Myristoylated Protein Kinase C Beta II Peptide Inhibitor Attenuates Hypoxia- Reoxygenation Injury in Human Umbilical Vein Endothelial Cells

Endothelial dysfunction as a result of ischemia/reperfusion (I/R) injury contributes to local organ damage in heart attack patients. In vascular cells, NADPH oxidase (NOX) and the mitochondrial electron transport chain are initiating sources of reactive oxygen species (ROS) during I/R injury. Protein kinase C beta II (PKCβII) is an attractive therapeutic target due to its phosphorylation of p66Shc to enhance mitochondrial-derived ROS production and p47 phox to promote ROS release from NOX. In previous studies, a cell-permeable myristoylated PKCβII peptide inhibitor (N-myr-SLNPEWNET; myr-PKCβII-) has been shown to improve post-reperfusion cardiac function and reduce infarct size in rat myocardial I/R injury. The decrease in myocardial I/R injury with myr-PKCβII- may in part be attributed to improved vascular endothelial function. Due to myr-PKCβII- peptide sequence being highly conserved among mammalian species, we hypothesize that myr-PKCβII- will confer protection by directly inhibiting ROS production from NOX and mitochondria in human umbilical endothelial cells (HUVECs) subjected to hypoxia/reoxygenation (H/R) mediated injury. HUVECs, cultured in gelatin-coated 96-well plates, were subjected to 24h hypoxia and 24h reoxygenation in a Billups-Rothenburg chamber with 1% O , 5% CO , and balance nitrogen. Myr-PKCβII- (20 μM) was administered at the beginning of the 24h reoxygenation period. Cell viability was assessed using tetrazolium-salt (WST-8) colorimetric assay with a microplate reader (450 nm) and normalized against the normoxia control group. Data were analyzed using Student- Newman-Keuls post-hoc analysis. At the 24h reoxygenation period, cell viability (%) was significantly reduced to 78±2% (n=5; p