View metadata, citation and similar papers at core.ac.uk brought to you by CORE CARDIOTHORACIC TRANSPLANTATION provided by Elsevier - Publisher Connector Adenosine A1 receptor activation attenuates lung ischemia–reperfusion injury Lucas G. Fernandez, MD, DSc, Ashish K. Sharma, PhD, MBBS, Damien J. LaPar, MD, MSc, Irving L. Kron, MD, and Victor E. Laubach, PhD Objectives: Ischemia–reperfusion injury contributes significantly to morbidity and mortality in lung transplant patients. Currently, no therapeutic agents are clinically available to prevent ischemia–reperfusion injury, and treatment strategies are limited to maintaining oxygenation and lung function. Adenosine can modulate inflam- matory activity and injury by binding to various adenosine receptors; however, the role of the adenosine A1 re- ceptor in ischemia–reperfusion injury and inflammation is not well understood. The present study tested the hypothesis that selective, exogenous activation of the A1 receptor would be anti-inflammatory and attenuate lung ischemia–reperfusion injury. Methods: Wild-type and A1 receptor knockout mice underwent 1 hour of left lung ischemia and 2 hours of re- perfusion using an in vivo hilar clamp model. An A1 receptor agonist, 2-chloro-N6-cyclopentyladenosine, was administered 5 minutes before ischemia. After reperfusion, lung function was evaluated by measuring airway resistance, pulmonary compliance, and pulmonary artery pressure. The wet/dry weight ratio was used to assess edema. The myeloperoxidase and cytokine levels in bronchoalveolar lavage fluid were measured to determine the presence of neutrophil infiltration and inflammation. Results: In the wild-type mice, 2-chloro-N6-cyclopentyladenosine significantly improved lung function and at- tenuated edema, cytokine expression, and myeloperoxidase levels compared with the vehicle-treated mice after ischemia–reperfusion. The incidence of lung ischemia–reperfusion injury was similar in the A1 receptor knock- out and wild-type mice; and 2-chloro-N6-cyclopentyladenosine had no effects in the A1 receptor knockout mice. In vitro treatment of neutrophils with 2-chloro-N6-cyclopentyladenosine significantly reduced chemotaxis. Conclusions: Exogenous A1 receptor activation improves lung function and decreases inflammation, edema, and neutrophil chemotaxis after ischemia and reperfusion. These results suggest a potential therapeutic appli- cation for A1 receptor agonists for the prevention of lung ischemia–reperfusion injury after transplantation. (J Thorac Cardiovasc Surg 2013;145:1654-9) Ischemia–reperfusion (IR) injury and its more severe form, through binding of 4 different G protein-coupled receptors: primary graft dysfunction, lead to significant morbidity and A1 receptor (A1R), A2AR, A2BR, and A3R, and adenosine mortality after lung transplantation, with mortality rates ap- receptor signaling typically entails second messenger path- proaching 40%. Ischemia is unavoidable during transplan- ways such as the cyclic adenosine monophosphate- tation, and the subsequent effect of reperfusion results in dependent protein kinase A pathway or the phospholipase significant cellular damage, oxidative stress, innate immune C pathway. responses, and lung inflammation.1,2 During organ All 4 adenosine receptors are expressed in the lungs of inflammation and IR injury, such as after transplantation, mice3 and humans.4 Studies from our laboratory have sug- adenosine is a retaliatory metabolite released from many gested that activation of A1RorA3R, by selective agonists, cell sources and serves largely as a protective agent with offers protection from lung IR injury in an isolated rabbit anti-inflammatory effects. Adenosine mediates its effects lung model.5 We have also demonstrated potent anti- inflammatory effects of A2AR activation in both a mouse lung IR model6 and a porcine lung transplant model.7 How- From the Department of Surgery, University of Virginia Health System, Charlottes- ever, our studies suggested a proinflammatory role for the ville, Va. 8 A2BR in the setting of lung IR. The role of the A1Rin This study was funded by National Institutes of Health, National Heart, Lung, and Blood Institute grant R01HL092953 (to I.L.K and V.E.L.). lung IR injury remains controversial and not well under- Disclosures: Authors have nothing to disclose with regard to commercial support. stood. The A1R is expressed largely on endothelial, epithe- Received for publication Oct 17, 2012; revisions received Dec 6, 2012; accepted for lial, and inflammatory cells and signals through Gi or G0 publication Jan 11, 2013; available ahead of print Feb 11, 2013. TX proteins. Its effects include inhibition of adenylyl cyclase, Address for reprints: Victor E. Laubach, PhD, Department of Surgery, University of þ Virginia Health System, PO Box 801359, Charlottesville, VA 22908 (E-mail: activation of K channels, inhibition of N-, P-, and [email protected]). Q-type Caþ channels, and activation of phospholipase Cb.9 0022-5223/$36.00 Copyright Ó 2013 by The American Association for Thoracic Surgery Earlier studies reported that A1R antagonism attenuates 10 http://dx.doi.org/10.1016/j.jtcvs.2013.01.006 lung inflammation after IR. However, more recent studies, 1654 The Journal of Thoracic and Cardiovascular Surgery c June 2013 Fernandez et al Cardiothoracic Transplantation Lung Weight/Dry Weight Ratio Abbreviations and Acronyms Using separate groups of mice (n ¼ 6/group), the left lung was excised after the reperfusion period, blotted dry, and immediately weighed and des- A1RÀ/¼congenic A1 receptor knockout ¼ iccated until a stable dry weight was reached. The lung wet/dry weight ratio BAL bronchoalveolar lavage was calculated as a measure of lung edema. CCPA ¼ 2-chloro-N6-cyclopentyladenosine FBS ¼ fetal bovine serum Analysis of Cytokines and Myeloperoxidase IR ¼ ischemia–reperfusion Cytokines were measured in BAL fluid using a mouse Bio-plex cytokine MPO ¼ myeloperoxidase assay (Bio-Rad Laboratories, Hercules, Calif), as previously reported.6,8 TNF-a ¼ tumor necrosis factor-a The myeloperoxidase (MPO) levels were measured in BAL fluid using WT ¼ wild-type a mouse MPO enzyme-linked immunosorbent assay kit (Hycult Biotech, Uden, The Netherlands). MPO is abundant in the azurophilic granules of polymorphonuclear neutrophils and was used as an indicator of neutrophil activation and infiltration into alveolar airspaces. including our own, have suggested that A1R activation has protective effects in several models of inflammatory lung Chemotaxis Assay 5,11,12 injury. Using A1R knockout mice and a specific In vitro migration was assessed in bone marrow–derived murine neu- trophils using a commercially available kit (QCM 5-mm Chemotaxis Cell pharmacologic A1R agonist, the present study tested the hypothesis that specific A R activation would be anti- Migration Assay; Millipore, Billerica, Mass). In brief, bone marrow cells 1 were harvested from mouse femurs and tibias by flushing with 10 mL of inflammatory and provide significant protection against phosphate-buffered saline containing 10% fetal bovine serum (FBS). The lung IR injury. suspended cells were centrifuged at 500g for 10 minutes. After red blood cell lysis for 2 minutes at room temperature, the cells were washed, METHODS counted, and resuspended in Roswell Park Memorial Institute buffer. Animals and Study Design Neutrophil isolation was then performed using a commercially available anti-Ly-6G mouse microbead kit (Miltenyi Biotech, Bergisch Gladbach, Adult male C57BL/6 wild-type (WT) mice (Jackson Laboratories, Bar Germany), according to the manufacturer’s instructions. The neutrophils Harbor, Me) and congenic A R knockout (A RÀ/À) mice 8 to 12 weeks 1 1 were resuspended at 2 3 106 cells/mL in Roswell Park Memorial Insti- old were used. The A RÀ/À mice13 were a gift of Dr Jurgen Schnermann 1 tute buffer with 0.5% FBS, and 250 mL of the cell suspension was incu- (Institute of Pharmacology and Toxicology, University of Tubingen,€ bated in a 5-mm insert, with or without A R agonist CCPA (10 ng/mL), Tubingen,€ Germany). The mice underwent either sham or IR surgery 1 for 30 minutes at 37C. Next, 500 mL of serum-free medium was then (n ¼ 6 mice/group). The mice were randomly allocated to the various con- added to the lower chamber, with or without chemoattractant (10% trol and experimental groups. They were treated with either vehicle (0.1% FBS), and the plate was incubated for 4 hours at 37 C in a carbon dioxide dimethyl sulfoxide in saline) or 2-chloro-N6-cyclopentyladenosine incubator. After the incubation period, the upper chamber was removed, (CCPA; Sigma Aldrich, St Louis, Mo) by intravenous injection 5 minutes and the insert was incubated in 400 mL of cell stain for 20 minutes at before ischemia. CCPA is a highly selective and potent A R agonist (affin- 1 room temperature. Nonmigratory cells were removed from the interior ity Ki value, 0.4 and 3900 nM for rat A R and A R, respectively), and 1 2A of the insert with a cotton swab, and the insert was transferred to CCPA is nearly 10,000-fold more selective for A R than for A R.14 1 2A a new well containing 200 mL of extraction buffer for 15 minutes. Color- The Institutional Animal Care and Use Committee at the University of Vir- imetric measurement of the reaction was performed at 570 nm in a plate ginia approved all animal procedures and protocols used in the present reader (Quant; Bio-Tek Instruments, Winooski, Vt). Two independent study, which conformed to the National Institute of Health guidelines in vitro experiments were performed. (Guide for the Care and Use of Laboratory Animals). Statistical Analysis Lung IR Model The results were analyzed using 2-way analysis of variance to determine The IR group underwent 1 hour of left lung ischemia and 2 hours of re- whether significant differences existed between the 2 groups. Comparisons perfusion (IR) using an established in vivo hilar clamp model.6,8 The sham between the 2 groups were analyzed using unpaired Student t test. Data are group underwent anesthesia, left thoracotomy (without the hilar clamp), expressed as the mean Æ standard deviation. and 2 hours of reperfusion. Analgesia was administered to all the mice after surgery.