A randomized pilot study of nitrate supplementation with beetroot juice in acute respiratory failure Publication date: 1 January 2020 Source: Nitric Oxide, Volume 94 Author(s): D. Clark Files, Timothy Heinrich, Katherine L. Shields, Nathan J. Love, Carly Brailer, Rita N. Bakhru, Lina Purcell, Lori Flores, Kevin Gibbs, Gary D. Miller, Peter E. Morris, Michael J. Berry Abstract Nitrate rich beetroot juice (BRJ) can enhance nitric oxide signaling, leading to improved physical function in healthy and diseased populations, but its safety and biologic efficacy have not been evaluated in a critically ill population. We randomized 22 previously functional acute respiratory failure patients to either BRJ or placebo daily until day 14 or discharge. We measured blood nitrate and nitrite levels and quantified strength and physical function at intensive care unit (ICU) and hospital discharge. Participants were predominantly male (54%), aged 68.5 years with an APACHE III score of 62. BRJ increased plasma nitrate (mean 219.2 μM increase, p = 0.002) and nitrite levels (mean 0.144 μM increase, p = 0.02). We identified no adverse events. The unadjusted and adjusted effect sizes of the intervention on the short physical performance battery were small (d = 0.12 and d = 0.17, respectively). In this pilot trial, administration of BRJ was feasible and safe, increased blood nitrate and nitrate levels, but had a small effect on physical function. Future studies could evaluate the clinical efficacy of BRJ as a therapy to improve physical function in survivors of critical illness.

Past, current and future perspectives of the use of inhaled NO as a primary therapeutic for the newborn Publication date: 1 January 2020 Source: Nitric Oxide, Volume 94 Author(s): Joseph B. Philips

Sapropterin reduces coronary artery malformation in offspring of pregestational diabetes mice Publication date: 1 January 2020 Source: Nitric Oxide, Volume 94 Author(s): Anish Engineer, Yong Jin Lim, Xiangru Lu, Mella Y. Kim, Kambiz Norozi, Qingping Feng Abstract Endothelial nitric oxide synthase (eNOS) and oxidative stress are critical to embryonic coronary artery development. Maternal diabetes increases oxidative stress and reduces eNOS activity in the fetal heart. Sapropterin (Kuvan®) is an orally active, synthetic form of tetrahydrobiopterin (BH4) and a co-factor for eNOS with antioxidant properties. The aim of the present study was to examine the effects of sapropterin on fetal coronary artery development during pregestational diabetes in mice. Diabetes was induced by streptozotocin to adult female C57BL/6 mice. Sapropterin (10 mg/kg/day) was orally administered to pregnant mice from E0.5 to E18.5. Fetal hearts were collected at E18.5 for coronary artery morphological analysis. Sapropterin treatment to diabetic dams reduced the incidence of coronary artery malformation in offspring from 50.0% to 20.6%. Decreases in coronary artery luminal diameter, volume and abundance in fetal hearts from diabetic mothers, were prevented by sapropterin treatment. Maternal diabetes reduced epicardial epithelial-to-mesenchymal transition (EMT) and expression of transcription and growth factors critical to coronary artery development including hypoxia-inducible factor 1a (Hif1a), Snail1, Slug, β-catenin, retinaldehyde dehydrogenase 2 (Aldh1a2), basic fibroblast growth factor (bFGF) and vascular endothelial group factor receptor 2 (Vegfr2) in E12.5 hearts. Additionally, eNOS phosphorylation was lower while oxidative stress was higher in E12.5 hearts from maternal diabetes. Notably, these abnormalities were all restored to normal levels after sapropterin treatment. In conclusion, sapropterin treatment increases eNOS activity, lowers oxidative stress and reduces coronary artery malformation in offspring of pregestational diabetes. Sapropterin may have therapeutic potential in preventing coronary artery malformation in maternal diabetes. Graphical abstract Oral sapropterin (Kuvan®) treatment inhibits oxidative stress, improves Akt/eNOS activity, and prevents coronary artery malformation (CAM) during pregestational diabetes.

Influence of hemoglobin and albumin on the NO donation effect of tetranitrosyl iron complex with thiosulfate Publication date: 1 January 2020 Source: Nitric Oxide, Volume 94 Author(s): Olesya Pokidova, Tatiana Rudneva, Bogdan Tretyakov, Raisa Kotelnikova, Alexander Kotelnikov, Sergey Aldoshin Abstract The effects of deoxyhemoglobin (Hb) and albumin on the NO-donor activity of the anionic tetranitrosyl iron complex with thiosulfate ligands (1) were studied for the first time. It was shown that Hb significantly stabilizes complex 1; in its presence, NO generation from the complex proceeds at a noticeably slower rate. A similar effect is observed when complex 1 is bound to albumin, in which case complex 1 decomposes 27 times slower than in the absence of albumin in the solution. The observed effects provide a prolonged action of complex 1 as NO-donor, which may enhance its potential pharmacological efficacy.

Sickle cell disease subjects and mouse models have elevated nitrite and cGMP levels in blood compartments Publication date: 1 January 2020 Source: Nitric Oxide, Volume 94 Author(s): Luis E.F. Almeida, Sayuri Kamimura, Celia M. de Souza Batista, Nicholas Spornick, Margaret Y. Nettleton, Elizabeth Walek, Meghann L. Smith, Julia C. Finkel, Deepika S. Darbari, Paul Wakim, Zenaide M.N. Quezado Abstract The hypothesis of decreased nitric oxide (NO) bioavailability in sickle cell disease (SCD) proposes that multiple factors leading to decreased NO production and increased consumption contributes to vaso-occlusion, pulmonary hypertension, and pain. The anion nitrite is central to NO physiology as it is an end product of NO metabolism and serves as a reservoir for NO formation. However, there is little data on nitrite levels in SCD patients and its relationship to pain phenotype. We measured nitrite in SCD subjects and examined its relationship to SCD pain. In SCD subjects, median whole blood, red blood cell and plasma nitrite levels were higher than in controls, and were not associated with pain burden. Similarly, Townes and BERK homozygous SCD mice had elevated blood nitrite. Additionally, in red blood cells and plasma from SCD subjects and in blood and kidney from Townes homozygous mice, levels of cyclic guanosine monophosphate (cGMP) were higher compared to controls. In vitro, hemoglobin concentration, rather than sickle hemoglobin, was responsible for nitrite metabolism rate. In vivo, inhibition of NO synthases and xanthine oxidoreductase decreased nitrite levels in homozygotes but not in control mice. Long-term nitrite treatment in SCD mice further elevated blood nitrite and cGMP, worsened anemia, decreased platelets, and did not change pain response. These data suggest that SCD in humans and animals is associated with increased nitrite/NO availability, which is unrelated to pain phenotype. These findings might explain why multiple clinical trials aimed at increasing NO availability in SCD patients failed to improve pain outcomes.

Effects of age, sex and diet on salivary nitrate and nitrite in infants Publication date: 1 January 2020 Source: Nitric Oxide, Volume 94 Author(s): Niklas Timby, Magnus Domellöf, Olle Hernell, Bo Lönnerdal, Carina Nihlen, Ingegerd Johanssson, Eddie Weitzberg Abstract The inorganic anions nitrate and nitrite are oxidation products from endogenous nitric oxide (NO) generation and constituents in our diet. A nitrate-nitrite-NO pathway exists in which nitrate can be serially reduced to bioactive NO. The first step of this pathway occurs in the oral cavity where oral bacteria convert salivary nitrate to nitrite, whereafter nitrite is reduced to NO systemically by several enzymatic and non-enzymatic pathways. Data are scarce regarding salivary levels and oral conversion capacity of these anions in infants. We measured salivary nitrate and nitrate in infants at 4 and 12 months of age and related values to age, sex, dietary pattern and oral microbiome. Saliva was collected from a total of 188 infants at 4 and 12 months of age. Salivary nitrate, nitrite and nitrite/nitrate ratio as a measure of oral nitrate-reducing capacity were analyzed by HPLC and related to age, sex, type of diet (breast milk or formula) and oral microbiome. There was no difference in salivary nitrate, nitrite or nitrite/nitrate ratio between boys and girls at any age. At 4 months levels of these parameters were lower than what has been described in adults but they had all increased significantly at 12 months of age. At 4 months of age salivary nitrite/nitrate ratio was lower in breast-fed compared to formula-fed infants, but these differences disappeared at 12 months. Several bacterial species were associated with oral nitrate reducing capacity including Prevotella, Veillonella, Alloprevotella and Leptotrichia. We conclude that in infants there is an increase in salivary nitrate and nitrite as well as in oral nitrate-reductase capacity during the first year of life. Differences observed at 4 months of age between breast-fed and formula-fed infants disappear at one year of age.

Modulation of endothelium-derived nitric oxide production and activity by taurine and taurine-conjugated bile acids Publication date: 1 January 2020 Source: Nitric Oxide, Volume 94 Author(s): Daniele M. Guizoni, Jean F. Vettorazzi, Everardo M. Carneiro, Ana Paula Davel Abstract Taurine is a semiessential amino acid found at high concentrations in mammalian plasma and cells, where it regulates cellular functions such as ion flux, controls cell volume and serves as a substrate for conjugated bile acids (BAs). Exogenous administration of both taurine and taurine-conjugated BAs have also been implicated in the modulation of cardiovascular functions. This brief review summarizes the role of taurine and taurine-conjugated BAs in vascular relaxation through the modulation of endothelium-derived nitric oxide (NO). The effects of taurine on vascular health are controversial. However, in the presence of cardiometabolic risk factors, it has been proposed that taurine can increase vascular NO levels by increasing eNOS expression, eNOS phosphorylation on Ser1177, NO bioavailability, the level of antioxidative defense, and the l-arginine/NOS inhibitor asymmetric dimethylarginine (ADMA) ratio. The taurine-conjugated BA-mediated activation of Farnesoid X receptor (FXR), G protein-coupled BA receptor (TGR5) and/or muscarinic 3 receptor (M3) was also reported to increase vascular NO production. FXR activation increases eNOS expression and may reduce ADMA formation, while TGR5 increases mobilization of Ca2+ and phosphorylation of eNOS and Akt in endothelial cells. Furthermore, taurine and taurine-conjugated BAs might regulate NO synthesis and activity by enhancing H2S generation. Several studies have demonstrated the beneficial effects of both taurine and taurine-conjugated BAs in reversing the endothelial dysfunction associated with diabetes, atherosclerosis, hypertension, obesity, malnutrition, and smoking. In addition, taurine-conjugated BAs have emerged as a potential treatment for portal hypertension. Despite these favorable findings, there is a need to further explore the mechanisms and signaling pathways underlying the endothelial effects of taurine and taurine-conjugated BAs. Here, we summarize the main findings regarding the effects of taurine and taurine-conjugated BAs on the endothelial dysfunction associated with altered NO metabolism in cardiovascular diseases.

Systemic vasoprotection by inhaled carbon monoxide is mediated through prolonged alterations in monocyte/macrophage function Publication date: 1 January 2020 Source: Nitric Oxide, Volume 94 Author(s): Andrew Leake, Karim Salem, Michael C. Madigan, Ghee Rye Lee, Ankur Shukla, Guiying Hong, Brian S. Zuckerbraun, Edith Tzeng Abstract Carbon monoxide (CO) is anti-inflammatory and protective in models of disease. Its actions in vitro are short-lived but are sustained in vivo. We hypothesize that systemic CO can mediate prolonged phenotype changes in vivo, with a focus on macrophages (Mφs). Mφs isolated from CO treated rats responded to lipopolysaccharide (LPS) with increased IL6, IL10 and iNOS expression but decreased TNF. Conditioned media (CM) collected from peritoneal Mφs isolated from CO treated rats stimulated endothelial cell (EC) proliferation versus CM from Mφs from air treated rats. This effect was mediated by Mφ released VEGF and HMGB1. Inhaled CO reduced LPS induced Mφ M1 inflammatory phenotype for up to 5 days. Mitochondrial oxygen consumption in LPS treated Mφs from CO treated mice was preserved compared to LPS treated Mφs from control mice. Finally, transient reduction of inflammatory cells at the time of inhaled CO treatment eliminated the vasoprotective effect of CO in a rodent carotid injury model. Thus, inhaled CO induces a prolonged mixed phenotype change in Mφs, and potentially other inflammatory cells, that contribute to vasoprotection. These findings demonstrate the ability of inhaled CO to modify Mφs in a sustained manner to mediate its therapeutic actions, supporting the translational potential of inhaled CO.

Effects of dietary nitrate supplementation on microvascular physiology at 4559 m altitude – A randomised controlled trial (Xtreme Alps) Publication date: 1 January 2020 Source: Nitric Oxide, Volume 94 Author(s): Andrew F. Cumpstey, Philip J. Hennis, Edward T. Gilbert-Kawai, Bernadette O. Fernandez, Daniel Grant, William Jenner, Matthieu Poudevigne, Helen Moyses, Denny ZH. Levett, Alexandra Cobb, Paula Meale, Kay Mitchell, Helmut Pöhnl, Monty G. Mythen, Michael PW. Grocott, Daniel S. Martin, Martin Feelisch Abstract Native highlanders (e.g. Sherpa) demonstrate remarkable hypoxic tolerance, possibly secondary to higher levels of circulating nitric oxide (NO) and increased microcirculatory blood flow. As part of the Xtreme Alps study (a randomised placebo-controlled trial of dietary nitrate supplementation under field conditions of hypobaric hypoxia), we investigated whether dietary supplementation with nitrate could improve NO availability and microvascular blood flow in lowlanders. Plasma measurements of nitrate, nitrite and nitroso species were performed together with measurements of sublingual (sidestream dark-field camera) and forearm blood flow (venous occlusion plethysmography) in 28 healthy adult volunteers resident at 4559 m for 1 week; half receiving a beetroot-based high-nitrate supplement and half receiving an identically-tasting low nitrate ‘placebo’. Dietary supplementation increased plasma nitrate concentrations 4-fold compared to the placebo group, both at sea level (SL; 19.2 vs 76.9 μM) and at day 5 (D5) of high altitude (22.9 vs 84.3 μM, p < 0.001). Dietary nitrate supplementation also significantly increased both plasma nitrite (0.78 vs. 0.86 μM SL, 0.31 vs. 0.41 μM D5, p = 0.03) and total nitroso product (11.3 vs. 19.7 nM SL, 9.7 vs. 12.3 nM D5, p < 0.001) levels both at sea level and at 4559 m. However, plasma nitrite concentrations were more than 50% lower at 4559 m compared to sea level in both treatment groups. Despite these significant changes, dietary nitrate supplementation had no effect on any measured read-outs of sublingual or forearm blood flow, even when environmental hypoxia was experimentally reversed using supplemental oxygen. In conclusion, dietary nitrate supplementation does not improve microcirculatory function at 4559 m.

Treatment with nitrite prevents reactive oxygen species generation in the corpora cavernosa and restores intracavernosal pressure in hypertensive rats Publication date: 1 January 2020 Source: Nitric Oxide, Volume 94 Author(s): Natália A. Gonzaga, Gabriel T. do Vale, Carla B.P. da Silva, Lucas C. Pinheiro, Letícia N. Leite, Fernando S. Carneiro, José E. Tanus-Santos, Carlos R. Tirapelli Abstract Hypertension is a risk factor for erectile dysfunction (ED) and both conditions are associated with oxidative stress. Given that nitrite is described to display antioxidant effects, we hypothesized that treatment with nitrite would exert antioxidant effects attenuating both reactive oxygen species (ROS) generation in the corpora cavernosa (CC) and ED induced by hypertension. Two kidney, one clip (2K1C) hypertension was induced in male Wistar rats. Treatment with sodium nitrite (15 mg/kg/day, p.o., gavage) was initiated two weeks after surgery to induce hypertension and maintained for four weeks. Nitrite abrogated both the decrease in intracavernosal pressure and endothelial dysfunction of the CC induced by hypertension. Treatment with nitrite decreased hypertension-induced ROS generation in the CC assessed in situ using the fluorescent dye dihidroethidium (DHE) and with the lucigenin assay. Western immunoblotting analysis revealed that nitrite prevented the increase in Nox1 expression in the CC from 2K1C rats. Decreased concentrations of hydrogen peroxide (H2O2) were found in the CC from hypertensive rats and treatment with nitrite prevented this response. Treatment with nitrite increased the fluorescence of DAF-2DA in the CC from sham-operated rats and restored nitric oxide (NO) levels in the CC from 2K1C rats. In summary, we found novel evidence that nitrite reversed the decrease in intracavernosal pressure induced by 2K1C hypertension. This response was partially attributed to the antioxidant effect of nitrite that blunted ROS generation and endothelial dysfunction in the CC. In addition, nitrite-derived NO may have promoted direct protective actions against hypertension-induced CC dysfunction.