Daniel P. Mulloy, MD, Ashish K. Sharma, MBBS, PhD, Lucas G

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Adenosine A3 Receptor Activation Attenuates Lung Ischemia-Reperfusion Injury Daniel P. Mulloy, MD, Ashish K. Sharma, MBBS, PhD, Lucas G. Fernandez, MD, Yunge Zhao, MD, PhD, Christine L. Lau, MD, Irving L. Kron, MD, Victor E. Laubach, PhD The Annals of Thoracic Surgery Volume 95, Issue 5, Pages 1762-1767 (May 2013) DOI: 10.1016/j.athoracsur.2013.01.059 Copyright © 2013 The Society of Thoracic Surgeons Terms and Conditions

Fig 1 Pulmonary neutrophil infiltration after ischemia-reperfusion (IR) is attenuated by A3R agonism. Wild-type (WT) lungs that underwent sham surgery or IR in the presence or absence of Cl-IB-MECA (MECA, 100 μg/kg) were fixed and sectioned. Immunostaining for neutrophils (pink color in representative images in panel A) was performed, and the average number of neutrophils per high-powered field (neutrophils/HPF) is shown in panel B. (*p < 0.05 vs sham; #p < 0.05 vs IR (n = 4 to 6/group); scale bar = 100 μm.) The Annals of Thoracic Surgery 2013 95, 1762-1767DOI: (10.1016/j.athoracsur.2013.01.059) Copyright © 2013 The Society of Thoracic Surgeons Terms and Conditions

Fig 2 The A3R agonism decreased activation of neutrophils from wild-type (WT) but not A3R-/- mice after hypoxia-reoxygenation. Myeloperoxidase in cell culture supernatant was measured after primary neutrophils from WT or A3R-/- mice were incubated in the presence of Cl-IB-MECA (MECA, 1 μM) or vehicle, and underwent 4 hours of normoxia (Norm) or 3 hours hypoxia and 1 hour reoxygenation (HR). (*p < 0.05 vs norm; #p < 0.05 vs HR [n = 8/group]). The Annals of Thoracic Surgery 2013 95, 1762-1767DOI: (10.1016/j.athoracsur.2013.01.059) Copyright © 2013 The Society of Thoracic Surgeons Terms and Conditions

Fig 3 The A3R agonism attenuated neutrophil chemotaxis in vitro. Neutrophils from wild-type (WT) mice were added to the upper wells of a trans-well migration assay, incubated with Cl-IB-MECA (MECA, 1 μM) or vehicle, and then exposed to media or media with 10% fetal bovine serum (FBS) in the lower wells. After incubating for 4 hours at 37°C, trans-migrated cells were quantified and presented as a chemotaxis index. (*p < 0.05 vs media + vehicle; #p < 0.05 vs 10% FBS + vehicle (n = 8/group). The Annals of Thoracic Surgery 2013 95, 1762-1767DOI: (10.1016/j.athoracsur.2013.01.059) Copyright © 2013 The Society of Thoracic Surgeons Terms and Conditions