atl-146e and Reperfusion-Injury

Lung reperfusion injury is a significant problem in cardiothoracic surgery. Previous studies have demonstrated that an adenosine A(2A) agonist can attenuate lung reperfusion injury in a lung transplantation model. There has been little work, however, examining its effects in the setting of nontransplant ischemia reperfusion. Our hypothesis was that an A(2A) agonist would attenuate lung reperfusion injury in a warm ischemia hilar clamping model.. Sprague Dawley rats underwent 90 min of left hilar clamping followed by 4 h of reperfusion. Group 1 (n = 13) received an intravenous infusion of 0.06 ug/kg/min of ATL-146e, which was started 10 min before reperfusion. Group 2 (n = 16) received an equivalent saline infusion. A third sham group (n = 14) received the same protocol as Group 2 but no lung ischemia.. Animals receiving ATL-146e showed significant improvements in oxygenation (Group 1: 447 +/- 26.02 mmHg versus Group 2: 223 +/- 24.46 mmHg (P < 0.001) as well as ventilation (pCO2 Group 1: 48.78 +/- 3.88 versus Group 2: 63.56 +/- 4.80 (P = 0.009)). Total protein in the bronchoalveolar lavage was significantly higher in the saline group compared with the adenosine as well as a higher proportion of neutrophils. Histological analysis demonstrated a significantly higher number of neutrophils in the IR group compared with the adenosine group.. ATL-146e, an adenosine analogue that is a specific agonist for the A(2A) receptor, attenuates reperfusion injury in an in vivo rat lung model. Arterial blood gas measurements demonstrate a statistically significant increase in oxygenation and improved ventilation.

Topics: Adenosine A2 Receptor Agonists; Animals; Anti-Inflammatory Agents; Constriction; Cyclohexanecarboxylic Acids; Disease Models, Animal; Lung; Lung Diseases; Male; Purines; Rats; Rats, Sprague-Dawley; Reperfusion Injury

Adenosine receptor activation at reperfusion has been shown to ameliorate ischemia-reperfusion injury of the spinal cord, but the effects of therapy given in response to ischemic injury are unknown. We hypothesized that adenosine receptor activation with ATL-146e would produce similar protection from ischemic spinal cord injury, whether given at reperfusion or in a delayed fashion.. Twenty-two New Zealand white rabbits were divided into three groups. All three groups, including the ischemia-reperfusion group (IR, n = 8), underwent 45 min of infrarenal aortic occlusion. The early treatment group (early, n = 8) received 0.06 mug/kg/min of ATL-146e for 3 h beginning 10 min prior to reperfusion. The delayed treatment group (delayed, n = 6) received ATL-146e starting 1 h after reperfusion. After 48 h, hind limb function was graded using the Tarlov score. Finally, lumbar spinal cord neuronal cytoarchitecture was evaluated.. Hemodynamic parameters were similar among the groups. Hind limb function at 48 h was significantly better in the early group (3.5 +/- 1.0) compared to the IR group (0.625 +/- 0.5, P < or = 0.01). There was a trend towards better hind limb function in the early group compared to the delayed group (2.4 +/- 1.1, P = 0.08). Hind limb function was similar between delayed and IR groups. Hematoxylin-eosin spinal cord sections demonstrated preservation of viable motor neurons in the early group compared to the delayed and IR groups.. Early therapy with ATL-146e provided better protection in this study; therefore, therapy should not be delayed until there is evidence of ischemic neurological deficit. This study suggests that adenosine receptor activation is most effective as a preventive strategy at reperfusion for optimal protection in spinal cord ischemia-reperfusion injury.

Topics: Analysis of Variance; Animals; Cyclohexanecarboxylic Acids; Disease Models, Animal; Hemodynamics; Purines; Rabbits; Receptor, Adenosine A2A; Recovery of Function; Reperfusion Injury; Spinal Cord; Statistics, Nonparametric

A(2A) adenosine receptor (A(2A)R)-expressing bone marrow (BM)-derived cells contribute to the renal protective effect of A(2A) agonists in renal ischemia-reperfusion injury (IRI). We performed IRI in mice lacking T and B cells to determine whether A(2A)R expressed in CD4+ cells mediate protection from IRI. Rag-1 knockout (KO) mice were protected in comparison to wild-type (WT) mice when subjected to IRI. ATL146e, a selective A(2A) agonist, did not confer additional protection. IFN-gamma is an important early signal in IRI and is thought to contribute to reperfusion injury. Because IFN-gamma is produced by kidney cells and T cells we performed IRI in BM chimeras in which the BM of WT mice was reconstituted with BM from IFN-gamma KO mice (IFN-gamma KO-->WT chimera). We observed marked reduction in IRI in comparison to WT-->WT chimeras providing additional indirect support for the role of T cells. To confirm the role of CD4+ A(2A)R in mediating protection from IRI, Rag-1 KO mice were subjected to ischemia-reperfusion. The protection observed in Rag-1 KO mice was reversed in Rag-1 KO mice that were adoptively transferred WT CD4+ cells (WT CD4+-->Rag-1 KO) or A(2A) KO CD4+ cells (A(2A) KO CD4+-->Rag-1 KO). ATL146e reduced injury in WT CD4+-->Rag-1 KO mice but not in A(2A) KO CD4+-->Rag-1 KO mice. Rag-1 KO mice reconstituted with CD4+ cells derived from IFN-gamma KO mice (IFN-gamma CD4+-->Rag-1 KO) were protected from IRI; ATL146e conferred no additional protection. These studies demonstrate that CD4+ IFN-gamma contributes to IRI and that A(2A) agonists mediate protection from IRI through action on CD4+ cells.

Topics: Adenosine A2 Receptor Agonists; Animals; B-Lymphocytes; Bone Marrow; CD4-Positive T-Lymphocytes; Cells, Cultured; Cyclohexanecarboxylic Acids; Cytoprotection; Homeodomain Proteins; Immunity, Innate; Interferon-gamma; Kidney; Lymphopenia; Mice; Mice, Inbred C57BL; Mice, Knockout; Purines; Receptors, Adenosine A2; Reperfusion Injury; T-Lymphocytes

ATL-146e protects the spinal cord from ischemia/reperfusion injury, presumably via adenosine A(2A) receptor activation, but this relationship remains unproven. We hypothesized that spinal cord functional and cytoarchitectural preservation from ATL-146e would be lost with simultaneous administration of the specific adenosine A(2A) antagonist ZM241385 (ZM), thus proving that adenosine A(2A) receptor activation is responsible for the protective effects of this compound.. New Zealand White rabbits underwent 45 minutes of infrarenal aortic cross-clamping. Groups (n = 10) included sham, ischemia, ischemia plus ATL-146e (ATL-146E), ischemia plus ZM, or ischemia with both compounds (agonist-antagonist). Tarlov scores were recorded every 12 hours. After 48 hours, the spinal cord was fixed for histology and microtubule-associated protein 2 immunohistochemistry.. Tarlov scores at 48 hours were significantly better in the sham and ATL-146E groups (5.0 and 3.9, respectively) compared with the other three groups (all < or =1.3; P < .001). On hematoxylin and eosin, neuronal viability was higher in the sham, ATL-146E, and agonist-antagonist groups compared with the control and ZM groups (P < .05). Microtubule-associated protein 2 expression was preserved in the sham and ATL-146E groups but was lost in the ATL + ZM, ZM241385, and control groups.. ATL-146e preserves the spinal cord in terms of both cytoarchitecture and function after reperfusion of the ischemic spinal cord, but this preservation is not completely blocked by competitive adenosine A(2A) receptor antagonism. Although ATL-146e does seem to partially function through activation of the adenosine A(2A) receptor, the neuroprotective mechanism may not be limited to this particular receptor.

Topics: Adenosine A2 Receptor Agonists; Adenosine A2 Receptor Antagonists; Animals; Cell Survival; Cyclohexanecarboxylic Acids; Disease Models, Animal; Microtubule-Associated Proteins; Neurons; Neuroprotective Agents; Paraplegia; Purines; Rabbits; Receptor, Adenosine A2A; Reperfusion Injury; Spinal Cord; Spinal Cord Ischemia; Time Factors; Triazines; Triazoles

The role of monocytes/macrophages in the pathogenesis of ischemia-reperfusion injury (IRI) is unknown. We sought to determine whether activation of macrophage adenosine 2A (A(2A)) receptors (A(2A)Rs) mediates tissue protection. We subjected C57Bl/6 mice infused with clodronate [dichloromethylene bisphosphonate (Cl(2)MBP)] to IRI (32 min of ischemia followed by 24 h of reperfusion) to deplete them of macrophages. IRI induced an elevation of plasma creatinine that was reduced with Cl(2)MBP (26% of control). Adoptive transfer of murine RAW 264.7 cells reconstituted injury, an effect blocked significantly by A(2A) agonists (27% of plasma creatinine from mice reconstituted with macrophages). Macrophages subjected to A(2A) knockout by small interfering RNA were adoptively transferred to macrophage-depleted mice and reconstituted injury (110% of control mice); however, the increase in plasma creatinine was blocked by A(2A) agonists (20% of vehicle treatment). Finally, the A(2A) agonist effect on IRI was blocked in macrophage-depleted A(2A)-knockout mice reconstituted with wild-type RAW 264.7 cells. RNase protection assays 24 h after IRI demonstrated that macrophages are required for IL-6 and TGF-beta mRNA induction. However, A(2A) agonist-mediated tissue protection is independent of IL-6 and TGF-beta mRNA. We conclude that the full extent of IRI requires macrophages and that A(2A) agonist-mediated tissue protection is independent of activation of macrophage A(2A)Rs.

Topics: Acute Kidney Injury; Adenosine A2 Receptor Agonists; Animals; Antimetabolites; Cell Line; Clodronic Acid; Cyclohexanecarboxylic Acids; Cytokines; Humans; Kidney; Liposomes; Macrophages; Mice; Mice, Inbred C57BL; Purines; Receptor, Adenosine A2A; Reperfusion Injury; RNA, Messenger; Transfection

Ischemia-reperfusion (IR) injury negatively impacts patient outcome in lung transplantation. Clinically, we observed that lung transplant patients with ischemia-reperfusion injury tend to have cardiac dysfunction. Previous studies have shown that ATL-146e (4-{3-[6-amino-9-(5-ethylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl}-cyclohexanecarboxylic acid methyl ester), a selective adenosine A2A receptor agonist, reduces lung inflammation after ischemia-reperfusion. We hypothesized that pulmonary ischemia-reperfusion causes secondary heart dysfunction and ATL-146e will improve this dysfunction.. We utilized an in vivo rabbit lung ischemia-reperfusion model. The Sham group underwent 120 minutes single lung ventilation. The IR and ATL groups underwent 90 minutes right lung ischemia with 30 minutes right lung reperfusion. The ATL-146e was given intravenously to the ATL group during reperfusion. Cardiac output and arterial blood gases were monitored, and neutrophil sequestration was measured by myeloperoxidase activity.. Upon reperfusion, cardiac output (mL/min) significantly dropped in the IR and ATL groups. By 15 minutes reperfusion, cardiac output in the ATL group improved significantly over the IR group and remained significant thereafter. Lung myeloperoxidase activity was significantly reduced by ATL-146e. Although never hypoxemic, arterial oxygenation was lower in the IR and ATL groups while central venous pressures and mean arterial pressures were similar among groups. A separate experiment demonstrated that reperfusion with the antioxidant N-(2-mercaptopropionyl)glycine prevented cardiac dysfunction.. Pulmonary ischemia-reperfusion causes cardiac dysfunction independent of preload, afterload, and oxygenation. The ATL-146e improves this dysfunction presumably by the antiinflammatory effects of adenosine A2A receptor activation on neutrophils. One likely mechanism involves the release of oxidants from the ischemic lung upon reperfusion, which has immediate negative effects on the heart.

Topics: Adenosine A2 Receptor Agonists; Animals; Antioxidants; Blood Pressure; Cardiac Output; Cyclohexanecarboxylic Acids; Heart; Lung; Neutrophil Activation; Oxygen; Peroxidase; Purines; Rabbits; Reactive Oxygen Species; Reperfusion Injury

Activation of the A2A adenosine receptor (A(2A)R) during reperfusion of various tissues has been found to markedly reduce ischemia-reperfusion injury. In this study, we used bone marrow transplantation (BMT) to create chimeric mice that either selectively lack or selectively express the A(2A)R on bone marrow-derived cells. Bolus i.p. injection of the selective A2A agonist, 4-[3-[6-amino-9-(5-cyclopropylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl]-piperidine-1-carboxylic acid methyl ester (ATL313; 3 microg/kg), at the time of reperfusion protects wild-type (wt) mice from liver ischemia-reperfusion injury. ATL313 also protects wt/wt (donor/recipient BMT mouse chimera) and wt/knockout chimera but produces modest protection of knockout/wt chimera as assessed by alanine aminotransferase activity, induction of cytokine transcripts (RANTES, IFN-gamma-inducible protein-10, IL-1alpha, IL-1-beta, IL-1Ralpha, IL-18, IL-6, and IFN-gamma), or histological criteria. ATL313, which is highly selective for the A(2A)R, produces more liver protection of chimeric BMT mice than 4-[3-[6-amino-9-(5-ethylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl]-cyclohexanecarboxylic acid methyl ester, which is rapidly metabolized in mice to produce 4-[3-[6-amino-9-(5-ethylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl]-cyclohexanecarboxylic acid, which has similar affinity for the A(2A)R and the proinflammatory A3 adenosine receptor. GFP chimera mice were created to show that vascular endothelial cells in the injured liver do not account for liver protection because they are not derived by transdifferentiation of bone marrow precursors. The data suggest that activation of the A(2A)R on bone marrow-derived cells is primarily responsible for protecting the liver from reperfusion injury.

Topics: Adenosine A2 Receptor Agonists; Alanine Transaminase; Animals; Bone Marrow Cells; Bone Marrow Transplantation; Cell Differentiation; Chimera; Cyclohexanecarboxylic Acids; Cytokines; Endothelium, Vascular; Liver; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Purines; Receptor, Adenosine A2A; Reperfusion Injury

Reperfusion injury continues to significantly affect patients undergoing lung transplantation. Isolated lung models have demonstrated that adenosine A 2A receptor activation preserves function while decreasing inflammation. We hypothesized that adenosine A 2A receptor activation by ATL-146e during the initial reperfusion period preserves pulmonary function and attenuates inflammation in a porcine model of lung transplantation.. Mature pig lungs preserved with Viaspan (Barr Laboratories, Pomona, NY) underwent 6 hours of cold ischemia before transplantation and 4 hours of reperfusion. Animals were treated with (ATL group, n = 7) and without (IR group, n = 7) ATL-146e (0.05 microg kg -1 . min -1 ATL-146e administered intravenously for 3 hours). With occlusion of the opposite pulmonary artery, the animal was maintained for the final 30 minutes on the allograft alone. Recipient lung physiology was monitored before tissue evaluation of pulmonary edema (wet-to-dry weight ratio), myeloperoxidase assay, and tissue tumor necrosis factor alpha by means of enzyme-linked immunosorbent assay.. When the ATL group was compared with the IR group, the ATL group had better partial pressure of carbon dioxide (43.8 +/- 4.1 vs 68.9 +/- 6.3 mm Hg, P < .01) and partial pressure of oxygen (272.3 +/- 132.7 vs 100.1 +/- 21.4 mm Hg, P < .01). ATL-146e-treated animals exhibited lower pulmonary artery pressures (33.6 +/- 2.1 vs 47.9 +/- 3.5 mm Hg, P < .01) and mean airway pressures (16.25 +/- 0.08 vs 16.64 +/- 0.15 mm Hg, P = .04). ATL-146e-treated lungs had lower wet-to-dry ratios (5.9 +/- 0.39 vs 7.3 +/- 0.38, P < .02), lower myeloperoxidase levels (2.9 x 10 -5 +/- 1.2 x 10 -5 vs 1.3 x 10 -4 +/- 4.0 x 10 -5 DeltaOD mg -1 . min -1 , P = .03), and a trend toward decreased lung tumor necrosis factor alpha levels (57 +/- 12 vs 96 +/- 15 pg/mL, P = .06). The ATL group demonstrated significantly less inflammation on histology.. Adenosine A 2A activation during early reperfusion attenuated lung inflammation and preserved pulmonary function in this model of lung transplantation. ATL-146e and similar compounds could play a significant role in improving outcomes of pulmonary transplantation.

Topics: Adenosine A2 Receptor Agonists; Animals; Blood Gas Analysis; Carbon Dioxide; Cyclohexanecarboxylic Acids; Disease Models, Animal; Drug Evaluation, Preclinical; Enzyme-Linked Immunosorbent Assay; Female; Inflammation; Lung; Lung Transplantation; Male; Neutrophil Activation; Organ Size; Oxygen; Peroxidase; Pulmonary Edema; Purines; Random Allocation; Receptor, Adenosine A2A; Reperfusion Injury; Respiratory Function Tests; Severity of Illness Index; Swine; Time Factors; Tumor Necrosis Factor-alpha

Ischemia-reperfusion (I/R) injury occurs as a result of restoring blood flow to previously hypoperfused vessels or after tissue transplantation and is characterized by inflammation and microvascular occlusion. We report here that 4-[3-[6-amino-9-(5-ethylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl]-cyclohexanecarboxylic acid methyl ester (ATL146e), a selective agonist of the A(2A) adenosine receptor (A(2A)AR), profoundly protects mouse liver from I/R injury when administered at the time of reperfusion, and protection is blocked by the antagonist ZM241385. ATL146e lowers liver damage by 90% as assessed by serum glutamyl pyruvic transaminase and reduces hepatic edema and MPO. Most protection remains if ATL146e treatment is delayed for 1 h but disappears when delayed for 4 h after the start of reperfusion. In mice lacking the A(2A)AR gene, protection by ATL1465e is lost and ischemic injury of short duration is exacerbated compared with wild-type mice, suggesting a protective role for endogenous adenosine. I/R injury causes induction of hepatic transcripts for IL-1alpha, IL-1beta, IL-1Ra, IL-6, IL-10, IL-18, INF-beta, INF-gamma, regulated on activation, normal T cell expressed, and presumably secreted (RANTES), major intrinsic protein (MIP)-1alpha, MIP-2, IFN-gamma-inducible protein (IP)-10, and monocyte chemotactic protein (MCP)-1 that are suppressed by administering ATL146e to wild-type but not to A(2A)AR knockout mice. RANTES, MCP-1, and IP-10 are notable as induced chemokines that are chemotactic to T lymphocytes. The induction of cytokines may contribute to transient lymphopenia and neutrophilia that occur after liver I/R injury. We conclude that most damage after hepatic ischemia occurs during reperfusion and can be blocked by A(2A)AR activation. We speculate that inhibition of chemokine and cytokine production limits inflammation and contributes to tissue protection by the A(2A)AR agonist ATL146e.

Topics: Adenosine A2 Receptor Agonists; Animals; Blood Cells; Chemokines; Cyclohexanecarboxylic Acids; Cytokines; Drug Administration Schedule; Edema; Gene Deletion; Ischemia; Leukocytes; Liver; Liver Circulation; Liver Diseases; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Purines; Radioligand Assay; Receptor, Adenosine A2A; Reperfusion Injury; RNA, Messenger; Triazines; Triazoles

Ischemia-reperfusion (I/R) has been shown to cause microvascular dysfunction and to alter the appearance of the glycocalyx in electron micrographs. We hypothesized that I/R injury might alter the structure and/or permeability of the glycocalyx. Prior work had shown a role for adenosine in protection from I/R injury, and, therefore, we also explored the idea that activation of the adenosine A(2A) receptor would attenuate I/R glycocalyx injury. Here, we report that I/R causes a rapid and dramatic decrease in the ability of the glycocalyx to exclude FITC-Dextran 70 (Dex70). Over a reperfusion period of 45 min, the glycocalyx dye exclusion zone for Dex70 decreased by one-half in capillaries and postcapillary venules, whereas the red blood cell exclusion zone was very slightly reduced in capillaries only. Pretreatment with the A(2A) agonist ATL-146e significantly inhibited the changes in both vessel types. The modifications of the glycocalyx appear to be an early step in the inflammatory cascade typically associated with reperfusion injury, and adenosine A(2A) receptor activation may play a role in protection from this injury.

Topics: Animals; Arterioles; Biotransformation; Capillaries; Cyclohexanecarboxylic Acids; Erythrocytes; Glycocalyx; Male; Mice; Mice, Inbred C57BL; Muscle, Skeletal; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Purines; Receptor, Adenosine A2A; Reperfusion Injury; Triazines; Triazoles

Activation of A2A adenosine receptors (A2ARs) protects kidneys from ischemia-reperfusion injury (IRI). A2ARs are expressed on bone marrow-derived (BM-derived) cells and renal smooth muscle, epithelial, and endothelial cells. To measure the contribution of A2ARs on BM-derived cells in suppressing renal IRI, we examined the effects of a selective agonist of A2ARs, ATL146e, in chimeric mice in which BM was ablated by lethal radiation and reconstituted with donor BM cells derived from GFP, A2AR-KO, or WT mice to produce GFP-->WT, A2A-KO-->WT, or WT-->WT mouse chimera. We found little or no repopulation of renal vascular endothelial cells by donor BM with or without renal IRI. ATL146e had no effect on IRI in A2A-KO mice or A2A-KO-->WT chimera, but reduced the rise in plasma creatinine from IRI by 75% in WT mice and by 60% in WT-->WT chimera. ATL146e reduced the induction of IL-6, IL-1beta, IL-1ra, and TGF-alpha mRNA in WT-->WT mice but not in A2A-KO-->WT mice. Plasma creatinine was significantly greater in A2A-KO than in WT mice after IRI, suggesting some renal protection by endogenous adenosine. We conclude that protection from renal IRI by A2AR agonists or endogenous adenosine requires activation of receptors expressed on BM-derived cells.

Topics: Adenosine; Animals; Bone Marrow Cells; Brain; Cell Transplantation; Chemokines; Cyclohexanecarboxylic Acids; Cytokines; Endothelium, Vascular; Genotype; Green Fluorescent Proteins; Kidney; Luminescent Proteins; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Phenotype; Purinergic P1 Receptor Agonists; Purines; Receptors, Purinergic P1; Reperfusion Injury; Transplantation Chimera

High pulmonary artery flow rates can result in severe reperfusion injury after lung transplantation. Our hypothesis was that selective activation of the adenosine A(2A) receptor with a highly specific analog (ATL-146e) would inhibit leukocyte activation and decrease reperfusion injury after high-flow reperfusion.. Using our isolated, ventilated, blood-perfused rabbit lung model, all groups (n = 8 per group) underwent lung harvest, 4 hours of cold storage, and blood reperfusion for 30 minutes. Measurements of pulmonary artery pressure (in millimeters of mercury), arterial oxygenation (in millimeters of mercury), myeloperoxidase, peak inspiratory pressure, and wet/dry weight ratio were obtained. Groups 1 (high flow) and 2 (high flow ATL-146e) underwent reperfusion at 120 mL/min for 30 minutes. Groups 3 (controlled high flow) and 4 (controlled high flow ATL-146e) underwent controlled reperfusion with an initial reperfusion of 60 mL/min for the first 5 minutes, followed by a rate of 120 mL/min for 25 minutes. During reperfusion, groups 2 and 4 received ATL-146e at 4 microg. kg(-1). min(-1).. ATL-146e significantly improved lung physiologic measurements under both high-flow (group 1 vs group 2) and controlled high-flow (group 3 vs group 4) conditions after 30 minutes.. The adenosine A(2A) receptor analogue ATL-146e significantly decreases the severity of reperfusion injury in the setting of both high-flow and controlled high-flow reperfusion.

Topics: Animals; Blood Pressure; Cyclohexanecarboxylic Acids; Disease Models, Animal; Enzyme Activation; Female; Lung; Male; Organ Size; Peroxidase; Pulmonary Artery; Pulmonary Wedge Pressure; Purines; Rabbits; Receptor, Adenosine A2A; Receptors, Purinergic P1; Reperfusion; Reperfusion Injury; Time Factors

Inflammation is likely a major contributor to spinal cord reperfusion injury after aortic reconstruction. Systemic 4-(3-[6-amino-9-(5-ethylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl)-cyclohexanecarboxylic acid methyl ester (ATL-146e), a selective adenosine A(2A) agonist, has been shown to reduce paralysis after spinal cord ischemia. We hypothesized that ATL-146e reduces cytokine production during spinal cord reperfusion, curtailing inflammation and decreasing spinal cord capillary platelet-endothelial cell adhesion molecule-1 (PECAM-1) expression.. New Zealand White rabbits sustained spinal cord ischemia with 45-minute cross-clamping of the infrarenal aorta. One group of animals received intravenous ATL-146e at 0.06 microg/kg/min for 3 hours during reperfusion, beginning after 30 minutes of ischemia. A second group received saline solution vehicle alone for 3 hours, serving as an ischemic control. A third group served as sham-operated animals, undergoing laparotomy with anesthesia. Serum was assayed with enzyme-linked immunosorbent assay for tumor necrosing factor-alpha (TNF-alpha). Animals were allowed to recover for 48 hours and were evaluated for hind-limb motor function with the Tarlov (0 to 5) scoring system. At necropsy, animals from each group yielded spinal cords for immunohistochemical staining for PECAM-1. Data are expressed as mean +/- standard error of the mean, with statistical analysis with Student t test and Kruskal-Wallis nonparametric test.. Markedly improved Tarlov scores were seen in rabbits with ATL-146e (P <.001) during spinal cord reperfusion as compared with ischemic control animals. A significant reduction was found in TNF-alpha in the sera of rabbits with ATL-146e infusion (P <.01) as compared with ischemic control animals. Significantly reduced endothelial PECAM-1 staining intensity (P <.05) was seen in microscopic spinal cord sections from rabbits with ATL-146e.. ATL-146e, an adenosine A(2A) agonist, reduces spinal cord reperfusion injury. The mechanism of the protection may involve a reduction in circulating TNF-alpha during a critical 3-hour reperfusion interval and reduction in spinal cord endothelial PECAM-1 upregulation.

Topics: Animals; Capillaries; Cyclohexanecarboxylic Acids; Disease Models, Animal; Platelet Endothelial Cell Adhesion Molecule-1; Purines; Rabbits; Reperfusion Injury; Spinal Cord; Spinal Cord Ischemia; Tumor Necrosis Factor-alpha

Activation of A(2A) adenosine receptors (A(2A)-AR) by ATL-146e (formerly DWH-146e) prevents inflammatory cell activation and adhesion. Recurrent ischemia-reperfusion (I/R) of the skin results in pressure ulcer formation, a major clinical problem. ATL-146e was evaluated in a novel reproducible rat model of pressure ulcer. A 9-cm(2) region of dorsal rat skin was cyclically compressed at 50 mmHg using a surgically implanted metal plate and an overlying magnet to generate reproducible tissue necrosis. Osmotic minipumps were implanted into 24 rats divided into four equal groups to infuse vehicle (control), ATL-146e (0.004 microg x kg(-1) x min(-1)), ATL-146e plus an equimolar concentration of A(2A) antagonist, ZM-241385, or ZM-241385 alone. Each group received 10 I/R cycles. In non-I/R-treated skin, ATL-146e has no effect on blood flow. I/R-treated skin of the ATL-146e group compared with the vehicle group had 65% less necrotic area, 31% less inhibition of average skin blood flow, and fewer extravasated leukocytes (23 +/- 3 vs. 49 +/- 6 per 500 microm(2)). These data suggest that ATL-146e, acting via an A(2A)-AR, reduces leukocyte infiltration and is a potent prophylactic for I/R injury in skin.

Topics: Animals; Cell Line; Cyclohexanecarboxylic Acids; Dermatitis; Drug Combinations; Humans; Ischemia; Leukocytes; Male; Necrosis; Pressure Ulcer; Purines; Rats; Rats, Sprague-Dawley; Receptor, Adenosine A2A; Receptors, Purinergic P1; Reference Values; Regional Blood Flow; Reperfusion Injury; Skin; Triazines; Triazoles

We previously demonstrated in rats and mice that agonists of A(2A)-adenosine receptors (A(2A)-ARs) reduce renal injury following ischemia-reperfusion. We now extend these studies and examine the effects of ATL-146e (formerly DWH-146e), an A(2A)-AR agonist, and rolipram, a type IV phosphodiesterase (PDE 4) inhibitor, on murine renal injury following ischemia-reperfusion.. C57BL/6 mice were treated with rolipram, ATL-146e, or both compounds combined and were subjected to renal ischemia for 32 minutes and reperfusion for 24 to 48 hours. In vitro studies were performed on suspended and adhering human neutrophils.. Continuous delivery of rolipram or ATL-146e during reperfusion reduced renal injury in a dose-dependent manner. Maximal protection was observed when ATL-146e was infused for six hours during reperfusion. Elevated plasma creatinine and myeloperoxidase activity produced by ischemia-reperfusion were reduced by rolipram (0.1 ng/kg/min) and ATL-146e (10 ng/kg/min) by up to approximately 60% and 70%, respectively. Co-infusion of both compounds produced a maximum reduction of plasma creatinine of approximately 90% and myeloperoxidase activity. In vitro studies on suspended and adhering human neutrophils demonstrated that selective stimulation of A(2A)-ARs by ATL-146e increased cAMP accumulation, reduced oxidative activity of activated neutrophils, and decreased activated neutrophil adherence. These responses were potentiated by rolipram.. We conclude that the combined infusion of ATL-146e and rolipram leads to enhanced renal tissue protection from ischemia-reperfusion by mechanisms that may include reduced neutrophil adherence/recruitment and release of reactive oxygen species.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Acute Kidney Injury; Animals; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic Nucleotide Phosphodiesterases, Type 4; Cyclohexanecarboxylic Acids; Dose-Response Relationship, Drug; Drug Therapy, Combination; Humans; Mice; Mice, Inbred C57BL; Neutrophils; Peroxidase; Phosphodiesterase Inhibitors; Purinergic P1 Receptor Agonists; Purines; Reactive Oxygen Species; Receptor, Adenosine A2A; Receptors, Purinergic P1; Reperfusion Injury; Respiratory Burst; Rolipram

We hypothesized that inflammation during spinal cord reperfusion worsens ischemic injury. ATL-146e, an adenosine A(2A) agonist with known anti-inflammatory properties, was used to test this hypothesis at varied intervals to determine the time course of reperfusion injury.. Forty rabbits underwent cross-clamping of the infrarenal aorta for 45 minutes. One group (n = 14 animals) received 0.06 microg/kg/min systemic ATL-146e over 3 hours, beginning after 30 minutes of ischemic time. A second group (n = 6 animals) received ATL-146e over 1.5 hours. A third group (n = 3 animals) received ATL-146e over 1 hour, and a fourth group (n = 17 animals) received saline solution. All animals were assessed at 48 hours for hind limb motor function (Tarlov scale, 0-5).. Animals that received ATL-146e for 3 hours (Tarlov score, 4.3 +/- 0.22; P <.001) or 1.5 hours (Tarlov score, 2.7 +/- 0.6; P <.05) had improved neurologic outcomes compared with rabbits that received saline solution (Tarlov score, 0.6 +/- 0.29). Animals that received ATL-146e for 1 hour (Tarlov score, 0.7 +/- 0.8) were not significantly different from those animals that received saline solution.. Systemic ATL-146e, given during reperfusion, results in time-dependent improvement in spinal cord function after ischemia. This implies that the mechanism of spinal reperfusion injury includes leukocyte-mediated inflammation at a critical post-ischemic time interval.

Topics: Adenosine; Animals; Aorta, Thoracic; Cyclohexanecarboxylic Acids; Disease Models, Animal; Drug Administration Schedule; Neurologic Examination; Purinergic P1 Receptor Agonists; Purines; Rabbits; Receptor, Adenosine A2A; Recovery of Function; Reperfusion Injury; Spinal Cord; Surgical Instruments

The adenosine A2A agonist ATL-146e (4-[3-[6-Amino-9-(5-ethylcarbamoyl-3,4-dihydroxytetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl]-cyclohexanecarboxylic acid methyl ester) has been shown to prevent reperfusion injury in multiple organ systems through inhibition of activated leukocyte-endothelial interaction. We hypothesized that systemic ATL-146e could reduce spinal cord reperfusion injury after aortic clamping.. Twenty-six rabbits underwent cross-clamping of the infrarenal aorta for 45 minutes. One group received intravenous ATL-146e for 3 hours during reperfusion. A second cohort received only vehicle and served as controls. Animals were assessed at 24 and 48 hours using the Tarlov (0 to 5) scoring system for hind limb function. To evaluate neuronal attrition, immunostaining of lumbar spinal cord sections was performed using anti-SMI 33 antibody against neurofilament.. Systemic ATL-146e was tolerated without hemodynamic lability. Animals that received ATL-146e had significantly improved neurologic outcomes 24 and 48 hours after spinal cord ischemia (p < 0.001). There was preservation of neuronal architecture in the ventral horn of spinal cord sections from animals receiving ATL-146e compared with control animals.. Intravenous ATL-146e given during reperfusion is tolerated without hemodynamic lability, and results in substantially improved spinal cord function after ischemia by preservation of ventral horn neurons.

Topics: Animals; Cell Survival; Cyclohexanecarboxylic Acids; Neurologic Examination; Neurons; Purinergic P1 Receptor Agonists; Purines; Rabbits; Receptor, Adenosine A2A; Reperfusion Injury; Spinal Cord; Spinal Cord Ischemia