Inhaled nitric oxide suppresses neuroinflammation in experimental ischemic stroke.

Ischemic stroke is a major global health issue and characterized by acute vascular dysfunction and subsequent neuroinflammation. However, the relationship between these processes remains elusive. In the current study, we investigated whether alleviating vascular dysfunction by restoring vascular nitric oxide (NO) reduces post-stroke inflammation. Mice were subjected to experimental stroke and received inhaled NO (iNO; 50 ppm) after reperfusion. iNO normalized vascular cyclic guanosine monophosphate (cGMP) levels, reduced the elevated expression of intercellular adhesion molecule-1 (ICAM-1), and returned leukocyte adhesion to baseline levels. Reduction of vascular pathology significantly reduced the inflammatory cytokines interleukin-1ß (Il-1ß), interleukin-6 (Il-6), and tumor necrosis factor-α (TNF-α), within the brain parenchyma. These findings suggest that vascular dysfunction is responsible for leukocyte adhesion and that these processes drive parenchymal inflammation. Reversing vascular dysfunction may therefore emerge as a novel approach to diminish neuroinflammation after ischemic stroke and possibly other ischemic disorders.

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. METHODS 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.

Antiinflammatory actions of inorganic nitrate stabilize the atherosclerotic plaque.

Reduced bioavailable nitric oxide (NO) plays a key role in the enhanced leukocyte recruitment reflective of systemic inflammation thought to precede and underlie atherosclerotic plaque formation and instability. Recent evidence demonstrates that inorganic nitrate (NO3-) through sequential chemical reduction in vivo provides a source of NO that exerts beneficial effects upon the cardiovascular system, including reductions in inflammatory responses. We tested whether the antiinflammatory effects of inorganic nitrate might prove useful in ameliorating atherosclerotic disease in Apolipoprotein (Apo)E knockout (KO) mice. We show that dietary nitrate treatment, although having no effect upon total plaque area, caused a reduction in macrophage accumulation and an elevation in smooth muscle accumulation within atherosclerotic plaques of ApoE KO mice, suggesting plaque stabilization. We also show that in nitrate-fed mice there is reduced systemic leukocyte rolling and adherence, circulating neutrophil numbers, neutrophil CD11b expression, and myeloperoxidase activity compared with wild-type littermates. Moreover, we show in both the ApoE KO mice and using an acute model of inflammation that this effect upon neutrophils results in consequent reductions in inflammatory monocyte expression that is associated with elevations of the antiinflammatory cytokine interleukin (IL)-10. In summary, we demonstrate that inorganic nitrate suppresses acute and chronic inflammation by targeting neutrophil recruitment and that this effect, at least in part, results in consequent reductions in the inflammatory status of atheromatous plaque, and suggest that this effect may have clinical utility in the prophylaxis of inflammatory atherosclerotic disease.

A double-blind, randomised, placebo-controlled parallel study to investigate the effect of sex and dietary nitrate on COVID-19 vaccine-induced vascular dysfunction in healthy men and women: protocol of the DiNOVasc-COVID-19 study.

BACKGROUND: Cardiovascular events, driven by endothelial dysfunction, are a recognised complication of COVID-19. SARS-CoV-2 infections remain a persistent concern globally, and an understanding of the mechanisms causing endothelial dysfunction, particularly the role of inflammation, nitric oxide, and whether sex differences exist in this response, is lacking. We have previously demonstrated important sex differences in the inflammatory response and its impact on endothelial function and separately that the ingestion of inorganic nitrate can protect the endothelium against this dysfunction. In this study, we will investigate whether sex or a dietary inorganic nitrate intervention modulates endothelial function and inflammatory responses after the COVID-19 vaccine. METHODS: DiNOVasc-COVID-19 is a double-blind, randomised, single-centre, placebo-controlled clinical trial. A total of 98 healthy volunteers (49 males and 49 females) will be recruited. Participants will be randomised into 1 of 2 sub-studies: part A or part B. Part A will investigate the effects of sex on vascular and inflammatory responses to the COVID-19 vaccine. Part B will investigate the effects of sex and dietary inorganic nitrate on vascular and inflammatory responses to the COVID-19 vaccine. In part B, participants will be randomised to receive 3 days of either nitrate-containing beetroot juice (intervention) or nitrate-deplete beetroot juice (placebo). The primary outcome for both sub-studies is a comparison of the change in flow-mediated dilatation (FMD) from baseline after COVID-19 vaccination. The study has a power of > 80% to assess the primary endpoint. Secondary endpoints include change from baseline in inflammatory and leukocyte counts and in pulse wave analysis (PWA) and pulse wave velocity (PWV) following the COVID-19 vaccination. DISCUSSION: This study aims to evaluate whether sex or dietary influences endothelial function and inflammatory responses in healthy volunteers after receiving the COVID-19 vaccine. TRIAL REGISTRATION: ClinicalTrials.gov NCT04889274. Registered on 5 May 2023. The study was approved by the South Central - Oxford C Research Ethics Committee (21/SC/0154).

Neuropilin-1 interacts with VE-cadherin and TGFBR2 to stabilize adherens junctions and prevent activation of endothelium under flow.

Linear and disturbed flow differentially regulate gene expression, with disturbed flow priming endothelial cells (ECs) for a proinflammatory, atheroprone expression profile and phenotype. Here, we investigated the role of the transmembrane protein neuropilin-1 (NRP1) in ECs exposed to flow using cultured ECs, mice with an endothelium-specific knockout of NRP1, and a mouse model of atherosclerosis. We demonstrated that NRP1 was a constituent of adherens junctions that interacted with VE-cadherin and promoted its association with p120 catenin, stabilizing adherens junctions and inducing cytoskeletal remodeling in alignment with the direction of flow. We also showed that NRP1 interacted with transforming growth factor-ß (TGF-ß) receptor II (TGFBR2) and reduced the plasma membrane localization of TGFBR2 and TGF-ß signaling. NRP1 knockdown increased the abundance of proinflammatory cytokines and adhesion molecules, resulting in increased leukocyte rolling and atherosclerotic plaque size. These findings describe a role for NRP1 in promoting endothelial function and reveal a mechanism by which NRP1 reduction in ECs may contribute to vascular disease by modulating adherens junction signaling and promoting TGF-ß signaling and inflammation.

Suppression of endothelial P-selectin expression contributes to reduced cell trafficking in females: an effect independent of NO and prostacyclin.

OBJECTIVE: Sex hormones underlie the lower incidence of cardiovascular disease in premenopausal women. Vascular inflammation is involved in the pathogenesis of several cardiovascular diseases and it has been reported that sex hormones modulate inflammatory responses but mechanisms responsible for these effects are not yet fully established. Herein, we assessed whether sex differences in leukocyte recruitment might exist and investigated the underlying mechanisms involved in this response. METHODS AND RESULTS: Treatment with interleukin-1ß (IL-1ß) or tumor necrosis factor-α caused leukocyte rolling, adhesion, and emigration in mesenteric postcapillary venules in vivo that was substantially reduced in female mice compared with male mice; this difference was abolished by ovariectomy and partially restored by estrogen replacement. Deletion of endothelial nitric oxide (NO) synthase or cyclooxygenase-1 alone or in combination did not alter the leukocyte recruitment in IL-1ß-treated females but significantly enhanced this response in male mice. Treatment of murine pulmonary endothelial cells with IL-1ß increased expression of P-selectin in male but not female cells. CONCLUSIONS: We have demonstrated a profound estrogen-dependent and NO and prostacyclin-independent suppression of leukocyte recruitment in females.

20-hydroxyeicosatetraenoic acid (20-HETE) is a pivotal endogenous ligand for TRPV1-mediated neurogenic inflammation in the skin.

BACKGROUND AND PURPOSE: Transient receptor potential cation channel subfamily V member 1 (TRPV1) is localized to sensory C-fibres and its opening leads to membrane depolarization, resulting in neuropeptide release and neurogenic inflammation. However, the identity of the endogenous activator of TRPV1 in this setting is unknown. The arachidonic acid metabolites 12-hydroperoxyeicosatetraenoyl acid (12-HpETE) and 20-hydroxyeicosatetraenoic acid (20-HETE) have emerged as potential endogenous activators of TRPV1. However, whether these lipids underlie TRPV1-mediated neurogenic inflammation remains unknown. EXPERIMENTAL APPROACH: We analysed human cantharidin-induced blister samples and inflammatory responses in TRPV1 transgenic mice. KEY RESULTS: In a human cantharidin-blister model, the potent TRPV1 activators 20-HETE but not 12-HETE (stable metabolite of 12-HpETE) correlated with arachidonic acid levels. Similarly, in mice, levels of 20-HETE (but not 12-HETE) and arachidonic acid were strongly positively correlated within the inflammatory milieu. Furthermore, LPS-induced oedema formation and neutrophil recruitment were substantially and significantly attenuated by pharmacological block or genetic deletion of TRPV1 channels, inhibition of 20-HETE formation or SP receptor neurokinin 1 (NK1 ) blockade. LPS treatment also increased cytochrome P450 ω-hydroxylase gene expression, the enzyme responsible for 20-HETE production. CONCLUSION AND IMPLICATIONS: Taken together, our findings suggest that endogenously generated 20-HETE activates TRPV1 causing C-fibre activation and consequent oedema formation. These findings identify a novel pathway that may be useful in the therapeutics of diseases/conditions characterized by a prominent neurogenic inflammation, as in several skin diseases.

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

BACKGROUND AND PURPOSE: 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 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 to NO, exerts potent BP lowering, but whether it might be useful in treating undesirable cardiac remodelling is not known. EXPERIMENTAL APPROACH: We analysed demographics in a nested age- and sex-matched case-control study of hypertensive patients with or without left ventricular hypertrophy (NCT03088514) and assessed the effects of dietary nitrate in mouse models of cardiac dysfunction. KEY RESULTS: Lower plasma nitrite concentrations and vascular dysfunction accompanied cardiac hypertrophy and fibrosis in patients. In mouse models of cardiac remodelling, restoration of circulating nitrite levels using dietary nitrate improved endothelial dysfunction through targeting the xanthine oxidoreductase-driven increase in levels of H2 O2 and superoxide, and decreased cardiac fibrosis through NO-mediated block of SMAD phosphorylation leading to improvements in cardiac structure and function. CONCLUSIONS AND IMPLICATIONS: Dietary nitrate offers easily translatable therapeutic options for delivery of NO and thereby treatment of cardiac dysfunction.

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

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).

Intracoronary nitrite suppresses the inflammatory response following primary percutaneous coronary intervention.

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 nitrite-treated 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. TRIAL REGISTRATION NUMBER: NCT01584453.

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.

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.

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.

"Repurposing" of Xanthine Oxidoreductase as a Nitrite Reductase: A New Paradigm for Therapeutic Targeting in Hypertension.

SIGNIFICANCE: In contrast to nitric oxide (NO), which has well-established, important effects in regulation of cardiovascular homeostasis, its oxidative metabolite nitrite has, until recently, been considered to be of minor functional significance. RECENT ADVANCES: However, this view of nitrite has been radically revised over the past 10 years with evidence now supporting a critical role for this anion as a storage form of NO. CRITICAL ISSUES: Importantly, while hypoxia and acidosis have been shown to play a pivotal role in the generation of nitrite to NO, a number of mammalian nitrite reductases have been identified that facilitate the reduction of nitrite. Critically, these nitrite reductases have been demonstrated to operate under physiological pH conditions and in normoxia, extending the functional remit of this anion from an ischemic mediator to an important regulator of physiology. One particular nitrite reductase that has been shown to operate under a wide range of environmental conditions is the enzyme xanthine oxidoreductase (XOR). FUTURE DIRECTIONS: In this review, we discuss the evidence supporting a role for XOR as a nitrite reductase while focusing particularly on its function in hypertension. In addition, we discuss the potential merit in exploiting this activity of XOR in the therapeutics of hypertension.

Dietary nitrate provides sustained blood pressure lowering in hypertensive patients: a randomized, phase 2, double-blind, placebo-controlled study.

UNLABELLED: Single dose administration of dietary inorganic nitrate acutely reduces blood pressure (BP) in normotensive healthy volunteers, via bioconversion to the vasodilator nitric oxide. We assessed whether dietary nitrate might provide sustained BP lowering in patients with hypertension. We randomly assigned 68 patients with hypertension in a double-blind, placebo-controlled clinical trial to receive daily dietary supplementation for 4 weeks with either dietary nitrate (250 mL daily, as beetroot juice) or a placebo (250 mL daily, as nitrate-free beetroot juice) after a 2-week run-in period and followed by a 2-week washout. We performed stratified randomization of drug-naive (n=34) and treated (n=34) patients with hypertension aged 18 to 85 years. The primary end point was change in clinic, ambulatory, and home BP compared with placebo. Daily supplementation with dietary nitrate was associated with reduction in BP measured by 3 different methods. Mean (95% confidence interval) reduction in clinic BP was 7.7/2.4 mm Hg (3.6-11.8/0.0-4.9, P<0.001 and P=0.050). Twenty-four-hour ambulatory BP was reduced by 7.7/5.2 mm Hg (4.1-11.2/2.7-7.7, P<0.001 for both). Home BP was reduced by 8.1/3.8 mm Hg (3.8-12.4/0.7-6.9, P<0.001 and P<0.01) with no evidence of tachyphylaxis over the 4-week intervention period. Endothelial function improved by ≈20% (P<0.001), and arterial stiffness was reduced by 0.59 m/s (0.24-0.93; P<0.01) after dietary nitrate consumption with no change after placebo. The intervention was well tolerated. This is the first evidence of durable BP reduction with dietary nitrate supplementation in a relevant patient group. These findings suggest a role for dietary nitrate as an affordable, readily-available, adjunctive treatment in the management of patients with hypertension (funded by The British Heart Foundation). CLINICAL TRIAL REGISTRATION URL: http://www.clinicaltrials.gov. Unique identifier: NCT01405898.

Endothelial C-type natriuretic peptide maintains vascular homeostasis.

The endothelium plays a fundamental role in maintaining vascular homeostasis by releasing factors that regulate local blood flow, systemic blood pressure, and the reactivity of leukocytes and platelets. Accordingly, endothelial dysfunction underpins many cardiovascular diseases, including hypertension, myocardial infarction, and stroke. Herein, we evaluated mice with endothelial-specific deletion of Nppc, which encodes C-type natriuretic peptide (CNP), and determined that this mediator is essential for multiple aspects of vascular regulation. Specifically, disruption of CNP leads to endothelial dysfunction, hypertension, atherogenesis, and aneurysm. Moreover, we identified natriuretic peptide receptor-C (NPR-C) as the cognate receptor that primarily underlies CNP-dependent vasoprotective functions and developed small-molecule NPR-C agonists to target this pathway. Administration of NPR-C agonists promotes a vasorelaxation of isolated resistance arteries and a reduction in blood pressure in wild-type animals that is diminished in mice lacking NPR-C. This work provides a mechanistic explanation for genome-wide association studies that have linked the NPR-C (Npr3) locus with hypertension by demonstrating the importance of CNP/NPR-C signaling in preserving vascular homoeostasis. Furthermore, these results suggest that the CNP/NPR-C pathway has potential as a disease-modifying therapeutic target for cardiovascular disorders.

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.

Sexual dimorphism in rodent models of hypertension and atherosclerosis.

Approximately one third of all deaths are attributed to cardiovascular disease (CVD), making it the biggest killer worldwide. Despite a number of therapeutic options available, the burden of CVD morbidity continues to grow indicating the need for continued research to address this unmet need. In this respect, investigation of the mechanisms underlying the protection that premenopausal females enjoy from cardiovascular-related disease and mortality is of interest. In this review, we discuss the essential role that rodent animal models play in enabling this field of research. In particular, we focus our discussion on models of hypertension and atherosclerosis.

Natriuretic peptide receptor-3 underpins the disparate regulation of endothelial and vascular smooth muscle cell proliferation by C-type natriuretic peptide.

BACKGROUND AND PURPOSE: C-type natriuretic peptide (CNP) is an endothelium-derived vasorelaxant, exerting anti-atherogenic actions in the vasculature and salvaging the myocardium from ischaemic injury. The cytoprotective effects of CNP are mediated in part via the G(i) -coupled natriuretic peptide receptor (NPR)3. As GPCRs are well-known to control cell proliferation, we investigated if NPR3 activation underlies effects of CNP on endothelial and vascular smooth muscle cell mitogenesis. EXPERIMENTAL APPROACH: Proliferation of human umbilical vein endothelial cells (HUVEC), rat aortic smooth muscle cells (RAoSMC) and endothelial and vascular smooth muscle cells from NPR3 knockout (KO) mice was investigated in vitro. KEY RESULTS: CNP (1 pM-1 µM) facilitated HUVEC proliferation and inhibited RAoSMC growth concentration-dependently. The pro- and anti-mitogenic effects of CNP were blocked by the NPR3 antagonist M372049 (10 µM) and the extracellular signal-regulated kinase (ERK) 1/2 inhibitor PD98059 (30 µM) and were absent in cells from NPR3 KO mice. Activation of ERK 1/2 by CNP was inhibited by Pertussis toxin (100 ng·mL⁻¹) and M372049 (10 µM). In HUVEC, ERK 1/2 activation enhanced expression of the cell cycle promoter, cyclin D1, whereas in RAoSMC, ERK 1/2 activation increased expression of the cell cycle inhibitors p21(waf1/cip1) and p27(kip1) . CONCLUSIONS AND IMPLICATIONS: A facet of the vasoprotective profile of CNP is mediated via NPR3-dependent ERK 1/2 phosphorylation, resulting in augmented endothelial cell proliferation and inhibition of vascular smooth muscle growth. This pathway may offer an innovative approach to reversing the endothelial damage and vascular smooth muscle hyperplasia that characterize many vascular disorders.