Shanti Velmurugan

Antiplatelet effects of dietary nitrate in healthy volunteers: involvement of cGMP and influence of sex.

Ingestion of vegetables rich in inorganic nitrate has emerged as an effective method, via the formation of a nitrite intermediate, for acutely elevating vascular NO levels. As such a number of beneficial effects of dietary nitrate ingestion have been demonstrated including the suggestion that platelet reactivity is reduced. In this study we investigated whether inorganic nitrate supplementation might also reduce platelet reactivity in healthy volunteers and have determined the mechanisms involved in the effects seen. We conducted two randomised crossover studies each in 24 (12 of each sex) healthy subjects assessing the acute effects of dietary nitrate (250 ml beetroot juice) or potassium nitrate capsules (KNO3, 8 mmol) vs placebo control on platelet reactivity. Inorganic nitrate ingested either from a dietary source or via supplementation raised circulating nitrate and nitrite levels in both sexes and attenuated ex vivo platelet aggregation responses to ADP and, albeit to a lesser extent, collagen but not epinephrine in male but not female volunteers. These inhibitory effects were associated with a reduced platelet P-selectin expression and elevated platelet cGMP levels. In addition, we show that nitrite reduction to NO occurs at the level of the erythrocyte and not the platelet. In summary, our results demonstrate that inorganic nitrate ingestion, whether via the diet or through supplementation, causes a modest decrease in platelet reactivity in healthy males but not females. Our studies provide strong support for further clinical trials investigating the potential of dietary nitrate as an adjunct to current antiplatelet therapies to prevent atherothrombotic complications. Moreover, our observations highlight a previously unknown sexual dimorphism in platelet reactivity to NO and intimate a greater dependence of males on the NO-soluble guanylate cyclase pathway in limiting thrombotic potential.

Dietary nitrate improves vascular function in patients with hypercholesterolemia: a randomized, double-blind, placebo-controlled study

Background: The beneficial cardiovascular effects of vegetables may be underpinned by their high inorganic nitrate content. Objective: We sought to examine the effects of a 6-wk once-daily intake of dietary nitrate (nitrate-rich beetroot juice) compared with placebo intake (nitrate-depleted beetroot juice) on vascular and platelet function in untreated hypercholesterolemics. Design: A total of 69 subjects were recruited in this randomized, double-blind, placebo-controlled parallel study. The primary endpoint was the change in vascular function determined with the use of ultrasound flow-mediated dilatation (FMD). Results: Baseline characteristics were similar between the groups, with primary outcome data available for 67 patients. Dietary nitrate resulted in an absolute increase in the FMD response of 1.1% (an ∼24% improvement from baseline) with a worsening of 0.3% in the placebo group (P < 0.001). A small improvement in the aortic pulse wave velocity (i.e., a decrease of 0.22 m/s; 95% CI: −0.4, −0.3 m/s) was evident in the nitrate group, showing a trend (P = 0.06) to improvement in comparison with the placebo group. Dietary nitrate also caused a small but significant reduction (7.6%) in platelet-monocyte aggregates compared with an increase of 10.1% in the placebo group (P = 0.004), with statistically significant reductions in stimulated (ex vivo) P-selectin expression compared with the placebo group (P < 0.05) but no significant changes in unstimulated expression. No adverse effects of dietary nitrate were detected. The composition of the salivary microbiome was altered after the nitrate treatment but not after the placebo treatment (P < 0.01). The proportions of 78 bacterial taxa were different after the nitrate treatment; of those taxa present, 2 taxa were responsible for >1% of this change, with the proportions of Rothia mucilaginosa trending to increase and Neisseria flavescens (P < 0.01) increased after nitrate treatment relative to after placebo treatment. Conclusions: Sustained dietary nitrate ingestion improves vascular function in hypercholesterolemic patients. These changes are associated with alterations in the oral microbiome and, in particular, nitrate-reducing genera. Our findings provide additional support for the assessment of the potential of dietary nitrate as a preventative strategy against atherogenesis in larger cohorts. This trial was registered at clinicaltrials.gov as NCT01493752.

Randomized Phase 2 Trial of Intracoronary Nitrite During Acute Myocardial Infarction

Rationale: Preclinical evidence demonstrates that inorganic nitrite, after its in situ conversion to nitric oxide, attenuates consequent myocardial reperfusion injury. Objective: We investigated whether intracoronary injection of nitrite during primary percutaneous coronary intervention might improve infarct size in ST-elevated myocardial infarction. Methods and Results: Patients undergoing primary percutaneous coronary intervention (n=80) were randomized to receive intracoronary (10 mL) sodium nitrite (1.8 &mgr;mol) or NaCl (placebo) before balloon inflation. The primary end point was infarct size assessed by measuring creatine kinase release. Secondary outcomes included infarct size assessed by troponin T release and by cardiac MRI on day 2. Baseline characteristics were similar between the groups. No evidence of differences in creatine kinase release (P=0.92), troponin T (P=0.85), or cardiac MRI–assessed infarct size (P=0.254) were evident. In contrast, there was an improvement in myocardial salvage index (P=0.05) and reduction in major adverse cardiac event at 1 year (2.6% versus 15.8%; P=0.04) in the nitrite group. In a 66-patient subgroup with thrombolysis in myocardial infarction ⩽1 flow, there was reduced serum creatine kinase (P=0.030) and a 19% reduction in cardiac MRI–determined infarct size (P=0.034) with nitrite. No adverse effects of nitrite were detected. Conclusions: In this phase II study, intracoronary nitrite infusion did not alter infarct size, although a trend to improved myocardial salvage index and a significant reduction in major adverse cardiac event was evident. In a subgroup of patients with thrombolysis in myocardial infarction flow ⩽1, nitrite reduced infarct size and major adverse cardiac event and improved myocardial salvage index, indicating that a phase III clinical trial assessing intracoronary nitrite administration as an adjunct to percutaneous coronary intervention in ST-elevated myocardial infarction patients is warranted. Clinical Trial Registration: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01584453.

Accelerated resolution of inflammation underlies sex differences in inflammatory responses in humans

BACKGROUND. Cardiovascular disease occurs at lower incidence in premenopausal females compared with age-matched males. This variation may be linked to sex differences in inflammation. We prospectively investigated whether inflammation and components of the inflammatory response are altered in females compared with males. METHODS. We performed 2 clinical studies in healthy volunteers. In 12 men and 12 women, we assessed systemic inflammatory markers and vascular function using brachial artery flow-mediated dilation (FMD). In a further 8 volunteers of each sex, we assessed FMD response to glyceryl trinitrate (GTN) at baseline and at 8 hours and 32 hours after typhoid vaccine. In a separate study in 16 men and 16 women, we measured inflammatory exudate mediators and cellular recruitment in cantharidin-induced skin blisters at 24 and 72 hours. RESULTS. Typhoid vaccine induced mild systemic inflammation at 8 hours, reflected by increased white cell count in both sexes. Although neutrophil numbers at baseline and 8 hours were greater in females, the neutrophils were less activated. Systemic inflammation caused a decrease in FMD in males, but an increase in females, at 8 hours. In contrast, GTN response was not altered in either sex after vaccine. At 24 hours, cantharidin formed blisters of similar volume in both sexes; however, at 72 hours, blisters had only resolved in females. Monocyte and leukocyte counts were reduced, and the activation state of all major leukocytes was lower, in blisters of females. This was associated with enhanced levels of the resolving lipids, particularly D-resolvin. CONCLUSIONS. Our findings suggest that female sex protects against systemic inflammation-induced endothelial dysfunction. This effect is likely due to accelerated resolution of inflammation compared with males, specifically via neutrophils, mediated by an elevation of the D-resolvin pathway. TRIAL REGISTRATION. ClinicalTrials.gov NCT01582321 and NRES: City Road and Hampstead Ethics Committee: 11/LO/2038. FUNDING. The authors were funded by multiple sources, including the National Institute for Health Research, the British Heart Foundation, and the European Research Council.

A ‘green’ diet-based approach to cardiovascular health? Is inorganic nitrate the answer?

Ingestion of fruit and vegetables rich in inorganic nitrate (NO(3)(-)) has emerged as an effective method for acutely elevating vascular nitric oxide (NO) levels through formation of an NO(2)(-) intermediate. As such a number of beneficial effects of NO(3)(-) and NO(2)(-) ingestion have been demonstrated including reductions in blood pressure, measures of arterial stiffness and platelet activity. The pathway for NO generation from such dietary interventions involves the activity of facultative oral microflora that facilitate the reduction of inorganic NO(3)(-), ingested in the diet, to inorganic NO(2)(-). This NO(2)(-) then eventually enters the circulation where, through the activity of one or more of a range of distinct NO(2)(-) reductases, it is chemically reduced to NO. This pathway provides an alternative route for in vivo NO generation that could be utilized for therapeutic benefit in those cardiovascular disease states where reduced bioavailable NO is thought to contribute to pathogenesis. Indeed, the cardiovascular benefits of NO(2)(-) and NO(3)(-) are now starting to be translated in patients in several clinical trials. In this review, we discuss recent evidence supporting the potential utility of delivery of NO(3)(-) or NO(2)(-) for the treatment of cardiovascular diseases.

Erratum to "Antiplatelet effects of dietary nitrate in healthy volunteers: Involvement of cGMP and influence of sex" [Free Radic. Biol. Med. 65 (2013) 1521-1532]

© 2015, Elsevier. Licensed under the Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International http://creativecommons.org/licenses/by-nc-nd/4.0/

Sex differences in the nitrate-nitrite-NO• pathway: Role of oral nitrate-reducing bacteria

Abstract Oral reduction of nitrate to nitrite is dependent on the oral microbiome and is the first step of an alternative mammalian pathway to produce nitric oxide in humans. Preliminary evidence suggests important sex differences in this pathway. We prospectively investigated sex‐differences following inorganic nitrate supplementation on nitrate/nitrite levels and vascular function, and separately examined sex differences in oral nitrate reduction, and oral microbiota by 16S rRNA profiling. At baseline, females exhibit higher nitrite levels in all biological matrices despite similar nitrate levels to males. Following inorganic nitrate supplementation, plasma nitrite was increased to a significantly greater extent in females than in males and pulse wave velocity was only reduced in females. Females exhibited higher oral bacterial nitrate‐reducing activity at baseline and after nitrate supplementation. Despite these differences, there were no differences in the composition of either the total salivary microbiota or those oral taxa with nitrate reductase genes. Our results demonstrate that females have augmented oral nitrate reduction that contributes to higher nitrite levels at baseline and also after inorganic nitrate supplementation, however this was not associated with differences in microbial composition (clinicaltrials.gov: NCT01583803). Graphical abstract Figure. No caption available. HighlightsSex differences are apparent in nitrate‐nitrite‐nitric oxide pathway under basal conditions.Females have higher nitrite levels at baseline and after nitrate supplementation.Nitrate reduction to nitrite is dependent on oral microbiota.Females have greater oral nitrate reduction capability.This difference is not due to differences in oral microbiome community composition.

Sex differences in the inflammatory response and inflammation-induced vascular dysfunction

Abstract Background Premenopausal women have a lower incidence of cardiovascular disease, although the exact mechanism underlying this protection is unclear. Both systemic and localised inflammation have a crucial role in the progression of cardiovascular disease, and much preclinical and observational data in human beings suggest that differences in inflammation between the sexes exist. We investigated whether inflammation, and which components of the inflammatory response, might be altered in women compared with men. Methods We performed two clinical studies with 24 and 32 healthy volunteers. In 12 men and 12 women (mean age 26·0 years [SD 5·7] and 24·7 [6·8], respectively), we assessed systemic inflammatory markers and vascular function using brachial artery flow-mediated dilation (FMD) determined with ultrasound. Responses were assessed before administration of typhoid vaccine and then at 8 h and 32 h afterwards. In another study, in 16 male and 16 female volunteers (mean age 27·4 years [SD 1·1] and 26·8 [1·1], respectively), inflammatory exudate and cellular recruitment were measured at 24 h (acute) and 72 h (resolution) in skin blisters induced with cantharidin. Ethics approval was given by NRES: City Road and Hampstead Ethics Committee (11/LO/2038) for both studies. Both studies are registered with ClinicalTrials.gov, number NCT01582321. Findings Typhoid vaccine caused a mild systemic inflammation, which was associated with a trend to decreased FMD in men and an increased response in women compared with baseline (p=0·006). By 24 h cantharidin induced a fluid-filled blister of a similar volume in both sexes; however, after 72 h blisters had resolved only in women (p=0·003). At 24 h there was a significant reduction in both monocyte (p=0·003) and lymphocyte count (p=0·011) in blisters in women compared with those in men. A generalised reduction in the activation state of all major leucocytes including neutrophils was evident in women. These differences in cell recruitment and activation were associated with higher proresolving mediators, including the D-resolvins, and a reduction in concentrations of the neutrophil chemoattractant leukotriene B4. Interpretation Our findings suggest that female sex protects against endothelial dysfunction induced by systemic inflammation. This effect is probably due to a rapid resolution of inflammation in women specifically targeting the neutrophil through elevation of the D-resolvin pathway. Funding KR receives doctoral research fellowship funding from the National Institute for Health Research. JD receives funding from the European Research Council under the European Unions Horizon 2020 research and innovation programme (grant agreement 677542), and is also supported by a Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (grant 107613/Z/15/Z).