atl-313 and Reperfusion-Injury

Adenosine A(2A) receptor activation potently attenuates lung ischemia-reperfusion injury. This study tests the hypothesis that adenosine A(2A) receptor activation attenuates ischemia-reperfusion injury by inhibiting CD4+ T cell activation and subsequent neutrophil infiltration.. An in vivo model of lung ischemia-reperfusion injury was used. C57BL/6 mice were assigned to either sham group (left thoracotomy) or 7 study groups that underwent ischemia-reperfusion (1 hour of left hilar occlusion plus 2 hours of reperfusion). ATL313, a selective adenosine A(2A) receptor agonist, was administered 5 minutes before reperfusion with or without antibody depletion of neutrophils or CD4+ T cells. After reperfusion, the following was measured: pulmonary function using an isolated, buffer-perfused lung system, T cell infiltration by immunohistochemistry, myeloperoxidase and proinflammatory cytokine/chemokine levels in bronchoalveolar lavage fluid, lung wet/dry weight, and microvascular permeability.. ATL313 significantly improved pulmonary function and reduced edema and microvascular permeability after ischemia-reperfusion compared with control. Immunohistochemistry and myeloperoxidase content demonstrated significantly reduced infiltration of neutrophils and CD4+ T cells after ischemia-reperfusion in ATL313-treated mice. Although CD4+ T cell-depleted and neutrophil-depleted mice displayed significantly reduced lung injury, no additional protection occurred when ATL313 was administered to these mice. Expression of tumor necrosis factor-alpha, interleukin 17, KC, monocyte chemotactic protein-1, macrophage inflammatory protein-1, and RANTES were significantly reduced in neutrophil- and CD4+ T cell-depleted mice and reduced further by ATL313 only in neutrophil-depleted mice.. These results demonstrate that CD4+ T cells play a key role in mediating lung inflammation after ischemia-reperfusion. ATL313 likely exerts its protective effect largely through activation of adenosine A(2A) receptors on CD4+ T cells and neutrophils.

Topics: Adenosine A2 Receptor Agonists; Animals; Capillary Permeability; CD4-Positive T-Lymphocytes; Chemokines; Cytokines; Immunohistochemistry; Lung Diseases; Male; Mice; Mice, Inbred C57BL; Neutrophil Infiltration; Piperidines; Reperfusion Injury; Respiratory Function Tests

Adenosine and the activation of specific adenosine receptors are implicated in the attenuation of inflammation and organ ischemia-reperfusion injury. We hypothesized that activation of A(1), A(2A), or A(3) adenosine receptors would provide protection against lung ischemia-reperfusion injury.. With the use of an isolated, ventilated, blood-perfused rabbit lung model, lungs underwent 18 hours of cold ischemia followed by 2 hours of reperfusion. Lungs were administered vehicle, adenosine, or selective A(1), A(2A), or A(3) receptor agonists (CCPA, ATL-313, or IB-MECA, respectively) alone or with their respective antagonists (DPCPX, ZM241385, or MRS1191) during reperfusion.. Compared with the vehicle-treated control group, treatment with A(1), A(2A), or A(3) agonists significantly improved function (increased lung compliance and oxygenation and decreased pulmonary artery pressure), decreased neutrophil infiltration by myeloperoxidase activity, decreased edema, and reduced tumor necrosis factor-alpha production. Adenosine treatment was also protective, but not to the level of the agonists. When each agonist was paired with its respective antagonist, all protective effects were blocked. The A(2A) agonist reduced pulmonary artery pressure and myeloperoxidase activity and increased oxygenation to a greater degree than the A(1) or A(3) agonists.. Selective activation of A(1), A(2A), or A(3) adenosine receptors provides significant protection against lung ischemia-reperfusion injury. The decreased elaboration of the potent proinflammatory cytokine tumor necrosis factor-alpha and decreased neutrophil sequestration likely contribute to the overall improvement in pulmonary function. These results provide evidence for the therapeutic potential of specific adenosine receptor agonists in lung transplant recipients.

Topics: Adenosine; Adenosine A1 Receptor Agonists; Adenosine A1 Receptor Antagonists; Adenosine A2 Receptor Agonists; Adenosine A2 Receptor Antagonists; Adenosine A3 Receptor Agonists; Adenosine A3 Receptor Antagonists; Animals; Blood Pressure; Disease Models, Animal; In Vitro Techniques; Lung; Lung Compliance; Lung Diseases; Perfusion; Peroxidase; Piperidines; Protective Agents; Pulmonary Artery; Pulmonary Edema; Rabbits; Receptor, Adenosine A1; Receptor, Adenosine A2A; Receptor, Adenosine A3; Reperfusion Injury; Tumor Necrosis Factor-alpha

Lung ischemia-reperfusion (IR) injury leads to significant morbidity and mortality which remains a major obstacle after lung transplantation. However, the role of various subset(s) of lung cell populations in the pathogenesis of lung IR injury and the mechanisms of cellular protection remain to be elucidated. In the present study, we investigated the effects of adenosine A2A receptor (A2AAR) activation on resident lung cells after IR injury using an isolated, buffer-perfused murine lung model.. To assess the protective effects of A2AAR activation, three groups of C57BL/6J mice were studied: a sham group (perfused for 2 hr with no ischemia), an IR group (1 hr ischemia + 1 hr reperfusion) and an IR+ATL313 group where ATL313, a specific A2AAR agonist, was included in the reperfusion buffer after ischemia. Lung injury parameters and pulmonary function studies were also performed after IR injury in A2AAR knockout mice, with or without ATL313 pretreatment. Lung function was assessed using a buffer-perfused isolated lung system. Lung injury was measured by assessing lung edema, vascular permeability, cytokine/chemokine activation and myeloperoxidase levels in the bronchoalveolar fluid.. After IR, lungs from C57BL/6J wild-type mice displayed significant dysfunction (increased airway resistance, pulmonary artery pressure and decreased pulmonary compliance) and significant injury (increased vascular permeability and edema). Lung injury and dysfunction after IR were significantly attenuated by ATL313 treatment. Significant induction of TNF-alpha, KC (CXCL1), MIP-2 (CXCL2) and RANTES (CCL5) occurred after IR which was also attenuated by ATL313 treatment. Lungs from A2AAR knockout mice also displayed significant dysfunction, injury and cytokine/chemokine production after IR, but ATL313 had no effect in these mice.. Specific activation of A2AARs provides potent protection against lung IR injury via attenuation of inflammation. This protection occurs in the absence of circulating blood thereby indicating a protective role of A2AAR activation on resident lung cells such as alveolar macrophages. Specific A2AAR activation may be a promising therapeutic target for the prevention or treatment of pulmonary graft dysfunction in transplant patients.

Topics: Adenosine A2 Receptor Agonists; Animals; Blood Platelets; Bronchoalveolar Lavage Fluid; Capillary Permeability; Chemokines; Cytokines; Lung; Lung Diseases; Mice; Mice, Inbred C57BL; Mice, Knockout; Neutrophil Infiltration; Neutrophils; Peroxidase; Piperidines; Receptor, Adenosine A2A; Reperfusion Injury; Respiratory Function Tests

Cardiopulmonary bypass has been shown to exert an inflammatory response within the lung, often resulting in postoperative pulmonary dysfunction. Several studies have shown that adenosine A(2A) receptor activation attenuates lung ischemia-reperfusion injury; however, the effect of adenosine A(2A) receptor activation on cardiopulmonary bypass-induced lung injury has not been studied. We hypothesized that specific adenosine A(2A) receptor activation by ATL313 would attenuate inflammatory lung injury after cardiopulmonary bypass.. Adult male Sprague-Dawley rats were randomly divided into 3 groups: 1) SHAM group (underwent cannulation + heparinization only); 2) CONTROL group (underwent 90 minutes of normothermic cardiopulmonary bypass with normal whole-blood priming solution; and 3) ATL group (underwent 90 minutes of normothermic cardiopulmonary bypass with ATL313 added to the normal priming solution).. There was significantly less pulmonary edema and lung injury in the ATL group compared with the CONTROL group. The ATL group had significant reductions in bronchoalveolar lavage interleukin-1, interleukin-6, interferon-gamma, and myeloperoxidase levels compared with the CONTROL group. Similarly, lung tissue interleukin-6, tumor necrosis factor-alpha, and interferon-gamma were significantly decreased in the ATL group compared with the CONTROL group. There was no significant difference between the SHAM and ATL groups in the amount of pulmonary edema, lung injury, or levels of proinflammatory cytokines.. The addition of a potent adenosine A(2A) receptor agonist to the normal priming solution before the initiation of cardiopulmonary bypass significantly protects the lung from the inflammatory effects of cardiopulmonary bypass and reduces the amount of lung injury. Adenosine A(2A) receptor agonists could represent a new therapeutic strategy for reducing the potentially devastating consequences of the inflammatory response associated with cardiopulmonary bypass.

Topics: Adenosine A2 Receptor Agonists; Animals; Blood Gas Analysis; Bronchoalveolar Lavage Fluid; Cardiopulmonary Bypass; Interferon-gamma; Interleukin-1; Interleukin-6; Lung; Lung Diseases; Male; Peroxidase; Piperidines; Random Allocation; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Tumor Necrosis Factor-alpha

Adenosine A2A receptor activation during reperfusion improves lung ischemia-reperfusion injury. In this study we sought to determine whether pretreatment of rabbits with a potent and selective adenosine A2A receptor agonist, ATL-313, before transplantation or whether adding ATL-313 to the preservation solution results in equivalent or additional protection compared with ATL-313 added during reperfusion.. An isolated, ventilated, ex vivo blood-perfused rabbit lung model was used. All groups underwent 2 hours of reperfusion after 18 hours of cold ischemia (4 degrees C). ATL-313 was administered 1 hour before ischemia intravenously, with the preservation solution, and/or during reperfusion.. Both pretreatment of donor animals with ATL-313 or adding ATL-313 just during reperfusion improved pulmonary function, but significantly greater improvement was observed when pretreatment and treatment during reperfusion were combined (all P < .05). Myeloperoxidase levels, bronchoalveolar lavage tumor necrosis factor alpha levels, and pulmonary edema were all maximally decreased in the combined treatment group. The administration of an equimolar amount of the potent and highly selective adenosine 2A receptor antagonist, ZM 241385, along with ATL-313, resulted in the loss of protection conferred by ATL-313.. Adenosine A2A receptor activation with ATL-313 results in the greatest protection against lung ischemia-reperfusion injury when given before ischemia and during reperfusion. Improved pulmonary function observed with adenosine A2A receptor activation was correlated with decreased bronchoalveolar lavage tumor necrosis factor alpha and decreased lung myeloperoxidase. The loss of protection observed with the concurrent administration of the adenosine A2A receptor antagonist, ZM 241385, supports that the mechanism of ATL-313 protection is specifically mediated via adenosine A2A receptor activation.

Topics: Adenosine A2 Receptor Agonists; Animals; Anti-Inflammatory Agents; Female; In Vitro Techniques; Lung Diseases; Lung Transplantation; Male; Models, Animal; Piperidines; Rabbits; Receptor, Adenosine A2A; Reperfusion Injury; Tissue and Organ Harvesting

Adenosine receptors (AR) and extracellular signal-regulated kinases (ERK) have been implicated in tissue protection and apoptosis regulation during ischemia/reperfusion (I/R) injury. This study tests the hypothesis that reduction of reperfusion lung injury after A2A AR activation is associated with attenuation of apoptosis, modulation of ERK activation, and alterations in antiapoptotic and proapoptotic protein expression (Bcl-2 and Bax, respectively). Experiments were performed in intact-chest, spontaneously breathing cats in which the arterial branch of the left lower lung lobe was occluded for 2 h and reperfused for 3 h (I/R group). Animals were treated with the selective A2A AR agonist ATL313 given 5 min before reperfusion alone or in combination with the selective A2A AR antagonist ZM241385. Western blot analysis showed significant reduction in expression of Bcl-2 and increase in expression of Bax after reperfusion, compared with control lungs. Phosphorylated ERK1/2 levels were also increased after reperfusion. Compared with the I/R group, ATL313 markedly (P < 0.01) attenuated indices of injury and apoptosis including the percentage of injured alveoli, wet-dry weight ratio, myeloperoxidase activity, in situ terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling-positive cells, and caspase 3 activity and expression. Furthermore, compared with reperfused lungs, in ATL313-pretreated lungs, Western blot analysis demonstrated substantial ERK1/2 activation, increased expression of Bcl-2, and attenuated expression of Bax. The protective effects of ATL313 were blocked by pretreatment with ZM241385. In summary, the present study shows that in vivo activation of A2A AR confers protection against reperfusion lung injury. This protection is associated with decreased apoptosis and involves ERK1/2 activation and alterations in antiapoptotic Bcl-2 and proapoptotic Bax proteins.

Topics: Adenosine A2 Receptor Agonists; Animals; Apoptosis; bcl-2-Associated X Protein; Cats; Extracellular Signal-Regulated MAP Kinases; Lung; Lung Injury; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Piperidines; Proto-Oncogene Proteins c-bcl-2; Pulmonary Alveoli; Receptor, Adenosine A2A; Reperfusion Injury; Triazines; Triazoles; Wounds and Injuries

The current studies investigated the in vitro and in vivo effect of adenosine 2A receptor (A(2A)R) agonists to attenuate allogenic immune activation. We performed MLRs with spleen T lymphocytes and APCs isolated from wild-type and A(2A)R knockout mice of both C57BL/6 and BALB/c background strains. Two-way MLR-stimulated T cell proliferation was reduced by ATL313, a selective A(2A)R agonist in a dose-responsive manner (approximately 70%; 10 nM), an effect reversed by the A(2A)R antagonist ZM241385 (100 nM). By one-way MLRs, we observed that ATL313's inhibitory effect was due to effects on both T cells and APCs. ATL313 suppressed the activation markers CD25 and CD40L and the release of inflammatory cytokines IFN-gamma, RANTES, IL-12P(70), and IL-2. ATL313 also increased negative costimulatory molecules programmed death-1 and CTLA-4 expressed on T cells. In lymphocytes activated with anti-CD3e mAb, ATL313 inhibited the phosphorylation of Zap70, an effect that was reversed by the protein kinase A inhibitor H-89. In skin transplants, allograft survival was enhanced with ATL313, an effect blocked by ZM241385. These results indicate that A(2A)R agonists attenuate allogenic recognition by action on both T lymphocytes and APCs in vitro and delayed acute rejection in vivo. We conclude that A(2A)R agonists may represent a new class of compounds for induction therapy in organ transplantation.

Topics: Adenosine A2 Receptor Agonists; Adenosine A2 Receptor Antagonists; Animals; Antibodies, Monoclonal; Antigen-Presenting Cells; Antigens, CD; Antigens, Differentiation; CD3 Complex; CD40 Ligand; CTLA-4 Antigen; Cytokines; Graft Rejection; Interferon-gamma; Interleukin-2 Receptor alpha Subunit; Isoantigens; Lymphocyte Activation; Mice; Mice, Knockout; Piperidines; Programmed Cell Death 1 Receptor; Receptor, Adenosine A2A; Reperfusion Injury; Skin Transplantation; T-Lymphocytes; Triazines; Triazoles; ZAP-70 Protein-Tyrosine Kinase