The role of Sphigosine-1-Phosphate in vascular permeability

Abstract

There is a shortfall of kidneys available for transplant, therefore “marginal” kidneys are increasingly used. These are particularly susceptible to ischaemia reperfusion injury (IRI) which can lead to loss of graft function. Ex vivo normothermic kidney perfusion (EVNKP) can be used for organ quality assessment and drug delivery. We hypothesised that delivery of a sphingosine-1-phosphate receptor (S1PR) agonist (CYM5442) could reduce endothelial leak in these “marginal” organs via activation of S1PR1, as S1P activation of S1PR2/3 increases endothelial permeability. Changes to S1PR expression were examined in Human Microvascular Endothelial Cells (HMEC1) and Human Umbilical Vein Endothelial Cells (HUVEC) using mimics of ischaemia and reperfusion. Hypoxia reduced S1PR1 expression and increased S1PR3 expression. Vascular Endothelial Growth Factor (VEGF) caused increases in S1PR1 expression in HMEC-1 and halved expression in HUVEC. Hydrogen peroxide treatment induced reactive oxygen species release and decreased S1PR1-4 gene expression. Treatment of HMEC-1 with CYM5442 increased Intercellular Adhesion Molecule (ICAM-1) and Vascular Adhesion Molecule (VCAM-1) expression and caused S1PR1 internalisation within 15- 30minutes of treatment. CYM5442 treatment caused decreases in permeability to Evans Blue dye, decreases in neutrophil chemotaxis and a reduction in vascular endothelial-cadherin (VE-cadherin) phosphorylation at Y658 and Y685. An S1PR3 agonist, CYM5541 caused a trend towards increased permeability and neutrophil chemotaxis. S1P concentrations were compared in human serum and perfusate used in EVNKP; perfusate contained significantly less S1P than serum. To test the barrier enhancing effects of CYM5442 ex vivo, 0.5-50mg Evans Blue dye was administered to a porcine EVNKP circuit and could be distinguished from untreated tissue at 25mg. Murine models of IRI were treated with CYM5442 or vehicle; CYM5442 treated mice displayed reduced permeability to Evans Blue dye. This study therefore suggests CYM5442 could reduce endothelial leak during organ transplantation, which could be evaluated using Evans Blue dye.