Abstract
Adenosine is the final product of ATP metabolism, mainly derived from the action of 5′-nucleotidase cleavage of AMP. Cellular production of adenosine is greatly enhanced in inflamed tissues, ischemic tissues, and under hypoxia, where ATP is released from damaged cells. Much evidence has been accumulated on adenosine anti-inflammatory effects mediated through A2A receptor activation; A2A adenosine receptor has also been shown to play a role in matrix deposition and wound healing in a damaged tissue, contributing to dermal tissue protection and repair. Fibroblast growth factor-2 (FGF-2) is a powerful mitogen for fibroblast; it is expressed by several inflammatory cell types and plays a pivotal role in angiogenesis, wound healing, gastric ulcer protection. Human recombinant FGF-2 has been shown to have anti-inflammatory effects. The purpose of the present work was to investigate on the anti-inflammatory effect of systemic administration of the adenosine A2A agonist 2-p-(2-carboxyethyl)phenethylamino-5′-N-ethylcarboxamidoadenosine hydrochloride hydrate (CGS21680) in the rat model of carrageenan-induced paw edema. We found that CGS21680 inhibits inflammation induced by carrageenan injection into the rat paw, and this effect is associated to the local reduction of cytokine levels and dermal increase of FGF-2 expression. Our results suggest that FGF-2 might be involved in the anti-inflammatory and tissue protective effect due to A2A receptor activation.
Introduction
Adenosine is an endogenous purine nucleoside mainly derived by the breakdown of ATP. Extracellular adenosine accumulates in inflamed tissues, ischemic tissues, and under hypoxia, where ATP is released from damaged cells (Gordon, 1986). Through the interaction with four types of receptors, designed as A1, A2A, A2B, and A3, adenosine contributes to the homeostatic regulation of many systems, such as nervous, cardiovascular, renal, gastrointestinal, and immune system (Jacobson and Gao, 2006; Fredholm, 2007; Antonioli et al., 2008a,b). Much evidence has been accumulated both in vitro and in vivo on adenosine anti-inflammatory effects mediated through A2A receptor activation (Palmer and Trevethick, 2008; Antonioli et al., 2013, 2014).
Interestingly, A2A adenosine receptor has also been shown to play a role in matrix deposition and wound healing in a damaged tissue, contributing to dermal tissue protection and repair (Montesinos et al., 1997, 2002; Chan et al., 2006; Cronstein, 2006). Mazzon and coworkers (2011) demonstrated the beneficial effect of systemic A2A agonist administration in the model of collagen-induced arthritis in mice. It has been shown that adenosine A2A receptor also stimulates matrix and collagen production in dermal fibroblasts (Chan et al., 2013).
The dual role of A2A receptor as anti-inflammatory and tissue repairer has driven the attention of several researchers toward the role of this receptor on skin cells (Burnstock et al., 2012). It has already shown that application of the adenosine A2A receptor agonist CGS21680 reduces pressure-induced skin ulcers in rats (Peirce et al., 2001) and inflammation and epidermal hyperplasia in mice following application of phorbol myristate acetate (Arasa et al., 2014), representing a promising therapeutic agent devoid of the atrophic effect of corticosteroids.
It is known that FGF-2 is a powerful mitogen for fibroblast; besides fibroblasts, other inflammatory cell types can express FGF-2, including mononuclear phagocytes, CD4+, and CD8+ T lymphocytes and mast cells (Artuc et al., 1999). FGF-2 exerts its biologic effects by interacting with four members of FGF-receptor (FGFR1–FGFR4), a family of tyrosine kinase receptors and plays a pivotal role in angiogenesis, wound healing, gastric ulcer protection (Bikfalvi et al., 1997; Pohle et al., 1999). Human recombinant FGF-2 has been shown to inhibit croton oil-induced ear swelling and carrageenan-induced paw edema in mice and to reduce peritonitis induced by carrageenan in mice and rats (Hu and Wu, 2001). Moreover, in a murine model of asthma, recombinant FGF-2 has been shown to reduce airway responsiveness, mucus production, and lung inflammation and also to reduce allergen-induced proliferation of T cells (Jeon et al., 2007).
Thus, it is evident that both adenosine, through A2A receptor, and FGF-2 are on the edge of inflammation and tissue regeneration.
In this study, we used the model of rat carrageenan-induced paw edema, an acute model of inflammation that we previously demonstrated to be sensible to the anti-inflammatory effect of CGS21680 (Caiazzo et al., 2016) and that involves inflammation of epidermis and derma to investigate the anti-inflammatory and tissue protective effect of systemic adenosine A2A receptor activation.
We demonstrate that systemic treatment with CGS21680 inhibits inflammation induced by carrageenan injection into the rat paw, and this effect is paralleled by local increase of FGF-2 expression, suggesting that FGF-2 might be involved in the anti-inflammatory and tissue protective effect due to A2A receptor activation.
Materials and Methods
Animals.
All experiments were performed on male Wistar rats (180–220 g; Calco (LC), Italy). Rats, slightly anesthetized with enflurane, received in the left hind paw 100 µl of λ-carrageenan (Sigma-Aldrich S.r.l., Milan, Italy) suspension (1% w/v). Paw volume was measured at the time zero and each hour for 6 hours by a hydropletismometer (Ugo Basile, Comerio, Varese, Italy).
Drug Treatments.
Rats were randomly assigned to groups of five each and treated, just before edema induction, with the A2A agonist CGS21680 (0.02–2 mg/kg i.p.; Tocris Bioscience, Bristol, UK); with the A2A antagonist, 4-(2-[7-amino-2-[2-furyl][1,2,4]triazolo[2,3-a][1,3,5]triazin-5-yl-amino]ethyl)phenol (ZM241385 3 and 6 mg/kg i.p.; Tocris Bioscience); with CGS21680 (2 mg/kg i.p.) plus ZM241385 (3 mg/kg i.p.); or with the vehicle (dimethyl sulfoxide; Sigma-Aldrich S.r.l.). The dose of ZM241385 was chosen based on Caiazzo et al. (2016). From different groups of animals, at different times following edema induction, paws were excised, cut, frozen in liquid nitrogen or fixed in 4% (v/v) buffered formalin (Carlo Erba Reagents S.r.l., Cornaredo, Milan, Italy), and stored for further histologic and biochemical analysis.
All procedures were performed according to the Italian and European regulations (DL 26/2014) on the protection of animals used for experimental and other scientific purposes and were approved by Italian Ministry of Health.
Myeloperoxidase Assay.
Myeloperoxidase (MPO) activity, an indicator of polymorphonuclear leukocyte accumulation, was measured in the inflamed paws excised after 3 hours from edema induction as previously described (Mullane et al., 1985). At the specified time following injection of carrageenan, tissues were weighed and each piece homogenized in a solution containing 0.5% (w/v) hexadecyltrimethylammonium bromide dissolved in 10 mM potassium phosphate buffer (pH 7) and centrifuged for 30 minutes at 20,000 g. An aliquot of the supernatant was then allowed to react with a solution of 1.6 mM tetramethylbenzidine (Sigma-Aldrich S.r.l.) and 0.1 mM H2O2. The rate of change in absorbance was measured spectrophotometrically at 650 nm. MPO activity was determined as the quantity of enzyme degrading 1 mmol of peroxide per minutes at 37°C and was expressed in milliunits per grams of wet tissue. As control, MPO activity was also measured on tissues obtained from contralateral, noninflamed, paws.
Morphologic Analysis.
Tissue samples removed as described above were immediately fixed in 4% (v/v) neutral formalin for 48 hours and embedded in paraffin by conventional techniques. For morphologic analysis, 7-μm-thick sections were stained with hematoxylin and eosin (Carlo Erba Reagents S.r.l.). The sections were analyzed by using a standard light microscope (10× objective) and photographed under low power. Images were taken by a Leica DFC320 video camera (Leica, Milan, Italy) connected to a Leica DM RB microscope using the Leica Application Suite software V 4.1.0. 2.7.
Picro Sirius Red Staining.
To visualize tissue collagen, Picro Sirius red staining and polarization microscopy were performed. Briefly, the paraffin sections (7 µM) were dewaxed and rehydrated. The sections were stained with Mayer’s hematoxylin to show the nuclei and then incubated with 0.1% (w/v) Sirius Red solution (Sigma-Aldrich) dissolved in saturated picric acid solution for 1 hour at room temperature. After washing with acidified water, slides were alcohol dehydrated, mounted in a resinous medium, and visualized through a light microscope (Leica Microscope) equipped with a digital camera (Leica DFC320). Polarization microscopy images were visualized through a light microscope (Leits Dialux) equipped with a polarization filter (10× objective).
Measurement of Tumor Necrosis Factor α and Interleukin-6 Levels in Rat Paws.
In tissue sample homogenates, prepared as described below, tumor necrosis factor alpha (TNF-α) and interleukin-6 levels were evaluated by an enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN). Results obtained were expressed as picograms of cytokine per milligrams of protein.
Western Blot Analysis.
Tissue samples from rats at different time points were defrosted, weighed, and homogenized with a Polytron (three cycles of 10 seconds at maximum speed). To extract proteins, 1 ml of buffer (β-glycerophosphate, 50 mM; sodium orthovanadate, 100 μM; MgCl2, 2 mM; EGTA, 1 mM; dithiothreitol, 1 mM; phenymethylsulphonyl fluoride, 1 mM; aprotinin, 10 μg/ml; leupeptin, 10 μg/ml) was added to 100 mg of tissue samples. After centrifugation at 2500 rpm for 30 minutes at 4°C, protein supernatant content was measured by Bradford reagent (Segrate (MI), Italy). Denaturated samples (50 𝜇g) were subjected to electrophoresis on SDS 15 or 12% polyacrylamide gel (for FGF-2 and A2A, respectively) and transferred onto a nitrocellulose membrane (Protran; (Sigma-Aldrich S.r.l., Milan, Italy). Membranes were blocked by incubation with nonfat dry milk (5% w/v) in phosphate-buffered saline (PBS) supplemented with 0.1% (v/v) Tween 20 for 1 hour at room temperature and then incubated with a rabbit polyclonal anti-FGF-2 antibody (1:200 dilution; Santa Cruz Biotechnology, Inc. Santa Cruz, CA) or with a mouse polyclonal anti-A2A antibody (1:1000 dilution; Novus Biologicals, Inc., Littleton, CO) overnight at 4°C on a shaker. Successively, membranes were washed and then incubated with the secondary antibody conjugated with horseradish peroxidase (HRP), anti-rabbit IgG-HRP (1:5000 dilution; Santa Cruz Biotechnology Inc.), or anti-mouse IgG-HRP (1:2000 dilution; Dako, Glostrup, Denmark) for 3 hours at room temperature. Immunoreactive bands were detected using the enhanced chemiluminescence detection kit and Image Quant 400 GE Healthcare software (GE Healthcare, Milan, Italy). Densitometric values for each band were determined using Quantity One software (Bio-Rad, Milan, Italy). Successively, to confirm the equal protein loading, membranes were stripped and incubated with rabbit polyclonal anti- glyceraldehyde-3-phosphate dehydrogenase antibody (1:500 dilution, Santa Cruz Biotechnology Inc.) and subsequently with anti-rabbit IgG-HRP (1:5000 dilution, Santa Cruz Biotechnology Inc.), both for 3 hours at room temperature.
Immunofluorescence Analysis.
Seven micrometer sections of paraffin-embedded paw tissue were deparaffinized, rehydrated, and heat induced for antigen retrieval by using Diva decloaker (Biocar Medical, Casablanca, Morocco). After blocking of endogenous peroxidase (0.3% H2O2, 30 minutes) and unspecific binding with avidin/biotin blocking kit (Vector Laboratories, Burlingame, CA), sections were incubated with rabbit polyclonal anti-FGF-2 (1:100 dilution; Santa Cruz Biotechnology Inc.) antibody in casein plus Triton X-100 0.3% overnight at 4°C. The next day, sections were washed with PBS plus Tween 0.05% before incubation with fluorescein isothiocyanate-conjugated antirabbit antibody (1:250 dilution; Jackson Immuno Research Laboratories, Inc., West Grove, PA) for 30 minutes at room temperature. Slides were then washed in PBS-Tween 0.05% and mounted in Vectashield Mounting Medium with 4′, 6-diamidino-2-phenylindole (1.5 µg/ml; Vector Laboratories). Images were observed using a fluorescence microscope Leica DM RB (Leica Microsystems, Wetzlar, Germany) equipped with appropriate standard filter and acquired using Leica Application Suite V 4.1.0 software
Inhibition of FGF-2 Signaling.
The effect of the A2A agonist CGS21680 (2 mg/kg i.p.) was also evaluated in groups of rats treated with the FGFR inhibitor, 3-(2,6-dichloro-3,5-dimethoxyphenyl)-1-(6-(4-(4-ethylpiperazin-1-yl) phenylamino) pyrimidin-4-yl)-1-methylurea (NVP-BGJ398, 30 mg/kg i.p.; Adipogen, San Diego, CA; Guagnano et al., 2011) or with an anti FGF-2 neutralizing antibody (clone bFM-1), 10 µg/paw (Merck Millipore, Vimodrone (MI), Italy; Even-Chen et al., 2017). All groups received the same amount of dimethyl sulfoxide that was the vehicle for CGS21680 and NVP-BGJ398. Anti FGF-2 neutralizing antibody (10 µg) diluted in distilled water was contained in 100 µl of carrageenan and injected into the paw.
Statistical Analysis.
All results were expressed as mean ± S.E.M. and analyzed by one-way analysis of variance followed by Dunnett’s or Bonferroni’s test for multiple comparisons, as appropriate. A value of P < 0.05 was taken as statically significant.
Results
Effect of CGS21680 Treatment on Carrageenan-Induced Edema.
Injection of carrageenan in the rat hind paw caused an edema peaking between 3 and 4 hours. Preliminary experiments showed that treatment with CGS21680 inhibited, in a dose-related manner, edema development, and MPO activity in the rat paw (Fig. 1, A and B), on the basis the dose of 2 mg/kg of CGS21680, was chosen for the entire study. The anti-inflammatory effect of CGS21680 (2 mg/kg i.p.) was reversed by coadministration with the A2A antagonist ZM241385 (3 mg/kg i.p.; Fig. 1C). On the contrary, ZM241385 (3 and 6 mg/kg i.p.) alone did not modify edema (data not shown).
Effect of CGS21680 (0.02–2 mg/kg i.p.) on carrageenan-induced rat paw edema and tissue MPO activity. Administration of CGS21680 (0.02–2 mg/kg i.p.) inhibited carrageenan-induced paw edema (A) and MPO activity (B) in inflamed rat paws. The effect of CGS21680 (2 mg/kg i.p.) was reversed by cotreatment with the A2A antagonist ZM241385 (3 mg /kg i.p.) (C). Values are expressed as mean ± S.E.M. (n = 10 for each group). Statistical significance was assessed by analysis of variance followed by Dunnett’s test; *P<0.05; **P<0.01 vs. vehicle [dimethyl sulfoxide (DMSO)]. ##P<0.01 vs. control (Ctrl; not injected paw).
Morphologic Analysis.
Morphologic examination of tissue sections stained with hematoxylin and eosin showed reduced cell infiltrate in paws from CGS21680 (2 mg/kg i.p.) treated rats; furthermore, in the same samples, there was no sign of edema in the dermal layer and it was evident that the cytoarchitecture of epidermis was intact, with dermal papillae well evident (Fig. 2C), compared with samples obtained by vehicle injected rats, characterized by flattening of epidermal dermal junction in inflamed tissue, suggesting a reduced epidermal dermal cohesion (Fig. 2B).
Effect of CGS21680 (2 mg/kg i.p.) on morphologic changes and collagen content in rat paw 3 hours after carrageenan injection. Morphologic evaluation of rat paws was performed by hematoxylin and eosin staining (H&E). (B) Paws from vehicle-treated rats show dermal edema, increased cell infiltration, and flattening of basal lamina (see arrows). (C) Systemic treatment with CGS21680 protects from morphologic changes (see arrows). Original magnification, 10×; scale bar, 200 µm. Picro Sirius red staining and polarizing microscopy were performed to visualize tissue collagen in rat paw. Tissue obtained from CGS21680-treated rats (F and I) show compact collagen fibers (see arrow) compared with tissues from vehicle-injected rats (E and H). Original magnification, 10×; scale bar, 200 µm.
Picro Sirius Staining.
Picro Sirius red staining of inflamed tissue to highlight collagen fibers showed that in tissue sections from inflamed animals dermal collagen fibers were loose (Fig. 2, E and H) compared with paws from control (Fig. 2, D and G) and from CGS21680 (Fig. 2, F and I) treated animals where dermal elastic fibers were well organized.
Cytokine Levels.
TNF-α and interleukin-6 levels into the inflamed tissues were significantly reduced by rat treatment with CGS21680 (2 mg/kg i.p.). This effect was reversed by coadministration of ZM241385 (3 mg/kg i.p.) (Fig. 3).
Effect of systemic administration of CGS21680 (2 mg/kg i.p.) on interleukin (IL)-6 and TNF-α content in rat inflamed paws 3 hours after carrageenan injection. CGS21680 reduced both IL-6 (A) and TNF-α (B) content in paw tissue and this effect was reversed by coadministration with the A2A antagonist ZM241385 (3 mg/kg i.p.). Values are expressed as mean ± S.E.M. (n = 6 for each group). Statistical significance was assessed by analysis of variance followed by Dunnett’s test; *P < 0.05; ***P < 0.001 vs. vehicle (DMSO).
FGF-2 and A2A Expression.
The time course of FGF-2 expression in rat paws showed a strong increased expression in the group treated with CGS21680, and the effect was evident at 3 and 4 hours following edema induction (Fig. 4, B and D). Conversely, in the vehicle-treated group, the slight increase in FGF-2 expression, observed at 1 and 2 hours following carrageenan injection, returned to control value at 3 and 4 hours when the inflammation peaked (Fig. 4, A and D). The time course of A2A expression showed an increased protein expression peaking between 3 and 4 hours (Fig. 5, A and D). This increased A2A expression was reduced to control values by CGS21680 (Fig. 5, B and D). Rat systemic treatment with CGS21680 (2 mg/kg i.p.) plus ZM241385 (3 mg/kg i.p.) reversed the effect of administration of CGS21680 alone on both FGF-2 and A2A tissue expression (Fig. 4; Fig. 5, C and D).
Representative Western blotting (A–C) and densitometric analysis (D) of the time course of FGF-2 expression in rat paws. Treatment with CGS21680 (2 mg/kg i.p.) strongly increased FGF-2 expression in rat paws at 3 and 4 hours after carrageenan injection (B and D). Treatment with ZM241385 (3 mg/kg i.p.) reversed the effect of CGS21680 (C and D). Values are expressed as mean ± S.E.M. of n = 5. Statistical significance was assessed by analysis of variance followed by Bonferroni’s test. #P< 0.05; ##P < 0.01; ###P < 0.001 vs. Ctrl ***P < 0.001 vs. vehicle (DMSO).
Representative Western blotting (A–C) and densitometric analysis (D) of the time course of A2A expression in rat paws. A2A expression increased at 3 and 4 hours after carrageenan injection (A and D) and it was reduced following rat treatment with CGS 21680 (2 mg/kg i.p.) (B and D). Treatment with ZM241385 (3 mg/kg i.p.) reversed the effect of CGS21680 (C and D). Values are expressed as mean ± S.E.M. of n = 5. Statistical significance was assessed by analysis of variance followed by Bonferroni’s test. #P < 0.05; ###P < 0.01 vs. Ctrl (not injected paw) and ***P < 0.001 vs. vehicle (DMSO).
Immunofluorescence Analysis for FGF-2.
On tissue obtained as described above, FGF-2 (18 kDa) was localized by immunofluorescence analysis. Immunolocalization of FGF-2 was increased in derma layer of paws from CGS21680 treated rats compared with control paws (Fig. 6).
Immunofluorescence detection of FGF-2 on rat paws excised 3 hours after carrageenan injection. Increased immunostaining for FGF-2 (green) was localized in derma of paws from CGS21680-treated rats (2 mg/kg i.p.) (C). Cell nuclei (blue) were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Original magnification, 20×; scale bar, 100 µm. (A) Ctrl; (B) vehicle.
Inhibition of FGF-2 Signaling.
Systemic administration to rats of FGFR inhibitor NVP-BGJ398 (30 mg/kg i.p.) or local administration of the anti-FGF-2 neutralizing antibody (10 µg/paw) reversed the anti-inflammatory effect of CGS21680. Both treatments did not have any effect on edema development (Fig. 7).
Effect of NVP-BJG398 (30 mg/kg i.p.) and of anti-FGF-2 neutralizing antibody (10 µg/paw) on carrageenan-induced rat paw edema. Both NVP-BJG398 (A) and anti-FGF-2 neutralizing antibody (B) reversed the anti-inflammatory effect of CGS21680 (2 mg/kg i.p.) and did not modify edema development. Values are expressed as mean ± S.E.M. (n = 5 for each group). Statistical significance was assessed by analysis of variance followed by Bonferroni’s test; (A) *P < 0.05; **P < 0.01; ***P < 0.001 vs. vehicle (DMSO). #P < 0.05; ##P < 0.01; ###P < 0.001 vs. CGS + NVP-BGJ398 (A) or vs. CGS + anti-FGF-2 (B).
Discussion
Carrageenan-induced rat paw edema is a classic model of acute inflammation, widely used to identify new therapeutic targets and to test the anti-inflammatory potential of new molecules. This model has been largely characterized; local changes of epidermis and derma following subcutaneous injection of carrageenan in the hind paw have also been described (Vinegar et al., 1987; Morris, 2003).
Adenosine A2A receptor has been described to be anti-inflammatory in several models of acute and chronic inflammation (Palmer and Trevethick, 2008; Antonioli et al., 2014) and to be involved in adenosine-mediated matrix production and wound healing (Valls et al., 2009). Several studies have reported the beneficial effect of topical application of A2A agonist on injured skin (Montesinos et al., 2002; Arasa et al., 2014); however, the mechanism at the basis is still unclear. However, there is also evidence that under pathologic conditions or following skin physical injury, such as radiation induced injury, A2A receptor activation is responsible for fibrosis (Perez-Aso et al., 2016). Here, we show that systemic administration of A2A agonist CGS21680 to rats inhibits paw inflammation induced by subcutaneous injection of carrageenan, increases FGF-2 dermal expression, and ameliorates skin cytoarchitecture.
First, we confirmed the functional role of A2A in the control of inflammation, by animal treatment with the A2A agonist CGS21680 or with the antagonist ZM241385. As previously demonstrated (Caiazzo et al., 2016), we found that systemic treatment with A2A agonist CGS21680 prevented edema development, whereas the antagonist ZM241385 did not have any effect (data not shown). Furthermore, CGS21680 inhibitory effect was prevented by coadministration with ZM241385. This finding confirms that the effect of CGS21680 was specific, through A2A receptor stimulation; nonetheless, A2A activation by endogenous adenosine seems not to offer protection against acute inflammation, because A2A antagonism did not exacerbate edema. This finding might suggest that CGS21680 would act through an indirect mechanism, involving stimulation of A2A receptor probably distant from the site of injury and activating anti-inflammatory mechanisms. A similar result was observed by Peirce et al. (2001), who demonstrated that systemic administration of ALT-146e, a selective A2A agonist, reduced skin ulceration induced by recurrent ischemia reperfusion in rats. The effect was reversed by the antagonist ZM241385; however, the antagonist alone did not exacerbate skin ulceration. Authors conclude that the A2A agonist probably acted outside the ischemic zone.
We also focused on skin morphologic changes following subcutaneous injection of carrageenan and found diffuse dermal edema paralleled by evident changes in epidermis cytoarchitecture that was absent following treatment with CGS21680. Furthermore, Picro Sirius red staining evidenced that dermal collagen fibers were more compact and allineated following rat treatment with CGS21680. It has been demonstrated that adenosine A2A receptor activation stimulates fibroblasts to produce collagen (Perez-Aso et al., 2013), and topical application of A2A agonist facilitates wound closure (Montesinos et al., 1997, 2002). These findings outline the importance of A2A receptor in matrix production, although the mechanism at the basis of A2A receptor protective effect on inflammation and tissue destruction is still unclear. It is known that extracellular matrix and fibroblast, as well as epidermal keratinocytes, are actively involved in the modulation of inflammation (Gilroy et al., 2004; Sorokin, 2010). Scheibner and co-workers (2009) demonstrated an anti-inflammatory role of A2A receptor through modulation of components of extracellular matrix. The same authors also demonstrate that transgenic mice, lacking A2A receptor, are more susceptible to bleomycin-induced lung fibrosis (Scheibner et al., 2009); this latter finding is in contrast with other papers demonstrating that lack of A2A receptor protect from bleomycin-induced fibrosis (Katebi et al., 2008) and that adenosine deaminase-deficient mice, a model of elevated tissue adenosine, develop spontaneous pulmonary and skin fibrosis (Fernández et al., 2008). It is conceivable that A2A receptor plays a different role on tissue regeneration or fibrosis depending on the duration of its stimulation by endogenous adenosine under acute or chronic tissue damage (Luo et al., 2016).
It is known that matrix can act as a reservoir for several growth factors, including fibroblast growth factor-2. FGF-2 is a molecule with autocrine and paracrine functions. Produced by several cell types and released by shedding vesicles (Taverna et al., 2008), FGF-2 plays a pivotal role in angiogenesis, wound healing, gastric ulcer protection, cell proliferation, and inflammation (Bikfalvi et al., 1997; Pohle et al., 1999). FGF-2 is a growth factor stimulating the production of collagen, the major constituent of extracellular matrix also actively involved in resolution of inflammation (Burgess, 2009). Experiments performed in Fgf-2 knockout mice have demonstrated that FGF-2 is required for epithelial repair and resolution of inflammation following intranasal instillation of bleomycin, but it is not required for pulmonary fibrosis (Guzy et al., 2015), according with previous data demonstrating a role for FGF-2 in epidermal repair (Ortega et al., 1998). Here, results obtained by Western blot analysis showed that increased tissue FGF-2 expression following rat treatment with CGS21680 compared with vehicle and even to control, not inflamed, paws and that coadministration with the A2A antagonist ZM241385 reversed the anti-inflammatory effect of CGS21680 and the increase in FGF-2 expression. This finding confirms that these effects are dependent upon adenosine A2A receptor activation. The functional role of FGF-2 signaling in the anti-inflammatory effect of adenosine A2A receptor activation was proved by evidence that NGJ-BVP398, a pan-specific FGFR inhibitor (Guagnano et al., 2011), reversed the effect of CGS21680. This result might also be suggestive of a physical interaction between adenosine A2A receptor and FGFR, as already demonstrated in vitro (Flajolet et al., 2008). In the end, evidence that the anti-inflammatory effect of systemic administration of CGS21680 was also reversed by the local injection into the paw of the neutralizing anti-FGF-2 further links the increase in tissue FGF-2 expression to the anti-inflammatory effect mediated by A2A activation. We also evaluated A2A receptor expression in rat inflamed paws to evaluate whether there was correspondence with FGF-2 expression. Results obtained by Western blot analysis showed that A2A receptor protein expression increased in the inflamed tissue rapidly following edema induction, with a peak at 3 to 4 hours, returning to control value 6 hours thereafter. Further analysis showed that the increased A2A expression was reduced following treatment with CGS21680. This finding would have suggested that the increased A2A expression in inflamed tissues might reflect cell, likely neutrophils, accumulation, and also considered that it was paralleled by an increased MPO activity and reduced by CGS21680 treatment.
In conclusion, we demonstrate that the adenosine A2A receptor agonist CGS21680 systemically administered to rats prevents paw edema development and cytokine increase in the rat paw injected with carrageenan by way of a mechanism dependent by receptor activation, because both effects are inhibited by the antagonist ZM241385. At the same time, A2A agonist cause an increased expression of FGF-2 into the rat paw that is also dependent upon receptor activation and likely related to the anti-inflammatory effect. Our results shed light on the role of adenosine A2A receptor in the modulation of an innate immune response and link its tissue protective effect to an increased expression of FGF-2.
Authorship Contributions
Participated in research design: Ialenti, Caiazzo, Cicala.
Conducted experiments: Caiazzo, Morello, Cicala.
Performed data analysis: Ialenti, Caiazzo, Morello, Cicala.
Wrote or contributed to the writing of the manuscript: Ialenti, Caiazzo, Carnuccio, Cicala.
Footnotes
- Received July 28, 2017.
- Accepted November 27, 2017.
↵1 A.I. and E.C. contributed equally to this work.
This work was supported by the grant of University of Naples Federico II (Research Program 2017–2019).
Abbreviations
- CGS21680
- 2-p-(2-carboxyethyl)phenethylamino-5′-N-ethylcarboxamidoadenosine hydrochloride hydrate
- FGF-2
- fibroblast growth factor-2
- FGFR
- FGF receptor
- HRP
- horseradish peroxidase
- MPO
- myeloperoxidase
- NVP-BGJ398
- 3-(2,6-dichloro-3,5-dimethoxyphenyl)-1-(6-(4-(4-ethylpiperazin-1-yl)phenylamino)pyrimidin-4-yl)-1-methylurea
- PBS
- phosphate-buffered saline
- TNF-α
- tumor necrosis factor alpha
- ZM241385
- 4-(2-[7-amino-2-[2-furyl][1,2,4]triazolo[2,3-a][1,3,5]triazin-5-yl-amino]ethyl)phenol
- Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics
References
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A2A Agonist Inhibits Inflammation and Increases Tissue FGF-2
