Background—We previously used adenosine A_2Areceptor (A2AR) knockout (KO) mice and bone marrow transplantation to show that the infarct-sparing effect of A2AR activation at reperfusion is primarily due to effects on bone marrow–derived cells. In this study we show that CD4⁺but not CD8⁺T lymphocytes contribute to myocardial ischemia/reperfusion injury.Method and Results—After a 45-minute occlusion of the left anterior descending coronary artery and reperfusion, T cells accumulate in the infarct zone within 2 minutes. Addition of 10 μg/kg of the A2AR agonist ATL146e 5 minutes before reperfusion produces a significant reduction in T-cell accumulation and a significant reduction in infarct size (percentage of risk area) measured at 24 hours. In Rag1 KO mice lacking mature lymphocytes, infarct size is significantly smaller than in C57BL/6 mice. Infarct size in Rag1 KO mice is increased to the level of B6 mice by adoptive transfer of 50 million CD4⁺T lymphocytes derived from C57BL/6 or A2AR KO but not interferon-γ KO mice. ATL146e completely blocked the increase in infarct size in Rag1 KO mice reconstituted with B6 but not A2AR KO CD4⁺T cells. The number of neutrophils in the reperfused heart at 24 hours after infarction correlated well with the number of lymphocytes and infarct size.Conclusions—These results strongly suggest that the infarct-sparing effect of A2AR activation is primarily due to inhibition of CD4⁺T-cell accumulation and activation in the reperfused heart.

Background— A _2A -adenosine receptor (A _2A AR) activation on reperfusion after ischemia reduces the size of myocardial infarction, but the mechanism of action has not been fully defined. Methods and Results— We created chimeric mice by bone marrow transplantation from A _2A AR-knockout or green fluorescent donor mice to irradiated congenic C57BL/6 (B6) recipients. In the GFP chimeras, we were unable to detect green fluorescent–producing cells in the vascular endothelium, indicating that bone marrow–derived cells were not recruited to endothelium at appreciable levels after bone marrow transplantation and/or acute myocardial infarction. Injection of 5 or 10 μg/kg of a potent and selective agonist of A _2A AR, ATL146e, had no effect on hemodynamic parameters but reduced infarct size in B6 mice after 45 minutes of left anterior descending artery occlusion followed by 24 hours of reperfusion to 42.5±3.0% and 39.3±4.7% of risk region, respectively, compared with 61.0±2.3% in vehicle-treated B6 mice ( P <0.05). Myocardial myeloperoxidase activity in the risk region measured at 4 hours after reperfusion was significantly reduced by ATL146e. The salutary effects of ATL146e were absent in A _2A AR-knockout mice or in mice treated with a selective A _2A AR antagonist, ZM241385. ATL146e also reduced infarct size and myeloperoxidase in B6/B6 (donor/recipient) chimeras ( P <0.05) but not in A _2A AR-knockout/B6 chimeras. In immunocompromised Rag-1–KO mice, infarct size was significantly reduced compared with B6 mice but was not further reduced by ATL146e. Conclusions— The results indicate that A _2A AR activation on bone marrow–derived cells, specifically T or B lymphocytes, is responsible for the infarct-sparing and antiinflammatory effects of ATL146e administered at the time of reperfusion after coronary occlusion.

Damage-associated molecular patterns, such as high-mobility group box 1 (HMGB1) and cell-free DNA (cfDNA), play critical roles in mediating ischemia-reperfusion injury (IRI). HMGB1 activates RAGE to exacerbate IRI, but the mechanism underlying cfDNA-induced myocardial IRI remains unknown. We hypothesized that the infarct-exacerbating effect of cfDNA is mediated by HMGB1 and receptor for advanced glycation end products (RAGE). C57BL/6 wild type mice, RAGE knockout (KO), and Toll-like receptor 9 KO mice underwent 20- or 40-minute occlusions of the left coronary artery followed by up to 60 minutes of reperfusion. Cardiac coronary perfusate was acquired from ischemic hearts without reperfusion. Exogenous mitochondrial DNA was acquired from livers of normal C57BL/6 mice. Myocardial infarct size (IS) was reported as percent risk region, as measured by 2,3,5-triphenyltetrazolium chloride and Phthalo blue (Heucotech, Fairless Hill, Pa) staining. cfDNA levels were measured by Sytox Green assay (Thermo Fisher Scientific, Waltham, Mass) and/or spectrophotometer. Free HMGB1 and cfDNA levels were increased in the ischemic myocardium during prolonged ischemia and subsequently in the plasma during reperfusion. In C57BL/6 mice undergoing 40'/60' IRI, deoxyribonuclease I, or anti-HMGB1 monoclonal antibody reduced IS by approximately half to 29.0% ± 5.2% and 24.3% ± 3.5% (P < .05 vs control 54.3% ± 3.4%). However, combined treatment with deoxyribonuclease I + anti-HMGB1 monoclonal antibody did not further attenuate IS (29.3% ± 4.9%). In C57BL/6 mice undergoing 20'/60' IRI, injection of 40'/5' plasma upon reperfusion increased IS by more than 3-fold (to 19.9 ± 4.3; P < .05). This IS exacerbation was abolished by pretreating the plasma with deoxyribonuclease I or by depleting the HMGB1 by immunoprecipitation, or by splenectomy. The infarct-exacerbating effect also disappeared in RAGE KO mice and Toll-like receptor 9 KO mice. Injection of 40'/0' coronary perfusate upon reperfusion similarly increased IS. The levels of HMGB1 and cfDNA were significantly elevated in the 40'/0' coronary perfusate and 40'/reperfusion (min) plasma but not in those with 10' ischemia. In C57BL/6 mice without IRI, 40'/5' plasma significantly increased the interleukin-1β protein and messenger RNA expression in the spleen by 30 minutes after injection. Intravenous bolus injection of recombinant HMGB1 (0.1 μg/g) or mitochondrial DNA (0.5 μg/g) 5 minutes before reperfusion did not exacerbate IS (P = not significant vs control). However, combined administration of recombinant HMGB1 + mitochondrial DNA significantly increased IS (P < .05 vs individual treated groups) and this infarct-exacerbating effect disappeared in RAGE KO mice and splenectomized C57BL/6 mice. The accumulation of cfDNA in the spleen after combined recombinant HMGB1 + mitochondrial DNA treatment was significantly more elevated in C57BL/6 mice than in RAGE KO mice. Both HMGB1 and cfDNA are released from the heart upon reperfusion after prolonged ischemia and both contribute importantly and interdependently to post-IRI by a common RAGE-Toll-like receptor 9-dependent mechanism. Depleting either of these 2 damage-associated molecular patterns suffices to significantly reduce IS by approximately 50%.

Background. Activation of the adenosineA2Breceptor (A2BR) can reduce myocardial ischemia/reperfusion (IR) injury. However, the mechanism underlying theA2BR-mediated cardioprotection is less clear. The present study was designed to investigate the potential mechanisms of cardioprotection mediated byA2BR.Methods and Results. C57BL/6 mice underwent 40-minute ischemia and 60-minute reperfusion. ATL-801, a potent selectiveA2BR antagonist, could not block ischemic preconditioning induced protection. BAY 60-6583, a highly selectiveA2BR agonist, significantly reduced myocardial infarct size, and its protective effect could be blocked by either ATL-801 or wortmannin. BAY 60-6583 increased phosphorylated Akt (p-Akt) levels in the heart at 10 min of reperfusion, and this phosphorylation could also be blocked by ATL-801 or wortmannin. Furthermore, BAY 60-6583 significantly increased M2 macrophages and decreased M1 macrophage and neutrophils infiltration in reperfused hearts, which also could be blocked by wortmannin. Meanwhile, confocal imaging studies showed that the majority of Akt phosphorylation in the heart was colocalized to CD206+ cells in both control and BAY 60-6583 pretreated hearts.Conclusion. Our results indicated that pretreatment with BAY 60-6583 protects the heart against myocardial IR injury by its anti-inflammatory effects, probably by modulating macrophages phenotype switching via a PI3K/Akt pathway.