P67 Slow release H2S donors protect human microvascular endothelial cells from oxidative stress induced cell death: Markedly increased potency by mitochondria-targeting

Abstract

Introduction Microvascular endothelial dysfunction (MED) is observed in diabetic and hypertensive patients, due at least in part to increased vascular oxidative stress and mitochondrial dysfunction. Lower levels of H 2 S were observed in diabetic subjects compared to age and BMI-matched controls and H 2 S levels negatively correlated with MED in vivo. This suggests that reduced vascular synthesis of H 2 S contributes to MED and that increasing H 2 S bioavailability may protect the vasculature from oxidative stress-induced injury. Methods Human microvascular endothelial cells (HMEC) were exposed to slow release H 2 S donors (SRHD; 100 μM) GYY4137, AP67, AP72 and two mitochondria-targeted H 2 S donors AP39 and AP123 (0.1 μM), before and after the induction of oxidative stress using SIN-1 (100 μM), H 2 O 2 (100 μM) and 4-HNE (10 μM). Cell viability (alamarBlue), intracellular oxidant generation (ROS; DHE, H 2 DCFDA, mitosoxRed) and mitochondrial membrane potential (MMP; TMRM) were determined. Western blotting and fluorescence assays were used for Akt, Erk1/2, caspases 3, 7 and 9 expression and activity. Phosphatidylserine externalisation and plasma membrane integrity were assessed by flow cytometry. Oxidative stress significantly reduced cell viability, MMP and increased ROS generation (ANOVA, p p p Conclusion These data suggest SHRD can inhibit and/or reverse oxidative stress-mediated cellular injury and highlight strategies which increase vascular H 2 S bioavailability, and in particular target mitochondria, represent a new therapeutic opportunity to limit MED.