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The discovery of nitric oxide and its role in almost every facet of human biology opened a new avenue for treatment through manipulation of its canonical signaling and by attempts to augment endogenous nitric oxide generation through provision of substrate and co-factors to the endothelial nitric oxide synthase complex. This has been particularly so in the cardiovascular system and it is well recognized that there is reduced bioavailable nitric oxide in patients with both cardiovascular risk factors and manifest vascular disease. However, these attempts have failed to deliver the expected benefits of such an approach. Recently, an alternative pathway for nitric oxide synthesis has been elucidated that can produce authentic nitric oxide from the 1 electron reduction of inorganic nitrite. Furthermore, it has long been known that symbiotic, facultative, oral microflora can facilitate the reduction of inorganic nitrate, that is ingested in the average diet in millimolar amounts, to inorganic nitrite itself. Thus, there exists an alternative reductive pathway from nitrate, via nitrite as an intermediate, to nitric oxide that provides a novel pathway that may be amenable to therapeutic manipulation. As such, various research groups have explored the utility of manipulation of this nitrate–nitrite–nitric oxide pathway in situations in which nitric oxide is known to have a prominent role. Animal and early-phase human studies of both inorganic nitrite and nitrate supplementation have shown beneficial effects in blood pressure control, platelet function, vascular health and exercise capacity. This review considers in detail the pathways of inorganic nitrate bioactivation and the evidence of clinical utility to date on the cardiovascular system.

Nitrite is protective against renal ischemia/reperfusion injury (IRI); an effect due to its reduction to nitric oxide (NO). In addition to other reductase pathways, endothelial NO synthase (eNOS) may also facilitate nitrite reduction in ischemic environments. We investigated the role of eNOS in sodium nitrite (60 microM, 10 ml/kg applied topically 1 min before reperfusion)-induced protection against renal IRI in C57/BL6 wild-type (WT) and eNOS knockout (eNOS KO) mice subjected to bilateral renal ischemia (30 min) and reperfusion (24h). Markers of renal dysfunction (plasma [creatinine] and [urea]), damage (tubular histology) and inflammation (cell recruitment) were elevated following IRI in WT mice; effects significantly reduced following nitrite treatment. Chemiluminescence analysis of cortical and medullary sections of the kidney demonstrated rapid (within 1 min) distribution of nitrite following application. Whilst IRI caused a significant (albeit substantially reduced compared to WT mice) elevation of markers of renal dysfunction and damage in eNOS KO mice, the beneficial effects of nitrite were absent or reduced, respectively. Moreover, nitrite treatment enhanced renal dysfunction in the form of increased plasma [creatinine] in eNOS KO mice. Confirmation of nitrite reductase activity of eNOS was provided by demonstration of nitrite (100 microM)-derived NO production by kidney homogenates of WT mice, that was significantly reduced by L-NMMA. L-NMMA was without effect using kidney homogenates of eNOS KO mice. These results support a role for eNOS in the pathways activated during renal IRI and also identify eNOS as a nitrite reductase in ischemic conditions; activity which in part underlies the protective effects of nitrite.

Within the human circulation physiological levels of nitrite can act as a source of NO and as such exert important vasodilator effects (Cosby et al., JCI 9 (2003) 1498–1505). Our previous studies have shown that purified red blood cells (RBCs) collected from healthy volunteers facilitate nitrite reduction at pH 7.4 and 6.8. Moreover, at pH 7.4, nitrite reduction was dependent upon activity of endothelial nitric oxide synthase (eNOS), whilst at pH 6.8 xanthine oxidoreductase (XOR) played an important role (Webb et al., Circ. Res. 103 (2008) 957–964). Whether such a profile is similarly evident in cardiovascular disease is unknown. Therefore, we investigated red blood cell (RBC) nitrite reductase activity in the spontaneously hypertensive rat (SHR) and Wistar Kyoto (WKY) controls and in patients with hypertension. Nitrite (10–300 μM) caused concentration-dependent NO production in the presence of RBCs. This effect was greater with RBCs collected from SHR compared to WKY at pH 7.4 but not 6.8 (48.5 ± 6.1% increase, P

Vegetables possessing high levels of inorganic nitrate provide a source of vasoprotective nitric oxide, via generation of nitrite intermediate, that attenuates ex-vivo stimulated platelet aggregation. We investigated whether potassium nitrate supplementation might replicate these effects. Twenty-four healthy volunteers (12 men and 12 women) consented to participate in a randomised double-blind cross-over study of potassium nitrate (KNO3, 8 mmol) vs. potassium chloride (KCl, 8 mmol placebo) capsules. Blood was collected before and 3 h following intervention. Ozone chemiluminescence assessment of plasma demonstrated elevations of circulating [nitrite] above baseline at 3 h (males 0.35 ± 0.49 vs 0.54 ± 0.02 μM, P KNO 3 supplementation causes a rise in circulating nitrite levels in both sexes but attenuates platelet reactivity of male but not female volunteers. Moreover, we find that nitrite activity on platelets requires the presence of whole blood. Our findings suggest that nitrate supplementation may provide an additional option in anti-platelet medication.

Background Percutaneous coronary intervention (PCI) is essential following myocardial infarction to restore cardiac perfusion. However, in the long term, the beneficial effects of PCI with stent insertion are limited by restenosis (associated with vascular injury and smooth muscle cell proliferation) and stent thrombosis (associated with inhibition of/delayed re-endothelialisation). Pre-clinical studies suggest that vascular injury and intimal hyperplasia are associated with lack of endogenous nitric oxide (NO) and that nitrite, via its conversion to NO decreases intimal hyperplasia following vascular injury (Alef et al., J. Clin. Invest. 121 (2011) 1646–1656). The aim of this study was to determine the effect of nitrite on the proliferation/viability of EC and VSMC in normoxia, hypoxia and ischaemia/reperfusion (I/R). Methods Human umbilical vein (HUVEC) and coronary artery (HCAEC) endothelial cells and aortic VSMC (HAVSMC) were incubated for 24 h in the absence or presence of nitrite (0.1–10 μM) under normoxic (21% O2) or hypoxic (1–5% O2) conditions. In a separate series of experiments, ECs were subjected to 24 h of 0.1% O2 followed by 48 h of normoxia to simulate I/R injury. Cell proliferation (cell count) and cell viability (flow cytometry with apoptosis assay-staining for annexin V/propidium iodide) were determined in control cells or cells treated with inhibitors of two potential nitrite reductases: xanthine oxidoreductase and endothelial nitric oxide synthase (eNOS) using allopurinol (100 μM) or l -NMMA (300 μM), respectively. Results Hypoxia attenuated proliferation of HUVECs with 5% O2 causing a 21.2 ± 2.8% and 1% O2 a 24.1 ± 3.1% reduction (P l -NMMA by ∼85% (P Conclusion These studies demonstrate that nitrite protects ECs from the damaging effects of ischaemia or I/R injury but exerts an opposing repressive influence over smooth muscle cell growth. These results suggest that in an environment where improved EC growth but repressed SMC growth is desired, such as in restenosis following stent implantation, raising circulating nitrite levels may be of therapeutic utility. D.A.J. and this work are funded by an NIHR Doctoral Fellowship. Disclosure Supported by NIHR Doctoral Research fellowship.

Within the healthy human circulation nitrite acts as a source of NO exerting important vasodilator and blood pressure (BP)-lowering effects. In vitro studies demonstrate enhanced nitrite-derived NO generation under pathological conditions, where conventional l -arginine-derived NO synthesis is impaired. Whether nitrite-induced hypotension and reduction to NO might be enhanced in hypertension (where conventional NO synthesis is impaired) is unknown. We compared the effects of nitrite on BP in vivo and vascular reactivity in vitro in Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR). Nitrite (1–30,000 nmol/kg) caused dose-dependent decrease in BP that was enhanced in SHR compared to WKY anaesthetised rats (max decrease of 32.0 ± 6.0 and 6.3 ± 1.9 mmHg respectively, P In sum, we show that nitrite-induced decreases in blood pressure are enhanced in hypertension and likely due to XOR-mediated reduction of nitrite to NO that does not occur at the level of the blood vessel wall.

In the healthy blood vessel tonic endothelium-derived nitric oxide (NO) plays an essential anti-inflammatory role, in part by inhibiting leukocyte–endothelial interactions. In atherosclerosis, a chronic inflammatory disorder characterised by leukocyte infiltration and plaque formation, NO bioavailability is reduced. This reduction characterises the pro-inflammatory endothelium that is thought to be key in precipitating plaque formation. Provision of nitrite ( NO 2 - ) to restore the levels of bioavailable NO, via vascular NO 2 - reduction, may be a therapeutic approach to augment bioavailable NO levels. Intravital microscopy of mouse cremaster venules was used to determine the effect of NO 2 - (1, 3 and 10 μM) on leukocyte rolling, adhesion and emigration in 6 and 12 week old wild type (WT) and Apolipoprotein (Apo) E knockout (KO) littermate mice, an atherosclerotic prone mouse model. Superfusion of NO 2 - caused a concentration-dependent reduction in leukocyte rolling (basal, 18.7 ± 2.9 cells/min, max effect 60 ± 10% inhibition, n = 6, P NO 2 - had no effect in age matched WT mice (basal rolling of 32.3 ± 9.5 cells/min in 6 week and 42.9 ± 9.1 cells/min in 12 week old mice). At both ages, leukocyte adhesion and emigration were comparable between the genotypes. In summary, our results suggest that in the ApoE KO mouse model NO 2 - reduction is enhanced resulting in an up-regulation of NO 2 - to NO conversion, leading to a reduction in leukocyte activation.

Highlights ► Inorganic nitrate supplementation does not alter endothelial function in healthy volunteers. ► Despite this, there was a reduction in blood pressure and improvement in arterial compliance. ► Improvements in vascular function are likely to contribute to the beneficial effects of inorganic nitrate on blood pressure., Ingestion of inorganic nitrate elevates blood and tissue levels of nitrite via bioconversion in the entero-salivary circulation. Nitrite is converted to NO in the circulation, and it is this phenomenon that is thought to underlie the beneficial effects of inorganic nitrate in humans. Our previous studies have demonstrated that oral ingestion of inorganic nitrate decreases blood pressure and inhibits the transient endothelial dysfunction caused by ischaemia–reperfusion injury in healthy volunteers. However, whether inorganic nitrate might improve endothelial function per se in the absence of a pathogenic stimulus and whether this might contribute to the blood pressure lowering effects is yet unknown. We conducted a randomised, double-blind, crossover study in 14 healthy volunteers to determine the effects of oral inorganic nitrate (8 mmol KNO3) vs. placebo (8 mmol KCl) on endothelial function, measured by flow-mediated dilatation (FMD) of the brachial artery, prior to and 3 h following capsule ingestion. In addition, blood pressure (BP) was measured and aortic pulse wave velocity (aPWV) determined. Finally, blood, saliva and urine samples were collected for chemiluminescence analysis of [nitrite] and [nitrate] prior to and 3 h following interventions. Inorganic nitrate supplementation had no effect on endothelial function in healthy volunteers (6.9 ± 1.1% pre- to 7.1 ± 1.1% post-KNO3). Despite this, there was a significant elevation of plasma [nitrite] (0.4 ± 0.1 μM pre- to 0.7 ± 0.2 μM post-KNO3, p