The Real-Time Measurements of Blood Nitric Oxide (NO) and Hydrogen Peroxide (H2O2) Levels under Acute Hyperglycemia

BACKGROUND: Vascular endothelial dysfunction is one of the earliest recognizable events under hyperglycemic conditions. It is characterized by decreased endothelium-derived nitric oxide (NO) and increased oxidative stress, such as superoxide and hydrogen peroxide (H2O2). However, the real-time changes in blood NO and H2O2 levels under acute hyperglycemia have not been evaluated

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 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

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

Mitoquinone (mitoQ) Exerts Antioxidant Effects Independent of Mitochondrial Targeted Effects in Phorbol-12-myristate-13-acetate (PMA) or N-formyl-L-methiony-L-leucyl-L-phenylalanine (fMLP) Stimulated Polymorphonuclear Leukocyte (PMN) Superoxide (SO) Release

MitoQ is a mitochondrial-targeted coenzyme Q antioxidant analog that dose-dependently restored cardiac function and reduced infarct size in isolated perfused rat hearts subjected to ischemia reperfusion (I/R). Moreover, mitoQ also dose-dependently attenuated PMA stimulated PMN superoxide (SO) release at the same concentration (10uM) as the cardioprotective dose. NADPH oxidase is the principle source of PMN SO release. We speculate that mitoQ may exert antioxidant effects independent of the mitochondria. Therefore, we hypothesized that inhibition of mitoQ on PMN-SO release will be similar as other coenzyme Q analogs: coenzyme Q1 and decylubiquinone without affecting cell viability. SO release was measured spectrophotometrically from isolated rat PMNs measured by the reduction of ferricytochrome c and were stimulated with 100nM PMA. The absorbance was measured at 550 nm up to 360sec. Positive control samples were given SO dismutase (SOD; 10ug/ml) which inhibited PMA induced SO release by \u3e90%. MitoQ significantly inhibited SO release by 56 + 3% (10uM, n=10 ,

The Role of Tetrahydrobiopterin and Dihydrobiopterin in Ischemia/Reperfusion Injury When Given at Reperfusion

Reduced nitric oxide (NO) bioavailability and increased oxidative stress are major factors mediating ischemia/reperfusion (I/R) injury. Tetrahydrobiopterin (BH4) is an essential cofactor of endothelial NO synthase (eNOS) to produce NO, whereas dihydrobiopterin (BH2) can shift the eNOS product profile from NO to superoxide, which is further converted to hydrogen peroxide (H2O2) and cause I/R injury. The effects of BH4 and BH2 on oxidative stress and postreperfused cardiac functions were examined in ex vivo myocardial and in vivo femoral I (20 min)/R (45 min) models. In femoral I/R, BH4 increased NO and decreased H2O2 releases relative to saline control, and these effects correlated with improved postreperfused cardiac function. By contrast, BH2 decreased NO release relative to the saline control, but increased H2O2 release similar to the saline control, and these effects correlated with compromised postreperfused cardiac function. In conclusion, these results suggest that promoting eNOS coupling to produce NO and decrease H2O2 may be a key mechanism to restore postreperfused organ function during early reperfusion

Effects of antiplatelet therapy on stroke risk by brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases: subgroup analyses of the RESTART randomised, open-label trial

Background Findings from the RESTART trial suggest that starting antiplatelet therapy might reduce the risk of recurrent symptomatic intracerebral haemorrhage compared with avoiding antiplatelet therapy. Brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases (such as cerebral microbleeds) are associated with greater risks of recurrent intracerebral haemorrhage. We did subgroup analyses of the RESTART trial to explore whether these brain imaging features modify the effects of antiplatelet therapy

Clinical outcomes and response to treatment of patients receiving topical treatments for pyoderma gangrenosum: a prospective cohort study

Background: pyoderma gangrenosum (PG) is an uncommon dermatosis with a limited evidence base for treatment. Objective: to estimate the effectiveness of topical therapies in the treatment of PG. Methods: prospective cohort study of UK secondary care patients with a clinical diagnosis of PG suitable for topical treatment (recruited July 2009 to June 2012). Participants received topical therapy following normal clinical practice (mainly Class I-III topical corticosteroids, tacrolimus 0.03% or 0.1%). Primary outcome: speed of healing at 6 weeks. Secondary outcomes: proportion healed by 6 months; time to healing; global assessment; inflammation; pain; quality-of-life; treatment failure and recurrence. Results: Sixty-six patients (22 to 85 years) were enrolled. Clobetasol propionate 0.05% was the most commonly prescribed therapy. Overall, 28/66 (43.8%) of ulcers healed by 6 months. Median time-to-healing was 145 days (95% CI: 96 days, ∞). Initial ulcer size was a significant predictor of time-to-healing (hazard ratio 0.94 (0.88;80 1.00); p = 0.043). Four patients (15%) had a recurrence. Limitations: No randomised comparator Conclusion: Topical therapy is potentially an effective first-line treatment for PG that avoids possible side effects associated with systemic therapy. It remains unclear whether more severe disease will respond adequately to topical therapy alone

Effects of antiplatelet therapy after stroke due to intracerebral haemorrhage (RESTART): a randomised, open-label trial

Background: Antiplatelet therapy reduces the risk of major vascular events for people with occlusive vascular disease, although it might increase the risk of intracranial haemorrhage. Patients surviving the commonest subtype of intracranial haemorrhage, intracerebral haemorrhage, are at risk of both haemorrhagic and occlusive vascular events, but whether antiplatelet therapy can be used safely is unclear. We aimed to estimate the relative and absolute effects of antiplatelet therapy on recurrent intracerebral haemorrhage and whether this risk might exceed any reduction of occlusive vascular events. Methods: The REstart or STop Antithrombotics Randomised Trial (RESTART) was a prospective, randomised, open-label, blinded endpoint, parallel-group trial at 122 hospitals in the UK. We recruited adults (≥18 years) who were taking antithrombotic (antiplatelet or anticoagulant) therapy for the prevention of occlusive vascular disease when they developed intracerebral haemorrhage, discontinued antithrombotic therapy, and survived for 24 h. Computerised randomisation incorporating minimisation allocated participants (1:1) to start or avoid antiplatelet therapy. We followed participants for the primary outcome (recurrent symptomatic intracerebral haemorrhage) for up to 5 years. We analysed data from all randomised participants using Cox proportional hazards regression, adjusted for minimisation covariates. This trial is registered with ISRCTN (number ISRCTN71907627). Findings: Between May 22, 2013, and May 31, 2018, 537 participants were recruited a median of 76 days (IQR 29–146) after intracerebral haemorrhage onset: 268 were assigned to start and 269 (one withdrew) to avoid antiplatelet therapy. Participants were followed for a median of 2·0 years (IQR [1·0– 3·0]; completeness 99·3%). 12 (4%) of 268 participants allocated to antiplatelet therapy had recurrence of intracerebral haemorrhage compared with 23 (9%) of 268 participants allocated to avoid antiplatelet therapy (adjusted hazard ratio 0·51 [95% CI 0·25–1·03]; p=0·060). 18 (7%) participants allocated to antiplatelet therapy experienced major haemorrhagic events compared with 25 (9%) participants allocated to avoid antiplatelet therapy (0·71 [0·39–1·30]; p=0·27), and 39 [15%] participants allocated to antiplatelet therapy had major occlusive vascular events compared with 38 [14%] allocated to avoid antiplatelet therapy (1·02 [0·65–1·60]; p=0·92). Interpretation: These results exclude all but a very modest increase in the risk of recurrent intracerebral haemorrhage with antiplatelet therapy for patients on antithrombotic therapy for the prevention of occlusive vascular disease when they developed intracerebral haemorrhage. The risk of recurrent intracerebral haemorrhage is probably too small to exceed the established benefits of antiplatelet therapy for secondary prevention