The Noncanonical Pathway for In Vivo Nitric Oxide Generation: The Nitrate-Nitrite-Nitric Oxide Pathway.

In contrast to nitric oxide, which has well established and important roles in the regulation of blood flow and thrombosis, neurotransmission, the normal functioning of the genitourinary system, and the inflammation response and host defense, its oxidized metabolites nitrite and nitrate have, until recently, been considered to be relatively inactive. However, this view has been radically revised over the past decade and more. Much evidence has now accumulated demonstrating that nitrite serves as a storage form of nitric oxide, releasing nitric oxide preferentially under acidic and/or hypoxic conditions but also occurring under physiologic conditions: a phenomenon that is catalyzed by a number of distinct mammalian nitrite reductases. Importantly, preclinical studies demonstrate that reduction of nitrite to nitric oxide results in a number of beneficial effects, including vasodilatation of blood vessels and lowering of blood pressure, as well as cytoprotective effects that limit the extent of damage caused by an ischemia/reperfusion insult, with this latter issue having been translated more recently to the clinical setting. In addition, research has demonstrated that the other main metabolite of the oxidation of nitric oxide (i.e., nitrate) can also be sequentially reduced through processing in vivo to nitrite and then nitrite to nitric oxide to exert a range of beneficial effects-most notably lowering of blood pressure, a phenomenon that has also been confirmed recently to be an effective method for blood pressure lowering in patients with hypertension. This review will provide a detailed description of the pathways involved in the bioactivation of both nitrate and nitrite in vivo, their functional effects in preclinical models, and their mechanisms of action, as well as a discussion of translational exploration of this pathway in diverse disease states characterized by deficiencies in bioavailable nitric oxide. SIGNIFICANCE STATEMENT: The past 15 years has seen a major revision in our understanding of the pathways for nitric oxide synthesis in the body with the discovery of the noncanonical pathway for nitric oxide generation known as the nitrate-nitrite-nitric oxide pathway. This review describes the molecular components of this pathway, its role in physiology, potential therapeutics of targeting this pathway, and their impact in experimental models, as well as the clinical translation (past and future) and potential side effects

Natural mutations of human XDH promote the nitrite (NO2 −)-reductase capacity of xanthine oxidoreductase: A novel mechanism to promote redox health?

Several rare genetic variations of human XDH have been shown to alter xanthine oxidoreductase (XOR) activity leading to impaired purine catabolism. However, XOR is a multi-functional enzyme that depending upon the environmental conditions also expresses oxidase activity leading to both O2·- and H2O2 and nitrite (NO2−) reductase activity leading to nitric oxide (·NO). Since these products express important, and often diametrically opposite, biological activity, consideration of the impact of XOR mutations in the context of each aspect of the biochemical activity of the enzyme is needed to determine the potential full impact of these variants. Herein, we show that known naturally occurring hXDH mutations do not have a uniform impact upon the biochemical activity of the enzyme in terms of uric acid (UA), reactive oxygen species (ROS) and nitric oxide ·NO formation. We show that the His1221Arg mutant, in the presence of xanthine, increases UA, O2·- and NO generation compared to the WT, whilst the Ile703Val increases UA and ·NO formation, but not O2·-. We speculate that this change in the balance of activity of the enzyme is likely to endow those carrying these mutations with a harmful or protective influence over health that may explain the current equipoise underlying the perceived importance of XDH mutations. We also show that, in presence of inorganic NO2−, XOR-driven O2·- production is substantially reduced. We suggest that targeting enzyme activity to enhance the NO2−-reductase profile in those carrying such mutations may provide novel therapeutic options, particularly in cardiovascular disease

Erratum: Antiplatelet effects of dietary nitrate in healthy volunteers: Involvement of cGMP and influence of sex (Free Radical Biology and Medicine (2013) 65 (1521-1532))

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Combined analysis of the safety of intra-coronary drug delivery during primary percutaneous coronary intervention for acute myocardial infarction: A study of three clinical trials.

Background: The local injection of novel cardioprotective study drugs prior to percutaneous coronary intervention could cause embolisation of thrombus, resulting in increased reperfusion injury and subsequent infarct size. The aim of this study was to assess the safety of the delivery of an intracoronary therapy delivered during primary percutaneous coronary intervention for acute myocardial infarction prior to the re-establishment of thrombolysis in myocardial infarction III flow. Methods: One hundred sixty-seven patients with acute myocardial infarction successfully reperfused through primary percutaneous coronary intervention and undergoing Cardiac MRI within the first week after reperfusion were studied. Patients either underwent the delivery of an intracoronary agent (IMP or placebo) prior to balloon dilatation (n = 80) or standard primary percutaneous coronary intervention procedure (n = 117). Results: Baseline characteristics were similar between the two groups. There were a similar number of successful procedures (IC IMP 75 (93.8%) vs. No IMP 114, (97.4%), p = 0.374), rates of no-reflow (IC IMP 1 (1.3%) vs. No IMP 2 (1.7%), p = 0.99) and levels of ST segment resolution (88.5% IC IMP vs. No IC IMP 87.0%, p = 0.669) between the two groups. Similar levels of microvascular obstruction were seen between the two groups with a trend to reduced infarct size, and improved ejection fractions in the IMP group. Lower MACE rates were seen in the IMP group. Conclusion: The local intracoronary infusion of potential cardioprotective agents prior to the restoration of TIMI flow in patients undergoing primary percutaneous coronary intervention for acute myocardial infarction appears to be safe and does not increase microvascular damage. This route should be considered when testing novel cardioprotective agents.The author would like to thank National Institute for Health Research, UK and Heart Cells Foundation for the financial suppo

Local delivery of nitric oxide prevents endothelial dysfunction in periodontitis

AIMS: Increased cardiovascular disease risk underlies elevated rates of mortality in individuals with periodontitis. A key characteristic of those with increased cardiovascular risk is endothelial dysfunction, a phenomenon synonymous with deficiencies of bioavailable nitric oxide (NO), and prominently expressed in patients with periodontitis. Also, inorganic nitrate can be reduced to NO in vivo to restore NO levels, leading us to hypothesise that its use may be beneficial in reducing periodontitis-associated endothelial dysfunction. Herein we sought to determine whether inorganic nitrate improves endothelial function in the setting of periodontitis and if so to determine the mechanisms underpinning any responses seen. METHOD AND RESULTS: Periodontitis was induced in mice by placement of a ligature for 14 days around the second molar. Treatment in vivo with potassium nitrate, either prior to or following establishment of experimental periodontitis, attenuated endothelial dysfunction, as determined by assessment of acetylcholine-induced relaxation of aortic rings, compared to control (potassium chloride treatment). These beneficial effects were associated with a suppression of vascular wall inflammatory pathways (assessed by quantitative-PCR), increases in the anti-inflammatory cytokine interleukin (IL)-10 and reduced tissue oxidative stress due to attenuation of xanthine oxidoreductase-dependent superoxide generation. In patients with periodontitis, plasma nitrite levels were not associated with endothelial function indicating dysfunction. CONCLUSION: Our results suggest that inorganic nitrate protects against, and can partially reverse pre-existing, periodontitis-induced endothelial dysfunction through restoration of nitrite and thus NO levels. This research highlights the potential of dietary nitrate as adjunct therapy to target the associated negative cardiovascular outcomes in patients with periodontitis

Inorganic nitrate attenuates cardiac dysfunction: roles for xanthine oxidoreductase and nitric oxide

Nitric oxide (NO) is a vasodilator and independent modulator of cardiac remodelling. Commonly, in cardiac disease (e.g. heart failure) endothelial dysfunction (synonymous with NO-deficiency) has been implicated in increased blood pressure (BP), cardiac hypertrophy and fibrosis. Currently no effective therapies replacing NO have succeeded in the clinic. Inorganic nitrate (NO3 - ), through chemical reduction to nitrite and then NO, exerts potent BP-lowering but whether it might be useful in treating undesirable cardiac remodelling is unknown. In a nested age- and sex-matched case-control study of hypertensive patients +/- left ventricular hypertrophy (NCT03088514) we show that lower plasma nitrite concentration and vascular dysfunction accompany cardiac hypertrophy and fibrosis in patients. In mouse models of cardiac remodelling, we also show that restoration of circulating nitrite levels using dietary nitrate improves endothelial dysfunction through targeting of xanthine oxidoreductase (XOR)-driven H2 O2 and superoxide, and reduces cardiac fibrosis through NO-mediated block of SMAD-phosphorylation leading to improvements in cardiac structure and function. We show that via these mechanisms dietary nitrate offers easily translatable therapeutic options for treatment of cardiac dysfunction

1, 9-Pyrazoloanthrones Downregulate HIF-1α and Sensitize Cancer Cells to Cetuximab-Mediated Anti-EGFR Therapy

Cetuximab, a monoclonal antibody that blocks the epidermal growth factor receptor (EGFR), is currently approved for the treatment of several types of solid tumors. We previously showed that cetuximab can inhibit hypoxia-inducible factor-1 alpha (HIF-1α) protein synthesis by inhibiting the activation of EGFR downstream signaling pathways including Erk, Akt, and mTOR. 1, 9-pyrazoloanthrone (1, 9 PA) is an anthrapyrazolone compound best known as SP600125 that specifically inhibits c-jun N-terminal kinase (JNK). Here, we report 1, 9 PA can downregulate HIF-1α independently of its inhibition of JNK. This downregulatory effect was abolished when the oxygen-dependent domain (ODD) of HIF-1α (HIF-1α-ΔODD, the domain responsible for HIF-1α degradation) was experimentally deleted or when the activity of HIF-1α prolyl hydroxylase (PHD) or the 26S proteasomal complex was inhibited, indicating that the 1, 9 PA downregulates HIF-1α by promoting PHD-dependent HIF-1α degradation. We found that the combination of 1, 9 PA and cetuximab worked synergistically to induce apoptosis in cancer cells in which cetuximab or 1, 9 PA alone had no or only weak apoptotic activity. This synergistic effect was substantially decreased in cancer cells transfected with HIF-1α-ΔODD, indicating that downregulation of HIF-1α was the mechanism of this synergistic effect. More importantly, 1, 9 PA can downregulate HIF-1α in cancer cells that are insensitive to cetuximab-induced inhibition of HIF-1α expression due to overexpression of oncogenic Ras (RasG12V). Our findings suggest that 1, 9 PA is a lead compound of a novel class of drugs that may be used to enhance the response of cancer cells to cetuximab through a complementary effect on the downregulation of HIF-1α

Intracoronary nitrite suppresses the inflammatory response following primary percutaneous coronary intervention

© 2016 BMJ Publishing Group Ltd & British Cardiovascular Society. Objective Recent work suggests that intracoronary nitrite reduces myocardial infarct size following primary percutaneous coronary intervention (PPCI) for acute myocardial infarction (AMI), although the exact mechanisms are unclear. We explored the effects of nitrite on reperfusion-induced inflammation, by assessing the levels of specific pro-inflammatory mediators, chemokines and adhesion molecules in plasma and circulating cell subtypes as exploratory end points in the NITRITE-AMI cohort. Methods Peripheral blood leucocyte subsets, cell adhesion molecules, high-sensitivity C reactive protein (hs-CRP), the monocyte and neutrophil chemoattractants CCL2 and CXCL1, CXCL5, respectively were measured in the blood of patients who received either intracoronary sodium nitrite (N=40) or placebo (N=40) during PPCI for AMI. Major adverse cardiac events were recorded at 3 years post-PPCI. Results In the placebo-treated patients, total circulating neutrophil numbers and levels of hs-CRP were raised postreperfusion and then decreased over time; in nitritetreated patients these changes were suppressed compared with placebo up to 6 months post-PPCI (p < 0.01). This effect was associated with reduced expression of neutrophil CD11b, plasma CXCL1, CXCL5 and CCL2 levels (p < 0.05). There were no differences in the number of other any other leucocyte population measured (monocytes and lymphocytes) or activation markers expressed by these cells between the treatment groups. These effects were associated with a reduction in both microvascular obstruction and infarct size. Conclusions Important reductions in neutrophil numbers and activation post-PPCI in patients with ST elevated myocardial infarction were associated with nitrite treatment, an effect we propose likely underlies, at least in part, the beneficial effects of nitrite upon infarct size