The effects of Pyrococcus furiosus superoxide reductase and protein kinase C peptide modulators on hypoxia/reoxygenation–induced cell death in human umbilical vein endothelial cells

Date of Award


Degree Type


Degree Name

Master of Science in Biomedical Sciences

First Advisor

Francis E Jenney, Jr, PhD

Second Advisor

Lindon H Young, PhD

Third Advisor

Kimberly Baker, PhD

Fourth Advisor



Endothelial dysfunction contributes to myocardial infarction and multiple organ failure following hemorrhagic shock. Reduced nitric oxide (NO) bioavailability is facilitated by high levels of reactive oxygen species (ROS), predominantly generated from uncoupled endothelial nitric oxide synthase (eNOS), NADPH oxidase (Nox) and mitochondria. Cell survival depends on the capacity to neutralize excessive ROS, such as superoxide, with antioxidant defense (e.g., superoxide dismutase; SOD). We aim to test two therapeutic approaches: antioxidant scavenging and selective inhibition of protein kinase C (PKC) pathways. Recombinant superoxide reductase from Pyrococcus furiosus (PfSOR), an anaerobic hyperthermophile, exhibits functional scavenging of superoxide at mesophilic temperatures and should retain metabolic activity in human cells. Due to having stronger electrostatic attraction for superoxide and requiring an electron donor, PfSOR should demonstrate superior dose-dependent protection via more efficient ROS scavenging compared to SOD. PKC epsilon and beta-II (PKCε and PKCβII) phosphorylate various protein targets that mediate ROS production. PKCε is known to phosphorylate eNOS at serine-1177 when it’s uncoupled during oxidative stress, resulting in superoxide instead of NO production. PKCβII phosphorylates p66Shc to stimulate mitochondrial-derived ROS and p47 phox to promote ROS release from Nox. We hypothesized that cell-permeable myristoylated peptide (myr-PKCε or myr-PKCβII) activator and inhibitor, previously shown to modulate NO release in various in vivo and ex vivo animal studies, will demonstrate similar translatable effects in human vascular cells. PKCε or PKCβII inhibition during oxidative stress should confer protection by preventing ROS production from their respective molecular targets. Cultured human umbilical vein endothelial cells (HUVECs) were subjected to variable durations of hypoxia/reoxygenation (H/R) and starvation media. Cell viability was assessed via colorimetric assay. Real-time NO release was measured with a calibrated electrode in normoxic conditions. PfSOR (1 U/ml and 10 U/ml) demonstrated superior HUVEC recovery against 2.5-hr H/R and starvation media-induced injury compared to SOD (p<0.05). Dose-dependent effects were inconclusive. Myr-PKCε inhibitor and activator demonstrated opposing effects on NO release in normoxia (p<0.05), but were equally protective of HUVECs subjected to 24-hr H/R (p<0.05). Myr-PKCβII inhibitor significantly increased HUVEC viability after 24-hr H/R (p<0.01), but had no significant effect on NO release.

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