Objective Ex vivo lung perfusion (EVLP) has been successful in the assessment of marginal donor lungs, including donation after cardiac death (DCD) donor lungs. EVLP also represents a unique platform for targeted drug delivery. We sought to determine if ischemia-reperfusion injury (IRI) would be decreased after transplantation of DCD donor lungs subjected to prolonged cold preservation and treated with an adenosine A2A receptor (A2AR) agonist during EVLP. Methods Porcine DCD donor lungs were preserved at 4°C for 12 hours and underwent EVLP for 4 hours. Left lungs were then transplanted and reperfused for 4 hours. Three groups (n=4/group) were randomized according to treatment with the A2AR agonist ATL-1223 or the dimethyl sulfoxide (DMSO) vehicle: DMSO (infusion of DMSO during EVLP and reperfusion), ATL-E (infusion of ATL-1223 during EVLP and DMSO during reperfusion), and ATL-E/R (infusion of ATL-1223 during EVLP and reperfusion). Final PaO2/FiO2 ratios were determined from samples obtained from the left superior and inferior pulmonary veins. Results Final PaO2/FiO2 ratios in the ATL-E/R group (430.1 ± 26.4 mmHg) were similar to final PaO2/FiO2 ratios in the ATL-E group (413.6 ± 18.8 mmHg), but both treated groups had significantly higher final PaO2/FiO2 ratios compared to the DMSO group (84.8 ± 17.7 mmHg). Low PO2 gradients during EVLP did not preclude superior postoperative oxygenation capacity. Conclusions Following prolonged cold preservation, treatment of DCD donor lungs with an A2AR agonist during EVLP enabled PaO2/FiO2 ratios above 400 mmHg after transplantation in a preclinical porcine model. Pulmonary function during EVLP was not predictive of outcomes after transplantation.

Background Ischemia-reperfusion injury is a major source of morbidity and mortality after lung transplantation. We previously demonstrated a proinflammatory role of adenosine A2B receptor (A2BR) in lung ischemia-reperfusion injury. The current study tests the hypothesis that A2BR antagonism is protective to ischemic lungs after in vivo reperfusion or ex vivo lung perfusion (EVLP). Methods Mice underwent lung ischemia-reperfusion with/without administration of ATL802, a selective A2BR antagonist. A murine model of EVLP was also utilized to evaluate rehabilitation of donation after circulatory death (DCD) lungs. DCD lungs underwent ischemia, cold preservation and EVLP with Steen solution with/without ATL802. A549 human type 2 alveolar epithelial cells were exposed to hypoxia-reoxygenation (3hr/1hr) with/without ATL802 treatment. Cytokines were measured in bronchoalveolar lavage fluid and culture media by ELISA. Results After ischemia-reperfusion, ATL802 treatment significantly improved lung function (increased pulmonary compliance; reduced airway resistance and pulmonary artery pressure) and significantly attenuated proinflammatory cytokine production, neutrophil infiltration, vascular permeability and edema. ATL802 also significantly improved the function of DCD lungs after EVLP (increased compliance and reduced pulmonary artery pressure). After hypoxia-reoxygenation, A549 cells exhibited robust production of IL-8, a potent neutrophil chemokine, which was significantly attenuated by ATL802. Conclusions These results demonstrate that A2BR antagonism attenuates lung ischemia-reperfusion injury and augments reconditioning of DCD lungs by EVLP. The protective effects of ATL802 may involve targeting of A2BRs on alveolar epithelial cells to prevent IL-8 production. A2BR may be a novel therapeutic target for mitigating ischemia-reperfusion injury to increase the success of lung transplantation.

Outcomes for lung transplantation are the worst of any solid organ, and ischemia-reperfusion injury (IRI) limits both short- and long-term outcomes. Presently no therapeutic agents are available to prevent IRI. Sphingosine 1-phosphate (S1P) modulates immune function through binding to a set of G protein-coupled receptors (S1PR1-5). Although S1P has been shown to attenuate lung IRI, the S1P receptors responsible for protection have not been defined. The present study tests the hypothesis that protection from lung IRI is primarily mediated through S1PR1 activation. Mice were treated with either vehicle, FTY720 (a nonselective S1P receptor agonist), or VPC01091 (a selective S1PR1 agonist and S1PR3 antagonist) before left lung IR. Function, vascular permeability, cytokine expression, neutrophil infiltration, and myeloperoxidase levels were measured in lungs. After IR, both FTY720 and VPC01091 significantly improved lung function (reduced pulmonary artery pressure and increased pulmonary compliance) vs. vehicle control. In addition, FTY720 and VPC01091 significantly reduced vascular permeability, expression of proinflammatory cytokines (IL-6, IL-17, IL-12/IL-23 p40, CC chemokine ligand-2, and TNF-α), myeloperoxidase levels, and neutrophil infiltration compared with control. No significant differences were observed between VPC01091 and FTY720 treatment groups. VPC01091 did not significantly affect elevated invariant natural killer T cell infiltration after IR, and administration of an S1PR1 antagonist reversed VPC01091-mediated protection after IR. In conclusion, VPC01091 and FTY720 provide comparable protection from lung injury and dysfunction after IR. These findings suggest that S1P-mediated protection from IRI is mediated by S1PR1 activation, independent of S1PR3, and that selective S1PR1 agonists may provide a novel therapeutic strategy to prevent lung IRI.

Background Despite the critical need for donor lungs, logistical and geographical barriers hinder lung utilization. We hypothesized that donation after circulatory death (DCD) lungs subjected to 6-hours of cold preservation after ex vivo lung perfusion (EVLP) would have similar outcomes after transplantation compared to lungs transplanted immediately after EVLP, and both would perform superiorly compared with lungs transplanted immediately after procurement. Methods Donor porcine lungs were procured after circulatory death and 15-minutes of warm ischemia. Three groups (n=5/group) were randomized: immediate left lung transplantation (Immediate), EVLP for 4-hours followed by transplantation (EVLP), or EVLP for 4-hours followed by 6-hours of cold preservation followed by transplantation (EVLP+Cold). Lungs were reperfused for 2-hours prior to obtaining pulmonary vein samples for PaO2/FiO2 calculations, airway pressures for compliance measurements, and wet/dry weight ratios. Results PaO2/FiO2 ratios in EVLP and EVLP+Cold groups were significantly improved compared with the Immediate group (429.7±51.8 and 436.7±48.2 versus 117.4±22.9 mmHg, respectively). Additionally, dynamic compliance was significantly improved in the EVLP and EVLP+Cold groups compared to Immediate group (26.2±4.2 and 27.9±3.5 versus 11.1±2.4 mL/cmH2O, respectively). There were no differences in oxygenation capacity or dynamic compliance between EVLP and EVLP+Cold groups. Inflammatory cytokine levels were significantly lower in EVLP and EVLP+Cold groups. Conclusions DCD lungs can be successfully transplanted up to 6-hours after EVLP. Cold preservation of lungs following ex vivo assessment and rehabilitation may improve organ allocation, even to distant recipients, without compromising allograft function.

Background Ex vivo lung perfusion (EVLP) enables assessment and rehabilitation of marginal donor lungs prior to transplantation. We previously demonstrated that adenosine A2A receptor (A2AR) agonism attenuates lung ischemia-reperfusion injury. The current study utilizes a novel murine EVLP model to test the hypothesis that A2AR agonist enhances EVLP-mediated rehabilitation of donation after circulatory death (DCD) lungs. Methods Mice underwent euthanasia and 60 min warm ischemia, and lungs were flushed with Perfadex and underwent cold static preservation (CSP, 60 min). Three groups were studied: no EVLP (CSP), EVLP with Steen solution for 60 min (EVLP), and EVLP with Steen solution supplemented with ATL1223, a selective A2AR agonist (EVLP+ATL1223). Lung function, wet/dry weight, cytokines and neutrophil numbers were measured. Microarrays were performed using the Affymetrix GeneChip Mouse Genome 430A 2.0 Array. Results EVLP significantly improved lung function versus CSP, which was further, significantly improved by EVLP+ATL1223. Lung edema, cytokines and neutrophil counts were reduced after EVLP and further, significantly reduced after EVLP+ATL1223. Gene array analysis revealed differential expression of 1,594 genes after EVLP, which comprise canonical pathways involved in inflammation and innate immunity including IL-1, IL-8, IL-6 and IL-17 signaling. Several pathways were uniquely regulated by EVLP+ATL1223 including the downregulation of genes involved in IL-1 signaling such as ADCY9, ECSIT, IRAK1, MAPK12 and TOLLIP. Conclusion EVLP modulates pro-inflammatory genes and reduces pulmonary dysfunction, edema and inflammation in DCD lungs, which are further reduced by A2AR agonism. This murine EVLP model provides a novel platform to study rehabilitative mechanisms of DCD lungs.