Ex Vivo Perfusion With Adenosine A2A Receptor Agonist Enhances Rehabilitation of Murine Donor Lungs After Circulatory Death

Abstract

Background: Ex vivo lung perfusion (EVLP) enables assessment and rehabilitation of marginal donor lungs before 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 minutes warm ischemia, and lungs were flushed with Perfadex and underwent cold static preservation (CSP, 60 minutes). Three groups were studied: no EVLP (CSP), EVLP with Steen solution for 60 minutes (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: Ex vivo lung perfusion 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 1594 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.

Conclusions: Ex vivo lung perfusion modulates proinflammatory 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.

Figure 1. Diagram of the murine EVLP system

An isolated, buffer-perfused mouse lung system (Hugo Sachs Elektronik, March-Huggstetten, Germany) was utilized. The right ventricular (RV) cannula is passed through the pulmonary valve into the pulmonary artery. A left atrial (LA) cannula drains perfusate into waste container. Lungs are perfused at a constant flow of 60 μl/g body wt/min. Lungs are ventilated with room air at 100 breaths/min at a tidal volume of 7 μl/g body weight with a positive end expiratory pressure of 2 cm H₂O using a positive pressure ventilator. The perfusate and lungs are maintained at 37°C by use of a circulating water bath as shown. Air bubbles are removed from the perfusate via a bubble trap as shown. Several differential pressure transducers (DPT) and a pneumotachometer are used to measure arterial pressure, tracheal pressure and respiratory flow via the PULMODYN data acquisition system (Hugo Sachs Elektronik).

Figure 2. EVLP-directed delivery of ATL1223 improves function and reduces edema in DCD lungs

Compared to lungs after cold static preservation (CSP), EVLP significantly increased pulmonary compliance and reduced pulmonary artery pressure and wet/dry weight (edema). ATL1223 treatment during EVLP provided further, significantly improved lung function and reduced edema. One-way ANOVA with post-hoc Tukey's multiple comparison test was performed to compare groups. Results are presented as mean ± SEM. *p<0.05 versus CSP; ^#p<0.05 versus EVLP; n=10-12/group (pulmonary compliance and pulmonary artery pressure); n=8-9/group (wet/dry weight).

Figure 3. EVLP-directed delivery of ATL1223 reduces proinflammatory cytokine production in DCD lungs

Compared to lungs after cold static preservation (CSP), expression of CXCL1, CCL2 and TNF-α were slightly, but not significantly, reduced by EVLP. ATL1223 treatment during EVLP resulted in significant reductions in expression of CXCL1, CCL2 and TNFα. One-way ANOVA with post-hoc Tukey's multiple comparison test was performed to compare groups. Results are presented as mean ± SEM. *p<0.05 versus CSP; ^#p<0.05 versus EVLP; n=4-7/group.

Figure 4. EVLP-directed delivery of ATL1223 depletes pulmonary neutrophils

(A) Representative lung sections (20× magnification) showing immunostaining for neutrophils (red staining). (B) Quantitation of the number of neutrophils per high-powered field (HPF) in immunostained lung sections. EVLP with ATL1223 resulted in significantly fewer neutrophils/HPF versus cold static preservation (CSP) and EVLP alone. One-way ANOVA with post-hoc Tukey's multiple comparison test was performed to compare groups. Results are presented as mean ± SEM. *p<0.05 versus all; n=5-6/group.

Figure 5. Differentially expressed genes among study groups as a function of EVLP-mediated rehabilitation strategy for DCD lungs

(A) Venn diagram showing numbers of differentially expressed genes in lungs from EVLP and EVLP+ATL1223 groups when compared to the cold static preservation (CSP) control group (FDR 1%). (B) Volcano plots depicting the gene expression profile of pairwise comparisons of indicated groups. Each dot represents a unique, differentially expressed probeset. Significant probesets [p-value ≤ 0.001 or –log10 (p-value) ≥ 3] are illustrated by red dots.

Figure 6. Differentially-expressed canonical pathways affected by EVLP

(A) Ten canonical pathways focused on inflammatory and innate immune responses (selected from the significant canonical pathways in SDC Table 1, p<0.05) are shown along the x-axis that are significantly different between EVLP versus CSP. (B) Ten canonical pathways focused on inflammatory and innate immune responses (selected from the significant canonical pathways in SDC Table 2, p<0.05) are shown along the x-axis that are significantly different between EVLP+ATL223 versus CSP. (C) The canonical pathways resulting from the evaluation of genes uniquely differentially expressed in EVLP+ALT1223 vs. CSP (801 genes, FDR 1%). The total number of genes associated with each pathway is listed above each bar. Green and red bars indicate the percentage (y axis) of those genes down- or up-regulated, respectively, as calculated by (# genes in a given pathway that meet cutoff criteria)/(total # genes that comprise that pathway). Pathways are presented left-to-right from highest to lowest significance (specific p values are provided in SDC Tables 1 and 2).