Abstract 2278: Isoform Specific PKC Responses Modulate ET-1 and Hypoxia Reperfusion Induced Injury

Abstract

Background: Hypoxia and reperfusion (H/R) injury is a predominant cause of allograft failure following heart transplantation. Several investigators have shown that endothelin-1 (ET) reduces myocardial function and survival via modulation of protein kinase C (PKC). We examined the role of ET and PKC modulation on human saphenous vein endothelial cells (HSVEC). Methods: HSVEC were submitted to 24 hours of hypoxia (PO 2 <0.1%) and 24 hour of reperfusion (PO 2 =21%). Normoxic controls were incubated at PO 2 =21%. Cells were incubated with either ET:100nM, BOS (ET antagonist):10μM, ET+BOS or PBS during H/R. Cells were also treated with PKCδ, PKCλ and PKCϵ inhibitors. Caspase activity was measured. Free radical (ROS) and NO production were measured. Protein expression and translocation of PKC isoforms and eNOS were assessed using Western Blot analysis. Results: Apoptosis (35% increase in caspase 3,8,9 activity) was observed following H/R compared to normoxia. BOS therapy prevented apoptosis while ET exposure increased caspase activity by 75%, p<0.01. H/R resulted in PKCδ and ϵ translocation while reducing PKCλ activity. BOS exposure prevented H/R induced PKCδ and ϵ activation and PKCλ inhibition while incubation with ET enhanced H/R induced PKC effects. PKCδ and ϵ inhibition reduced caspase activity after H/R and abrogated ET induced apoptosis, p<0.01. PKCλ inhibition enhanced apoptosis. NO production was impaired following H/R, p<0.05. BOS therapy restored NO homeostasis while ET exposure worsened NO production compared to H/R alone, p<0.05. PKCδ and ϵ inhibition improved NO production while PKCλ inhibition worsened NO release. H/R driven ROS production was inhibited by BOS and enhanced by ET exposure, p<0.01. PKCλ inhibition raised ROS production while PKCδ and ϵ inhibition reduced ROS release, p<0.01. eNOS protein expression was reduced by H/R while BOS treatment prevented downregulation. Conclusions: Our findings revealed that ET plays a crucial role in cellular injury following H/R via specific PKC isoforms. ET via PKC modulation leads to endothelial dysfunction and cell death. Therefore, targeted isoform specific PKC modulation as well as BOS may prove to be optimal strategy to prevent endothelial injury during allograft storage and transplantation.