Abstract 1789: Limiting Endothelial Cell Hypoxic Injury: The Role Of Isoform Specific PKC Modulation
Background: Hypoxia and reperfusion (H/R) injury is a major determinate of mortality after cardiac transplantation. Several studies have shown that endothelin-1 (ET) and certain protein kinase C (PKC) isoforms reduce myocardial function. We examined the effect of ET and PKC modulation on H/R induced endothelial dysfunction and apoptosis. We also investigated the ability of the ET antagonist, Bosentan (BOS), to offer endothelial cell protection during H/R.
Methods: Human saphenous vein endothelial cells were submitted to 24 hours of hypoxia (PO2 <0.1%) and 24 hours of reperfusion (PO2 = 21%). Normoxic controls were incubated at PO2 = 21%. Cells were also incubated with either ET: 100nM, BOS: 10μM, ET+BOS or PBS (control) during hypoxia. Cells were also treated with PKCδ, PKCλ and PKCε inhibitors. Apoptosis was determined by assessing caspase activity. NO production was determined by measuring nitrate/nitrite levels in culture medium. Free radical (ROS) production was also measured.
Results: Induction of apoptosis was observed following H/R (35% increase compared to normoxia) and enhanced by ET (75% increase) exposure while BOS treatment prevented apoptosis (p < 0.01). NO production was impaired (40% decrease compared to normoxia) following H/R (p < 0.05) and was further reduced by ET-1 exposure (60% decrease). ROS production was increased after H/R and enhanced by ET (p < 0.001). BOS therapy was able to improve NO homeostasis and abolished both ET and H/R induced ROS production (p < 0.001). Treatment with PKCδ and PKCϵ inhibitors reduced apoptosis and abrogated the effect of ET on cell death (p < 0.001), NO production and ROS release. In contrast, PKCλ inhibition enhanced apoptosis, worsened NO release and increased ROS production.
Conclusions: Our study revealed that ET exposure worsens H/R injury and that specific PKC isoforms play crucial roles in cellular injury following H/R. ET and PKCλ inhibition increased ROS production and impaired NO homeostasis leading to apoptosis, while BOS treatment, PKCδ and PKCϵ inhibition abrogates both H/R and ET induced endothelial injury. PKC modulation as well as BOS may prove to be NOVEL therapies to prevent endothelial injury during allograft storage and following cardiac transplantation.