Ex-vivo lung perfusion : a platform for assessment and reconditioning of human donor lungs

Abstract

Background: Although lung transplantation is regarded as an effective therapy for patients with end-stage lung disease, the demand for donor organs exceeds the overall donor organ supply. Ex-vivo lung perfusion (EVLP) has emerged as a technique to objectively assess and recondition donor lungs that have been deemed unsuitable for transplantation. The cellular and molecular events occurring during EVLP have not been well characterised, and how these processes differ between lungs that are deemed useable compared with those that are discarded is unknown. The aims of this project were three-fold. Firstly, to identify potential predictive biomarkers during EVLP that could distinguish donor lungs that can be reconditioned and predict clinical outcomes. Secondly, to investigate the differences in the inflammatory profiles of donor lungs following donation after brain death (DBD) versus donation after circulatory death (DCD). Finally, to assess EVLP as a therapeutic platform by examining the physiological and immunological changes in donor lungs undergoing EVLP supplemented with the known vasodilatory agent and phosphodiesterase-5 inhibitor, sildenafil. Methods: In retrospective studies of the DEVELOP-UK cohort and prospective studies using a research EVLP model, markers of inflammation, tissue injury and angiogenesis in perfusate, bronchoalveolar lavage (BAL), and lung tissue were examined. Laboratory techniques including ELISA, MSD and immunohistochemistry were used. Results: RNA-sequencing demonstrated an unbiased profile of the transcriptome of human donor lungs prior to, and following EVLP, comparing changes in gene expression between lungs deemed suitable for transplantation, and those discarded on the basis of standard physiological parameters. During EVLP, we observed an increase in a number of immune pathway genes. In the DEVELOP-UK sample cohort significant differences were detected in the inflammatory profiles prior to commencing EVLP between DBD and DCD donor lungs. DBD donor lungs were reported to have significantly higher concentrations of lung tissue C-reactive protein (CRP), IL-12p70, IL-4 and IL-6 compared with DCD iv donor lungs. Conversely, the levels of acute phase cytokines, IL-1β and IL-1α, were significantly higher in the perfusate from DCD lungs during EVLP. EVLP was shown to be an effective and reproducible therapeutic platform which could allow for the testing of agents with the potential for improving the function of lungs turned down for transplantation. Sildenafil was hypothesised to improve pulmonary physiology during EVLP and lead to improved oxygenation and pulmonary vascular homeostasis, however this did not occur. Sildenafil did not have any significant effects on the physiology and immune profile of human donor lungs during EVLP. Discussion: In order for EVLP is to be effectively utilised in clinical practice as a platform for the assessment, reconditioning and potential treatment of donor lungs, an increased understanding of the cellular and molecular events occurring is required. In this study, we aimed to address this knowledge gap by performing an unbiased analysis of the lung transcriptome prior to and following EVLP. RNA-sequencing can be used to study the pro-inflammatory changes within the lung transcriptome during EVLP, with subsequent correlation to potential protein biomarkers in perfusate. We reported significant differences in the inflammatory profiles of DBD versus DCD lungs. Finally, administration of sildenafil into the vascular compartment of the EVLP circuit failed to produce the hypothesised beneficial effects in human donor lung physiology and function.