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. 2008 Jun;172(6):1675-82.
doi: 10.2353/ajpath.2008.070952. Epub 2008 May 8.

Pharmacological blockade of A2A receptors prevents dermal fibrosis in a model of elevated tissue adenosine

Patricia Fernández  1 , Sean Trzaska, Tuere Wilder, Luis Chiriboga, Michael R Blackburn, Bruce N Cronstein, Edwin S L Chan
Affiliations

Pharmacological blockade of A2A receptors prevents dermal fibrosis in a model of elevated tissue adenosine

Patricia Fernández et al. Am J Pathol. 2008 Jun.

Abstract

Adenosine is a potent modulator of inflammation and tissue repair. We have recently reported that activation of adenosine A(2A) receptors promotes collagen synthesis by human dermal fibroblasts and that blockade or deletion of this receptor in mice protects against bleomycin-induced dermal fibrosis, a murine model of scleroderma. Adenosine deaminase (ADA) is the principal catabolic enzyme for adenosine in vivo, and its deficiency leads to the spontaneous development of pulmonary fibrosis in mice. The aim of this study was to characterize further the contributions of endogenous adenosine and adenosine A(2A) receptors to skin fibrosis. Taking advantage of genetically modified ADA-deficient mice, we herein report a direct fibrogenic effect of adenosine on the skin, in which increased collagen deposition is accompanied by increased levels of key mediators of fibrosis, including transforming growth factor beta1, connective tissue growth factor, and interleukin-13. Pharmacological treatment of ADA-deficient mice with the A(2A) receptor antagonist ZM-241385 prevented the development of dermal fibrosis in this model of elevated tissue adenosine, by reducing dermal collagen content and expression of profibrotic cytokines and growth factors. These data confirm a fibrogenic role for adenosine in the skin and reveal A(2A) receptor antagonists as novel therapeutic agents for the modulation of dermal fibrotic disorders.

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Figures

Figure 1
Figure 1
Adenosine levels are increased in skin of ADA-deficient mice. Skin biopsies were cut and incubated for 4 hours at 37°C, 5%CO2. Culture supernatants were concentrated, and adenosine levels were assessed by high-pressure liquid chromatography. Shown here are differences in adenosine levels between ADA-deficient mice (ADA-KO) and their wild-type littermates (ADA-WT). P < 0.01, n = 5 per group.
Figure 2
Figure 2
ADA-deficient mice develop dermal fibrosis, which is prevented by A2A receptor antagonist treatment. Shown here are differences in (A) thickness, (B) skin-fold thickness, (C) breaking tension, and (D) hydroxyproline content among ADA-deficient (ADA-KO), wild-type control (ADA-WT) mice, and ADA-deficient mice treated with the adenosine A2AR antagonist ZM-241385 (ADA KO + ZM). E: Histological sections of the different groups were stained with hematoxylin & eosin and picrosirius red viewed under polarized microscopy. P < 0.05, P < 0.01, n = 5 per group.
Figure 3
Figure 3
Effects of adenosine on skin IL-13, TGFβ1, and IL-6 levels. IL-13 (A) and active TGFβ1 (B) levels were significantly increased in skin homogenates of ADA-deficient mice when compared with their wild-type control mice and treatment of ADA-deficient mice with the adenosine A2AR antagonist ZM-241385 significantly reduced levels of these profibrotic cytokines. A similar pattern was found with IL-6 levels (C) although none of the observed changes reached statistical significance. Skin biopsies (6 mm) were homogenated and protein was measured by BCA method. Cytokine levels were determined by ELISA. P < 0.05, P < 0.01, n = 5 per group.
Figure 4
Figure 4
Effects of adenosine on skin CTGF expression. A: Immunohistochemistry was performed to determine CTGF expression in skin sections and photomicrographs were taken at original magnification ×400. Arrows indicate positive staining B: Computerized image analysis was used to quantify CTGF staining in sections among the different groups, P < 0.01, n = 5 per group. C: Western blot of skin homogenates was performed to corroborate differences on CTGF expression and a representative immunoblot is shown.
Figure 5
Figure 5
α smooth muscle actin positive cells are increased in skin sections of ADA-deficient mice and normalized by A2AR antagonist treatment. A: Immunohistochemistry was performed to identify α-SMA-positive cells on skin sections, and photomicrographs were taken at original magnification ×400. B: Computerized image analysis was used to quantify α-SMA staining in sections among the different groups. (P < 0.01, n = 5 per group). C: Western blot analysis of skin homogenates was performed to corroborate differences on α-SMA expression and a representative immunoblot is shown.
Figure 6
Figure 6
Effects of adenosine on skin PDGF expression. Western blot was performed to determine PDGF-A expression in skin homogenates. PDGF-A dimers were immunodetected and band intensities were quantified and normalized to β-actin level. A representative immunoblot is shown.
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