Pharmacological blockade of adenosine A2A receptors diminishes scarring.
ABSTRACT Adenosine A2A receptor (A(2A)R) stimulation promotes wound healing and is required for the development of fibrosis in murine models of scleroderma and cirrhosis. Nonetheless, the role of A(2A)R in the formation of scars following skin trauma has not been explored. Here, we examined the effect of pharmacological blockade of A(2A)R, with the selective adenosine A(2A)R-antagonist ZM241385 (2.5 mg/ml), in a murine model of scarring that mimics human scarring. We found that application of the selective adenosine A(2A)R antagonist ZM241385 decreased scar size and enhanced the tensile strength of the scar. Within the scar itself, collagen alignment and composition (marked reduction in collagen 3), but not periostin, biglycan, or fibronectin accumulation, was improved by application of ZM241385. Moreover, A(2A)R blockade reduced the number of myofibroblasts and angiogenesis but not macrophage infiltration in the scar. Taken together, our work strongly suggests that pharmacological A(2A)R blockade can be used to diminish scarring while improving the collagen composition and tensile strength of the healed wound.-Perez-Aso, M., Chiriboga, L., Cronstein, B. N. Pharmacological blockade of adenosine A(2A) receptors diminishes scarring.
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ABSTRACT: Adenosine has an important role in inflammation and tissue remodeling and promotes dermal fibrosis by adenosine receptor (A2AR) activation. Adenosine may be formed intracellularly from adenine nucleotides or extracellularly through sequential phosphohydrolysis of released ATP by nucleoside triphosphate diphosphohydrolase (CD39) and ecto-5'-nucleotidase (CD73). Because the role of these ecto-enzymes in fibrosis appears to be tissue specific, we determined whether these ectonucleotidases were directly involved in diffuse dermal fibrosis. Wild-type and mice globally deficient in CD39 knockout (CD39KO), CD73 (CD73KO), or both (CD39/CD73DKO) were challenged with bleomycin. Extracellular adenosine levels and dermal fibrosis were quantitated. Adenosine release from skin cultured ex vivo was increased in wild-type mice after bleomycin treatment but remained low in skin from CD39KO, CD73KO, or CD39/CD73DKO bleomycin-treated mice. Deletion of CD39 and/or CD73 decreased the collagen content, and prevented skin thickening and tensile strength increase after bleomycin challenge. Decreased dermal fibrotic features were associated with reduced expression of the profibrotic mediators, transforming growth factor-β1 and connective tissue growth factor, and diminished myofibroblast population in CD39- and/or CD73-deficient mice. Our work supports the hypothesis that extracellular adenosine, generated in tandem by ecto-enzymes CD39 and CD73, promotes dermal fibrogenesis. We suggest that biochemical or biological inhibitors of CD39 and/or CD73 may hold promise in the treatment of dermal fibrosis in diseases such as scleroderma.American Journal Of Pathology 12/2013; 183(6):1740-1746. · 4.60 Impact Factor
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ABSTRACT: The nucleoside adenosine is a known regulator of immunity and inflammation that mediates, at least in part, the anti-inflammatory effect of methotrexate, an immunosuppressive agent widely used to treat autoimmune inflammatory diseases. Adenosine A2A receptors play a key role in the inhibition of the inflammatory process besides promoting wound healing. Therefore, we aimed to determine the topical effect of a selective agonist, CGS-21680, on a murine model of skin hyperplasia with a marked inflammatory component. Pretreatment with either CGS-21680 (5 μg per site) or the reference agent dexamethasone (200 μg/site) prevented the epidermal hyperplasia and inflammatory response induced by topical application of 12-O-tetradecanoylphorbol-13-acetate (TPA, 2 nmol/site) for three consecutive days. The histological analysis showed that both CGS-21680 and dexamethasone produced a marked reduction of inflammatory cell infiltrate, which correlated with diminished myeloperoxidase (MPO) activity in skin homogenates. Both treatments reduced the levels of the chemotactic mediators LTB4 and CXCL-1, and the inflammatory cytokine TNF-α, through the suppression of NFκB phosphorylation. The immunohistochemical analysis of the hyperproliferative markers cytokeratin 6 (CK6) and Ki67 revealed that while both agents inhibit the number of proliferating cells in the epidermis, CGS-21680 treatment promoted dermal fibroblasts proliferation. Consistently, increased collagen deposition in dermis was observed in tissue sections from agonist-treated mice. Our results showed that CGS 21680 efficiently prevents phorbol-induced epidermal hyperplasia and inflammation in mice without the deleterious atrophic effect of topical corticosteroids.This article is protected by copyright. All rights reserved.Experimental Dermatology 05/2014; · 3.58 Impact Factor
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ABSTRACT: Chronic musculoskeletal pain involves connective tissue remodeling triggered by inflammatory mediators, such as bradykinin. Fibroblast cells signaling involve changes in intracellular Ca2+ ([Ca2+]i). ATP has been related to connective tissue mechanotransduction, remodeling and chronic inflammatory pain, via P2 purinoceptors activation. Here, we investigated the involvement of ATP in bradykinin-induced Ca2+ signals in human subcutaneous fibroblasts. Bradykinin, via B2 receptors, caused an abrupt rise in [Ca2+]i to a peak that declined to a plateau, which concentration remained constant until washout. The plateau phase was absent in Ca2+-free medium; [Ca2+]i signal was substantially reduced after depleting intracellular Ca2+ stores with thapsigargin. Extracellular ATP inactivation with apyrase decreased the [Ca2+]i plateau. Human subcutaneous fibroblasts respond to bradykinin by releasing ATP via connexin and pannexin hemichannels, since blockade of connexins, with 2-octanol or carbenoxolone, and pannexin-1, with 10Panx, attenuated bradykinin-induced [Ca2+]i plateau, whereas inhibitors of vesicular exocytosis, such as brefeldin A and bafilomycin A1, were inactive. The kinetics of extracellular ATP catabolism favors ADP accumulation in human fibroblast cultures. Inhibition of ectonucleotidase activity and, thus, ADP formation from released ATP with POM-1 or by Mg2+ removal from media reduced bradykinin-induced [Ca2+]i plateau. Selective blockade of the ADP-sensitive P2Y12 receptor with AR-C66096 attenuated bradykinin [Ca2+]i plateau, whereas the P2Y1 and P2Y13 receptor antagonists, respectively MRS 2179 and MRS 2211, were inactive. Human fibroblasts exhibited immunoreactivity against connexin-43, pannexin-1 and P2Y12 receptor. Bradykinin induces ATP release from human subcutaneous fibroblasts via connexin and pannexin-1-containing hemichannels leading to [Ca2+]i mobilization through the cooperation of B2 and P2Y12 receptors.Cell Communication and Signaling 09/2013; 11(1):70. · 5.09 Impact Factor