The Role of Endothelial Nitric Oxide Synthase (eNOS) Coupling Status During Acute Hyperglycemia as Determined by Real-time Measurements of Blood Nitric Oxide And Hydrogen Peroxide in Rat

Acute hyperglycemia can impair vascular endothelial function in non-diabetic subjects in addition to diabetic patients. Decreased endothelial-derived nitric oxide (NO) bioavailability and increased concentrations of reactive oxygen species (ROS), such as superoxide (SO) and hydrogen peroxide (H202), are the major characteristics of vascular endothelial dysfunction. Normally, vascular endothelial function depends on NO production from coupled endothelial NO synthase (eNOS) in the presence of 5,6,7,8- tetrahydrobiopterin (BH4). By contrast, 7,8-dihydrobiopterin (BH2, Oxidized form of BH4) and/or lack ofL-arginine (coupled eNOS substrate) causes eNOS uncoupling to produce SO, which can be quickly converted to H202. The role of eNOS uncoupling in acute hyperglycemia induced vascular dysfunction in vivo is unclear. In this study; we hypothesized that acute hyperglycemia (200 mg/dL) would increase H202 and decrease NO release in blood relative to saline control. By contrast, BH4 or L-arginine would attenuate the acute hyperglycemia-induced blood NO and H20 2 changes. However, BH2 will exacerbate the acute hyperglycemia-induced blood NO and H202levels. To test the hypothesis, blood NO or H20 2 levels were measured simultaneously using calibrated NO or H20 2 micro sensors ( 100 )lm; WPI Inc.) by placing them into the femoral veins of male Sprague-Dawley rats. The electrical traces from microsensors were recorded at baseline and throughout 3 hours of infusion with saline or 20% D-glucose with or without a drug (BH4,BH2, or L-arginine) and converted into a concentration based on a calibration curve. Acute hyperglycemia (200 mg/dL by i.v. 20% D-glucose) significantly increased H20 2 (n=6) and reduced NO (n=6) blood levels in comparison to the saline group (n=7, p\u3c0.05). BH2 (MW=239.23 g/mol, 4mg/kg) exacerbated hyperglycemia,- induced increased H20 2 levels (n=7) and decreased NO levels (n=6) (p\u3c0.05). By contrast, BH4 (n=6, MW=314.20 glmol, 6.5mglkg), significantly reduced blood H20 2 levels and increased blood NO levels during acute hyperglycemia compared to saline control (p\u3c0.05). Moreover, L-arginine (MW=21 0.66 glmol, 600mglkg) had similar effects on H20 2 (n=5) and NO (n=6) blood levels as BH4, showing significant reduction of blood H202 and enhancement ofblood NO compared to saline control (p\u3c0.05). In summary, uncoupled eNOS serves as a significant mechanism mediating acute hyperglycemiainduced vascular dysfunction and oxidative stress. Therefore, promotion of eNOS coupling may be effective in protecting vascular endothelial function from hyperglycemic insult

Effects of Mitochondria-Targeted Antioxidants on Real-time Blood Nitric Oxide and Hydrogen Peroxide Release in Hind Limb Ischemia and Reperfusion

In the body, reperfusion of ischemic tissue with blood causes the release of reactive oxygen species (ROS), in part, from damaged mitochondria leading to endothelial and organ dysfunction. Endothelial dysfunction occurs within 5 min of reperfusion, is common to all vascular beds, and is characterized by increased hydrogen peroxide (H2O2) and decreased nitric oxide (NO) levels in the blood that further exacerbate reperfusion injury. Previous studies have shown that promoting endothelial NO synthase coupling during reperfusion increases blood NO and decreases blood H2O2 levels in hind limb I/R and attenuates myocardial I/R injury (1). This study specifically examines the effects mitochondria-targeted antioxidants, mitoquinone (mitoQ; Fig. 1), a cell permeable coenzyme Q analogue or SS-31 ((D-Arg)-Dmt-Lys-Phe-Amide; Genemed Synthesis, San Antonio, TX) (Fig.1), a cell permeable peptide, on inhibiting H2O2 release and increasing NO bioavailability in hind limb I/R. MitoQ (2) and SS-31 (3,4) are able to concentrate into the inner mitochondrial membrane via an electrical potential gradient or selective diffusion respectively, where they attenuate superoxide and subsequent H2O2 production thus allowing a concurrent increase in NO bioavailability

Effects of Mitochondrial-Targeted Antioxidants on Real-Time Blood Nitric Oxide and Hydrogen Peroxide Release in Acute Hyperglycemia Rats

Acute hyperglycemia in non-diabetic subjects can impair vascularendothelial function, causing decreased endothelium-derived nitric oxide (NO) release and increased reactive oxygen species (ROS), such assuperoxide and hydrogen peroxide (H2O2). Hyperglycemia may induce mitochondrial dysfunction leading to ROS production and exacerbation of vascular endothelial dysfunction. We investigated whether mitochondrial-targeted antioxidants mitigate acute hyperglycemia-induced oxidative stress and reduced blood NO. To test this hypothesis, blood NO or H2O2 levels were measured simultaneously using NO or H2O2 microsensors (100 µm) which were placed into the femoral veins of anesthesized male Sprague-Dawley rats. Acute hyperglycemia was induced by infusion 20% D-glucose intravenously with or without mitochondria-targeted antioxidants (mitoquinone: mitoQ, MW=1714 g/mol, 2.3 mg/Kg; SS-31: (D-Arg)-Dmt-Lys-Phe-Amide, MW=640g/mol, 2.7 mg/Kg) for 3 hours. We found that acute hyperglycemia (200 mg/dL) significantly increased blood H2O2 by 3.0±0.5 M (n=7) and reduced blood NO by 68.0±13.5 nM (n=9) compared to the saline group at end of infusion (both p\u3c0.05). MitoQ significantly attenuated hyperglycemia– induced H2O2 levels by 2.5±0.2 M (n=7) and increased blood NO levels by 59.3±9.7 nM (n=5) (both p\u3c0.05 compared to hyperglycemia). Similarly, SS-31 significantly reduced hyperglycemia-induced blood H2O2 level by 4.0±0.6 M (n=5) and enhanced blood NO levels by 52.8±7.7 nM (n=6) at end of infusion (both p\u3c0.05 compared to hyperglycemia). In summary, acute hyperglycemia induces mitochondria-derived ROS which in turn contribute to vascular endothelial dysfunction. Therefore, mitochondria-targeted antioxidants are useful to attenuate acute hyperglycemia-induced vascular endothelial dysfunction and oxidative stress

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

Varespladib and cardiovascular events in patients with an acute coronary syndrome: the VISTA-16 randomized clinical trial

IMPORTANCE: Secretory phospholipase A2(sPLA2) generates bioactive phospholipid products implicated in atherosclerosis. The sPLA2inhibitor varespladib has favorable effects on lipid and inflammatory markers; however, its effect on cardiovascular outcomes is unknown. OBJECTIVE: To determine the effects of sPLA2inhibition with varespladib on cardiovascular outcomes. DESIGN, SETTING, AND PARTICIPANTS: A double-blind, randomized, multicenter trial at 362 academic and community hospitals in Europe, Australia, New Zealand, India, and North America of 5145 patients randomized within 96 hours of presentation of an acute coronary syndrome (ACS) to either varespladib (n = 2572) or placebo (n = 2573) with enrollment between June 1, 2010, and March 7, 2012 (study termination on March 9, 2012). INTERVENTIONS: Participants were randomized to receive varespladib (500 mg) or placebo daily for 16 weeks, in addition to atorvastatin and other established therapies. MAIN OUTCOMES AND MEASURES: The primary efficacy measurewas a composite of cardiovascular mortality, nonfatal myocardial infarction (MI), nonfatal stroke, or unstable angina with evidence of ischemia requiring hospitalization at 16 weeks. Six-month survival status was also evaluated. RESULTS: At a prespecified interim analysis, including 212 primary end point events, the independent data and safety monitoring board recommended termination of the trial for futility and possible harm. The primary end point occurred in 136 patients (6.1%) treated with varespladib compared with 109 patients (5.1%) treated with placebo (hazard ratio [HR], 1.25; 95%CI, 0.97-1.61; log-rank P = .08). Varespladib was associated with a greater risk of MI (78 [3.4%] vs 47 [2.2%]; HR, 1.66; 95%CI, 1.16-2.39; log-rank P = .005). The composite secondary end point of cardiovascular mortality, MI, and stroke was observed in 107 patients (4.6%) in the varespladib group and 79 patients (3.8%) in the placebo group (HR, 1.36; 95% CI, 1.02-1.82; P = .04). CONCLUSIONS AND RELEVANCE: In patients with recent ACS, varespladib did not reduce the risk of recurrent cardiovascular events and significantly increased the risk of MI. The sPLA2inhibition with varespladib may be harmful and is not a useful strategy to reduce adverse cardiovascular outcomes after ACS. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT01130246. Copyright 2014 American Medical Association. All rights reserved

Omecamtiv mecarbil in chronic heart failure with reduced ejection fraction, GALACTIC‐HF: baseline characteristics and comparison with contemporary clinical trials

Aims: The safety and efficacy of the novel selective cardiac myosin activator, omecamtiv mecarbil, in patients with heart failure with reduced ejection fraction (HFrEF) is tested in the Global Approach to Lowering Adverse Cardiac outcomes Through Improving Contractility in Heart Failure (GALACTIC‐HF) trial. Here we describe the baseline characteristics of participants in GALACTIC‐HF and how these compare with other contemporary trials. Methods and Results: Adults with established HFrEF, New York Heart Association functional class (NYHA) ≥ II, EF ≤35%, elevated natriuretic peptides and either current hospitalization for HF or history of hospitalization/ emergency department visit for HF within a year were randomized to either placebo or omecamtiv mecarbil (pharmacokinetic‐guided dosing: 25, 37.5 or 50 mg bid). 8256 patients [male (79%), non‐white (22%), mean age 65 years] were enrolled with a mean EF 27%, ischemic etiology in 54%, NYHA II 53% and III/IV 47%, and median NT‐proBNP 1971 pg/mL. HF therapies at baseline were among the most effectively employed in contemporary HF trials. GALACTIC‐HF randomized patients representative of recent HF registries and trials with substantial numbers of patients also having characteristics understudied in previous trials including more from North America (n = 1386), enrolled as inpatients (n = 2084), systolic blood pressure < 100 mmHg (n = 1127), estimated glomerular filtration rate < 30 mL/min/1.73 m2 (n = 528), and treated with sacubitril‐valsartan at baseline (n = 1594). Conclusions: GALACTIC‐HF enrolled a well‐treated, high‐risk population from both inpatient and outpatient settings, which will provide a definitive evaluation of the efficacy and safety of this novel therapy, as well as informing its potential future implementation

Pulmonary symptoms and diagnoses are associated with HIV in the MACS and WIHS cohorts.

BackgroundSeveral lung diseases are increasingly recognized as comorbidities with HIV; however, few data exist related to the spectrum of respiratory symptoms, diagnostic testing, and diagnoses in the current HIV era. The objective of the study is to determine the impact of HIV on prevalence and incidence of respiratory disease in the current era of effective antiretroviral treatment.MethodsA pulmonary-specific questionnaire was administered yearly for three years to participants in the Multicenter AIDS Cohort Study (MACS) and Women's Interagency HIV Study (WIHS). Adjusted prevalence ratios for respiratory symptoms, testing, or diagnoses and adjusted incidence rate ratios for diagnoses in HIV-infected compared to HIV-uninfected participants were determined. Risk factors for outcomes in HIV-infected individuals were modeled.ResultsBaseline pulmonary questionnaires were completed by 907 HIV-infected and 989 HIV-uninfected participants in the MACS cohort and by 1405 HIV-infected and 571 HIV-uninfected participants in the WIHS cohort. In MACS, dyspnea, cough, wheezing, sleep apnea, and incident chronic obstructive pulmonary disease (COPD) were more common in HIV-infected participants. In WIHS, wheezing and sleep apnea were more common in HIV-infected participants. Smoking (MACS and WIHS) and greater body mass index (WIHS) were associated with more respiratory symptoms and diagnoses. While sputum studies, bronchoscopies, and chest computed tomography scans were more likely to be performed in HIV-infected participants, pulmonary function tests were no more common in HIV-infected individuals. Respiratory symptoms in HIV-infected individuals were associated with history of pneumonia, cardiovascular disease, or use of HAART. A diagnosis of asthma or COPD was associated with previous pneumonia.ConclusionsIn these two cohorts, HIV is an independent risk factor for several respiratory symptoms and pulmonary diseases including COPD and sleep apnea. Despite a higher prevalence of chronic respiratory symptoms, testing for non-infectious respiratory diseases may be underutilized in the HIV-infected population

Proteomics of Coagulopathy Following Injury Reveals Limitations of Using Laboratory Assessment to Define Trauma-Induced Coagulopathy to Predict Massive Transfusion

OBJECTIVE: Trauma-induced coagulopathy (TIC) is provoked by multiple mechanisms and is perceived to be one driver of massive transfusions (MT). Single laboratory values using prothrombin time (INR) or thrombelastography (TEG) are used to clinically define this complex process. We used a proteomics approach to test whether current definitions of TIC (INR, TEG, or clinical judgement) are sufficient to capture the majority of protein changes associated with MT. METHODS: Eight level-I trauma centers contributed blood samples from patients available early after injury. TIC was defined as INR >1.5 (INR-TIC), TEG maximum amplitude 10 units of red blood cells in 24 hours or > 4 units RBC/hour during the first 4 hr. SomaLogic proteomic analysis of 1,305 proteins was performed. Pathways associated with proteins dysregulated in patients with each TIC definition and MT were identified. RESULTS: Patients (n=211) had a mean injury severity score of 24, with a MT and mortality rate of 22% and 12%, respectively. We identified 578 SOMAscan analytes dysregulated among MT patients, of which INR-TIC, TEG-TIC, and Clin-TIC patients showed dysregulation only in 25%, 3%, and 4% of these, respectively. TIC definitions jointly failed to show changes in 73% of the protein levels associated with MT, and failed to identify 26% of patients that received a massive transfusion. INR-TIC and TEG-TIC patients showed dysregulation of proteins significantly associated with complement activity. Proteins dysregulated in Clin-TIC or massive transfusion patients were not significantly associated with any pathway. CONCLUSION: These data indicate there are unexplored opportunities to identify patients at risk for massive bleeding. Only a small subset of proteins that are dysregulated in patients receiving MT are statistically significantly dysregulated among patients whose TIC is defined based solely on laboratory measurements or clinical assessment

Proteomics of Coagulopathy Following Injury Reveals Limitations of Using Laboratory Assessment to Define Trauma-Induced Coagulopathy to Predict Massive Transfusion

Objective:. Trauma-induced coagulopathy (TIC) is provoked by multiple mechanisms and is perceived to be one driver of massive transfusions (MT). Single laboratory values using prothrombin time (INR) or thrombelastography (TEG) are used to clinically define this complex process. We used a proteomics approach to test whether current definitions of TIC (INR, TEG, or clinical judgment) are sufficient to capture the majority of protein changes associated with MT. Methods:. Eight level I trauma centers contributed blood samples from patients available early after injury. TIC was defined as INR >1.5 (INR-TIC), TEG maximum amplitude 10 units of red blood cells in 24 hours or >4 units RBC/hour during the first 4 hours. SomaLogic proteomic analysis of 1305 proteins was performed. Pathways associated with proteins dysregulated in patients with each TIC definition and MT were identified. Results:. Patients (n = 211) had a mean injury severity score of 24, with a MT and mortality rate of 22% and 12%, respectively. We identified 578 SOMAscan analytes dysregulated among MT patients, of which INR-TIC, TEG-TIC, and Clin-TIC patients showed dysregulation only in 25%, 3%, and 4% of these, respectively. TIC definitions jointly failed to show changes in 73% of the protein levels associated with MT, and failed to identify 26% of patients that received a massive transfusion. INR-TIC and TEG-TIC patients showed dysregulation of proteins significantly associated with complement activity. Proteins dysregulated in Clin-TIC or massive transfusion patients were not significantly associated with any pathway. Conclusion:. These data indicate there are unexplored opportunities to identify patients at risk for massive bleeding. Only a small subset of proteins that are dysregulated in patients receiving MT are statistically significantly dysregulated among patients whose TIC is defined based solely on laboratory measurements or clinical assessment