Adenosine A2A Receptor Agonist, 2-p-(2-Carboxyethyl)phenethylamino-5′-N- ethylcarboxamidoadenosine hydrochloride hydrate (CGS21680), Inhibits Inflammation and Increases Fibroblast Growth Factor 2 (FGF-2) Tissue Expression in Carrageenan Induced Rat Paw Edema

Armando Ialenti1, Elisabetta Caiazzo1, Silvana Morello, Rosa Carnuccio, Carla Cicala Department o Pharmacy, School of Medicine, University of Naples Federico II, Naples, Italy (A.I., E.C., R.C., C.C.)
Department of Pharmacy, University of Salerno, Fisciano, SA, Italy (S.M.)

Running title: A2A agonist inhibits inflammation and increases tissue FGF2

Corresponding author: Carla Cicala Ph.D. Department of Pharmacy, University of Naples Federico II, Via D. Montesano 49, 80131 Naples, Italy. Phone: (+39) 081678455
E-mail address: [email protected] Number of text pages: 27 Number of tables: 0
Number of 7 Number of references: 42 Number Words:
Abstract: 205

Introduction: 548

Discussion: 1156

List of non-standard abbreviations:

ADA, adenosine deaminase; CGS21680, 2-p-(2-Carboxyethyl)phenethylamino-5′-N- ethylcarboxamidoadenosine hydrochloride hydrate; DAPI, 4′,6-diamidino-2-phenylindole; DMSO, dimethyl sulfoxide; DTT, dithiothreitol; ELISA, enzyme-linked immunosorbent assay; ECL, enhanced chemiluminescence; FGF-2, fibroblast growth factor-2; FGFR, FGF Receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HRP, horseradish peroxidase; IL-6, Interleukin-6; 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; PMN, polymorphonuclear, PMSF, phenymethylsulphonyl fluoride; TNF-α, tumor necrosis factor alpha; TPA, phorbol myristate acetate; ZM241385, 4- (2-[7-amino-2-[2-furyl][1,2,4]triazolo[2,3-a][1,3,5]triazin-5-yl-amino]ethyl)phenol.
Recommended section: Inflammation, immunopharmacology and asthma

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 antinflammatory 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 shown to have antinflammatory effects. The purpose of the present work was to investigate on the antinflammatory 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 antinflammatory and tissue protective effect due to A2Areceptor 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 et al., 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 (Antonioli et al., 2008a; 2008b; Jacobson et al., 2006; Fredholm et al., 2007). Much evidence has been accumulated both in vitro and in vivo on adenosine antinflammatory effects mediated through A2A receptor activation (Palmer and Trevethick, 2008; Antonioli et al., 2013, Antonioli et al., 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) have 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 (TPA) (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 biological 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 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 have previously demonstrated to be sensible to the antinflammmatory effect of CGS21680 (Caiazzo et al., 2016), and that involves inflammation of epidermis and derma, to investigate on the antinflammatory 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 antinflammatory and tissue protective effect due to A2A receptor activation.

Materials and methods

Animals. All experiments were performed on male Wistar rats (Charles River, 180 – 220 g). Rats, slightly anaesthetized 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 h by a hydropletismometer (Ugo Basile, Comerio, VA, Italy).
Drug treatments. Rats were randomly assigned to groups of 5 each and treated, just before edema induction, with the A2A agonist, CGS21680 (0.02 – 2 mg/kg i.p.; Tocris Bioscience, Bristol, U.K); 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, Bristol, U.K); with CGS21680 (2 mg/kg i.p.) plus ZM241385 (3 mg/kg i.p.) or with the vehicle (dimethyl sulfoxide, DMSO; Sigma-Aldrich S.r.l., Milan, Italy). The dose of ZM241385 was chosen based on Caiazzo et al., 2016. From different groups of animals, at different time 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 histological 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 (PMN) 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 min at 20,000 x g. An aliquot of the supernatant was then allowed to react with a solution of 1.6 mM tetramethylbenzidine (Sigma-Aldrich S.r.l., Milan, Italy) 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 min at 37°C and was expressed in milli-units per g of wet tissue. As control, MPO activity was also measured on tissues obtained from contralateral, non-inflamed, paws.
Morphological analysis. Tissue samples removed as described above were immediately fixed in 4 % (v/v) neutral formalin for 48 h and embedded in paraffin by conventional techniques. For morphological analysis, sections 7 μm thick were stained with hematoxylin and eosin (Carlo Erba Reagents S.r.l., Cornaredo, Milan, Italy). The sections were analyzed by using a standard light microscope (x 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. In order to visualize tissue collagen, Picro Sirius red staining and polarization microscopy were performed. Briefly, the paraffin sections (7 µM) were de-waxed and rehydrate. 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, Milan, Italy) dissolved in saturated picric acid solution for one 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 trough a light microscope (Leits Dialux) equipped with a polarization filter (x 10 objective).

Measurement of tumor necrosis factor alpha and interleukin – 6 levels in rat paws. In tissue sample homogenates, prepared as described below, tumor necrosis factor alpha (TNF- α) and interleukin (IL)-6 levels were evaluated by an enzyme-linked immunosorbent assay (ELISA; R&D System). Results obtained were expressed as picogram of cytokine per mg 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 s at maximum speed). In order to extract proteins, 1 ml of buffer (β-glycerophosphate, 50 mM; sodium orthovanadate, 100 μM; MgCl2, 2mM; EGTA, 1mM; dithiothreitol (DTT), 1 mM; phenymethylsulphonyl fluoride (PMSF), 1mM; aprotinin, 10 μg/ml; leupeptin, 10 μg/ml) was added to 100 mg of tissue samples. After centrifugation at 2500 rpm for 30 min at 4°C, protein supernatant content was measured by Bradford reagent (Bio-Rad Laboratories). Denaturated samples (50