Abstract 1572: Hypoxia And Reperfusion: Role Of Protein kinase C And eNOS In Free Radical Generation
Background: The main determinate of morbidity and mortality after cardiac transplantation remains hypoxia and reperfusion (H/R) injury. Reactive oxygen species (ROS) and protein kinase C (PKC) have been shown to play a key role in allograft dysfunction. We examined the effect of PKC modulation on H/R induced ROS production. We also aimed to determine whether ROS are generated through eNOS.
Methods: Human saphenous vein endothelial cells (HSVEC) were submitted to 24 hours of hypoxia (PO2 <0.1%) and 1 or 24 hours of reperfusion. Normoxic controls were incubated at PO2=21%. HSVEC were incubated with either PKCδ, PKCλ or PKCϵ inhibitors during hypoxia or reperfusion. Cell viability was quantified by XTT labeling. ROS production was measured. Antioxidant therapy was achieved by incubation with sepiapterin (Sep) and L-NAME. Protein expression and translocation (membrane cytosolic ratios (M/C)) of PKC isoforms and eNOS were assessed using Western Blot analysis.
Results: HSVEC exposure to hypoxia resulted in PKCδ,ϵ activation while reducing PKCλ activity. Following reperfusion PKCδ activity was further increased compared to normoxia while PKCϵ,λ translocation demonstrated a gradual return towards normoxic levels p<0.01. ROS generation was observed following hypoxia with a further reperfusion time dependent increase in ROS p<0.01. PKCλ inhibition caused a rise in ROS production following H/R p<0.001. PKCϵ inhibition resulted in ROS reduction after H/R while PKCδ inhibition lowered ROS formation only during reperfusion p<0.01. Sep and L-NAME decreased ROS production following H/R p<0.01. H/R downregulated eNOS protein expression by 30%±3% and increased the eNOS M/C ratio by 21%±4% p<0.01. Cell viability was reduced by 26±2% following H/R. PKCδ,ϵ inhibition improved cell viability after H/R while PKCλ inhibition enhanced cellular injury. Therapy with Sep and L-NAME limited the effects of H/R on cell viability.
Conclusions: Our study demonstrated that H/R led to ROS production via specific PKC isoform and eNOS dependent pathways. H/R generated ROS through eNOS activation and uncoupling as demonstrated by inhibition with Sep and L-NAME. PKC as well as eNOS modulation may prove to be novel therapies to limit endothelial injury after heart transplantation.