Adenosine A2B receptor agonist improves epidermal barrier integrity in a murine model of epidermal hyperplasia.

Adenosine regulates multiple physiological processes through the activation of four receptor subtypes, of which the A2B adenosine receptor (A2BAR) has the lowest affinity for adenosine. Being the adenosine receptor subtype most prominently expressed in epidermis, we recently described the antiproliferative and anti-inflammatory effect of the selective A2BAR agonist BAY60-6583 (BAY) in human keratinocytes stimulated with 12-O-tetradecanoylphorbol-13-acetate (TPA), so we sought to establish the effect of topical application of BAY in a model of murine epidermal hyperplasia. Topical application of BAY (1 or 10 µg/site) prevented the inflammatory reaction and skin lesions induced by TPA, minimizing hyperproliferation and acanthosis, as well as the expression of specific markers of proliferative keratinocytes. On the other hand, pre-treatment with the selective A2BAR antagonist, PSB-1115 (PSB, 5 or 50 µg/site) reversed these beneficial effects. Additionally, BAY application normalized the expression of epidermal barrier proteins, whose integrity is altered in inflammatory skin diseases, while treatment with the antagonist alone worsened it. Our results, besides confirming the anti-inflammatory and antiproliferative effects of the A2BAR agonist, further demonstrate a role of A2BAR activation to preserve the epidermal barrier. Therefore, the activation of A2BAR may constitute a possible new pharmacological target for the treatment of skin inflammatory diseases such as psoriasis.

Trends in Scientific Production on Pharmaceutical Follow-up andthe Dader Method / Tendencias en la producción científica sobre el seguimiento farmacéutico y el método Dader

Objective: Pharmacotherapeutic Follow-up is the Professional Pharmaceutical Care Service aimed at detecting Drug-Related Problems for the prevention and resolution of negative medicine outcomes. The Dader Method is considered a clear and simple tool to develop Pharmacotherapeutic Follow-up. This research aims to analyze the evolution of the international scientific production related to Pharmacotherapeutic Follow-up and the Dader Method to show the current situation of this Professional Pharmacy Assistance Service. In addition, from the data obtained, we give a critical perspective on the implementation of the Dader Method in Community Pharmacy, considering its advantages and disadvantages based on the published scientific literature. Methods: Using bibliometrics tools, indicators were obtained to analyze the international production of scientific articles on Pharmacotherapeutic Followup and the Dader Method during the period (1999-2022) through the Scopus database. Results: The results showed a growth in the international scientific production of publications on Pharmacotherapeutic Follow-up, obtaining 30,287 papers, placing the United States, the United Kingdom, Australia, Canada and Spain as the five most productive countries. The publication of 83 papers on the Dader Method places Spain with the highest number of publications, followed by other Spanish or Portuguese speaking countries, among which Brazil and Colombia have the most prominent number of published papers in Latin America. The most frequent international journal covering the topic of Pharmacotherapeutic Follow-up was the American Journal of Health- Pharmacy (12.4%), while on the Dader Method, the journal Pharmaceutical Care Spain (21.7%) is in the first position, followed by Farmacia Hospitalaria (8.4%). Conclusion: The publications on the Dader method highlighs the greater productivity of the University of Granada and the author María José Faus Dáder. The inclusion of patients in the PTF service using the Dader Method, is more frequent in the hospital context, and is based on the presence of defined chronic pathologies (mainly diabetes), polymedication or specialized care follow-up, with elderly population being the most represented in all cases.(AU)

Annexin A2-Mediated Plasminogen Activation in Endothelial Cells Contributes to the Proangiogenic Effect of Adenosine A2A Receptors.

Adenosine A2A receptor mediates the promotion of wound healing and revascularization of injured tissue, in healthy and animals with impaired wound healing, through a mechanism depending upon tissue plasminogen activator (tPA), a component of the fibrinolytic system. In order to evaluate the contribution of plasmin generation in the proangiogenic effect of adenosine A2A receptor activation, we determined the expression and secretion of t-PA, urokinase plasminogen activator (uPA), plasminogen activator inhibitor-1 (PAI-1) and annexin A2 by human dermal microvascular endothelial cells stimulated by the selective agonist CGS-21680. The plasmin generation was assayed through an enzymatic assay and the proangiogenic effect was studied using an endothelial tube formation assay in Matrigel. Adenosine A2A receptor activation in endothelial cells diminished the release of PAI-1 and promoted the production of annexin A2, which acts as a cell membrane co-receptor for plasminogen and its activator tPA. Annexin A2 mediated the increased cell membrane-associated plasmin generation in adenosine A2A receptor agonist treated human dermal microvascular endothelial cells and is required for tube formation in an in vitro model of angiogenesis. These results suggest a novel mechanism by which adenosine A2A receptor activation promotes angiogenesis: increased endothelial expression of annexin A2, which, in turn, promotes fibrinolysis by binding tPA and plasminogen to the cell surface.

Cyanocobalamin Ultraflexible Lipid Vesicles: Characterization and In Vitro Evaluation of Drug-Skin Depth Profiles.

Atopic dermatitis (AD) and psoriasis are the most common chronic inflammatory skin disorders, which importantly affect the quality of life of patients who suffer them. Among other causes, nitric oxide has been reported as part of the triggering factors in the pathogenesis of both conditions. Cyanocobalamin (vitamin B12) has shown efficacy as a nitric oxide scavenger and some clinical trials have given positive outcomes in its use for treating skin pathologies. Passive skin diffusion is possible only for drugs with low molecular weights and intermediate lipophilicity. Unfortunately, the molecular weight and hydrophilicity of vitamin B12 do not predict its effective diffusion through the skin. The aim of this work was to design new lipid vesicles to encapsulate the vitamin B12 to enhance its skin penetration. Nine prototypes of vesicles were generated and characterized in terms of size, polydispersity, surface charge, drug encapsulation, flexibility, and stability with positive results. Additionally, their ability to release the drug content in a controlled manner was demonstrated. Finally, we found that these lipid vesicle formulations facilitated the penetration of cyanocobalamin to the deeper layers of the skin. The present work shows a promising system to effectively administer vitamin B12 topically, which could be of interest in the treatment of skin diseases such as AD and psoriasis.

Microneedle-Based Delivery: An Overview of Current Applications and Trends.

Microneedle arrays (MNA) are considered as one of the most promising resources to achieve systemic effects by transdermal delivery of drugs. They are designed as a minimally invasive, painless system which can bypass the stratum corneum, overcoming the potential drawbacks of subcutaneous injections and other transdermal delivery systems such as chemical enhancers, nano and microparticles, or physical treatments. As a trendy field in pharmaceutical and biomedical research, its applications are constantly evolving, even though they are based on very well-established techniques. The number of molecules administered by MNA are also increasing, with insulin and vaccines administration being the most investigated. Furthermore, MNA are being used to deliver cells and applied in other organs and tissues like the eyes and buccal mucosae. This review intends to offer a general overview of the current state of MNA research, focusing on the strategies, applications, and types of molecules delivered recently by these systems. In addition, some information about the materials and manufacturing processes is presented and safety data is discussed.

Improved effectiveness from individualized dosing of self-administered biologics for the treatment of moderate-to-severe psoriasis: a 5-year retrospective chart review from a Spanish University Hospital.

Background: Biologics for moderate-to-severe psoriasis are expensive and treatment substitutions may vastly increase cost. Moreover, administration regimens in routine practice may differ from recommended guidelines.Objectives: To evaluate long-term effectiveness, regimen, drug-survival, and efficiency of self-administered biologics in clinical practice.Methods: We performed a 5-year retrospective study in 72 patients (44 ± 14 years old) with moderate-to-severe psoriasis at the University Hospital La Plana (Vila-real, Spain), treated with subcutaneous biologics. We determined the effectiveness (PASI 75 or PASI < 5), and drug-survival using Kaplan-Meier estimates, and analyzed reasons for treatment interruption, drug substitution patterns, and costs.Results: Etanercept was less effective (45%) than ustekinumab (85%) and adalimumab (71%). In 15% of patients, optimal responses were maintained despite dose intervals lengthening. Drug-survival was significantly lower for etanercept than for the other biologics (p < .005). Most adalimumab and etanercept discontinuations were due to adverse events or lack of effectiveness; for ustekinumab the causes were unrelated to drug effects. Ustekinumab was 100% effective as a secondary biologic.Conclusion: Ustekinumab was the safest and most efficient treatment. Etanercept showed the highest treatment failure rate, incurring higher costs. Dosage individualization according to patient needs improves the therapy efficiency, reducing therapeutic failure and derived costs.

Methodological Approach to Use Fresh and Cryopreserved Vessels as Tools to Analyze Pharmacological Modulation of the Angiogenic Growth.

The sprouting of new vessels is greatly influenced by the procedure chosen. We sought to optimize the experimental conditions of the angiogenic growth of fresh and cryopreserved vessels cultured in Matrigel with the aim to use this system to analyze the pharmacological modulation of the process. Segments of second-order branches of rat mesenteric resistance arteries, thoracic aorta of rat or mouse, and cryopreserved rat aorta and human femoral arteries were cultured in Matrigel for 7-21 days in different mediums, as well as in the absence of endothelial or adventitia layer. Quantification of the angiogenic growth was performed by either direct measurement of the mean length of the neovessels or by calcein AM staining and determination of fluorescence intensity and area. Fresh and cryopreserved arterial rings incubated in Matrigel exhibited a spontaneous angiogenic response that was strongly accelerated by fetal calf serum. Addition of vascular endothelial growth factor, fibroblast growth factor, endothelial growth factor, or recombinant insulin-like growth factor failed to increase aortic sprouting, unless all were added together. Removal of adventitia, but not the endothelial layer, abrogated the angiogenic response of aortic rings. Determination of the mean neovessel length is an easy and accurate method to quantify the angiogenic growth devoid of confounding factors, such as inclusion of other cellular types surrounding the neovessels. Activity of a α1-adrenoceptor agonist (phenylephrine) and its inhibition by a selective antagonist (prazosin) were analyzed to prove the usefulness of the Matrigel system to evaluate the pharmacological modulation of the angiogenic growth.

Apremilast, a novel phosphodiesterase 4 (PDE4) inhibitor, regulates inflammation through multiple cAMP downstream effectors.

INTRODUCTION: This work was undertaken to delineate intracellular signaling pathways for the PDE4 inhibitor apremilast and to examine interactions between apremilast, methotrexate and adenosine A2A receptors (A2AR). METHODS: After apremilast and LPS incubation, intracellular cAMP, TNF-α, IL-10, IL-6 and IL-1α were measured in the Raw264.7 monocytic murine cell line. PKA, Epac1/2 (signaling intermediates for cAMP) and A2AR knockdowns were performed by shRNA transfection and interactions with A2AR and A2BR, as well as with methotrexate were tested in vitro and in the murine air pouch model. Statistical differences were determined using one or two-way ANOVA or Student's t test. The alpha nominal level was set at 0.05 in all cases. A P value of < 0.05 was considered significant. RESULTS: In vitro, apremilast increased intracellular cAMP and inhibited TNF-α release (IC50=104nM) and the specific A2AR-agonist CGS21680 (1µM) increased apremilast potency (IC50=25nM). In this cell line, apremilast increased IL-10 production. PKA, Epac1 and Epac2 knockdowns prevented TNF-α inhibition and IL-10 stimulation by apremilast. In the murine air pouch model, both apremilast and MTX significantly inhibited leukocyte infiltration, while apremilast, but not MTX, significantly inhibited TNF-α release. The addition of MTX (1 mg/kg) to apremilast (5 mg/kg) yielded no more inhibition of leukocyte infiltration or TNF-α release than with apremilast alone. CONCLUSIONS: The immunoregulatory effects of apremilast appear to be mediated by cAMP through the downstream effectors PKA, Epac1, and Epac2. A2AR agonism potentiated TNF-α inhibition by apremilast, consistent with the cAMP-elevating effects of that receptor. Because the A2AR is also involved in the anti-inflammatory effects of MTX, the mechanism of action of both drugs involves cAMP-dependent pathways and is therefore partially overlapping in nature.

Promotion of Wound Healing by an Agonist of Adenosine A2A Receptor Is Dependent on Tissue Plasminogen Activator.

Impaired wound healing, as it occurs in diabetes mellitus or long-term corticoid treatment, is commonly associated with disability, diminished quality of life, and high economic costs. Selective agonists of the A2A receptor subtype of adenosine, an endogenous regulator of inflammation, promote tissue repair in animal models, both healthy and with impaired healing. Plasmin-mediated proteolysis of fibrin and other matrix proteins is essential for cell migration at sites of injury. Since adenosine A2A receptor activation increases plasminogen activator release from macrophages and mast cells, we studied the effect of a selective agonist, CGS-21680, on full-thickness excisional wound closure in wild-type, urokinase plasminogen activator (uPA)-deficient, and tissue plasminogen activator (tPA)-deficient mice. Wound closure was impaired in tPA- and uPA-deficient mice as compared with wild-type mice, and topical application of CGS-21680 significantly increased the rate at which wounds closed in wild-type mice and uPA-deficient mice, but not in tPA-deficient mice. Immunostaining of tissue sections showed that tPA was present in endothelial cells and histiocytes by day 3 post-wound and also by day 6. In contrast, uPA was more prominent in these cell types only by day 6 post-wound. Our results confirm that plasminogen activation contributes to wound repair and are consistent with the hypothesis that adenosine A2A receptor activation promotes wound closure by a mechanism that depends upon tPA, but not uPA. Moreover, our results suggest that topical adenosine A2A receptor agonists may be useful in promotion of wound closure in patients with impaired wound healing.

ß-Adrenoceptors differentially regulate vascular tone and angiogenesis of rat aorta via ERK1/2 and p38.

ß-Adrenoceptors (ß-ARs) modulate ERK1/2 and p38 in different cells, but little is known about the contribution of these signaling pathways to the function of ß-ARs in vascular tissue. Immunoblotting analysis of rat aortic rings, primary endothelial (ECs) and smooth muscle cells (SMCs) isolated from aorta showed that ß-AR stimulation with isoprenaline activated p38 in aortic rings and in both cultured cell types, whereas it had a dual effect on ERK1/2 phosphorylation, decreasing it in ECs while increasing it in SMCs. These effects were reversed by propranolol, which by itself increased p-ERK1/2 in ECs. Isoprenaline ß-AR mediated vasodilation of aortic rings was potentiated by the ERK1/2 inhibitor, U0126, in the presence or absence of endothelium or L-NAME, whereas inhibition of p38 had no impact. Isoprenaline moderately decreased sprouting from aorta rings in the Matrigel angiogenesis assay; conversely propranolol not only prevented isoprenaline inhibition, but stimulated angiogenesis. ERK1/2 inhibition decreased angiogenesis, while a dramatic stimulation was observed by p38 blockade. Our results suggest that ERK1/2 activation after ß-ARs stimulation in the smooth muscle hinders the vasodilator effect of isoprenaline, but in the endothelium ß-ARs decreases ERK1/2 and increases p38 activity reducing therefore angiogenesis.

NF-κB and STAT3 inhibition as a therapeutic strategy in psoriasis: in vitro and in vivo effects of BTH.

Benzo[b]thiophen-2-yl-3-bromo-5-hydroxy-5H-furan-2-one (BTH) is a simple and interesting synthetic derivative of petrosaspongiolide M, a natural compound isolated from a sea sponge with demonstrated potent anti-inflammatory activity through inhibition of the NF-κB signaling pathway. In the present study, we report the in vitro and in vivo pharmacological effect of BTH on some parameters related to the innate and adaptive response in the pathogenesis of psoriasis. BTH inhibited the release of some of the key psoriatic cytokines such as tumor necrosis factor α, IL-8, IL-6, and CCL27 through the downregulation of NF-κB in normal human keratinocytes. Moreover, it impaired signal transducers and activators of transcription 3 (STAT3) phosphorylation and translocation to the nucleus, which resulted in decreased keratinocyte proliferation. These results were confirmed in vivo in two murine models of psoriasis: the epidermal hyperplasia induced by 12-O-tetradecanoylphorbol-13-acetate and the imiquimod-induced skin inflammation model. In both cases, topical administration of BTH prevented skin infiltration and hyperplasia through suppression of NF-κB and STAT3 phosphorylation. Our results confirm the pivotal role of both transcriptional factors in skin inflammation, as occurs in psoriasis, and highlight the potential of small molecules as therapeutic agents for the treatment of this skin disease, with BTH being a potential candidate for future drug research.

Potential antipsoriatic effect of chondroitin sulfate through inhibition of NF-κB and STAT3 in human keratinocytes.

Chondroitin sulfate (CS) is a natural glycosaminoglycan, formed by the 1-3 linkage of d-glucuronic acid to N-acetylgalactosamine, present in the extracellular matrix. It is used as a slow acting disease modifying agent in the treatment of osteoarthritis, and part of its beneficial effects are due to its antiinflammatory properties that result from an inhibitory effect on NF-κB signaling pathway. This ability raises the hypothesis that CS might be effective in other chronic inflammatory processes such as psoriasis, in which a deregulation of NF-κB is a key feature. In addition, psoriasis is characterized by an upregulation of STAT3 signaling pathway that is related to the epidermal hyperplasia. In the present study we report the pharmacological modulation of the NF-κB and STAT3 signaling pathways by CS in normal human keratinocytes. CS inhibited NF-κB activation and the release of some of the key psoriatic cytokines such as TNFα, IL-8, IL-6 and CCL27. Moreover, it impaired STAT3 translocation to the nucleus and significantly reduced STAT3 transcriptional activity by a mechanism that was independent from STAT3 phosphorylation. Our results confirm the interest of CS as a candidate for future drug research in the therapeutics of psoriasis given the need of more effective and safer oral medications for these patients.

Adenosine receptor agonists for promotion of dermal wound healing.

Wound healing is a dynamic and complex process that involves a well-coordinated, highly regulated series of events including inflammation, tissue formation, revascularization and tissue remodeling. However, this orderly sequence is impaired in certain pathophysiological conditions such as diabetes mellitus, venous insufficiency, chronic glucocorticoid use, aging and malnutrition. Together with proper wound care, promotion of the healing process is the primary objective in the management of chronic poorly healing wounds. Recent studies have demonstrated that A(2A) adenosine receptor agonists promote wound healing in normal and diabetic animals and one such agonist, Sonedenoson, is currently being evaluated as a prospective new therapy of diabetic foot ulcers. We will review the mechanisms by which adenosine receptor activation affects the function of the cells and tissues that participate in wound healing, emphasizing the potential beneficial impact of adenosine receptor agonists in diabetic impaired healing.

The antiinflammatory mechanism of methotrexate depends on extracellular conversion of adenine nucleotides to adenosine by ecto-5'-nucleotidase: findings in a study of ecto-5'-nucleotidase gene-deficient mice.

OBJECTIVE: Evidence from in vitro, in vivo, and clinical studies indicates that adenosine mediates, at least in part, the antiinflammatory effects of methotrexate (MTX), although the biochemical events involved have not been fully elucidated. This study was undertaken to investigate whether MTX exerts antiinflammatory effects in mice that lack ecto-5'-nucleotidase (ecto-5'-NT) (CD73) and are unable to convert AMP to adenosine extracellularly, in order to determine whether adenosine is generated intracellularly and transported into the extracellular space or is generated from the extracellular dephosphorylation of AMP to adenosine. METHODS: Male CD73 gene-deficient mice and age-matched wild-type mice received intraperitoneal injections of saline or MTX (1 mg/kg/week) for 5 weeks. Air pouches were induced on the back by subcutaneous injection of air; 6 days later, inflammation was induced by injection of carrageenan. RESULTS: Fewer leukocytes, but higher levels of tumor necrosis factor alpha (TNFalpha), accumulated in the air pouches of vehicle-treated CD73-deficient mice compared with those of wild-type mice. As expected, MTX treatment reduced the number of leukocytes and TNFalpha levels in the exudates and increased exudate adenosine concentrations in wild-type mice. In contrast, MTX did not reduce exudate leukocyte counts or TNFalpha levels or increase exudate adenosine levels in CD73-deficient mice. CONCLUSION: These results demonstrate that the antiinflammatory actions of MTX are mediated, at least in part, by increased release of adenine nucleotides that are hydrolyzed extracellularly to adenosine via an ecto-5'-NT-dependent pathway.

Suppression of inflammation by low-dose methotrexate is mediated by adenosine A2A receptor but not A3 receptor activation in thioglycollate-induced peritonitis.

Prior studies demonstrate that adenosine, acting at one or more of its receptors, mediates the anti-inflammatory effects of methotrexate in animal models of both acute and chronic inflammation. Both adenosine A2A and A3 receptors contribute to the anti-inflammatory effects of methotrexate treatment in the air pouch model of inflammation, and the regulation of inflammation by these two receptors differs at the cellular level. Because different factors may regulate inflammation at different sites we examined the effect of low-dose weekly methotrexate treatment (0.75 mg/kg/week) in a model of acute peritoneal inflammation in adenosine A2A receptor knockout mice and A3 receptor knockout mice and their wild-type littermates. Following intraperitoneal injection of thioglycollate there was no significant difference in the number or type of leukocytes, tumor necrosis factor alpha (TNF-alpha) and IL-10 levels that accumulated in the thioglycollate-induced peritoneal exudates in adenosine A2A knockout mice or wild-type control mice. In contrast, there were more leukocytes, TNF-alpha and IL-10 in the exudates of the adenosine A3 receptor-deficient mice. Low-dose, weekly methotrexate treatment increased the adenosine concentration in the peritoneal exudates of all mice studied, and reduced the leukocyte accumulation in the wild-type mice and A3 receptor knockout mice but not in the A2A receptor knockout mice. Methotrexate reduced exudate levels of TNF-alpha in the wild-type mice and A3 receptor knockout mice but not the A2A receptor knockout mice. More strikingly, IL-10, a critical regulator of peritoneal inflammation, was increased in the methotrexate-treated wild-type mice and A3 knockout mice but decreased in the A2A knockout mice. Dexamethasone, an agent that suppresses inflammation by a different mechanism, was similarly effective in wild-type mice, A2A mice and A3 knockout mice. These findings provide further evidence that adenosine is a potent regulator of inflammation that mediates the anti-inflammatory effects of methotrexate. Moreover, these data provide strong evidence that the anti-inflammatory effects of methotrexate and adenosine are mediated by different receptors in different inflammatory loci, an observation that may explain why inflammatory diseases of some organs but not of other organs respond to methotrexate therapy.

Genetically based resistance to the antiinflammatory effects of methotrexate in the air-pouch model of acute inflammation.

OBJECTIVE: Low-dose methotrexate (MTX), a mainstay in the treatment of rheumatoid arthritis, is effective in only 60-70% of patients, a finding mirrored by poor antiinflammatory efficacy in some animal models, most notably collagen-induced arthritis. To determine whether genetic factors or the model itself is responsible for the poor response to MTX, we directly compared the responses of 4 inbred mouse strains to MTX in the air-pouch model of acute inflammation. METHODS: The exudate leukocyte count and adenosine concentration were determined in inbred mice treated with MTX (0.75 mg/kg intraperitoneally every week for 4 weeks) or vehicle 4 hours after injection of carrageenan into the air pouch using previously described methods. Quantitative trait locus mapping was performed using an in silico, or computer-based, method to identify loci potentially associated with each phenotype. RESULTS: MTX significantly reduced the exudate leukocyte count in C57BL/6J and BALB/cJ mice, but not DBA/1J (the strain used in the collagen-induced arthritis model) or DBA/2J mice. In a parallel manner, MTX increased adenosine concentration in inflammatory exudates of C57BL/6J and BALB/cJ mice, but not DBA/1J or DBA/2J mice. Antiinflammatory and adenosine responses to MTX in DBA/1J x C57BL/6J F(1) and F(2) offspring were most consistent with single genetic loci being responsible for each phenotype. In silico mapping identified partially overlapping loci containing candidate genes involved in both responses. CONCLUSION: Genetic factors contribute to the antiinflammatory efficacy of MTX, and a single locus involved in MTX-induced adenosine up-regulation is likely responsible for the observed resistance to MTX in DBA/1J mice.

Adenosine A2A receptor stimulation increases angiogenesis by down-regulating production of the antiangiogenic matrix protein thrombospondin 1.

Topical adenosine A2A receptor agonists promote wound healing by, among other effects, increasing microvessel formation. Results of representational display analysis of human umbilical vein endothelial cells suggested that A2A receptor occupancy modulates expression of the antiangiogenic matrix protein thrombospondin 1 (TSP1). We therefore determined whether A2A receptor occupation stimulates angiogenesis by modulating TSP1 secretion. Human microvascular endothelial cells (HMVEC) were treated with medium alone, 2-p-[2-carboxyethyl] phenethyl-amino-5'-N-ethylcarboxamido-adenosine (CGS-21680), or 2-[2-(4-chlorophenyl)ethoxy]adenosine (MRE0094), selective A2A receptor agonists. TSP1 protein secretion was down-regulated after treatment with the A2A agonists CGS-21680 or MRE0094 in a dose-dependent manner (EC50 = 6.65 nM and 0.23 microM respectively). The selective A2A receptor antagonist 4-[2-[7-amino-2-(2-furyl)[1,2,4]triazolo-[2,3-a][1,3,5]triazin-5-ylamino]ethyl]phenol (ZM241385) but not the A1 and A2B receptor antagonists diphenylcyclopentylxanthine, enprofylline, and N-(4-acetylphenyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)phenoxy]acetamide (MRS1706) completely abrogated the A2A receptor agonist-mediated effect on TSP1. Vascular tube formation by HMVEC was increased by adenosine A2A receptor agonists in a dose-dependent fashion (EC50 = 0.1 microM for both), and this effect was reversed by the A2A antagonist. Moreover, in the presence of antibodies to TSP1 and CD36, the receptor for TSP1, the adenosine A2A receptor agonists stimulated no increase in vascular tube formation. These results indicate that the angiogenic effects of adenosine A2A receptor activation are, at least in part, caused by the suppression of TSP1 secretion.

An interaction between genetic factors and gender determines the magnitude of the inflammatory response in the mouse air pouch model of acute inflammation.

The widely used mouse air pouch model of acute inflammation is inducible in a variety of inbred strains, but the potential influence of genetic background and gender on inflammation severity has never been examined. We directly compared the degree of inflammation induced in the air pouch model across four commonly utilized inbred strains in both male and female mice. We then applied an in silico mapping method to identify loci potentially associated with determining inflammation severity for each gender. Air pouches were induced by subcutaneous injection 3 (3 cc) and 5 (1.5 cc) days prior to the experiment. 4h after carrageenan injection, exudates were retrieved and leukocyte concentration quantified using a hemocytometer. The in silico mapping method was applied as described below. The strain order for mean leukocyte count/mL in inflamed exudates differed between genders. In males, the order was C57BL/6J > BALB/cByJ > DBA/2J > DBA/1J, while in females the order was BALB/cByJ > DBA/2J > C57BL/6J > DBA/1J. The difference in inflammation severity between genders reached significance only in C57BL/6J mice. Independent in silico analysis based on phenotypic data from male versus female mice identified distinct sets of loci as potentially associated with the exudate count reached. We conclude that the degree of inflammation induced in the mouse air pouch model of inflammation is strain-specific and, therefore, genetically based, and the pattern of interstrain differences is altered in male relative to female mice. The loci identified by in silico mapping likely contain genes with differential roles in determining this phenotype between genders.

Adenosine A2A receptor occupancy stimulates expression of proteins involved in reverse cholesterol transport and inhibits foam cell formation in macrophages.

Transport of cholesterol out of macrophages is critical for prevention of foam cell formation, the first step in the pathogenesis of atherosclerosis. Proteins involved in this process include cholesterol 27-hydroxylase and adenosine 5'-triphosphate-binding cassette transporter A1 (ABCA1). Proinflammatory cytokines and immune complexes (IC) down-regulate cholesterol 27-hydroxylase and impede cholesterol efflux from macrophages, leading to foam cell formation. Prior studies have suggested occupancy of the anti-inflammatory adenosine A2A receptor (A2AR) minimizes early atherosclerotic changes in arteries following injury. We therefore asked whether A2AR occupancy affects macrophage foam cell formation in response to IC and the cytokine interferon-gamma. We found that the selective A2AR agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamido-adenosine (CGS-21680) inhibited foam cell formation in stimulated THP-1 human macrophages, and the effects of CGS-21680 were reversed by the selective A2AR antagonist 4-(2-[7-amino-2-(2-furyl) [1, 2, 4]triazolo[2,3-a] [1, 3, 5]triazin-5-ylamino]ethyl)phenol. In confirmation of the role of A2AR in prevention of foam cell formation, CGS-21680 also inhibited foam cell formation in cultured murine peritoneal macrophages but did not affect foam cell formation in A2AR-deficient mice. Agents that increase foam cell formation also down-regulate cholesterol 27-hydroxylase and ABCA1 expression. Therefore, we determined the effect of A2AR occupancy on expression of these reverse cholesterol transport (RCT) proteins and found that A2AR occupancy stimulates expression of message for both proteins. These results indicate that one mechanism for the antiatherogenic effects of adenosine is stimulation of the expression of proteins involved in RCT. These findings suggest a novel approach to the development of agents that prevent progression of atherosclerosis.

Adenosine A(2A) receptor activation promotes wound neovascularization by stimulating angiogenesis and vasculogenesis.

Recent reports indicate that circulating endothelial progenitor cells (EPCs) may be recruited to sites of neovascularization where they differentiate into endothelial cells (EC). As we have previously demonstrated that adenosine A(2A) agonists promote neovascularization in wounds, we sought to determine whether adenosine A(2A) receptor agonist-augmented wound healing involves vessel sprouting (angiogenesis) or EPC recruitment (vasculogenesis) or both. Four weeks after bone marrow reconstitution from donor FVB/N Tie2GFP transgenic mice, two full-thickness excisional wounds were performed on the dorsum of FVB/N wild-type mice and treated with either an A(2A) receptor agonist (CGS-21680) or vehicle alone. Vessel density, as measured by CD31 staining, and density of EPC-derived vessels, as measured by GFP expression, were quantified in a blinded fashion using two-color fluorescence microscopy. We observed nearly a threefold increase in CD31-positive vessels and a more than 10-fold increase in GFP-positive cells in A(2A) agonist-treated 3-day old wounds, but by 6 days after wounding the differences between A(2A) agonist-treated and vehicle-treated wounds were no longer statistically significant. In conclusion, this is the first evidence that an exogenous agent such as an adenosine A(2A) receptor agonist increases neovascularization in the early stages of wound repair by increasing both EPC recruitment (vasculogenesis) and local vessel sprouting (angiogenesis).