The role of endothelial nitric oxide synthase (eNOS) uncoupling in acute hyperglycemia – induced oxidative stress and vascular endothelial dysfunction by measuring blood nitric oxide and hydrogen peroxide in real-time

Acute hyperglycemia can impair vascular endothelial function in non-diabetic subjects in addition to diabetic patients. Decreased eNOS derived nitric oxide (NO) bioavailability and increased reactive oxygen species (ROS), such as superoxide (SO) and hydrogen peroxide (H2O2), are the major characteristics of vascular endothelial dysfunction. Furthermore, eNOS can change from coupled to an uncoupled status resulting in SO production instead of NO production. The role of eNOS uncoupling in acute hyperglycemia induced vascular dysfunction is unclear in vivo. In this study we hypothesized that acute hyperglycemia (200 mg/dL) would increase H2O2 and decrease NO release in blood relative to saline control. By contrast, 5,6,7,8-tetrahydrobiopterin (BH4, an essential cofactor of coupled eNOS) (MW=241.247 g/mol, 6.5 mg/kg) or L-arginine (the substrate of coupled eNOS) (MW=210.66 g/mol, 600 mg/kg) would attenuate acute hyperglycemia-induced blood NO/H2O2 change. However, 7,8-dihydrobiopterin (BH2, an oxidized form of BH4 and serves as a cofactor for uncoupled eNOS) (MW=239.231 g/mol, 4 mg/kg) will exacerbate acute hyperglycemia-induced blood NO/H2O2 change. Blood NO or H2O2 levels were measured simultaneously using calibrated NO or H2O2 microsensors (100 µm WPI Inc.) by placing them into the femoral veins of male Sprague-Dawley rats. The electrical traces were recorded at baseline and throughout 3 hours of infusion with saline or 20% D-glucose with or without a drug and converted into concentration based on the calibration curve. We found that acute hyperglycemia (200 mg/dL) significantly increased H2O2 (n=6) and reduced NO (n=6) blood levels compared to the saline group (n=7, p2 exacerbated hyperglycemia– induced increased H2O2 levels (n=7) and decreased NO levels (n=4) (p4 (n=6), significantly attenuated hyperglycemia– induced increased H2O2 levels and decreased NO levels (p2O2 (n=5) and NO (n=6) blood levels as BH4, showing significant reduction of blood H2O2 and enhancement of blood NO (p2O2 and reduced NO blood levels. Uncoupled eNOS serves as a significant source mediating acute hyperglycemia-induced vascular dysfunction. Therefore, promotion of eNOS coupling may be effective in protecting vascular endothelial function from hyperglycemic insult

The Role of Protein Kinase C Epsilon in the Regulation of Endothelial Nitric Oxide Synthase (eNOS) during Oxidative Stress caused by Extracorporeal Shock Wave Lithotripsy (ESWL)

BACKGROUND: Clinical ESWL treatment to ablate kidney stones can cause acute to chronic damage in renal microvasculature leading to decreased renal blood flow and hypertension. Shockwaves can stimulate endothelial cells to release superoxide resulting in decreased nitric oxide (NO) bioavailability and increased oxidative stress, causing vascular endothelial dysfunction in the kidney. When the dihydrobiopterin:tetrahydrobiopterin ratio is increased during oxidative stress such as ESWL, eNOS becomes uncoupled and produces superoxide instead of NO. Superoxide is converted to hydrogen peroxide (H2O2) by superoxide dismutase. Protein kinase C epsilon (PKC-ε) is known to positively regulate endothelial NO synthase (eNOS) activity. In order to establish controls for the effects of PKC-ε activator and inhibitor, the effect of ESWL was tested by the comparison of ESWL-treated rats to those with no ESWL exposure, both with a saline infusion. We hypothesized that the PKC-ε peptide inhibitor (Myr-EAVSLKPT, MW = 1054.6) would decrease ESWL-induced H2O2 release and decreased the attenuation of NO release compared to ESWL-saline control rats. PKC-ε activator (Myr-NDAPIGYD, MW = 1098.5) was expected to show no effect on H2O2 or NO release, displaying a similar trend to ESWL-saline control rats

The Effects of Modulating Endothelial Nitric Oxide Synthese (eNOS) Activity and Coupling in Extracorporeal Shock Wave Lithotripsy (ESWL)

Introduction: ESWL is a clinical therapy to break down kidney and uretal stones into smaller fragments that are more easily eliminated through the urinary tract. High-energy shock waves are focused on the stone to cause shear stress and cavitation bubbles which synergistically ablate the stones. While ESWL is the preferred treatment for kidney stones over invasive surgeries, the repetitive shock waves necessary to break up the stones may also cause damage to the renal vasculature endothelium and that can lead to chronic hypertension [1]. Previous studies have found that ESWL can cause endothelial dysfunction which is characterized decreased nitric oxide (NO) bioavailability and increased production of reactive oxygen species (ROS) such as superoxide (O2-) [2]. Normally, endothelial nitric oxide synthase (eNOS) is in a coupled state which forms NO in the presence of essential cofactor tetrahydrobiopterin (BH4) and molecular oxygen. Oxidative stress, such as that caused by ESWL-induced ROS, can cause BH4 to be oxidized to dihydrobiopterin (BH2). When the BH2:BH4 ratio is increased, eNOS becomes uncoupled and produces O2- instead of NO [2, 3] (Figure 1). O2- is short-lived and converted to hydrogen peroxide (H2O2) in blood by superoxide dismutase. Protein kinase C epsilon (PKCε) has previously been found to regulate eNOS activity via phosphorylation at serine-1177. Cell-permeable PKCε peptide activator (PKCε+) increases eNOS activity while PKCε inhibitor (PKCε-) reduces eNOS activity [2]. Using a combination of eNOS cofactors BH4 or BH2 with eNOS activity regulators PKCε+ or PKCε-, we can explore the role of modulating eNOS to reduce oxidative stress and endothelial dysfunction caused by ESWL

Gp91ds-tat, a Selective NADPH Oxidase Peptide Inhibitor, Increases Blood Nitric Oxide (NO) Bioavailability in Bind Limb Ischemia and Reperfusion (I/R)

I/R injury induces cell death and organ dysfunction in part due to a burst of reactive oxygen species that occurs upon the reintroduction of oxygen into the ischemic area, leading to endothelial dysfunction: decreased blood NO and increased hydrogen peroxide (H2O2 ) levels. We’ve previously shown in isolated rat hearts subjected to I/R injury, gp91ds-tat attenuated cardiac contractile dysfunction and reduced infarct size compared to controls presumably by the inhibition of NADPH oxidase induced superoxide release. Superoxide can quench NO via the formation of peroxynitrite and also be converted to H2O2 in blood. We attempted to confirm this hypothesis using a rat hind limb I/R model that permitted real time measurements of changes in blood NO and H2O2. NO or H2O2 microsensors were inserted into both femoral veins in anesthetized male rats. One limb’s femoral artery/vein is subjected to I(30min)/R(45min) while the other served as a non-ischemic sham. Preliminary results show blood NO release significantly increased by the end of reperfusion in gp91ds-tat treated rats (1.2 mg/kg, MW 2452g/mol, n=5) compared to saline treated rats (n=3;

The Role of Endothelial Nitric Oxide Synthase (eNOS) Uncoupling on Leukocyte-Endothelial Interactions in Rat Mesenteric Postcapillary Venules

BACKGROUND: Endothelial derived nitric oxide (NO) is essential in the regulation of blood pressure and attenuates leukocyte-endothelial interactions associated with vascular injury. Endothelial NO synthase (eNOS) is coupled to L-arginine in the presence of tetrahydrobiopetrin (BH4) to produce NO. However, when BH4 is oxidized to dihydrobiopetrin (BH2) under conditions of oxidative stress, the ratio of BH2 to BH4 is increased causing the uncoupling of eNOS to use molecular oxygen as a substrate, instead of L-arginine, to produce superoxide

The Role of Autophagy During Myocardial Ischemia/Reperfusion Injury

Autophagy is a housekeeping process to remove damaged cytoplasmic constituents. However, a debate persists on whether autophagy is beneficial or detrimental when an ischemic/reperfusion (I/R) insult occurs in the heart. This study tested the effects of autophagy enhancers (e.g. rapamycin and trehalose) and autophagy inhibitor (e.g. 3-methyladenine) on heart function and infarct size after global I (30 minutes) and R (45 minutes) when given prior to ischemia (pre-treatment) or at the beginning of reperfusion (post-treatment). We found that Rapamycin (25nM) pre-treatment and post-treatment significantly restored final left ventricular developed pressure (LVDP) to 75.4±9.1% and 60±5% of initial baseline respectively (both n=5, p\u3c0.05), compared to I/R group (n=9) that recovered to 35±5.5% of initial baseline. Likewise, trehalose (5mM) pre-treatment and post-treatment also significantly restored final LVDP to 61.4±3.7% (n=6) and 69.1±2.7% (n=5) of initial baseline respectively, compared to I/R group. However, 3-methyladenine (1mM) pre-treatment (n=6) and post-treatment (n=5) showed similar reduction in final LVDP to 24.7±9.1% and 33.4±12.8 % of initial baseline respectively, as I/R group. Moreover, infarction percentage was significantly reduced by rapamycin pre-treatment and post-treatment (14 ± 2.8% and 21.4 ± 5.3%, respectively; both p\u3c0.05); and trehalose pre-treatment and post-treatment (19.2 ± 3% and 15.2% ± 3, respectively; both p\u3c0.05), but not by 3-methyladenine pre-treatment and post-treatment (26±2% and 28±4.1%, respectively) when compared to I/R group (38.6±4.3%). The data suggests that autophagy enhancement before ischemia or at reperfusion is beneficial for reducing I/R injury

The Effects of Dihydrobiopterin and Tetrahydrobiopterin on Hydrogen Peroxide and Nitric Oxide Release During Extracorporeal Shockwave Lithotripsy

Extracorporeal shockwave lithrotripsy (ESWL) is an effective, non-invasive clinical therapy utilized to break up stones in the kidney and urinary tract. A lithotripter generates high-energy acoustic pulses and propagates those shock waves through a lens on a region that focuses on the location of the stone, in turn breaking up the stone. The successive pulses generate shearing forces and cavitation bubbles. Cavitation bubbles are the formation and implosion of liquid free zones. The cavitation bubbles implode rapidly to create their own shockwaves that also put pressure on the stone. After treatment, fragmentation of the stone allows the debris to be cleared by the flow of the urinary tract. The problem is that to break up the kidney stone, it requires many repetitive shock waves that not only hit the kidney stone but also the surrounding tissue. Although lithotripsy provides a safer alternative to invasive treatments for removing harmful stones, ESWL may cause prolonged vasoconstriction after ESWL treatment, reducing renal blood flow, and subsequent endothelial dysfunction, which may cause kidney damage leading to acute to chronic hypertension clinically. ESWL-induced vascular oxidative stress and further endothelial dysfunction may be mediated by reduced levels of endothelial-derived nitric oxide (NO) and/or increased reactive oxygen species. Previous studies have shown that ESWL can induce oxidative stress, which can cause an increase in blood hydrogen peroxide (H2O2) and a decrease in endothelial-derived NO bioavailability release. Under normal conditions, tetrahydrobiopterin (BH4) is the cofactor to promote eNOS coupling, and endothelial-derived NO is produced. When the dihydrobiopterin (BH2) to tetrahydrobiopterin (BH4) ratio is increased during oxidative stress, such as ESWL, BH2 promotes eNOS uncoupling and produces superoxide (SO) instead of NO. (1,2) (Figure 1) SO is then later converted to H2O2 by superoxide dismutase. BH4 and BH2 bind to eNOS with equal affinity, therefore the ratio will determine whether eNOS principally produces NO or SO

Effects of NADPH oxidase inhibitor apocynin on real-time blood hydrogen peroxide release in femoral artery/vein ischemia and reperfusion

Background: Vascular endothelial dysfunction can initiate oxidative stress during ischemiaJreperfusion (IIR). Endothelial dysfunction is characterized by an increase in blood hydrogen peroxide (H20 ]) and a decrease in endothelial-derived nitric oxide (NO) bioavailability. Previous studies using Go 6983, a broad-spectrum protein kinase C inhibitor that can inhibit NADPH oxidase activity, has attenuated blood H20 ] levels during femoralliR in vivo. This study examines the effects of apocynin, a direct NADPH oxidase inhibitor, on real-time blood H20 ] levels in femoral I1R in vivo. H20 ] microsensors (100 Mm) were inserted into both femoral veins in anesthetized rats

The effects of mitoquinone pretreatment on doxorubicin-induced acute cardiac dysfunction

Introduction: Doxorubicin (DOX) is a widely used anti-cancer drug notorious for its irreversible cardiac toxicity. Currently, Dexrazoxane is the only FDA-approved treatment for this toxicity. However, Dexrazoxane still bears some serious adverse events, and developing new strategies to mitigate DOX-induced heart damage is critical. Our lab has shown that pretreatment of the H9c2 myoblast cells with mitoquinone (MitoQ), a mitochondrial-targeted antioxidant, and significantly improved cells’ resiliency to DOX. This study aimed to determine if MitoQ pretreatment can preserve cardiac function against DOX-induced damage in isolated rat hearts. Objectives: The effects of DOX and MitoQ on cardiac function were evaluated in isolated rat hearts. Moreover, the benefits of MitoQ pretreatment on DOX-induced cardiac dysfunction were also assessed. Methods: Langendorff heart preparation was performed after anesthesia of male SD rats (275-325 g). Hearts were isolated and retrograde perfused with Krebs’ buffer at a constant pressure of 80 mmHg with 37 ⁰C and pH of 7.35-7.45. Cardiac parameters, including left ventricle end-systolic pressure (LVESP), left ventricle end-diastolic pressure (LVEDP), left ventricular developed pressure (LVDP=LVESP-LVEDP), maximal rate of rise of LVP (dP/dt(max)), and heart rate (HR), were measured by a pressure transducer placed in the left ventricle of the rat heart. After obtaining a stable initial cardiac function, DOX (20 µM or 25 µM) or MitoQ (0.1-0.5 or 1-2.5 µM) were infused into the heart for 60 min. to determine the individual drug\u27s effects on the cardiac function. Moreover, another set of hearts was pretreated with MitoQ (0.25-0.5 or 1-2.5 µM) for 10-15 min before giving DOX (25 µM) to evaluate if MitoQ pretreatment would mitigate DOX-induced cardiac dysfunction. Cardiac functions were recorded every 5 min. throughout the experiments. The ratio between the final and initial recordings was calculated and compared among experimental groups. Results: Acute infusion of DOX into the isolated hearts dose-dependently reduced some cardiac parameters. Higher dose DOX (25 μM, n=5) induced a higher reduction in the ratios of LVESP, LVDP, and dP/dt(max) to 0.39±0.05, 0.35±0.06, and 0.26±0.05 than those of lower dose DOX infusion (20 μM, n=2; 0.77±0.01, 0.75±0.01, and 0.57±0.01), respectively. DOX had no effects on LVEDP and HR. Moreover, lower doses of MitoQ (0.1-0.5 μM, n=6) only slightly reduced HR to 0.77±0.01 without affecting other parameters. By contrast, higher doses of MitoQ (1-5 μM, n=4) reduced the ratios of LVESP, LVDP, dP/dt(max), and HR to 0.72±0.12, 0.51±0.18, and 0.45±0.17 0.65±0.07, respectively. Interestingly, MitoQ pretreatment before DOX (25 µM) exhibited better cardiac function accompanied by reduced HR than DOX alone. Higher MitoQ (1-2.5 µM) pretreatment improved the ratios of cardiac LVESP, LVDP, and dP/dt(max) to 0.67±0.14, 0.65±0.16, and 0.40±0.09, which were higher than those of lower dose MitoQ (0.25-0.5 μM, n=3; 0.49±0.11, 0.44±0.11, and 0.36±0.08), respectively. Conclusion: The preliminary data suggest that infusion of DOX into the heart acutely attenuated cardiac systolic function. Higher doses of MitoQ, not lower doses, also suppressed cardiac function. MitoQ pretreatment mitigated DOX-induced heart dysfunction. Acknowledgement: The project is funded by CCDA at PCOM