Towards donor lung recovery—gene expression changes during ex vivo lung perfusion of human lungs

Abstract

We and others have demonstrated that acellular normothermic ex vivo lung perfusion of high-risk donor lungs can result in posttransplant outcomes equivalent to that of contemporaneous lung transplantation using standard donor lungs. However, the mechanism of this effect remains unclear. Given the restoration of cellular metabolic activity during normothermic perfusion, one possibility is that of lung healing via natural innate recovery mechanisms. We explored this by examining the gene expression changes occurring in human lungs during ex vivo lung perfusion. Human lungs clinically rejected for transplantation were perfused for 12 hours of EVLP with biopsies taken at the start, at 1 hour, at 3 hours, and then every 3 hours thereafter to 12 hours. Temporal changes were identified in 2585 genes using the Short Time-series Expression Miner and used for pathway analysis. Despite increases in endothelial markers of inflammation, circulating leukocyte cell-specific gene expression fell over 12 hours of ex vivo lung perfusion (EVLP), suggesting an interrupted inflammation response secondary to washout of circulating leukocytes. Analysis of these gene changes suggests lung recovery follows specific stages: cellular death, cellular preservation, cellular reorganization, and cellular invasion. EVLP may improve posttransplant lung function by washout of leukocytes and facilitating innate mechanisms of repair.

Influence of ex vivo perfusion on the biomolecular profile of rat lungs

“…Occurrence of a substantial inflammatory response associated with EVLP at signaling, transcription, and protein synthesis levels, despite careful avoidance of any injurious stimulus assured by the model, is major evidence in this research. The presence of EVLP-induced inflammation is consistent with previous data that documented the enhanced production of cytokines and chemokines in perfusates (14,(28)(29)(30) and lung tissue (31)(32)(33)(34). The current study confirms and expands these results, demonstrating a progressive increase in cytokines, chemokines, and adhesion molecules in the perfusion fluid associated with their enhanced transcription in lung tissue.…”

Treatment with 3-aminobenzamide during ex vivo lung perfusion of damaged rat lungs reduces graft injury and dysfunction after transplantation

“…We found that EVLP reduced the amount of infiltrating PMNs after LTx in comparison to lung grafts not exposed to EVLP, despite an increase in the expression of various adhesion molecules (including P and E‐selectin, as well as VCAM‐1). This suggests that EVLP primarily reduced the pool of PMNs from the donor lung, in line with data from previous investigators, which could reflect the mechanical removal of marginated PMNs during EVLP . It seems intriguing at first glance that, despite an increased expression of inflammatory mediators, EVLP‐treated lungs displayed such reduction of inflammatory cell infiltration and improved histological status after LTx.…”

“…It has been indeed frequently reported that EVLP upregulates the expression of many inflammatory genes, due to the activation of pro‐inflammatory signaling pathways, including NF‐κB, STAT‐3, and MAP kinases, following ex vivo reperfusion of the graft at normothermia, that is, at full metabolic activity. This apparent paradox has been recently worked out by Yeung et al, who showed that EVLP increased endothelial markers of inflammation, but at the same time reduced the expression of leukocyte‐specific inflammatory genes, due to the washout of circulating leukocytes during EVLP . It is therefore plausible that the improved histological status and reduced PMN infiltration after EVLP noted in our study resulted from such interruption of leukocyte‐dependent inflammatory response.…”

“…This apparent paradox has been recently worked out by Yeung et al, who showed that EVLP increased endothelial markers of inflammation, but at the same time reduced the expression of leukocyte-specific inflammatory genes, due to the washout of circulating leukocytes during EVLP. 48 It is therefore plausible that the improved histological status and reduced PMN infiltration after EVLP noted in our F I G U R E 7 mRNA expression levels of adhesion molecules after LTx. Lung expression levels of the mRNA of (A) P-selectin (SelP), (B) E-selectin (SelE), (C) VCAM-1, and (D) ICAM-1 after LTx in the 3 groups of animals (n = 9-11 rats/ group).…”

“…This suggests that EVLP primarily reduced the pool of PMNs from the donor lung, in line with data from previous investigators, 47 which could reflect the mechanical removal of marginated PMNs during EVLP. 48 It seems intriguing at first glance that, despite an increased expression of inflammatory mediators, EVLP-treated lungs displayed such reduction of inflammatory cell infiltration and improved histological status after LTx. It has been indeed frequently reported that EVLP upregulates the expression of many inflammatory genes, due to the activation of pro-inflammatory signaling pathways, including NF-κB, STAT-3, and MAP kinases, 9,35 following ex vivo reperfusion of the graft at normothermia, that is, at full metabolic activity.…”