The Effects of Modulating Endothelial Nitric Oxide Synthase (eNOS) Activity and Coupling in Coronary, Hindlimb, Renal, and Mesenteric Vascular Inflammation Models

Ischemia/reperfusion (I/R) injury is initiated in part by vascular endothelial dysfunction, which is characterized by reduced endothelial-derived nitric oxide (NO) and/or increased oxidative stress, followed by inflammation. When the tetrahydrobiopterin (BH4) to dihydrobiopterin (BH2, oxidized form of BH4) ratio is reduced, eNOS can become uncoupled shifting production of NO to superoxide (SO). Protein kinase C epsilon activator (PKCε+) enhances eNOS activity while PKCε inhibitor (PKCε-) reduces eNOS activity. The effects of PKCε+ or PKCε- combined with BH4 or BH2 were studied in rat myocardial and hindlimb I/R, rat renal lithotripsy, and rat mesenteric inflammation models. Promoting eNOS coupling using PKCε+ with BH4 or inhibiting uncoupled eNOS activity using PKCε- significantly increased blood NO and decreased blood H2O2 levels in reperfused femoral and renal veins, reduced BH2-induced leukocyte-endothelial interactions in mesenteric postcapillary venules, and improved post-reperfused cardiac function associated with reduced leukocyte heart tissue infiltration when compared to controls. In contrast, PKCε+ with BH2 had opposite effects. These results suggest that enhancing coupled eNOS or inhibiting uncoupled eNOS activities can attenuate the I/R-induced vascular endothelial dysfunction, inflammation, and organ damage. This study was supported by NHLBI Grant 2R15HL-76235-02 and the CCDA at PCOM

Protein kinase C beta II (PKC ßII) peptide inhibitor exerts cardioprotective effects in myocardial ischemia/reperfusion injury

During myocardial ischemia/reperfusion (I/R), the generation of reactive oxygen species (ROS) contributes to the post-reperfused cardiac injury and contractile dysfunction. Activation of Protein Kinase C beta II (PKC βII) has been associated with increased ROS release from myocardial I/R tissue, decreased endothelial-derived nitric oxide, and increased infarct size. We tested the hypothesis that using a cell permeable PKC βII peptide inhibitor (PKC βII-) (N-myr-SLNPEWNET, MW=1300 g/mol, 10µM) will attenuate infarct size and improve post-reperfused cardiac function compared to untreated controls in isolated perfused rat hearts subjected to I(30min)/R(45 or 90 min). The 90 min reperfusion group (n=9) showed significantly less recovery to the initial baselines in left ventricular developed pressure (LVDP) (38±6%) and maximal rate of LVDP (+dP/dtmax ) (28±4%), both p˂0.01. The 45 min reperfusion group (n=9) also showed significantly compromised LVDP (46±6%) and +dP/dtmax (35±4%) compared to initial baseline but to a lesser extent than the 90 min group. Conversely, PKC βII- treated hearts significantlyimproved cardiac function compared to controls (all p\u3c0.05). Similarly, 90 min reperfusion (n=7) showed a reduced recovery in LVDP (57±7%) and +dP/dtmax (48±5%) compared to 45 min reperfusion (LVDP: 70±6%; +dP/dtmax: 55±6%; n=7). Furthermore, PKC βII- treated hearts showed significant reduction in infarct size (24±3% and 29±3% for 45 and 90 min reperfusion, respectively) compared to controls (43±2% and 46±3% for 45 and 90 min reperfusion, respectively; [p˂0.01]). The results suggest that PKC βII- is effective in improving cardiac function and reducing infarct size and aids in clinical myocardial infarction/organ transplantation patient recovery

Protein Kinase C (PKC) Delta Activator Attenuates NG-Nitro-L-Arginine-Methyl-Ester (L-NAME) Induced Leukocyte-Endothelial Interactions in Rat Mesenteric Post-Capillary Venules

Vascular endothelial dysfunction has been reported as the initial and critical step leading to inflammation-related diseases. It is characterized by reduced endothelial-derived nitric oxide (NO) and/or increased oxidative stress (i.e., superoxide (SO)), which leads to increased leukocyte-endothelial interactions. Endothelial dysfunction is attenuated by enhancing NO generation via endothelial NO synthase (eNOS) or by reducing SO release via inhibition of NADPH oxidase. PKC δ negatively regulates NADPH oxidase and reduces SO release, which will attenuate quenching of NO. The role of PKC δ on leukocyte endothelial interactions is unclear. This study examined these interactions, which are characterized by leukocyte rolling, adherence, and transmigration in rat mesenteric postcapillary venules via intravital microscopy. We found that superfusion of L-NAME (MW=270, 50μM, n=5) significantly augmented inflammation by inhibiting NO production via eNOS compared to Krebs’ buffer control (p\u3c0.01, n=5). Conversely, PKC δ activator (Myr MRAAEDPM, MW=1130, 10 μM, n=4), a cell-permeable peptide, significantly attenuated L-NAME induced leukocyte-endothelial interactions (p\u3c0.05). The preliminary data suggest that PKC δ activation may be an important mechanism in attenuating leukocyte-endothelial interactions induced by endothelial dysfunction. This study was supported by the Center for Chronic Disorders of Aging at PCOM

Comparing the effectiveness of TAT and Myristoylation of gp91ds on Leukocyte Superoxide (SO) Release

[No abstract is available

Protein Kinase C Beta II (PKC ßII) Peptide Inhibitor Exerts Cardioprotective Effects in Myocardial Ischemia/Reperfusion Injury

Coronary heart disease is the leading cause of death worldwide, and is primarily attributable to the detrimental effects of tissue infarct after an ischemic insult, The most effective therapeutic intervention for reducing infarct size associated with myocardial ischemia injury is timely and effective reperfusion of blood flow back to the ischemic heart tissue. However, the reperfusion of blood itself can induce additional cardiomyocyte death that can account for up to 50% of the final infarction size. Currently, there are no effective clinical pharmacologic treatments to limit myocardial ischemia/reperfusion (MI/R) injury in heart attack patients. Reperfusion injury is initiated by decreased endothelial derived nitric oxide (NO) which occurs within 5 min of reperfusion, and may in part be explained by PKC ßII mediated activation of NADPH oxidase, which occurs upon cytokine release during MI/. PKC ßII activity is increased in animal models of MI/R and known to exacerbate tissue injury. PKC ßII is known to increase NADPH oxidase activity in leukocytes, endothelial cells and cardiac myocytes via phox47 phosphorylation, and decrease endothelial NO synthase (eNOS) activity via phosphorylation of Thr 495. NADPH oxidase produces superoxide (SO) and quenches endothelial derived NO in cardiac endothelial cells. Moreover, PKC ßII phosphorylation of p66Shc at Ser 36 leads to increased mitochondrial reactive active oxygen species (ROS) production, opening of the mitochondrial permeability transition pore (MPTP), and pro-apoptotic factors leading to cell death and increased infarct size. Therefore, using a pharmacologic agent that inhibits the rapid release of PKC ßII mediated ROS, would attenuate endothelial dysfunction and downstream pro apoptotic pathways when given during reperfusion and should be an ideal candidate to attenuate MI/R injury. PKC ßII peptide inhibitor mechanism of action is to inhibit PKC ßII translocation to cellular substrates such as eNOS, NADPH oxidase, and mitochondrial p66Shc protein that increase ROS leading to opening of the MPTP which in turn leads to consequent release of proapoptotic factors into the cytosol. We\u27ve previously shown that PKC ßII peptide inhibitor restored post-reperfused cardiac function and reduced polymorphornuclear leukocyte (PMN) infiltration in isolated rat hearts subjected to MI(20min)/R(45min) reperfused with PMNs. In addition, the use of PKC ßII peptide inhibitor (10-20 µM) correlated with the inhibition of SO release from isolated leukocytes suggesting that this dose range maybe effective in attenuating ROS production. We extended our research in the current study by using a MI (30min)/R (90min) isolated perfused rat heart model. A cell permeable PKC ßII peptide inhibitor (10-20 µM) was given at the beginning of reperfusion for five minutes. Post-reperfused cardiac function and infarct size were measured and compared to untreated control MI/R hearts