATCCCTGCCTCTAC-3′ (forward) and 5′-CCTGTTGCTGTAGCCAAAT-3′ (reverse). The thermal cycling conditions included an initial 95°C for 300 seconds, and then 95°C for 60 seconds, 58°C for 45 seconds, and 72°C for 45 seconds for 40 cycles. For each assay, standards, no-template controls, and no-RT controls were included to verify the quality and cDNA specificity of the primers. The initial number of copies for each template was calculated using Mx3005P software, with results normalized to GAPDH.
Quantification of collagen production by 14C-proline incorporation and Sircol assay.
Primary human dermal fibroblasts in early passage were grown to near confluence prior to treatment. Cells were treated with ascorbic acid (50 μg/ml for 24 hours) and then pulsed with 14C-labeled proline (Moravek, Brea, CA) to which β-aminopropionitrile was added (50 μg/ml) for 24 hours. The adenosine A2A receptor agonist CGS-21680 (at concentrations of 0–10 μM) was incubated with primary human dermal fibroblasts for 24 hours (37°C in 5% CO2) with or without the addition of adenosine receptor antagonists (ZM241385, DPCPX, or enprofylline at 10 μM each). Supernates were collected following antagonist treatment and collagen was extracted by the addition of ethanol (3:1 volume:volume). The precipitate was then redissolved in 1% sodium dodecyl sulfate (SDS) following centrifugation at 6,000g for 5 minutes and electrophoresed on 7% polyacrylamide gels. Collagen was identified as a high molecular weight, collagenase-sensitive protein visible on SDS–polyacrylamide gel electrophoresis (SDS-PAGE). Radioactivity was quantified following a 21-day PhosphorImager exposure using ImageQuant software version 5.0 (Molecular Dynamics, Sunnyvale, CA), in which band intensity was determined using Kodak 1-dimensional software version 2.0.1, with results adjusted to the relative density of protein in Coomassie blue–stained gels. Collagen production on supernates was also quantitated using the Sircol collagen assay (Biocolor, Belfast, Northern Ireland) following the manufacturer's instructions.
Measurement of ERK activation.
Activation of ERK-1 and ERK-2 was measured as previously described (4). Briefly, ERK activity was measured as the ability of dermal fibroblast lysates to phosphorylate MBPp, a synthetic peptide substrate containing the specific sequence PRTP (underline indicates the substitution) of myelin basic protein that is phosphorylated by the serine/threonine kinase ERK. Cells were treated with or without CGS-21680 in the presence or absence of MEK-1 inhibitors UO126 (10 μM), PD098059 (10 μM), or the p38 MAPK inhibitor SB203580 (10 μM). Reactions were quenched by addition of 50 μl lysis buffer (20 mM Tris, pH 7.4, 1 mM Na-EGTA, 2 mM sodium vanadate, 25 mM sodium fluoride, 0.5% Triton X-100, 2 mM phenylmethylsulfonyl fluoride, 10 units/ml aprotinin, and 10 μg/ml each of chymostatin, antipain, and pepstatin) per well on a 6-well plate, and placed on a platform shaker at 4°C for 15 minutes. Cell lysates were scraped from the plates, transferred to labeled Eppendorf centrifuge tubes, and centrifuged at 12,000 revolutions per minute for 10 minutes at room temperature.
An equal volume of each supernatant was transferred onto new tubes and loading buffer was added. Tubes were subjected to heat at 100°C for 3 minutes and samples were frozen overnight for Western blot analysis. Activation-specific phosphorylation of ERK was determined by immunoblotting lysates of dermal fibroblasts with antiserum specific for phosphorylated, active-state ERK-1/2. Anti-pERK antibody was bound by125I-labeled protein A, with detection by PhosphorImaging (Molecular Dynamics). The immunoblots were then stripped. After several washes in Tris buffered saline–Tween 20, the blots were blocked again and probed using anti–ERK-1/2 antisera, which were nonreactive to the phosphorylated state (Santa Cruz Biotechnology). ERK activation was calculated as the ratio of pERK to total ERKs, with results expressed as the percentage of values obtained in either control or stimulated conditions.
Determination of MMP activity by gelatin zymography.
Supernates were fractionated on 10% SDS-PAGE with 10% gelatin (Bio-Rad, Hercules, CA). Protein samples (15 μl/lane) were mixed with double volume of 1× zymogram sample buffer (Bio-Rad). The gel was incubated with renaturation buffer (Bio-Rad) for 30 minutes at room temperature, and then incubated in developing buffer (Bio-Rad) overnight at 37°C with gentle agitation. The gel was stained with 0.5% Coomassie blue in 40% methanol and 10% acetic acid for 1 hour at room temperature, and destained with 40% methanol and 10% acetic acid until clear bands appeared against the blue background. MMP controls were obtained from Sigma.
Primary human dermal fibroblasts were incubated with different treatments in Dulbecco's modified Eagle's medium (Gibco, Grand Island, NY). Western blotting was performed to examine expression of the adenosine A2A receptor (membrane cell preparations), type I collagen, and MMP-14 proteins (whole cell lysates). Whole cell lysates were collected using RIPA lysis buffer (150 mM NaCl, 10 mM Tris, pH 7.2, 0.1% SDS, 1.0% Triton X-100) containing protease/phosphatase inhibitor cocktail (Sigma). Protein samples (20 μg/lane) were mixed with 5× loading buffer (50 mM Tris HCl, pH 6.8, 2% SDS, 0.1% bromphenol blue, 10% glycerol) and 2-mercaptoethanol (96 mM) and heated for 5 minutes. Lysates were fractionated on SDS-PAGE and transferred onto a nitrocellulose membrane (Bio-Rad). The nitrocellulose membrane was blocked for 2 hours at 4°C in blocking solution (3% bovine serum albumin in 1× Tween 20–Tris buffered saline [consisting of 20 mM Tris HCl, pH 7.4, 150 mM NaCl, and 0.1% Tween 20]). After blocking, the membrane was incubated with primary antibody (1:500 dilution for type I collagen, 1:1,000 for A2A receptor, and 1:5,000 for MMP-14) and incubated for 2 hours at 37°C with gentle shaking on a platform shaker. After incubation with secondary antibody, proteins were visualized using an enhanced chemiluminescence kit (Amersham Biosciences, Little Chalfont, UK).
Quantification of dermal hydroxyproline content.
Hydroxyproline content in tissue specimens was measured colorimetrically as described previously, with modifications (5). Tissue specimens were dried and hydrolyzed in 6N HCl at 110°C for 24 hours. Hydrolysates were filtered and neutralized to pH 7 with NaOH. Two hundred microliters of each sample was mixed with 500 μl of chloramine-T solution (1.4% chloramine-T, 10% N-propanol, and 80% citrate-acetate buffer). The mixture was incubated for 20 minutes at room temperature. Five hundred microliters of Ehrlich's solution was added and the samples were incubated at 65°C for 18 minutes. Absorbance was measured at 560 nm. Standard curves (0–10 μg) were generated for each experiment using reagent hydroxyproline as a standard. Results were expressed as μg of hydroxyproline per mg of tissue.
Morphometric dermal measurements in bleomycin-treated mice.
A2A receptor–deficient mice (6, 7) and their WT littermates were injected with bleomycin (10 mg/ml, comprising 0.1 ml subcutaneously on alternate days) for 18 days, and the animals were killed at the end of the experimental period. The backs of the animals were shaved prior to morphometric measurements. Skinfold (pinch) thickness was measured using skin calipers on the same area over the middle to upper back of the mice. Skin thickness was measured on 6-mm punch biopsy specimens obtained from the upper back away from previous sites of injection. Breaking strength of the skin was measured on the 6-mm punch biopsy specimens using a tensiometer (Series EG2 digital force gauge; Mark-10, Copiague, NY), and the point of maximal stress before tearing of the biopsy specimen was recorded. All measurements were undertaken in a blinded manner. These studies were approved by the Institutional Animal Care and Use Committee of New York University School of Medicine.
Administration of adenosine A2A receptor antagonist to mice with bleomycin-induced fibrosis.
Male C57BL/6 mice were treated with the A2A receptor antagonist ZM241385 (50 mg/kg twice per day intraperitoneally, administered in carrier consisting of 15% DMSO, 15% Cremophor EL, and 70% water to a total injection volume of 0.1 ml) or carrier alone (control), starting 3 days prior to fibrosis induction with bleomycin (10 mg/ml, 0.1 ml subcutaneously on alternate days for 18 days). This group was compared with male mice treated with phosphate buffered saline (PBS) alone (without bleomycin). We have previously observed that breaking tension and hydroxyproline content were greater in the skin of male C57BL/6 mice than their female counterparts, consistent with findings by other investigators (8).
Unless otherwise stated, data were analyzed by one-way analysis of variance (ANOVA), and significance of differences between groups was determined by Dunn's multiple comparison test when one-way ANOVA demonstrated significant intergroup differences (P < 0.007). All statistical analyses were performed with GraphPad Prism software version 4.02 (GraphPad Software, San Diego, CA).
Expression of adenosine receptors on primary human dermal fibroblasts.
Almost all cells express adenosine receptors, although not all adenosine receptors are expressed on all cells. We have previously found message for adenosine A2A, A2B, and A3 receptors in a fibroblast cell line (CCD-25sk) (1). In the present study, we detected the presence of adenosine A1, A2A, and A2B receptor message in normal primary human dermal fibroblasts by RT-PCR (Figure 1A). These cells did not express the adenosine A3 receptor. We also confirmed, on Western blot, the expression of adenosine A2A receptor in cell membrane preparations, with THP-1 cells (9) as a positive control (Figure 1B).
Induction of increased collagen production by primary human dermal fibroblasts with adenosine A2A receptor agonist CGS-21680.
Since previous studies indicated that CGS-21680 promotes wound healing, we investigated the effect of this adenosine A2A receptor agonist on collagen production by primary human dermal fibroblasts in the presence or absence of selective adenosine receptor antagonists. Treatment with adenosine A2A receptor agonist CGS-21680 dramatically increased dermal fibroblast collagen production in a dose-dependent manner, to as much as 2,264 ± 1,104% of control values (mean ± SEM of 3 samples) at 10 μM (P < 0.001 versus control by two-way ANOVA), as assessed by 14C-proline incorporation (Figure 2A). The CGS-21680–induced increase in collagen production was almost completely abrogated by coincubation with the adenosine A2A receptor antagonist ZM241385 (1 μM at 37°C in 5% CO2), but not the A1 receptor antagonist DPCPX or the A2B receptor antagonist enprofylline.
The amount of soluble 14C-proline–labeled collagen detected was very low in basal conditions, and therefore the magnitude of the change in collagen production induced by A2A receptor occupancy may have been exaggerated. Thus, to further confirm the role of adenosine A2A receptors in stimulating collagen production by adult human dermal fibroblasts, we quantitated cell-associated type I collagen by Western blot analysis. Treatment with CGS-21680 increased the production of type I collagen in dermal fibroblast lysates by up to 151 ± 21% (mean ± SEM of control values) in a dose-dependent manner (ranging 0–10 μM for 24 hours at 37°C in 5% CO2), and this increase was reduced by coincubation with the adenosine A2A receptor antagonist ZM241385 (P < 0.04 by two-way ANOVA), but not with the A1 receptor antagonist DPCPX or the A2B receptor antagonist MRS1706 (1 μM for each) (Figure 2C).
As further confirmation, we also measured total collagen production in the supernates using the Sircol assay. Treatment with CGS-21680 (at 10 μM) again enhanced the production of total collagen in supernates, and this increase was blocked by the addition of ZM241385 (1 μM) (Figure 2B). Taken together, these results indicate that the CGS-21680–induced increase in collagen was due, at least in part, to enhancement of collagen synthesis.
The increase in total collagen protein following stimulation with CGS-21680 was accompanied by an increase in Col1a1 and Col3a1 mRNA expression. At 10 hours, CGS-21680 significantly increased Col1a1 message expression (145.9 ± 15.0% of control values; P < 0.05 versus controls) to a level similar to that achieved following treatment with TGFβ (151.3 ± 4.9% of control values; P < 0.01 versus controls) (mean ± SEM of 3 samples each). A similar increase in Col3a1 message was observed, although this increase did not achieve statistical significance (with CGS-21680, 123.6 ± 11.1% of control values, and with TGFβ, 125.6 ± 2.5% of control values [both n = 3]; P not significant [NS] and P < 0.05 versus controls, respectively). The magnitude of the A2A receptor–mediated increase in collagen production measured both by Western blotting and by Sircol assay paralleled the magnitude of the increase in collagen message detected.
Suppression of CGS-21680–induced collagen production by primary human dermal fibroblasts with MEK-1 inhibitors.
Because adenosine A2A receptors are known to signal through MAPKs in some cell types (10, 11), we determined whether MAPK inhibitors diminished the effect of adenosine A2A receptor ligation on collagen production. CGS-21680–induced collagen production was determined after treatment of dermal fibroblasts with 2 inhibitors of MEK-1 (the upstream activator of ERK), UO126 and PD098059. Both UO126 and PD098059 suppressed the CGS-21680–mediated increase in collagen production by 60 ± 6% and 44 ± 7% of control values, respectively (mean ± SEM of 4 samples each; both P < 0.001 versus control). In contrast, the selective p38 MAPK inhibitor SB203580 did not affect CGS-21680–stimulated collagen production (106 ± 11% of control values [n = 4]; P NS versus control) (Figure 3A). Therefore, the CGS-21680–induced increase in collagen production by dermal fibroblasts occurs, at least in part, through the MEK-1/2/ERK pathway.
Promotion of ERK phosphorylation by adenosine A2A receptor ligation in primary human dermal fibroblasts.
To confirm that the inhibition of ERK was involved in reversal of the A2A receptor–mediated stimulation of collagen production, we determined the effects of adenosine A2A receptor ligation on ERK activation, with results expressed as the mean ± SEM levels of ERK phosphorylation. CGS-21680 increased ERK-1 phosphorylation by 91 ± 1% and ERK-2 phosphorylation by 43 ± 2% above control values (each n = 3), and these increases were blocked by PD098059 and UO126 (both P < 0.001 versus CGS-21680 alone) (Figure 3B). The increase in ERK phosphorylation induced by CGS-21680 was reversed in the presence of 1 μM ZM241385 (109.3 ± 6.0% of control values [n = 3]; P NS versus control). Furthermore, treatment with 1 μM ZM241385 alone did not alter the basal level of ERK phosphorylation (108.3 ± 6.1% of control values [n = 3]; P NS versus control). These data confirm that stimulation of the adenosine A2A receptor leads to phosphorylation and activation of ERK in primary human dermal fibroblasts, and this process functions, at least in part, as a required signaling step for stimulation of increased collagen production.
Suppression of MMP expression and activity by adenosine A2A receptor stimulation.
To determine whether the A2A receptor–mediated increase in collagen production was due to an increase in collagen synthesis alone or whether there was also an effect on matrix breakdown, we examined CGS-21680–induced alteration of MMP production, by gelatin zymography. MMP-9 activity in the supernates of primary human dermal fibroblasts was suppressed in a concentration-dependent manner by as much as 44 ± 10% of control values (mean ± SEM of 3 samples; P < 0.05 versus control) (Figure 4A). This effect appeared to be selective, since concentrations of CGS-21680 up to 1 μM had little effect on MMP-2 activity. At a CGS-21680 concentration of 10 μM, MMP-2 activity was decreased by 32 ± 13% of control values (n = 3; P < 0.05 versus control). The expression of the plasma membrane–associated metalloproteinase MMP-14 was also reduced in a concentration-dependent manner, by as much as 48 ± 7% of control values (n = 4; P < 0.01 versus control) (Figure 4B). These observations suggest that adenosine A2A receptor stimulation increases the apparent collagen production by dermal fibroblasts by, at least in part, suppressing the expression/activity of MMPs.
Protection of adenosine A2A receptor–deficient mice from bleomycin-induced dermal fibrosis.
Adenosine is released from stressed cells and tissues. Since adenosine A2A receptor occupancy stimulates collagen production in vitro, and pharmacologic activation of A2A receptors increases production of matrix, primarily collagen, in healing wounds, we hypothesized that endogenously released adenosine may play a role in pathologic forms of fibrosis. We therefore studied an inducible model of scleroderma that is characterized by diffuse dermal fibrosis, achieved by subcutaneous administration of bleomycin in adenosine A2A receptor–knockout mice and their WT littermates.
Following treatment with bleomycin, WT mice displayed greater thickness of punch biopsy skin specimens than did the vehicle (PBS)–treated WT littermate mice (mean ± SEM skin thickness 0.46 ± 0.02 mm versus 0.37 ± 0.01 mm, respectively [n = 5]; P < 0.01) and greater skinfold thickness (0.83 ± 0.02 mm versus 0.63 ± 0.03 mm, respectively [n = 5]; P < 0.05). These results were consistent with the observed higher dermal tensile strength, an indicator of the presence and quantity of collagen, in the bleomycin-treated WT mice compared with the vehicle-treated WT mice (279 ± 9 gm versus 210 ± 11 gm, respectively [n = 5]; P < 0.01) (Figure 5A) and higher dermal hydroxyproline content (27.4 ± 2.3 μg/mg versus 16.8 ± 1.1 μg/mg, respectively [n = 5]; P < 0.05) (Figure 5B). These findings were also consistent with the observations on morphometric analysis. The adenosine A2A receptor–knockout mice injected with bleomycin showed no statistically significant differences in any of the above parameters when compared with the adenosine A2A receptor–knockout mice injected with PBS (n = 5; P NS for comparisons of skin thickness, skinfold thickness, tensile strength, and hydroxyproline content) (Figures 5A and B).
In accordance with these findings, qualitative analysis of histologic sections of murine skin also demonstrated reduced skin thickness and decreased collagen density in adenosine A2A receptor–knockout mice compared with their WT counterparts after induction of fibrosis with bleomycin (Figure 5C). Thus, adenosine A2A receptor–deficient mice are protected against the dermal sclerosing effects of bleomycin in this murine model of scleroderma.
Protection against bleomycin-induced dermal fibrosis with adenosine A2A receptor antagonist ZM241385.
To further verify the role of the adenosine A2A receptor in the pathogenesis of dermal fibrosis in vivo, we examined the effects of the adenosine A2A receptor antagonist ZM241385 in the murine model of bleomycin-induced dermal fibrosis. ZM241385 antagonist–treated mice with bleomycin-induced dermal fibrosis displayed significantly reduced punch biopsy skin thickness compared with their control mice (mean ± SEM 0.36 ± 0.01 mm versus 0.42 ± 0.01 mm, respectively [n = 5]; P < 0.01), and also had lower skinfold thickness (0.71 ± 0.01 mm versus 0.84 ± 0.02 mm, respectively [n = 5]; P < 0.01). These results were consistent with the higher dermal tensile strength, measured as breaking strength (296 ± 11 gm versus 167 ± 17 gm [n = 5]; P < 0.01) (Figure 6A), and higher dermal hydroxyproline content (26.2 ± 1.0 μg/mg versus 20.9 ± 1.0 μg/mg [n = 5]; P < 0.01) (Figure 6B) in the control mice compared with the ZM241385 antagonist–treated mice given bleomycin. Histologic analysis of skin from the animals treated with PBS or bleomycin plus ZM241385 (Figure 6C) confirmed that pharmacologic blockade of adenosine A2A receptors attenuates the development of bleomycin-induced dermal fibrosis in this murine model of scleroderma. These results suggest that A2A receptor antagonism may be useful in the treatment of dermal fibrosis as seen in scleroderma.
The results reported herein demonstrate that occupancy of adenosine A2A receptors on primary human fibroblasts stimulates the production of collagen via a MEK-1/MAPK–mediated pathway. Interestingly, occupancy by the A2A receptor not only promotes the increased expression of collagen but also may diminish its degradation by down-regulating the expression of MMPs. The regulation of collagen production by adenosine A2A receptors is also important in vivo, since deletion or pharmacologic blockade of adenosine A2A receptors prevents fibrosis in a murine model of scleroderma. These findings are consistent with our previous observations of the beneficial effects of adenosine A2A receptor agonists in wound healing (1–3), but demonstrate that overstimulation of this receptor may be deleterious.
In the widely accepted murine model of human scleroderma used herein, in which dermal fibrosis was induced by bleomycin (12, 13), mice lacking the adenosine A2A receptor, in contrast to their otherwise genetically identical WT littermates, were protected against the sclerosing effects of this dermal fibrosis–inducing agent. Potential compensatory mechanisms cannot explain the discrepancies seen, since similar protective effects were observed in mice treated with an adenosine A2A receptor antagonist. The adenosine A2A receptor–mediated promotion of dermal collagen production occurs via ERK activation, since A2A receptor ligation directly activated ERK on primary human dermal fibroblasts, and inhibition of its upstream kinase, MEK-1, suppressed the adenosine-mediated collagen induction.
In response to oxidative stress or hypoxia, many cells and tissues release adenosine. Moreover, it has been previously demonstrated that primary human dermal fibroblasts and other fibroblasts release increasing amounts of adenosine under stimulated conditions, such as following exposure to H2O2, stimulated polymorphonuclear neutrophils, or methotrexate treatment (14–17). Bleomycin, a widely used fibrosing agent, promotes the generation of reactive oxygen species and resultant induction of oxidative stress (18–22), and antioxidants attenuate bleomycin-induced fibrosis, at least in the lung (23). Cellular release of adenosine is known to be increased under conditions of oxidative stress. This occurs, in part, as an adaptive mechanism, since we and other investigators have shown that adenosine receptor occupancy suppresses the generation of reactive oxygen species by stimulated neutrophils and other cells (24–27). However, when the oxidative insult results from nonsuppressible causes, such as a toxin or ongoing immunologic reaction, adenosine release may be maladaptive and can promote fibrosis, as shown herein. Thus, our results suggest that adenosine is released in response to bleomycin-induced generation of oxygen radicals in the skin, and the adenosine released promotes dermal fibrosis via occupancy of adenosine A2A receptors.
Unlike cardiac fibroblasts, primary human dermal fibroblasts do not express adenosine A3 receptors (28). We have observed a similar role for adenosine and its A2A receptor in the generation of hepatic fibrosis, and we have found that persistent adenosine release resulting from oxidative stress, hypoxia, or other stimuli may be a more general stimulus for fibrosis (Chan ES, et al: unpublished data). As a model for scleroderma, sclerotic skin induced by injection with bleomycin exhibits many histologic features akin to human disease, such as a predominantly mononuclear cell infiltrate that includes T lymphocytes, monocytes/macrophages, and mast cells (29–31). Important differences do exist, such as relative suppression of Smad-7 up-regulation in this model, and results must be interpreted in light of this knowledge (32).
The association between adenosine A2A receptor activation and dermal fibrosis is a novel finding, since, to date, there have been no reports of this relationship. However, evidence of a systemic disturbance in adenosine metabolism in scleroderma has been noted previously. Decreased adenosine uptake by endothelial cells has been observed in normal, clinically uninvolved skin in patients with systemic sclerosis but not in patients with primary Raynaud's disease (33). Meunier et al previously reported that adenosine deaminase activity is increased in the plasma of patients with systemic sclerosis (34). Furthermore, when these patients with systemic sclerosis were stratified into groups according to disease activity, a clear correlation between adenosine deaminase levels and disease activity could be demonstrated. Sasaki and Nakajima reported similar findings and found that serum adenosine deaminase activity in patients with progressive systemic sclerosis correlated well with the serum antinuclear antibody titers (35).
Since adenosine deaminase is a major catabolic enzyme for adenosine (36), it is possible that the increase in adenosine deaminase activity is an adaptive mechanism to counteract the sclerosing properties of circulating and tissue adenosine in this disease. Blackburn et al (37) generated mice deficient in adenosine deaminase, and as a result of the enzyme deficiency, the mice displayed high levels of adenosine in plasma and tissue. Moreover, these mice died prematurely from extensive pulmonary fibrosis, consistent with the role of adenosine in the pathogenesis of tissue fibrosis, although it is unclear whether adenosine receptors play a causative role in this process (37, 38).
Direct quantification of tissue adenosine levels in systemic sclerosis is difficult because of the short plasma and tissue half-lives (<10 seconds) of adenosine itself and because of the widespread presence of adenosine deaminase. Adenosine A2B receptors have also been reported to play a role in modulating collagen production. Adenosine A2B receptor overexpression decreased collagen synthesis in cardiac fibroblasts, while high concentrations of adenosine receptor agonists may increase collagen production (28). Similarly, suppression of collagen production in cardiac fibroblasts via A2B receptor activation has been reported by Dubey et al (39). A2B receptor activation, however, inhibits collagenase expression in fibroblast-like synoviocytes (40). The A2B receptor may indeed complement the physiologic role of the A2A receptor in modulating matrix production in various cell types.
Disturbances in cytokine and growth factor profiles have been noted in scleroderma, as has been reported previously in many other autoimmune diseases (41). In the murine model studied herein, bleomycin was shown to promote dermal fibrosis by enhancing the expression of extracellular matrix proteins such as Col1a1, as well as growth factors known to be associated with fibrous tissue production such as TGFβ and connective tissue growth factor (CTGF) (13). These growth factors are also key influences on dermal fibrogenesis in scleroderma (42, 43). We have previously demonstrated that inflammatory cytokines regulate the expression of adenosine A2A receptors in both human vascular endothelial cells and monocytoid cells (9, 44). We have also observed that TGFβ up-regulates adenosine A2A receptor mRNA expression in primary human dermal fibroblasts (Khoa ND, et al: unpublished observations), suggesting that TGFβ may also enhance the sensitivity of adenosine A2A receptors and thereby further promote dermal fibrosis. Although its effect on production of TGFβ and CTGF has not been studied, adenosine may also play a role in stimulating the production of other fibrogenic cytokines. Indeed, Blackburn and colleagues reported that tissue levels of interleukin-13, a profibrogenic cytokine, were increased in the lung by adenosine (37).
At present, there are no known long-term consequences regarding adenosine A2A receptor antagonism in humans, although such information should become available soon, since adenosine A2A receptor antagonists are currently undergoing phase III clinical trials for the treatment of Parkinson's disease. Observations in adenosine A2A receptor–knockout mice have suggested possible side effects from this treatment, such as hypertension, that may be important to consider in patients with systemic sclerosis, even if they are not seen in patients with Parkinson's disease (45). In the event that hypertension should occur, it is likely that clinicians will have to be particularly vigilant about treating hypertension in scleroderma patients in the setting of an adenosine A2A receptor antagonist. A2A receptor–knockout mice have been reported to be more aggressive, although this characteristic may differ with the source of the mice. We have not observed more overtly aggressive behavior in our A2A-knockout mice, which were generated in a different laboratory; however, we did not perform formal psychometric testing on these mice.
In conclusion, we have demonstrated a novel mechanism for dermal fibrosis mediated by the adenosine A2A receptor, both in vitro and in vivo. This occurs via activation of ERK, and inhibition of its upstream activating kinase MEK-1 abrogates the adenosine A2A receptor–mediated increase in dermal collagen production. These findings are relevant to dermal fibrogenesis as a whole but are particularly interesting in relation to scleroderma, since no effective treatment for its dermal manifestations exists at present. These results also parallel our findings in murine models of cirrhosis, in which adenosine A2A receptor antagonism has also been observed to be beneficial (46). Our results suggest that selective adenosine A2A receptor antagonists may hold promise in the treatment of dermal fibrosis in diseases such as scleroderma.