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 2 , 5% CO 2 , 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<0.05) in the non-treated H/R group compared to normoxia controls (n=5). Myr-PKCβII- significantly improved HUVEC survival (95±4%; n=5) compared to non-treated H/R controls (n=5; p<0.01) which were not significantly different from normoxia controls. The data suggest that PKCβII inhibition promotes cell survival possibly due to directly attenuating NOX and mitochondrial derived ROS in cells subjected to H/R conditions. Further studies are needed to determine cell survival potential under more severe H/R mediated injury.

Previously, naltrindole (NTI; selective delta opioid receptor antagonist) was shown to improve post-reperfused cardiac function and reduced infarct size when given prior to ischemia (I)/ reperfusion (R) in ex-vivo rat hearts. Conversely, naloxone (NX, broad-spectrum opioid antagonist) and nor-binaltrophine (BNI, selective kappa receptor antagonist) were similar to control hearts. In this study, the effects of NTI derivatives naltriben (NTB, delta receptor antagonist) and guanidonaltrindole (GNTI, kappa receptor antagonist) were compared to NTI, BNI, and NX. Isolated hearts from male SD rats (300g) were subjected to global I(30min)/R(45min). Treatments were given 5 min before I (preconditioning) and during the first 5 min of R. Left ventricular (LV) cardiac function was measured using a pressure transducer. At the end of reperfusion, infarcted heart tissue was compared to total tissue weight. Data were evaluated using ANOVA. As shown in Table 1, NTI, NTB, and GNTI significantly improved post-reperfused cardiac function and reduced infarct size compared to control hearts. NTI and NTB elicited direct effects on cardiac function when given during preconditioning in contrast to all other study groups and were the most robust at reducing infarct size and restoring post reperfusion cardiac function. The negative inotropic effects of NTI and NTB were correlated with a decrease in the rise of ischemic pressure. GNTI also elicited significant improvement in post-reperfused cardiac function and reduction of infarct size compared to BNI which suggests a separate cardioprotective mechanism that this NTI derivative may exert in contrast to kappa opioid receptor inhibition. Results suggest that NTI and derivatives, GNTI and NTB, are cardioprotective against I/R injury resulting in reduced ischemic peak pressure (NTI/NTB) and infarct size. In future studies, we will examine the mechanism of the protective effects of NTI and derivatives in hearts subjected to I/R injury.

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.

Acute kidney injury (AKI) due to ischemia‐reperfusion (I/R) insult involves oxidative stress and inflammation leading to rapid renal decline and remains a significant cause of post‐operative mortality. Myristoylated protein kinase C beta II peptide inhibitor (N‐myr‐SLNPEWNET; myr‐PKCβII‐) is known to attenuate myocardial I/R injury in ex vivo rat hearts. We hypothesized that myr‐PKCβII‐ would attenuate severe renal I/R injury that is characterized by elevated serum creatinine (Cr) and a decrease in glomerular filtration rate (GFR). We predict that treatment with myr‐PKCβII‐ will improve these indices of kidney function compared to a scrambled control peptide (N‐myr‐WNPESLNTE; myr‐PKCβII‐scram). Renal pedicles of anesthetized male C57BL/6J mice (25–30g) were clamped bilaterally for 20 min or 19 min. Five minutes before unclamping, 2.0 mg/kg (20 µM serum) myr‐PKCβII‐ or myr‐PKCβII‐scram were given i.v. into the tail vein. Cr (mg/dL) was measured at baseline, 24h, 72h, and 96h post‐injury. GFR (µl/min) was determined with fluorescein‐isothiocyanate (FITC)‐Sinistrin renal clearance. Data were evaluated by unpaired Student’s t‐test. Following 20‐min renal ischemia (Fig 1.), myr‐PKCβII‐ (n=9) significantly reduced Cr at 24h and 72h post‐injury compared to myr‐PKCβII‐scram control (n=8, p<0.05). However, there were three fatalities prior to 96h. In 19‐min ischemia, there were no fatalities up to 96h post‐injury. However, Cr levels of the myr‐PKCβII‐scram control (n=8) in 19‐min I/R were significantly lower than 20‐min I/R at all time points post‐injury (all p<0.01): 24h (0.54 ± 0.10 vs 1.59 ± 0.06 mg/dL), 72h (0.24 ± 0.06 vs 1.28 ± 0.25 mg/dL), and 96h (0.17 ± 0.03 vs 0.72 ± 0.15 mg/dL). Myr‐PKCβII‐ did not significantly reduce Cr (Fig. 1) nor improve GFR (Fig. 2) following 19‐min I/R. Results suggest that 20‐min renal ischemia was more severe, which was indicated by a 5‐fold increase of peak Cr levels at 72h post‐injury compared to 19‐min ischemia and the unanticipated fatalities of three mice. Myr‐PKCβII‐ attenuated renal injury following 20‐min renal ischemia, but not at the milder 19‐min model in which peak Cr levels were too low to detect therapeutic benefit. The large difference in injury severity between 20‐min and 19‐min renal ischemia emphasizes the temporal relationship between renal function and ischemic duration. Future studies will characterize myr‐PKCβII‐ protection against AKI in 19‐min bilateral renal ischemia based on biomarkers indicating proximal renal tubule damage (e.g. NGAL and Kim‐1). A more optimal ischemic duration between 19 min and 20 min will be investigated for future experiments.

Protein kinase C beta II (PKCβII) activation promotes polymorphonuclear (PMN) superoxide (SO) production by phosphorylating serine and threonine amino acid residues on NADPH oxidase (NOX‐2). In previous studies, cell‐permeable myristic acid conjugated PKCβII inhibitor (myr‐PKCβII‐) significantly attenuated PMN SO release induced by phorbol 12‐myristate 13‐acetate (PMA), a diacylglycerol mimetic. Myr‐PKCβII‐ was determined to be superior to unconjugated peptides and nontreated controls, suggesting enhanced intracellular delivery of cargo. We hypothesize that the simple diffusion of myr‐conjugation combined with the endocytotic mechanism of trans‐activator of transcription (Tat) would optimize the intracellular delivery of PKCβII‐ cargo compared to myr‐conjugation alone. In this study, we tested the concentration‐dependent effects of a dual myr‐Tat conjugated PKCβII‐ (myr‐Tat‐PKCβII‐; N‐myr‐Tat‐CC‐SLNPEWNET) on intracellular delivery compared to myr‐PKCβII‐, scrambled myr‐Tat‐PKCβII‐ (myr‐Tat‐PKCβII‐ scram), unconjugated PKCβII‐, and 0.5% dimethyl sulfoxide (DMSO) vehicle control group. Rat PMNs were incubated for 15 min at 37°C with either unconjugated PKCβII‐ (20μM), myr‐Tat‐PKCβII‐ (2μM, 5μM, 7.5μM, 10μM, and 20μM), or myr‐Tat‐PKCβII‐scram (2μM, 5μM, 7.5μM, 10μM, and 20μM). PMN SO release was calculated by the change in absorbance at 550 nm over 390 sec via ferricytochrome c reduction after PMA stimulation (100nM). The efficacy of intracellular drug delivery was evaluated by the magnitude of PMA‐induced PMN SO release attenuation with the PKCβII‐ cargo. Data were analyzed with ANOVA Fisher’s PLSD post‐hoc analysis. Myr‐Tat‐PKCβII‐ 5μM (n=12, 0.392±0.04), 7.5μM (n=11, 0.397±0.05), 10μM (n=5, 0.211±0.05) and 20μM (n=5, 0.121±0.02) demonstrated a concentration‐dependent increase in intracellular delivery compared to DMSO vehicle control (n=84, 0.496±0.02, all p<0.05). Myr‐PKCβII‐ only significantly increased intracellular delivery at the 20μM concentration (n=27, 0.303±0.02, p<0.05) compared to DMSO vehicle control. Intracellular delivery of myr‐Tat‐PKCβII‐ 2μM (n=10, 0.436±0.06) and all concentrations of myr‐Tat‐PKCβII‐scram were not significantly different from DMSO vehicle controls. Results suggest that myr‐Tat dual conjugation is superior to myr‐conjugation alone at intracellular delivery of cell impermeant cargo. Future studies will investigate the concentration‐dependent effects of PKCβII‐ peptide conjugates on PMA‐induced PKCβII activity and translocation to membrane targets, such as NOX‐2, using immunocytochemistry and western blot analysis.