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Background Ex-vivo lung perfusion (EVLP) is a novel technique to assess, and potentially repair marginal lungs that may otherwise be rejected for transplantation. Adenosine has been shown to protect against lung ischemia-reperfusion injury through its A2A receptor. We hypothesized that combining EVLP with adenosine A2A receptor agonist treatment would enhance lung functional quality and increase donor lung usage. Methods Eight bilateral pig lungs were harvested and flushed with cold Perfadex. After 14 hours storage at 4°C, EVLP was performed for 5 hours on two explanted lung groups: 1) Control group lungs (n=4), were perfused with Steen Solution and Dimethyl sulfoxide (DMSO), and 2) treated group lungs (n=4) received 10μM CGS21680, a selective A2A receptor agonist, in a Steen Solution-primed circuit. Lung histology, tissue cytokines, gas analysis and pulmonary function were compared between groups. Results Treated lungs demonstrated significantly less edema as reflected by wet-dry weight ratio (6.6 vs. 5.2, p<0.03) and confirmed by histology. In addition, treated lung demonstrated significantly lower levels of interferon gamma (45.1 vs. 88.5, p<0.05). Other measured tissue cytokines (interleukin (IL) 1 beta, IL-6, and IL-8) were lower in treatment group, but values failed to reach statistical significance. Oxygenation index was improved in the treated group (1.5 vs. 2.3, p<0.01) as well as mean airway pressure (10.3 vs. 13 p<0.009). Conclusions EVLP is a novel and efficient way to assess and optimize lung function and oxygen exchange within donor lungs, and the use of adenosine A2A agonist potentiates its potential. EVLP with the concomitant administration of A2A agonist may enhance donor lung quality and could increase the donor lung pool for transplantation.

Objectives Adenosine A2A receptor (A2AR) activation following lung transplantation attenuates ischemia-reperfusion (IR) injury by reducing inflammation. However, the effect of A2AR activation in donor lungs prior to transplant remains ill-defined. This study compares the efficacy of three different treatment strategies for A2AR agonist in a clinically relevant porcine lung transplantation model. Methods Mature porcine lungs underwent six hours cold ischemia prior to allotransplantation and four hours reperfusion. Five groups (n=6/group) were evaluated based upon treatment with ATL-1223, a selective A2AR agonist: Sham (thoracotomy alone), IR (transplant alone), ATL-D (donor pretreatment via ATL-1223 bolus), ATL-R (recipient treatment via ATL-1223 infusion), and ATL-D/R (combination of both ATL-1223 treatments). Lung function and injury were compared. Results Blood oxygenation was significantly higher among ATL-D, ATL-R, and ATL-D/R groups versus IR (392.0±52.5, 428.9±25.5, 509.4±25.1 vs. 77.2±17.0 mmHg, respectively, p<0.001). ATL-1223-treated groups had lower pulmonary artery pressures (ATL-D=30.5±1.8, ATL-R=30.2±3.3, ATL-D/R=29.3±4.5 vs. IR=45.2±2.1 mmHg, p<0.001) and lower mean airway pressures versus IR (ATL-D=9.1±0.8, ATL-R=9.1±2.6, ATL-D/R=9.6±1.3 vs. IR=21.1 mmHg, p<0.001). Similarly, ATL-1223-treated groups had significantly lower lung wet/dry weight, proinflammatory cytokine expression and lung injury scores by histology compared to IR. Importantly, all parameters of lung function and injury in ATL-1223-treated groups were similar to Sham (all p>0.05). Conclusions Pretreatment of donor lungs with ATL-1223 was as efficacious as other treatment strategies in protecting against IR injury. If necessary, supplemental treatment of recipients with ATL-1223 may provide additional protection. These results support the development of pharmacologic A2AR agonists for use in human clinical trials for lung transplantation.

Background Development of bronchiolitis obliterans syndrome (BOS) following lung transplantation confers increased patient morbidity and mortality. Fibrocytes are circulating, bone marrow derived mesenchymal cell progenitors that influence tissue repair and fibrosis. Accumulated evidence has implicated fibrocytes in chronic pulmonary inflammatory processes. We investigated the correlation of circulating fibrocyte number with BOS development in lung transplant patients. Methods We prospectively quantified circulating fibrocyte levels among human lung transplant patients. Patients were stratified according to the development of BOS as indicated by predicted forced expiratory volume in one second (FEV1). Fibrocyte activity was analyzed by flow cytometry (CD45+, collagen 1+) in a blinded manner related to clinical presentation. Results A total of 39 patients previously underwent double (33.3%), left (25.6%), or right (41.0%) lung transplantation. Average patient age was similar between BOS and non-BOS patients (58.3±3.9 vs. 60.3±2.0 years, p=0.67). Males accounted for 61.5% of all patients. Chronic obstructive lung disease was the most common indication for lung transplantation (41.0%). Median FEV1 was lower among BOS patients compared to non-BOS patients (1.08 vs. 2.18 L/sec, p=0.001). Importantly, circulating fibrocyte number was increased in BOS patients compared to non-BOS patients (8.91×105 vs. 2.96×105 cells/ml, p=0.03) by flow cytometry and incrementally increased with advancing BOS stage (P=0.02). Conclusions Increased circulating fibrocyte levels correlate with the development of bronchiolitis obliterans syndrome following lung transplantation. Increasing fibrocyte levels positively correlates with advancing BOS stage. Quantification of circulating fibrocytes could serve as a novel biomarker and possible therapeutic target for BOS development in lung transplant patients.

Objectives Adenosine is produced in response to ischemia or inflammation and protects tissues from injury. There are four adenosine receptors, which play a critical role in the physiological negative-feedback mechanism for limitation and termination of tissue-specific and systemic inflammatory responses. Accumulating evidence has focused on the anti-inflammatory and immunosuppressive role of the adenosine 2A receptor (A2AR), and we have previously reported on its’ role in the development of bronchiolitis obliterans (BO) following lung transplantation. However, few studies have reported on the role of the adenosine 2B receptor (A2BR) in BO. Data suggests that the A2BR has pro-inflammatory and profibrotic roles. We hypothesized that adenosine signaling through the A2BR is involved in the development of BO. Methods A murine heterotopic tracheal model across a total alloantigeneic mismatch was used to study A2BR signaling in BO. Tracheal transplants consisted of Balb/c donor tracheas transplanted into wild-type or A2BR knockout C57BL/6 recipients. Transplanted tracheas were removed 3, 7, 12, and 21 days after transplantation. The luminal obliteration was evaluated through hematoxylin and eosin staining and the cellular infiltration (macrophage, neutrophil, CD3+ and Foxp3+ regulatory T cell) was detected by immunohistochemical staining. Results In comparison to allografts in wild type recipients, tracheas transplanted into A2BR knockout mice displayed less BO development on day 21. A2BR knockout mice had an increase in CD3+ T cells and CD4+/CD25+/Foxp3+ regulatory T cells when compared to wild type on day 7. By day 12, more CD3+ T cells were present in the wild-type trachea compared to the A2BR KO, but the percentage of CD4+/CD25+/Foxp3+ regulatory T cells remained higher in the tracheas of A2BR KO mice. Conclusions A2BR stimulation may promote the development of BO via inhibiting CD4+/CD25+/Foxp3+ regulatory T cell infiltration.