Myristoylated Protein Kinase C Beta II Peptide Inhibitor Improves Cell Survival in Cultured Human Umbilical Vein Endothelial Cells Subjected to Hypoxia/Reoxygenation
Authors: Lloyd Michael , Dean Tameka , Heller Gordon , Verwoert Alexis , Martorana Rose , Andre Claudine , Thompson Chantel , Major Ahsal , Jenney Francis , Chen Qian , Barsotti Robert , Young Lindon 
Published: April 2020

Endothelial dysfunction as a result of ischemia/reperfusion (I/R) injury is known to contribute to damage in myocardial infarction and organ transplant patients. In vascular cells, NADPH oxidase (NOX) and the mitochondrial electron transport chain are primary sources of reactive oxygen species (ROS) during I/R injury resulting in reduced nitric oxide bioavailability. Protein kinase C beta II (PKCβII) is an attractive therapeutic target due to its regulation of these downstream mediators of ROS production. PKCβII phosphorylates p66Shc to enhance mitochondrial‐derived ROS production and p47 phox to promote ROS release from NOX. We have previously shown that myristoylated PKCβII peptide inhibitor (N‐myr‐SLNPEWNET; myr‐PKCβII‐) improved post‐reperfusion cardiac function and reduced infarct size in rat myocardial I/R injury compared to non‐treated controls. The decrease in myocardial I/R injury with myr‐PKCβII‐treatment may be attributed to improved vascular endothelial function. The myristoylated peptide sequence (cargo sequence) targets a highly conserved peptide sequence among mammalian species and inhibits PKCβII translocation to protein substrates (e.g. NOX and p66Shc) after second messenger activation. We hypothesize that myr‐PKCβII‐ will confer protection by directly inhibiting ROS production from NOX and mitochondria in human umbilical vein endothelial cells (HUVECs) subjected to hypoxia/reoxygenation (H/R) injury. HUVECs were cultured in gelatin‐coated 96‐well plates and subjected to 24h hypoxia and 24h reoxygenation in Billups‐Rothenburg hypoxia chamber with 1% O2, 5% CO2, and balanced nitrogen. Myr‐PKCβII‐ (20 mM) treatment 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 end of the 24h reoxygenation period, cell viability (%) was significantly reduced to 78±2% (p<0.05; n=5) in the non‐treated H/R group compared to normoxia controls (n=5). Myr‐PKCβII‐significantly improved HUVEC survival (95±4%; p<0.01; n=5) compared to non‐treated H/R controls and was not significantly changed compared to normoxia controls. The data suggests that PKCβII inhibition promotes cell survival subjected to H/R possibly due to directly attenuating NOX and mitochondrial derived ROS. Further studies are needed to control for the myristoylated acid conjugation of the peptide cargo.

Support or Funding Information

This research was supported by the Division of Research, Department of Biomedical Sciences, and the Center for Chronic Disorders of Aging at the Philadelphia College of Osteopathic Medicine. Current license is supported by Young Therapeutics, LLC.