Andrew J. Webb

Acute Blood Pressure Lowering, Vasoprotective, and Antiplatelet Properties of Dietary Nitrate via Bioconversion to Nitrite

Diets rich in fruits and vegetables reduce blood pressure (BP) and the risk of adverse cardiovascular events. However, the mechanisms of this effect have not been elucidated. Certain vegetables possess a high nitrate content, and we hypothesized that this might represent a source of vasoprotective nitric oxide via bioactivation. In healthy volunteers, approximately 3 hours after ingestion of a dietary nitrate load (beetroot juice 500 mL), BP was substantially reduced (&Dgr;max −10.4/8 mm Hg); an effect that correlated with peak increases in plasma nitrite concentration. The dietary nitrate load also prevented endothelial dysfunction induced by an acute ischemic insult in the human forearm and significantly attenuated ex vivo platelet aggregation in response to collagen and ADP. Interruption of the enterosalivary conversion of nitrate to nitrite (facilitated by bacterial anaerobes situated on the surface of the tongue) prevented the rise in plasma nitrite, blocked the decrease in BP, and abolished the inhibitory effects on platelet aggregation, confirming that these vasoprotective effects were attributable to the activity of nitrite converted from the ingested nitrate. These findings suggest that dietary nitrate underlies the beneficial effects of a vegetable-rich diet and highlights the potential of a “natural” low cost approach for the treatment of cardiovascular disease.

Nitrate and nitrite in biology, nutrition and therapeutics

Inorganic nitrate and nitrite from endogenous or dietary sources are metabolized in vivo to nitric oxide (NO) and other bioactive nitrogen oxides. The nitrate-nitrite-NO pathway is emerging as an important mediator of blood flow regulation, cell signaling, energetics and tissue responses to hypoxia. The latest advances in our understanding of the biochemistry, physiology and therapeutics of nitrate, nitrite and NO were discussed during a recent 2-day meeting at the Nobel Forum, Karolinska Institutet in Stockholm.

Inorganic Nitrate Supplementation Lowers Blood Pressure in Humans. Role for Nitrite-Derived NO

Ingestion of dietary (inorganic) nitrate elevates circulating and tissue levels of nitrite via bioconversion in the entero-salivary circulation. In addition, nitrite is a potent vasodilator in humans, an effect thought to underlie the blood pressure–lowering effects of dietary nitrate (in the form of beetroot juice) ingestion. Whether inorganic nitrate underlies these effects and whether the effects of either naturally occurring dietary nitrate or inorganic nitrate supplementation are dose dependent remain uncertain. Using a randomized crossover study design, we show that nitrate supplementation (KNO3 capsules: 4 versus 12 mmol [n=6] or 24 mmol of KNO3 (1488 mg of nitrate) versus 24 mmol of KCl [n=20]) or vegetable intake (250 mL of beetroot juice [5.5 mmol nitrate] versus 250 mL of water [n=9]) causes dose-dependent elevation in plasma nitrite concentration and elevation of cGMP concentration with a consequent decrease in blood pressure in healthy volunteers. In addition, post hoc analysis demonstrates a sex difference in sensitivity to nitrate supplementation dependent on resting baseline blood pressure and plasma nitrite concentration, whereby blood pressure is decreased in male volunteers, with higher baseline blood pressure and lower plasma nitrite concentration but not in female volunteers. Our findings demonstrate dose-dependent decreases in blood pressure and vasoprotection after inorganic nitrate ingestion in the form of either supplementation or by dietary elevation. In addition, our post hoc analyses intimate sex differences in nitrate processing involving the entero-salivary circulation that are likely to be major contributing factors to the lower blood pressures and the vasoprotective phenotype of premenopausal women.

Nitrite-Derived Nitric Oxide Protects the Rat Kidney against Ischemia/Reperfusion Injury In Vivo: Role for Xanthine Oxidoreductase

In normal conditions, nitric oxide (NO) is oxidized to the anion nitrite, but in hypoxia, this nitrite may be reduced back to NO by the nitrite reductase action of deoxygenated hemoglobin, acidic disproportionation, or xanthine oxidoreductase (XOR). Herein, is investigated the effects of topical sodium nitrite administration in a rat model of renal ischemia/reperfusion (I/R) injury. Rats were subjected to 60 min of bilateral renal ischemia and 6 h of reperfusion in the absence or presence of sodium nitrite (30 nmol) administered topically 1 min before reperfusion. Serum creatinine, serum aspartate aminotransferase, creatinine clearance, fractional excretion of Na(+), and plasma nitrite/nitrate concentrations were measured. The nitrite-derived NO-generating capacity of renal tissue was determined under acidic and hypoxic conditions by ozone chemiluminescence in homogenates of kidneys that were subjected to sham, ischemia-only, and I/R conditions. Nitrite significantly attenuated renal dysfunction and injury, an effect that was abolished by previous treatment of rats with the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazole-1-oxyl-3-oxide (2.5 mumol intravenously 5 min before ischemia and 50 nmol topically 6 min before reperfusion). Renal tissue homogenates produced significant amounts of NO from nitrite, an effect that was attenuated significantly by the xanthine oxidoreductase inhibitor allopurinol. Taken together, these findings demonstrate that topically administered sodium nitrite protects the rat kidney against I/R injury and dysfunction in vivo via the generation, in part, of xanthine oxidoreductase-catalyzed NO production. These observations suggest that nitrite therapy might prove beneficial in protecting kidney function and integrity during periods of I/R such as those encountered in renal transplantation.

Mechanisms Underlying Erythrocyte and Endothelial Nitrite Reduction to Nitric Oxide in Hypoxia: Role for Xanthine Oxidoreductase and Endothelial Nitric Oxide Synthase

Reduction of nitrite (NO2−) provides a major source of nitric oxide (NO) in the circulation, especially in hypoxemic conditions. Our previous studies suggest that xanthine oxidoreductase (XOR) is an important nitrite reductase in the heart and kidney. Herein, we have demonstrated that conversion of nitrite to NO by blood vessels and RBCs was enhanced in the presence of the XOR substrate xanthine (10 &mgr;mol/L) and attenuated by the XOR inhibitor allopurinol (100 &mgr;mol/L) in acidic and hypoxic conditions only. Whereas endothelial nitric oxide synthase (eNOS) inhibition had no effect on vascular nitrite reductase activity, in RBCs L-NAME, L-NMMA, and l-arginine inhibited nitrite-derived NO production by >50% (P<0.01) at pH 7.4 and 6.8 under hypoxic conditions. Western blot and immunohistochemical analysis of RBC membranes confirmed the presence of eNOS and abundant XOR on whole RBCs. Thus, XOR and eNOS are ideally situated on the membranes of RBCs and blood vessels to generate intravascular vasodilator NO from nitrite during ischemic episodes. In addition to the proposed role of deoxyhemoglobin, our findings suggest that the nitrite reductase activity within the circulation, under hypoxic conditions (at physiological pH), is mediated by eNOS; however, as acidosis develops, a substantial role for XOR becomes evident.

Brief Communication: Rituximab in HIV-Associated Multicentric Castleman Disease

Context Castleman disease is a rare lymphoproliferative condition. Risk for the condition is elevated in people with HIV infection. Case reports and series suggest that rituximab shows some therapeutic promise in patients previously treated with chemotherapy, but data on initial therapy with rituximab are lacking. Contribution This uncontrolled case series suggests that initial treatment with rituximab can achieve better overall and disease-free survival than that anticipated in untreated patients. Laboratory measures improved with therapy. Caution The absence of a control group precludes definitive assessment of the efficacy or safety of rituximab in treating HIV-associated Castleman disease. The Editors Multicentric Castleman disease is a rare lymphoproliferative disorder that is increasingly occurring in people with HIV infection. It is associated with Kaposi sarcoma, sharing an etiologic agent, Kaposi sarcomaassociated herpesvirus (KSHV), also known as human herpesvirus-8 (1, 2). The gold-standard therapy for HIV-associated multicentric Castleman disease is yet to be established. The use of an anti-CD20 monoclonal antibody, rituximab, to target KSHV-infected plasmablasts in multicentric Castleman disease is a novel and potentially beneficial approach. It has been the subject of case reports and clinical series, in which patients were often pretreated with chemotherapy and follow-up was brief (310). We investigated the efficacy and safety of rituximab as initial monotherapy and correlate clinical findings with immune subset, plasma cytokine, and HIV and KSHV virologic variables. Methods Between 2003 and 2006, 21 patients (18 men) with multicentric Castleman disease were treated prospectively in a nonrandomized, open-label, phase II study with 4 infusions of rituximab at a standard dose of 375 mg per m2 of body surface area at weekly intervals. All biopsy specimens were reviewed and confirmed to be plasmablastic variants of multicentric Castleman disease with no microlymphoma, as defined by previous studies (11, 12). The plasmablasts showed immunoglobulin light chain restriction, were KSHV latent nuclear antigenpositive, and expressed CD20 on immunohistochemistry. Patients were recruited from 3 HIV and cancer centers, where local ethics review committees approved the study and patients gave informed consent. Toxicity was recorded at each visit and was graded by using the Common Terminology Criteria for Adverse Events, version 3.0 (13). We measured plasma KSHV DNA viral load at diagnosis and at 1 and 3 months after rituximab therapy by using Lightcycler quantitative polymerase chain reaction (Roche, Lewis, United Kingdom) on DNA extracted from whole blood using primers specific to KSHV ORF-7 gene, as described elsewhere (14). We assessed progression-free and overall survival by using the KaplanMeier method (15) and used the Wilcoxon rank-sum test to assess the statistical significance of changes in hematologic, biochemical, and immunologic variables. Summaries of data that were not normally distributed are presented as medians with interquartile ranges. All P values are 2-sided (Statview, version 4.57, Abacus Concepts, Berkeley, California). Role of the Funding Source Support for the cytokine assays was provided by St. Stephens AIDS Trust, a national charity supporting clinical research in HIV/AIDS, which had no role in the design, conduct, or reporting of this review or in the decision to publish the manuscript. Results We enrolled 21 patients with a histologically confirmed plasmablastic variant of multicentric Castleman disease without microlymphoma. Their median age was 37 years (range, 31 to 69 years), 9 (43%) patients had a previous AIDS-defining diagnosis, and 13 (62%) patients were receiving highly active antiretroviral therapy (HAART) at diagnosis of multicentric Castleman disease. The median CD4 cell count at diagnosis was 0.30109 cells/L (range, 0.08 to 0.73109 cells/L). Four patients had a plasma HIV-1 viral load less than 50 copies/mL, and 5 other patients had a viral load less than 400 copies/mL (Table 1). Table 1. Hematologic, Biochemical, and Immunologic Variables at Presentation and Change from Baseline 1 Month after Completion of Rituximab Therapy* At diagnosis, the median duration of symptoms was 4 months (range, 0.5 to 24 months), all patients had significant lymphadenopathy, 20 (95%) patients had fever of unknown origin, 18 of 20 (90%) patients had splenomegaly (1 had had splenectomy), and 11 (52%) patients had cutaneous Kaposi sarcoma. Ninety-five percent of the patients had an increased erythrocyte sedimentation rate (ESR) (>20 mm/h), 82% had an increased C-reactive protein (CRP) level (>10 mg/L), 67% were anemic (hemoglobin level <100 g/L), 67% were hypoalbuminemic (serum albumin level <30 g/L), and 14% were thrombocytopenic (platelet count <100109 cells/L). All patients had polyclonal hypergammaglobulinemia, and 2 patients had a serum IgG monoclonal paraprotein band (Table 1). One patient who was receiving intensive care at diagnosis died of progressive disease before completing the rituximab course. All 20 remaining patients achieved resolution of symptoms and fever by the end of rituximab treatment. Of the 21 patients, 14 (67%) had a partial response and 6 (29%) had stable disease according to the radiologic Response Evaluation Criteria in Solid Tumors. The median follow-up was 12 months (range, 1 to 49 months). The 2-year overall survival rate was 95% (95% CI, 86% to 100%), and the relapse-free survival rate was 92% (CI, 75% to 100%) at 1 year and 79% (CI, 52% to 100%) at 2 years. One month after completion of rituximab therapy, 0 of 20 patients were anemic, 0 of 20 were thrombocytopenic, 11 of 17 had an increased ESR, 2 of 16 had an increased CRP level, and 1 of 20 had hypoalbuminemia. Hemoglobin level, platelet count, and serum albumin level increased, whereas ESR and CRP level decreased, 1 month after rituximab treatment (Table 1). Quantitative polymerase chain reaction for KSHV was available for 11 patients at diagnosis and was detectable in 9 patients (median, 700 copies/mL; range, 0 to 400000 copies/mL). One month after treatment, only 2 of 10 (20%) patients had detectable KSHV (Table 1). In both cases, the titer was only 100 copies/mL. Three months after rituximab therapy, only 1 of 10 (10%) patients had detectable KSHV DNA, and once again the titer was only 100 copies/mL (P= 0.018). Serum IgG and IgM levels decreased 1 month after rituximab therapy, but IgA levels did not change (Table 1). Similarly, the CD19 cell count decreased, which persisted at 3 months (median decrease from baseline, 104 cells/mL; interquartile range, 14 to 350 cells/mL; P= 0.002), but the CD19 cell count had recovered to prerituximab levels by 12 months. We performed immune subset analysis on the 13 patients who were already receiving HAART at the time of rituximab therapy. The CD4, CD8, CD56 (natural killer) cell subsets, or HIV viral load did not change during this period. No Common Terminology Criteria for Adverse Events grade 3 or 4 toxicities were recorded with rituximab therapy; however, Kaposi sarcoma progressed during rituximab therapy in 4 of 11 (36%) patients who had cutaneous Kaposi sarcoma at diagnosis. We also measured 15 plasma cytokines: interleukin (IL)1, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12 p40/p70, IL-13, IL-15, IL-17, interferon-, interferon-, tumor necrosis factor-, and granulocyte macrophage colony-stimulating factor, before and after rituximab therapy and again 3 months after the completion of rituximab therapy. Most patients had elevated plasma cytokine levels at presentation, and the proportion with increased levels declined on completion of therapy (Table 2). Table 2. Proportion of Patients with Elevated Plasma Cytokine Levels at Diagnosis and Changes during Treatment* Discussion Rituximab therapy seems to be a promising first-line treatment for HIV-associated multicentric Castleman disease: Patients completing 4 weekly infusions achieved a clinical and biochemical remission within 1 month, and the radiologic response rate was 67%. Plasma KSHV viral load significantly decreased in individuals with this measurement (P= 0.018). The 2-year overall survival rate was 95% (CI, 86% to 100%), and the relapse-free survival rate was 79% (CI, 52% to 100%). This compares favorably with the median survival of 14 months recorded for 20 patients from the pre-HAART era (16). The clinical response to rituximab occurred within 1 month of completing therapy, and normalization of acute-phase inflammatory markers, such as ESR, CRP, and albumin, occurred by this point. Plasma KSHV DNA viral load was measured before, during, and after treatment and decreased dramatically with treatment and increased at relapse. The high plasma titers of KSHV reflect lytic replication, which is not a feature of Kaposi sarcoma but correlates with disease activity in multicentric Castleman disease. Indeed, KSHV-infected B-lymphocytes from lymph nodes in patients with multicentric Castleman disease are known to express KSHV lytic gene products (17, 18). Rituximab produced a decrease in CD19-positive B-lymphocytes, as would be expected, but was well tolerated in patients with HIV-related multicentric Castleman disease, with no grade 3 or 4 toxicities. In addition, rituximab did not seem to cause exacerbation of HIV infection, with no adverse effect on the immune T-cell subsets, including CD4 cell count or HIV viral load. However, Kaposi sarcoma progressed in 36% of patients with this disease, a phenomenon that has been recorded previously (4). The reason for this is unclear, but the rapid decrease in B-lymphocytes observed with rituximab therapy may play a role in the progression of Kaposi sarcoma (19). Because rituximab has also been associated with an increased risk for death from infection in AIDS-related non-Hodgkin lymphoma (20), the data we present should provide reassurance to clinicians. A recent trial i

Enhanced Vasodilator Activity of Nitrite in Hypertension Critical Role for Erythrocytic Xanthine Oxidoreductase and Translational Potential

Elevation of circulating nitrite (NO2 −) levels causes vasodilatation and lowers blood pressure in healthy volunteers. Whether these effects and the underpinning mechanisms persist in hypertension is unknown. Therefore, we investigated the consequences of systemic nitrite elevation in spontaneously hypertensive rats and conducted proof-of-principle studies in patients. Nitrite caused dose-dependent blood pressure–lowering that was profoundly enhanced in spontaneously hypertensive rats versus normotensive Wistar Kyoto controls. This effect was virtually abolished by the xanthine oxidoreductase (XOR) inhibitor, allopurinol, and associated with hypertension-specific XOR-dependent nitrite reductase activity localized to the erythrocyte but not the blood vessel wall. To determine whether these pathways translate to human hypertension, we investigated the effects of elevation of circulating nitrite levels in 15 drug naïve grade 1 hypertensives. To elevate nitrite, we used a dose of dietary nitrate (≈3.5 mmol) that elevated nitrite levels ≈1.5-fold (P<0.01); a rise shown previously to exert no significant blood pressure–lowering effects in normotensives. This dose caused substantial reductions in systolic (≈12 mm Hg) and diastolic blood pressures (P<0.001) and pulse wave velocity (P<0.05); effects associated with elevations in erythrocytic XOR expression and XOR-dependent nitrite reductase activity. Our observations demonstrate the improved efficacy of inorganic nitrate and nitrite in hypertension as a consequence of increased erythrocytic XOR nitrite reductase activity and support the concept of dietary nitrate supplementation as an effective, but simple and inexpensive, antihypertensive strategy.Elevation of circulating nitrite (NO2−) levels causes vasodilatation and lowers blood pressure in healthy volunteers. Whether these effects and the underpinning mechanisms persist in hypertension is unknown. Therefore, we investigated the consequences of systemic nitrite elevation in spontaneously hypertensive rats and conducted proof-of-principle studies in patients. Nitrite caused dose-dependent blood pressure–lowering that was profoundly enhanced in spontaneously hypertensive rats versus normotensive Wistar Kyoto controls. This effect was virtually abolished by the xanthine oxidoreductase (XOR) inhibitor, allopurinol, and associated with hypertension-specific XOR-dependent nitrite reductase activity localized to the erythrocyte but not the blood vessel wall. To determine whether these pathways translate to human hypertension, we investigated the effects of elevation of circulating nitrite levels in 15 drug naive grade 1 hypertensives. To elevate nitrite, we used a dose of dietary nitrate (≈3.5 mmol) that elevated nitrite levels ≈1.5-fold ( P <0.01); a rise shown previously to exert no significant blood pressure–lowering effects in normotensives. This dose caused substantial reductions in systolic (≈12 mm Hg) and diastolic blood pressures ( P <0.001) and pulse wave velocity ( P <0.05); effects associated with elevations in erythrocytic XOR expression and XOR-dependent nitrite reductase activity. Our observations demonstrate the improved efficacy of inorganic nitrate and nitrite in hypertension as a consequence of increased erythrocytic XOR nitrite reductase activity and support the concept of dietary nitrate supplementation as an effective, but simple and inexpensive, antihypertensive strategy. # Novelty and Significance {#article-title-61}

Inorganic nitrate and the cardiovascular system

Fruit and vegetable-rich diets reduce blood pressure and risk of ischaemic stroke and ischaemic heart disease. While the cardioprotective effects of a fruit and vegetable-rich diet are unequivocal, the exact mechanisms of this effect remain uncertain. Recent evidence has highlighted the possibility that dietary nitrate, an inorganic anion found in large quantities in vegetables (particularly green leafy vegetables), may have a part to play. This beneficial activity lies in the processing in vivo of nitrate to nitrite and thence to the pleiotropic molecule nitric oxide. In this review, recent preclinical and clinical evidence identifying the mechanisms involved in nitrate bioactivity, and the evidence supporting the potential utility of exploitation of this pathway for the prevention and/or treatment of cardiovascular diseases are discussed.

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.

Inhibition of p38 Mitogen-Activated Protein Kinase Improves Nitric Oxide–Mediated Vasodilatation and Reduces Inflammation in Hypercholesterolemia

Background— Oxidized low-density lipoprotein reduces endothelial nitric oxide production (an important mediator of vasoregulation) and activates p38 mitogen-activated protein kinase (MAPK), a mediator of vascular inflammation. Animal models of vascular stress have previously predicted improvements in vascular function after p38 MAPK inhibition. We hypothesized that a selective p38&agr;/&bgr; MAPK inhibitor (losmapimod; GW856553) would improve compromised nitric oxide–mediated vasoregulation in patients with hypercholesterolemia. Methods and Results— Untreated hypercholesterolemic patients (low-density lipoprotein cholesterol >4.1 mmol/L) were randomized to receive losmapimod 7.5 mg (n=27) or placebo (n=29) twice daily for 28 days. Patients with known vascular disorders (eg, diabetes mellitus, coronary heart disease) were excluded. Forearm blood flow was measured by venous occlusion plethysmography in response to serial intra-arterial infusion of acetylcholine, sodium nitroprusside, and NG-monomethyl-l-arginine (L-NMMA). Acetylcholine and L-NMMA responses were significantly impaired (P=0.01 and P=0.03) compared with responses in control subjects (n=12). In hypercholesterolemic patients treated with losmapimod, responses to acetylcholine were improved by 25% (95% confidence interval, 5 to 48; P=0.01), to sodium nitroprusside by 20% (95% confidence interval, 3 to 40; P=0.02), and to L-NMMA by 10% (95% confidence interval, −1 to 23; P=0.07) compared with placebo. C-reactive protein was reduced by 57% (95% confidence interval, −81 to −6%; P<0.05) in patients treated with losmapimod compared with placebo. Conclusions— Losmapimod improves nitric oxide–mediated vasodilatation in hypercholesterolemic patients, which is consistent with findings in previous translational animal models. These data support the hypothesis that attenuating the inflammatory milieu by inhibiting p38 MAPK activity improves NO activity. This suggests p38 MAPK as a novel target for patients with cardiovascular disease. Clinical Trial Registration— URL: http://clinicaltrials.gov. Unique identifier: NCT00474864.