Modulation of endothelial permeability during ex vivo lung perfusion

Abstract

Introduction: Ex vivo lung perfusion (EVLP) enables donor-lung assessment, but extended periods of perfusion cause lung injury and inflammation. Development of novel perfusate formulations could enable longer and advanced assessment strategies. EVLP also offers the opportunity for therapeutic administration pre-transplantation. S1P-receptor-1 (S1PR1) activation reduces endothelial permeability. It has been shown to reduce oedema formation during ischaemia reperfusion injury (IRI) and has the potential to improve endothelial integrity of marginal donor-lungs. Our aim was to validate a novel perfusate solution and then assess the therapeutic effects of S1PR1 agonist CYM5442 administration on vascular permeability during EVLP of human lungs declined for transplantation. Methods: RNA-sequencing and gene-set enrichment analysis (GSEA) was carried out on porcine lung biopsies post-EVLP to identify IRI-associated gene sets and validate novel perfusate modified Steen. In vitro, trans-endothelial electrical resistance (TEER) of pulmonary endothelial cell monolayers was assessed following treatment with models of IRI (SIN-1 and H2O2), CYM5442 or co-treatment. CYM5442 was tested ex vivo; human lung pairs were split, before administration of CYM5442 or vehicle to each single lung and 6 hours perfusion. Then permeability to Evan’s blue dye and wet/dry ratio was assessed. Results: Modified Steen showed significant negative enrichment of inflammatory gene-sets compared with Steen, but cold static storage (CSS) and EVLP was still associated with significant positive enrichment. In vitro, SIN-1 significantly decreased TEER of human pulmonary endothelial monolayers (p=0.0114), which was rescued by co-treatment with CYM5442 (p=0.0179). During EVLP, CYM5442 significantly reduced bronchoalveolar lavage Evan’s blue concentration (p=0.0070) and wet/dry ratio (p=0.0258; N=2). Conclusion: Despite use of a novel perfusate with anti-inflammatory properties, CSS and EVLP caused activation of inflammatory pathways, reinforcing the need for therapeutic intervention. In vitro and ex vivo permeability testing identified the potential of CYM5442 to improve barrier integrity. Targeting S1PR1 during EVLP could improve endothelial integrity of unsuitable donor-lungs prior to transplantation.