Adenosine Promotes Wound Healing and Mediates Angiogenesis in Response to Tissue Injury Via Occupancy of A2A Receptors
2002; Elsevier BV; Volume: 160; Issue: 6 Linguagem: Inglês
10.1016/s0002-9440(10)61151-0
ISSN1525-2191
AutoresM. Carmen Montesinos, Avani Desai, Jiang‐Fan Chen, Herman Yee, Michael A. Schwarzschild, J. Stephen Fink, Bruce N. Cronstein,
Tópico(s)Wound Healing and Treatments
ResumoRecent evidence indicates that topical application of adenosine A2A receptor agonists, unlike growth factors, increases the rate at which wounds close in normal animals and promotes wound healing in diabetic animals as well as growth factors, yet neither the specific adenosine receptor involved nor the mechanism(s) by which adenosine receptor occupancy promotes wound healing have been fully established. To determine which adenosine receptor is involved and whether adenosine receptor-mediated stimulation of angiogenesis plays a role in promotion of wound closure we compared the effect of topical application of the adenosine receptor agonist CGS-21680 (2-p-[2-carboxyethyl]phenethyl-amino-5′-N-ethylcarboxamido-adenosine) on wound closure and angiogenesis in adenosine A2A receptor knockout mice and their wild-type littermates. There was no change in the rate of wound closure in the A2A receptor knockout mice compared to their wild-type littermates although granulation tissue formation was nonhomogeneous and there seemed to be greater inflammation at the base of the wound. Topical application of CGS-21680 increased the rate of wound closure and increased the number of microvessels in the wounds of wild-type mice but did not affect the rate of wound closure in A2A receptor knockout mice. Similarly, in a model of internal trauma and repair (murine air pouch model), endogenously produced adenosine released into areas of internal tissue injury stimulates angiogenesis because there was a marked reduction in blood vessels in the walls of healing air pouches of A2A receptor knockout mice compared to their wild-type controls. Inflammatory vascular leakage and leukocyte accumulation in the inflamed air pouch were similarly reduced in the A2A receptor knockout mice reflecting the reduced vascularity. Thus, targeting the adenosine A2A receptor is a novel approach to promoting wound healing and angiogenesis in normal individuals and those suffering from chronic wounds. Recent evidence indicates that topical application of adenosine A2A receptor agonists, unlike growth factors, increases the rate at which wounds close in normal animals and promotes wound healing in diabetic animals as well as growth factors, yet neither the specific adenosine receptor involved nor the mechanism(s) by which adenosine receptor occupancy promotes wound healing have been fully established. To determine which adenosine receptor is involved and whether adenosine receptor-mediated stimulation of angiogenesis plays a role in promotion of wound closure we compared the effect of topical application of the adenosine receptor agonist CGS-21680 (2-p-[2-carboxyethyl]phenethyl-amino-5′-N-ethylcarboxamido-adenosine) on wound closure and angiogenesis in adenosine A2A receptor knockout mice and their wild-type littermates. There was no change in the rate of wound closure in the A2A receptor knockout mice compared to their wild-type littermates although granulation tissue formation was nonhomogeneous and there seemed to be greater inflammation at the base of the wound. Topical application of CGS-21680 increased the rate of wound closure and increased the number of microvessels in the wounds of wild-type mice but did not affect the rate of wound closure in A2A receptor knockout mice. Similarly, in a model of internal trauma and repair (murine air pouch model), endogenously produced adenosine released into areas of internal tissue injury stimulates angiogenesis because there was a marked reduction in blood vessels in the walls of healing air pouches of A2A receptor knockout mice compared to their wild-type controls. Inflammatory vascular leakage and leukocyte accumulation in the inflamed air pouch were similarly reduced in the A2A receptor knockout mice reflecting the reduced vascularity. Thus, targeting the adenosine A2A receptor is a novel approach to promoting wound healing and angiogenesis in normal individuals and those suffering from chronic wounds. Adenosine, a potent endogenous physiological mediator, regulates a wide variety of physiological processes. Adenosine mediates its physiological effects via interaction with one or more of four known cell-surface receptors (A1, A2A, A2B, and A3). Previous studies have demonstrated that topical application of adenosine A1 or A2A receptor agonists promotes healing of full thickness dermal wounds although the adenosine receptors involved and the mechanism for this effect have not been fully established.1Montesinos MC Gadangi P Longaker M Sung J Levine J Nilsen D Reibman J Li M Jiang CK Hirschhorn R Recht PA Ostad E Levin RI Cronstein BN Wound healing is accelerated by agonists of adenosine A2 (G alpha s-linked) receptors.J Exp Med. 1997; 186: 1615-1620Crossref PubMed Scopus (180) Google Scholar, 2Sun LL Xu LL Nielsen TB Rhee P Burris D Cyclopentyladenosine improves cell proliferation, wound healing, and hair growth.J Surg Res. 1999; 87: 14-24Abstract Full Text PDF PubMed Scopus (22) Google Scholar Among the pharmacological properties that may contribute to its effect in wound healing adenosine is reported to be angiogenic, based on in vitro studies. Adenosine, acting at A2 receptors, stimulates endothelial cell migration, proliferation, and secretion of vascular endothelial growth factor in vitro.3Meininger CJ Schelling ME Granger HJ Adenosine and hypoxia stimulate proliferation and migration of endothelial cells.Am J Physiol. 1988; 255: H554-H562PubMed Google Scholar, 4Des Rosiers C Nees S Functional evidence for the presence of adenosine A2-receptors in cultured coronary endothelial cells.NaunynSchmiedebergs Arch Pharmacol. 1987; 336: 94-98Crossref PubMed Scopus (35) Google Scholar, 5Ethier MF Chander V Dobson Jr, JG Adenosine stimulates proliferation of human endothelial cells in culture.Am J Physiol. 1993; 265: H131-H138PubMed Google Scholar, 6Schiele JO Schwabe U Characterization of the adenosine receptor in microvascular coronary endothelial cells.Eur J Pharmacol. 1994; 269: 51-58Crossref PubMed Scopus (26) Google Scholar, 7Fischer S Sharma HS Karliczek GF Schaper W Expression of vascular permeability factor/vascular endothelial growth factor in pig cerebral microvascular endothelial cells and its upregulation by adenosine.Mol Brain Res. 1995; 28: 141-148Crossref PubMed Scopus (84) Google Scholar, 8Sexl V Mancusi G Baumgartner-Parzer S Schutz W Freissmuth M Stimulation of human umbilical vein endothelial cell proliferation by A2-adenosine and beta 2-adrenoceptors.Br J Pharmacol. 1995; 114: 1577-1586Crossref PubMed Scopus (69) Google Scholar, 9Takagi H King GL Robinson GS Ferrara N Aiello LP Adenosine mediates hypoxic induction of vascular endothelial growth factor in retinal pericytes and endothelial cells.Invest Ophthalmol Vis Sci. 1996; 37: 2165-2176PubMed Google Scholar, 10Takagi H King GL Ferrara N Aiello LP Hypoxia regulates vascular endothelial growth factor receptor KDR/Flk gene expression through adenosine A2 receptors in retinal capillary endothelial cells.Invest Ophthalmol Vis Sci. 1996; 37: 1311-1321PubMed Google Scholar, 11Ethier MF Dobson Jr, JG Adenosine stimulation of DNA synthesis in human endothelial cells.Am J Physiol. 1997; 272: H1470-H1479PubMed Google Scholar, 12Lutty GA Mathews MK Merges C McLeod DS Adenosine stimulates canine retinal microvascular endothelial cell migration and tube formation.Curr Eye Res. 1998; 17: 594-607Crossref PubMed Google Scholar, 13Grant MB Tarnuzzer RW Caballero S Ozeck MJ Davis MI Spoerri PE Feoktistov I Biaggioni I Shryock JC Belardinelli L Adenosine receptor activation induces vascular endothelial growth factor in human retinal endothelial cells.Circ Res. 1999; 85: 699-706Crossref PubMed Scopus (151) Google Scholar Adenosine is also angiogenic in the chorioallantoic membrane angiogenesis assay and it is found prominently at the edge of forming vasculature during retinal vasculogenesis and oxygen-induced retinopathy in vivo, where adenosine A2A and/or A2B receptors and 5′-nucleotidase are highly expressed.13Grant MB Tarnuzzer RW Caballero S Ozeck MJ Davis MI Spoerri PE Feoktistov I Biaggioni I Shryock JC Belardinelli L Adenosine receptor activation induces vascular endothelial growth factor in human retinal endothelial cells.Circ Res. 1999; 85: 699-706Crossref PubMed Scopus (151) Google Scholar, 14Dusseau JW Hutchins PM Hypoxia-induced angiogenesis in chick chorioallantoic membranes: a role for adenosine.Respir Physiol. 1988; 71: 33-44Crossref PubMed Scopus (101) Google Scholar, 15Lutty GA Merges C McLeod DS 5′ nucleotidase and adenosine during retinal vasculogenesis and oxygen-induced retinopathy.Invest Ophthalmol Vis Sci. 2000; 41: 218-229PubMed Google Scholar, 16Taomoto M McLeod DS Merges C Lutty GA Localization of adenosine A2a receptor in retinal development and oxygen-induced retinopathy.Invest Ophthalmol Vis Sci. 2000; 41: 230-243PubMed Google Scholar, 17Grant MB Davis MI Caballero S Feoktistov I Biaggioni I Belardinelli L Proliferation, migration, and ERK activation in human retinal endothelial cells through A(2B) adenosine receptor stimulation.Invest Ophthalmol Vis Sci. 2001; 42: 2068-2073PubMed Google Scholar In addition to the effects of adenosine on angiogenesis we and others have established that adenosine is a potent regulator of inflammation, the first stage of the wound-healing process.18Montesinos MC Cronstein BN Role of P1 receptors in inflammation.in: Williams M.P.A.M. Handbook of Experimental Pharmacology, vol. 151/II. Purinergic and Pyrimidinergic Signalling II. Cardiovascular, Respiratory, Immune, Metabolic and Gastrointestinal Tract Function. Springer, Berlin2001: 303-321Google Scholar The capacity of adenosine to suppress inflammation via occupancy of A2 receptors was first noted for neutrophils19Cronstein BN Kramer SB Weissmann G Hirschhorn R Adenosine: a physiological modulator of superoxide anion generation by human neutrophils.J Exp Med. 1983; 158: 1160-1177Crossref PubMed Scopus (402) Google Scholar, 20Cronstein BN Rosenstein ED Kramer SB Weissmann G Hirschhorn R Adenosine; a physiologic modulator of superoxide anion generation by human neutrophils. Adenosine acts via an A2 receptor on human neutrophils.J Immunol. 1985; 135: 1366-1371PubMed Google Scholar, 21Cronstein BN Levin RI Philips MR Hirschhorn R Abramson SB Weissmann G Neutrophil adherence to endothelium is enhanced via adenosine A1 receptors and inhibited via adenosine A2 receptors.J Immunol. 1992; 148: 2201-2206PubMed Google Scholar and subsequently adenosine was shown to regulate the inflammatory functions of many other cell types including macrophages, endothelial cells, and lymphocytes.22Cronstein BN Adenosine and its receptors during inflammation.in: Serhan CN Ward PA Molecular and Cellular Basis of Inflammation. Humana Press, Totowa1998: 259-274Google Scholar The results reported here confirm the role of adenosine A2A receptors in promotion of wound healing after topical application of the selective A2A receptor agonist CGS-21680 and suggest a mechanism by which adenosine A2A receptor occupancy promotes wound healing. Evidence from experiments performed in adenosine A2A receptor knockout mice clearly demonstrate that the adenosine receptor involved in promotion of wound healing is the A2A receptor. We report further that interaction of exogenous adenosine A2A receptor agonists with their receptors increases angiogenesis in the healing wounds as compared to vehicle controls. Moreover, endogenously released adenosine interacts with A2A receptors to promote angiogenesis in an internal wound as well. These observations provide further evidence that agents that activate adenosine A2A receptors may be useful for promotion of wound healing and suggest that promotion of angiogenesis plays a major role in the mechanism by which adenosine A2A receptor agonists promote wound healing. CGS-21680 (2-p-[2-carboxyethyl]phenethyl-amino-5′-N-ethylcarboxamido-adenosine), carrageenan (type I), thioglycollate medium (FTG), and Evans Blue were obtained from Sigma Chemical Co. (St. Louis, MO). All other materials were the highest quality that could be obtained. BALB/c mice were purchased from Taconic Farms (Germantown, NY). Mice with a targeted disruption of the gene for the adenosine A2A receptor have been described in detail elsewhere.23Chen JF Huang Z Ma J Zhu J Moratalla R Standaert D Moskowitz MA Fink JS Schwarzschild MA A(2A) adenosine receptor deficiency attenuates brain injury induced by transient focal ischemia in mice.J Neurosci. 1999; 19: 9192-9200Crossref PubMed Google Scholar The mice used in these experiments were derived from four original heterozygous breeding pairs. Mice were housed in the NYU animal facility, fed regular mouse chow, and given access to drinking water ad libitum. All procedures described below were reviewed and approved by the Institutional Animal Care and Use Committee of NYU Medical Center and performed under the supervision of the facility veterinary staff. DNA was extracted from the tips of mouse tails using a standard protocol. Briefly, tail tips were lysed in 500 μl of lysis buffer (100 mmol/L NaCl, 20 mmol/L Tris-HCl, pH 8.0, 10 mmol/L ethylenediaminetetraacetic acid, 0.5% sodium dodecyl sulfate, 400 μg/ml proteinase K) overnight at 55°C. To the lysed tips 300 μl of a saturated solution of NaCl was added and, after 10 minutes on ice, tubes were centrifuged (16,000 × g, 4°C for 10 minutes). Genomic DNA present in the supernatant was precipitated by the addition of 800 μl of isopropanol. Precipitates were washed once with 70% ethanol, vacuum-dried, and resuspended in 30 μl of TE buffer. Using a method originally worked out by the laboratory of Dr. M. Sitkovsky (personal communication) the genomic DNA was then subjected to PCR using the following primers 5′-AGCCAGGGGTTACATCTGTG-3′ (upstream) and 5′-TACAGACAGCCTCGACATGTG-3′ (downstream), which detect a 163-bp band for the wild-type A2A allele; and 5′-AGACAATCGGCTGCTCTGAT-3′ (upstream) and 5′-CAAGCTCTTCAGCAATATCACG-3′ (downstream), which detect a 618-bp band for the mutated A2A allele. To perform the PCR, 0.3 μg of genomic DNA was used in 30 μl of final reaction. The PCR was performed in a GeneAmp PCR System 2400 thermal cycler (Perkin-Elmer; Branchburg, NJ) under the following conditions: 95°C for 2 minutes followed by 40 cycles (94°C for 1 minute, 55°C for 20 seconds, and 72°C for 1 minute) and a final extension of 72°C for 10 minutes. Two sterile, full-thickness excisional wounds of 10 mm in diameter were created on the dorsum of anesthetized 12- to 15-week-old male and female mice using a template and scissors in a genotype-blind manner. Wounds were treated daily with topical application of 20 μl of either the adenosine agonist CGS-21680 (250 μg/ml) or vehicle [1.5% w/v carboxymethylcellulose in phosphate-buffered saline (PBS)]. Mice were kept individually caged to minimize licking of wounds. To determine rate of closure, wound outlines were traced onto clear plastic sheets and the area of the wounds was measured by digitization of the tracings with a WACOM artZ II graphics tablet (Wacom Co., Ltd., Taiwan) and SigmaScan Pro software (Jandel Scientific, San Rafael, CA). Wounds were considered completely healed when the wound area was entirely re-epithelialized, and the surface of the wound was smooth, homogenous in color, and without residual defects. Some animals were killed on the stated day by CO2 poisoning. Wounds were then excised and fixed in 10% neutral-buffered formalin for at least 3 days and then processed by usual histopathological tissue processing. Paraffin-embedded tissue sections (5-μm thick) were stained with hematoxylin and eosin (H&E) and reviewed. To induce an air pouch, 10- to 15-week-old mice were injected subcutaneously on the back with 3 ml of air. After 2 days the pouches were reinflated with 1.5 ml of air. On day 6, inflammation was induced by injection of 1 ml of a suspension of carrageenan (2% w/v in calcium- and magnesium-free PBS) into the air pouch, as we have previously described.24Cronstein BN Naime D Ostad E The antiinflammatory mechanism of methotrexate: increased adenosine release at inflamed sites diminishes leukocyte accumulation in an in vivo model of inflammation.J Clin Invest. 1993; 92: 2675-2682Crossref PubMed Scopus (614) Google Scholar After 4 hours the mice were sacrificed by CO2 narcosis, the pouches were flushed with 2 ml of PBS, and the exudates harvested. Aliquots were diluted 1:1 with methylene blue (0.01% w/v in PBS) and cells were counted in a standard hemocytometer chamber (American Optical, Buffalo, NY). Air pouches were induced as described above. On day 6, 0.1 ml of Evans Blue dye (6.25 mg/ml) was injected intraperitoneally 1 hour before induction of inflammation by injection of carrageenan. After 4 hours, animals were sacrificed, air pouch exudates were harvested, and blood obtained by cardiac puncture was subjected to centrifugation and 1:50 dilution in PBS. The extravasation index was calculated as the ratio of the absorbances of the cell-free exudates and diluted serum at 620 nm. Air pouches were induced as described above in a similar group of mice. On day 6, animals were sacrificed and air pouches harvested and fixed in 10% neutral-buffered formalin for at least 3 days and then processed by usual histopathological tissue processing. Paraffin-embedded tissue sections (5-μm thick) were stained with H&E and reviewed. Thioglycollate peritonitis was induced by intraperitoneal injection of 0.5 ml of a sterile solution of thioglycollate medium (10% w/v in PBS). After 4 hours the animals were sacrificed by CO2 and their peritoneal cavities were lavaged with 3 ml of cold PBS. The peritoneal area was massaged before withdrawing the lavage fluid. Exudates were maintained at 4°C until aliquots were diluted 1:1 with methylene blue (0.01% w/v in PBS) and cells were counted in a standard hemocytometer chamber. Immunohistochemistry was performed on formalin-fixed paraffin-embedded tissues sections (5-μm thick) with appropriate positive and negative controls for Factor VIII and CD34. Antibodies, Factor VIII, and CD34 (Ventana Medical Systems, Tucson, AZ), were applied from prediluted dispensers on an automated immunostainer (NexES, Ventana Medical Systems) followed by washing and application of immunoperoxidase-labeled anti-goat antibodies. The positively stained cells were visualized by addition of immunoperoxidase substrate diaminobenzidine and the sections were counterstained with hematoxylin. The stained sections were reviewed for overall thickness of the dermal wound or induced air pouch, thickness of the granulation tissue layer, and a count of the blood vessels, as has been previously reported.25Choi HJ Hyun MS Jung GJ Kim SS Hong SH Tumor angiogenesis as a prognostic predictor in colorectal carcinoma with special reference to mode of metastasis and recurrence.Oncology. 1998; 55: 575-581Crossref PubMed Scopus (151) Google Scholar, 26Vermeulen PB Van den Eynden GG Huget P Goovaerts G Weyler J Lardon F van Marck E Hubens G Dirix LY Prospective study of intratumoral microvessel density, p53 expression.Br J Cancer. 1999; 79: 316-322Crossref PubMed Scopus (136) Google Scholar, 27Tokunaga T Nakamura M Oshika Y Abe Y Ozeki Y Fukushima Y Hatanaka H Sadahiro S Kijima H Tsuchida T Yamazaki H Tamaoki N Ueyama Y Thrombospondin 2 expression is correlated with inhibition of angiogenesis and metastasis of colon cancer.Br J Cancer. 1999; 79: 354-359Crossref PubMed Scopus (120) Google Scholar Tissue sections were reviewed at low magnification (×2) and counting of positive-stained endothelial cells was performed blindly on five random fields at a higher magnification (×40). The results are expressed as the mean (±SEM) of the values obtained from the areas studied. Differences between groups were analyzed by means of one-way and two-way analyses of variances using SigmaStat (SPSS, Inc., Chicago, IL). We have previously demonstrated, by use of receptor-specific antagonists and agonists, that occupancy of adenosine A2A receptors increases the rate at which wounds heal but others have reported that A1 receptors are involved in promotion of wound healing.1Montesinos MC Gadangi P Longaker M Sung J Levine J Nilsen D Reibman J Li M Jiang CK Hirschhorn R Recht PA Ostad E Levin RI Cronstein BN Wound healing is accelerated by agonists of adenosine A2 (G alpha s-linked) receptors.J Exp Med. 1997; 186: 1615-1620Crossref PubMed Scopus (180) Google Scholar, 2Sun LL Xu LL Nielsen TB Rhee P Burris D Cyclopentyladenosine improves cell proliferation, wound healing, and hair growth.J Surg Res. 1999; 87: 14-24Abstract Full Text PDF PubMed Scopus (22) Google Scholar To test the hypothesis that occupancy of A2A receptors plays a role in the normal process of wound healing, we compared rates of dermal wound closure and final healing time in adenosine A2A receptor knockout mice and their wild-type littermates. The rate of wound healing was similar in A2A knockout mice and their wild-type littermates (50% of wounds closed between days 12 and 13 or between days 13 and 14, respectively; Figure 1 and Table 1) consistent with our previous observation that A2A receptor antagonists do not interfere with the rate of wound closure.1Montesinos MC Gadangi P Longaker M Sung J Levine J Nilsen D Reibman J Li M Jiang CK Hirschhorn R Recht PA Ostad E Levin RI Cronstein BN Wound healing is accelerated by agonists of adenosine A2 (G alpha s-linked) receptors.J Exp Med. 1997; 186: 1615-1620Crossref PubMed Scopus (180) Google Scholar In contrast, application of the adenosine A2A receptor agonist CGS-21680 increased the rate of wound closure in wild-type mice (50% of wounds closed between days 11 and 12, P < 0.001, versus vehicle-treated; Figure 1 and Table 1) but not in A2A knockout mice (50% of wounds closed between days 12 and 13, NS versus vehicle-treated).Table 1Adenosine A2A Receptor Agonist Promotes Wound Healing50% wound closure, day50% of wounds healed, dayWild type4.313.6 Vehicle treated (n = 30)Wild type2.9*P < 0.05 versus vehicle-treated wild-type mice, 2-way analysis of variance (Tukey test).11.8†P < 0.001 versus vehicle-treated wild-type mice, 2-way analysis of variance (Tukey test). CGS-21680 treated (n = 32)A2A receptor knockout3.312.2 Vehicle treated (n = 38)A2A receptor knockout3.212.0 CGS-21680 treated (n = 40)Wounds (12-mm in diameter) were created on the dorsum of mice on day 0, the wound area determined, and expressed as the percentage of wound closure, as described in Materials and Methods. As a measure of the rate of wound closure the day at which 50% of the area of the original wound remained open is presented here (50% wound closure). A measure of the time at which complete wound closure occurred in 50% of the animals in any group was extrapolated from the plot in Figure 1 and is expressed as the time at which 50% of the wounds had completely healed.* P < 0.05 versus vehicle-treated wild-type mice, 2-way analysis of variance (Tukey test).† P < 0.001 versus vehicle-treated wild-type mice, 2-way analysis of variance (Tukey test). Open table in a new tab Wounds (12-mm in diameter) were created on the dorsum of mice on day 0, the wound area determined, and expressed as the percentage of wound closure, as described in Materials and Methods. As a measure of the rate of wound closure the day at which 50% of the area of the original wound remained open is presented here (50% wound closure). A measure of the time at which complete wound closure occurred in 50% of the animals in any group was extrapolated from the plot in Figure 1 and is expressed as the time at which 50% of the wounds had completely healed. Wound closure, as described above, represents primarily re-epithelialization and developing granulation tissue. In previous studies, we had observed that adenosine A2A receptor agonist treatment also affected granulation tissue formation.1Montesinos MC Gadangi P Longaker M Sung J Levine J Nilsen D Reibman J Li M Jiang CK Hirschhorn R Recht PA Ostad E Levin RI Cronstein BN Wound healing is accelerated by agonists of adenosine A2 (G alpha s-linked) receptors.J Exp Med. 1997; 186: 1615-1620Crossref PubMed Scopus (180) Google Scholar We therefore compared the histological appearance of healing dermal wounds in the A2A knockout and wild-type control mice and observed a marked defect in granulation tissue formation in the A2A knockout mice. Three days after wound formation there was a thick layer of granulation tissue consisting of edematous matrix infiltrated by large numbers of fibroblasts, macrophages, and infiltrating neutrophils in the wild-type control mice. In contrast, 3 days after wound formation, granulation tissue was almost completely absent in the A2A receptor knockout mice (Figure 2A). In the center of the healing wounds in knockout mice there was edematous, loose connective tissue and nonpolarized cells in addition to an infiltrate of inflammatory cells (neutrophils). A marked difference in granulation tissue persisted even by 6 days after wound formation (Figure 2B). There was a uniform layer of granulation tissue at the base of the wounds in the wild-type control mice. In contrast, granulation tissue was spotty and nonuniform at the base of the wounds in A2A receptor knockout mice. In some areas granulation tissue was well organized and highly cellular whereas in other areas the granulation tissue consisted of fibroblasts and other cells lacking polarity and loose extracellular matrix. In other areas there appeared to be a relative increase in inflammatory cells as well. As expected from the macroscopic wound evaluations, the extent of re-epithelialization did not differ between wild-type controls and A2A knockout mice (Figure 2). CGS-21680 treatment of wounds enhanced fibroblast infiltration, matrix density, and re-epithelialization in wild-type mice but it had no effect in knockout mice (Figure 2, A and B). Previous studies have indicated that adenosine, acting at A2A receptors, promotes angiogenesis in in vitro models,3Meininger CJ Schelling ME Granger HJ Adenosine and hypoxia stimulate proliferation and migration of endothelial cells.Am J Physiol. 1988; 255: H554-H562PubMed Google Scholar, 4Des Rosiers C Nees S Functional evidence for the presence of adenosine A2-receptors in cultured coronary endothelial cells.NaunynSchmiedebergs Arch Pharmacol. 1987; 336: 94-98Crossref PubMed Scopus (35) Google Scholar, 5Ethier MF Chander V Dobson Jr, JG Adenosine stimulates proliferation of human endothelial cells in culture.Am J Physiol. 1993; 265: H131-H138PubMed Google Scholar, 6Schiele JO Schwabe U Characterization of the adenosine receptor in microvascular coronary endothelial cells.Eur J Pharmacol. 1994; 269: 51-58Crossref PubMed Scopus (26) Google Scholar, 7Fischer S Sharma HS Karliczek GF Schaper W Expression of vascular permeability factor/vascular endothelial growth factor in pig cerebral microvascular endothelial cells and its upregulation by adenosine.Mol Brain Res. 1995; 28: 141-148Crossref PubMed Scopus (84) Google Scholar, 9Takagi H King GL Robinson GS Ferrara N Aiello LP Adenosine mediates hypoxic induction of vascular endothelial growth factor in retinal pericytes and endothelial cells.Invest Ophthalmol Vis Sci. 1996; 37: 2165-2176PubMed Google Scholar, 10Takagi H King GL Ferrara N Aiello LP Hypoxia regulates vascular endothelial growth factor receptor KDR/Flk gene expression through adenosine A2 receptors in retinal capillary endothelial cells.Invest Ophthalmol Vis Sci. 1996; 37: 1311-1321PubMed Google Scholar, 11Ethier MF Dobson Jr, JG Adenosine stimulation of DNA synthesis in human endothelial cells.Am J Physiol. 1997; 272: H1470-H1479PubMed Google Scholar, 28Li J Fenton RA Wheeler HB Powell CC Peyton BD Cutler BS Dobson Jr, JG Adenosine A2a receptors increase arterial endothelial cell nitric oxide.J Surg Res. 1998; 80: 357-364Abstract Full Text PDF PubMed Scopus (96) Google Scholar a critical component of granulation tissue. To determine whether angiogenesis was similarly compromised in the wounds of the A2A receptor knockout mice and whether application of an A2A receptor agonist promotes angiogenesis in vivo we examined the vascularity of wounds in knockout and wild-type control mice. In all of the sections of wounds stained for Factor VIII there was high background staining for Factor VIII because of presumed leakage of plasma into the tissue at the site of the wound. Nonetheless, individual cells and blood vessels were clearly differentiated from the background staining by the immunostaining for Factor VIII. In the 3-day-old wounds of wild-type mice there was a 15-fold increase in the number of Factor VIII-positive endothelial cells in the CGS-21680-treated wounds (50 ± 8 endothelial cells/mm2 versus 3 ± 2 endothelial cells/mm2, n = 19 and 14, respectively, P < 0.001; Figure 3, Figure 4). The CGS-21680-induced increase in vascularity persisted in the 6-day-old wounds although the disparity was not as great as at 3 days (119 ± 8 endothelial cells/mm2 versus 67 ± 9 endothelial cells/mm2, n = 18, P < 0.005; Figure 3, Figure 4). In the knockout mice, Factor VIII-positive endothelial cells were hardly found in the edematous 3-day-old wounds, and were not distributed uniformly in the 6-day-old wounds; most vessels were found in groups or aligned in the middle of the granulation tissue along a band of well-organized granulation tissue and no vessels were observed in the areas where granulation tissue was unorganized or where there was marked edema. Consequently, we were unable to adequately assess uniformly the number of vessels in the wounds of the knockout mice (Figure 3).Figure 4Number of Factor VIII-positive endothelial cells in the granulation tissue of untreated and CGS-21680-treated excisional wounds in wild-type mice. The number of Factor VIII-positive endothelial ce
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