Editorial Harnessing the Nitrate–Nitrite–Nitric Oxide Pathway for Therapy of Heart Failure With Preserved Ejection Fraction Rebecca Vanderpool, PhD; Mark T. Gladwin, MD
N
itrate accumulates in the plasma from oral intake of foods rich in nitrate such as green leafy vegetables and root plants such as beet root or from the intravascular oxidation of nitric oxide (NO), produced by the NO synthase enzymes, to nitrate by oxyhemoglobin. Nitrate is then concentrated in the saliva and reacts with oral commensal bacteria, which contain nitrate reductase enzymes.1 Humans do not possess nitrate reductase enzymes and thus require these bacteria for conversion of nitrate to nitrite. Nitrite is then swallowed and systemically absorbed so that it can be further reduced via single-electron transfer reactions with hemoglobin, myoglobin, neuroglobin, and molybdopterin-containing enzymes (such as xanthine oxidase, aldehyde oxidase, and mARC).2–7 Now referred to as the nitrate-nitrite-NO pathway, this involves a series of oxygen-independent and NO synthase–independent single-electron transfer reactions [Figure (A)].
Articles see p 371 and p 381 Nitrate and nitrite have now been shown to regulate blood pressure, hypoxic vasodilation, mitochondrial efficiency, and exercise performance.2,10–14 Infusions of nitrite have been shown to directly vasodilate the forearm arterial and venous circulation, with significant potentiation during hypoxia.10,14 Systemic infusions of nitrite in dogs exhibit both arteriolar and venous dilation, with more potent effects on the systemic vascular resistance, increasing cardiac output.15 Numerous normal human volunteer studies have now demonstrated that nitrate supplementation can increase exercise efficiency, as ⋅ defined by total oxygen consumption (Vo2) for total work 11 performed (Watts). In preclinical studies, nitrite has been shown to exhibit therapeutic efficacy in mouse, rat, and sheep models of pulmonary hypertension and mouse models of heart failure.16,17 Additionally, a recent study indicates that dietary supplementation of nitrate reverses the features of metabolic syndrome in endothelial NO synthase–deficient mice.18 Considering the known association between features of the The opinions expressed in this article are not necessarily those of the editors or of the American Heart Association. From the Pittsburgh Heart, Lung, Blood and Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, PA (R.V., M.T.G.); and Division of Pulmonary, Allergy and Critical Care Medicine, UPMC and University of Pittsburgh, Pittsburgh, PA (R.V., M.T.G.). Correspondence to Mark T. Gladwin, MD, Pittsburgh Heart, Lung, Blood and Vascular Medicine Institute, Division of Pulmonary, Allergy and Critical Care Medicine, Department of Medicine, University of Pittsburgh, E1244 BST 200 Lothrop St, Pittsburgh, PA 15213. E-mail [email protected] (Circulation. 2015;131:334-336. DOI: 10.1161/CIRCULATIONAHA.114.014149.) © 2014 American Heart Association, Inc. Circulation is available at http://circ.ahajournals.org DOI: 10.1161/CIRCULATIONAHA.114.014149
metabolic syndrome and heart failure with preserved ejection fraction (HFpEF), as well as the secondary development of pulmonary vascular disease and exercise intolerance in this disease, a potential therapeutic role for nitrate and nitrite for HFpEF is being considered and evaluated. In this issue of Circulation, Zamani and colleagues8 enrolled 17 subjects with HFpEF in a double-blind, placebocontrolled, crossover trial of a single dose of 12.9 mmol nitrate in 140 mL beet root juice or nitrate-depleted placebo beet root juice to address this question. The rationale for studying HFpEF was based on a number of pathogenic features that might be responsive to NO therapeutics, including abnormal diastolic left ventricular function, reduced exercise tolerance, reduced vasodilatory reserve during exercise, and abnormal skeletal muscle perfusion and function [Figure (A)]. The diagnosis of HFpEF was defined by clinical signs and symptoms of HFpEF on echocardiography, an increased ratio of the early mitral inflow velocity (E) to septal tissue Doppler velocity (e′) >8, and indirect or direct evidence of elevated left ventricular filling pressures. The primary end point of the trial was exercise efficiency, defined by the ratio of total work performed to total oxygen consumed, measured by supine cycle ergometer 2 hours after consumption of the beet root juice or placebo. A number of additional analyses were performed at maximal effort and constant-intensity cardiopulmonary exercise testing, including the evaluation of cardiac function by echocardiography, skeletal muscle deoxygenation during exercise in the calf using near-infrared spectroscopy, forearm postocclusive oxygen consumption rates and reactive hyperemia measured by Doppler ultrasound, and levels of plasma NO metabolites. Although there was no observed significant change in exercise efficiency, the primary end point of the trial, both exercise oxygen consumption and total work performed significantly increased. In addition, the exercise duration significantly increased by almost 1 minute, which was associated with significant reductions in aortic augmentation index, increases in cardiac output, and decreases in calculated systemic vascular resistance. There were no significant changes in near-infrared spectroscopy and postocclusive oxygen consumption or hyperemic flows, although trends for improvements were observed. These findings suggest that nitrate improves exercise capacity in HFpEF by reducing systemic vascular resistance and left ventricular pulsatile afterload and enhancing cardiac output and oxygen delivery to exercising muscles [Figure (B)]. Although these findings are provocative and the trial was well designed, a major limitation is a reliance on secondary end points, with a primary end point that is not consistent with a number of studies on the published effects of nitrate in normal volunteers.11 A central effect observed with nitrate
Downloaded from http://circ.ahajournals.org/ by guest on April 24, 2015 334
Vanderpool and Gladwin Harnessing the Nitrate-Nitrite-NO Pathway 335
Figure. A, The nitrate–nitrite–nitric oxide (NO) pathway is a series of oxygen-independent and NO synthase–independent single-electron transfer reactions that ultimately facilitate vasodilation. NO therapeutics (nitrate [NO3−] or nitrite [NO2−]) are potentially effective at reversing pathological features of heart failure with preserved ejection fraction (HFpEF). B, Findings from Zamani et al8 suggest that nitrate improves exercise capacity in HFpEF by reducing systemic vascular resistance (SVR) and left ventricular (LV) pulsatile afterload and enhancing cardiac output and oxygen delivery to exercising muscles. C, Webb et al9 found that nitrite significantly dilated both conduit and peripheral arteries. The mechanism by which nitrite dilates conduit arteries is still unknown. ETC indicates electron transport chain; and oxyHb, oxyhemoglobin.
repletion in prior studies was the effect on improved exercise efficiency, characterized by an increase in work performed for any level of oxygen consumption.11 On the other hand, the finding of an increase in exercise duration has been reported, particularly in disease states such as chronic obstructive pulmonary disease.19 The study also uses noninvasive measurements of hemodynamics and a single oral dose of nitrate, with no long-term follow-up to evaluate long-term effects on both exercise capacity or signs and symptoms of heart failure and the metabolic syndrome.
How Does Nitrite Vasodilate? Implications for HFpEF Therapy A second study by Omar and colleagues9 published in this issue of Circulation explored in more detail the pharmacological properties of nitrite in the human circulation, reporting for the first time that nitrite vasodilates not only the arteriolar and venous circulation, as previously described, but also the conduit blood vessels, an effect similar to that observed with nitroglycerin. This effect was associated with a reduction in central systolic blood pressure, augmentation index, and pulsed-wave velocity, which represent hemodynamic effects that might show therapeutic promise in the setting of HFpEF [Figure (C)]. Interestingly, although the effects of nitrite on arteriolar blood flow (measured by venous occlusion strain-gauge plethysmography) were potentiated by hypoxia, as previously described and quite distinct from the effects of nitroglycerin,14 the vasodilatory effects on the conduit arteries were actually inhibited under hypoxia and hyperoxia conditions. The mechanism for this normoxic nitrite bioactivation remains uncertain, but the lack of significant deoxygenation of hemoglobin or myoglobin and the dominant oxidase activities of molybdopterin enzymes under normoxic conditions
suggest that these pathways are unlikely to account for this conduit vasodilatory effect. Omar and colleagues9 infused 2 drugs to test the mechanisms for nitrite bioactivation in conduit arteries. The first was reloxifene, an inhibitor of aldehyde oxidize, classically considered an oxidase that generates superoxide but recently shown also to possess nitrite reductase activity under hypoxic conditions.20,21 Infusion of the aldehyde oxidase inhibitor actually increased nitrite-dependent conduit artery vasodilation, suggesting that this enzyme is in fact generating superoxide, which can scavenge NO. Reduced production of superoxide likely increases NO bioavailability from nitrite and increases vasodilator potency, similar to previous observations with inhibition of xanthine oxidase during nitrite infusions in the human forearm.10 The authors next tested infusions of acetazolamide, an inhibitor of carbonic anhydrase, also proposed to be a nitrite reductase (anhydrase).22 This inhibitor also increased the potency of nitrite. Although Aamand and colleagues22 have proposed that classic inhibitors of carbonic anhydrase can paradoxically increase nitrite reduction by this enzyme, we have recently shown that acetazolamide and dorzolamide reduce nitrite conversion to NO by carbonic anhydrase23, raising questions about this pathway in nitrite reduction. Clearly, carbonic anhydrase and aldehyde oxidase are major enzymes mediating nitrite conversion to NO, and more work is required to explore pathways for the observed conduit artery normoxic vasodilation. Nitrite is an inorganic salt (NO2−) and must be clearly differentiated from the organic nitrates such as nitroglycerin that are characterized by covalently bound nitro (NO2-R) groups adducted to carbon-based small molecules. The latter species require enzymatic metabolism to form NO and nitrite, and this enzymatic process is subject to tolerance. Studies in humans and primates suggest that nitrite is not subject to enzymatic
Downloaded from http://circ.ahajournals.org/ by guest on April 24, 2015
336 Circulation January 27, 2015 tolerance and continues to produce sustained vasodilation with long-term exposure.10 This lack of enzymatic tolerance suggests potential greater therapeutic potential for nitrite as a long-term therapy for HFpEF. These new studies now clarify that nitrite drives pharmacological arteriolar, venous, and conduit artery vasodilation, all effects that may prove beneficial as a long-term therapy for HFpEF. More study is required to understand how nitrite dilates conduit arteries under normoxic conditions and to test therapeutic effects of more long-term treatments with nitrate or nitrite in patients with HFpEF.
Disclosures Dr Gladwin is a coinventor on a National Institutes of Health government patent for the use of nitrite salts in cardiovascular diseases. Dr Gladwin consults with Aires/MAST Pharmaceuticals on the development of a phase II proof-of-concept trial using inhaled nitrite for pulmonary hypertension. Dr Vanderpool reports no conflicts.
References 1. Lundberg JO, Weitzberg E, Gladwin MT. The nitrate-nitrite-nitric oxide pathway in physiology and therapeutics. Nat Rev Drug Discov. 2008;7:156–167. doi: 10.1038/nrd2466. 2. Cosby K, Partovi KS, Crawford JH, Patel RP, Reiter CD, Martyr S, Yang BK, Waclawiw MA, Zalos G, Xu X, Huang KT, Shields H, Kim-Shapiro DB, Schechter AN, Cannon RO 3rd, Gladwin MT. Nitrite reduction to nitric oxide by deoxyhemoglobin vasodilates the human circulation. Nat Med. 2003;9:1498–1505. doi: 10.1038/nm954. 3. Shiva S, Huang Z, Grubina R, Sun J, Ringwood LA, MacArthur PH, Xu X, Murphy E, Darley-Usmar VM, Gladwin MT. Deoxymyoglobin is a nitrite reductase that generates nitric oxide and regulates mitochondrial respiration. Circ Res. 2007;100:654–661. doi: 10.1161/01. RES.0000260171.52224.6b. 4. Tiso M, Tejero J, Basu S, Azarov I, Wang X, Simplaceanu V, Frizzell S, Jayaraman T, Geary L, Shapiro C, Ho C, Shiva S, Kim-Shapiro DB, Gladwin MT. Human neuroglobin functions as a redox-regulated nitrite reductase. J Biol Chem. 2011;286:18277–18289. doi: 10.1074/jbc.M110.159541. 5. Zhang Z, Naughton DP, Blake DR, Benjamin N, Stevens CR, Winyard PG, Symons MC, Harrison R. Human xanthine oxidase converts nitrite ions into nitric oxide (NO). Biochem Soc Trans. 1997;25:524S. 6. Sparacino-Watkins CE, Tejero J, Sun B, Gauthier MC, Thomas J, Ragireddy V, Merchant BA, Wang J, Azarov I, Basu P, Gladwin MT. Nitrite reductase and nitric-oxide synthase activity of the mitochondrial molybdopterin enzymes mARC1 and mARC2. J Biol Chem. 2014;289:10345– 10358. doi: 10.1074/jbc.M114.555177. 7. Kim-Shapiro DB, Gladwin MT. Mechanisms of nitrite bioactivation. Nitric Oxide. 2014;38:58–68. doi: 10.1016/j.niox.2013.11.002. 8. Zamani P, Rawat D, Prithvi S-K, Geraci S, Bhuva R, Konda P, Doulias P-T, Ischiropoulos H, Townsend R, Margulies K, Cappola T, Poole D, Chirinos J. Effect of inorganic nitrate on exercise capacity in heart failure with preserved ejection fraction. Circulation. 2015;131:371–380. 9. Omar S, Fok H, Tilgner K, Nair A, Hunt J, Jiang B, Taylor P, Chowienczyk P, Webb A. Paradoxical normoxia-dependent selective actions of inorganic nitrite in human muscular conduit arteries and related selective actions on central blood pressures. Circulation. 2015;131:381–389. 10. Dejam A, Hunter CJ, Tremonti C, Pluta RM, Hon YY, Grimes G, Partovi K, Pelletier MM, Oldfield EH, Cannon RO 3rd, Schechter AN, Gladwin
MT. Nitrite infusion in humans and nonhuman primates: endocrine effects, pharmacokinetics, and tolerance formation. Circulation. 2007;116:1821– 1831. doi: 10.1161/CIRCULATIONAHA.107.712133. 11. Jones AM. Dietary nitrate supplementation and exercise performance. Sports Med. 2004;44(suppl 1):S35–S45. 12. Larsen FJ, Schiffer TA, Borniquel S, Sahlin K, Ekblom B, Lundberg JO, Weitzberg E. Dietary inorganic nitrate improves mitochondrial efficiency in humans. Cell Metab. 2011;13:149–159. doi: 10.1016/j. cmet.2011.01.004. 13. Kapil V, Haydar SM, Pearl V, Lundberg JO, Weitzberg E, Ahluwalia A. Physiological role for nitrate-reducing oral bacteria in blood pressure control. Free Radic Biol Med. 2013;55:93–100. doi: 10.1016/j. freeradbiomed.2012.11.013. 14. Maher AR, Milsom AB, Gunaruwan P, Abozguia K, Ahmed I, Weaver RA, Thomas P, Ashrafian H, Born GV, James PE, Frenneaux MP. Hypoxic modulation of exogenous nitrite-induced vasodilation in humans. Circulation. 2008;117:670–677. doi: 10.1161/CIRCULATIONAHA.107.719591. 15. Minneci PC, Deans KJ, Shiva S, Zhi H, Banks SM, Kern S, Natanson C, Solomon SB, Gladwin MT. Nitrite reductase activity of hemoglobin as a systemic nitric oxide generator mechanism to detoxify plasma hemoglobin produced during hemolysis. Am J Physiol Heart Circ Physiol. 2008;295:H743–H754. doi: 10.1152/ajpheart.00151.2008. 16. Sparacino-Watkins CE, Lai YC, Gladwin MT. Nitrate-nitrite-nitric oxide pathway in pulmonary arterial hypertension therapeutics. Circulation. 2012;125:2824–2826. doi: 10.1161/CIRCULATIONAHA.112.107821. 17. Baliga RS, Milsom AB, Ghosh SM, Trinder SL, Macallister RJ, Ahluwalia A, Hobbs AJ. Dietary nitrate ameliorates pulmonary hypertension: cytoprotective role for endothelial nitric oxide synthase and xanthine oxidoreductase. Circulation. 2012;125:2922–2932. doi: 10.1161/ CIRCULATIONAHA.112.100586. 18. Carlström M, Larsen FJ, Nyström T, Hezel M, Borniquel S, Weitzberg E, Lundberg JO. Dietary inorganic nitrate reverses features of metabolic syndrome in endothelial nitric oxide synthase-deficient mice. Proc Natl Acad Sci U S A. 2010;107:17716–17720. doi: 10.1073/ pnas.1008872107. 19. Berry MJ, Justus NW, Hauser JI, Case AH, Helms CC, Basu S, Rogers Z, Lewis MT, Miller GD. Dietary nitrate supplementation improves exercise performance and decreases blood pressure in COPD patients [published online ahead of print October 27, 2014]. Nitric Oxide. doi: 10.1016/j. niox.2014.10.007. http://www.sciencedirect.com/science/article/pii/ S1089860314004650. 20. Li H, Cui H, Kundu TK, Alzawahra W, Zweier JL. Nitric oxide production from nitrite occurs primarily in tissues not in the blood: critical role of xanthine oxidase and aldehyde oxidase. J Biol Chem. 2008;283:17855– 17863. doi: 10.1074/jbc.M801785200. 21. Li H, Kundu TK, Zweier JL. Characterization of the magnitude and mechanism of aldehyde oxidase-mediated nitric oxide production from nitrite. J Biol Chem. 2009;284:33850–33858. doi: 10.1074/jbc.M109.019125. 22. Aamand R, Dalsgaard T, Jensen FB, Simonsen U, Roepstorff A, Fago A. Generation of nitric oxide from nitrite by carbonic anhydrase: a possible link between metabolic activity and vasodilation. Am J Physiol Heart Circ Physiol. 2009;297:H2068–H2074. doi: 10.1152/ajpheart.00525.2009. 23. Liu C, Wajih N, Liu X, Basu S, Janes J, Marvel M, Keggi C, Helms CC, Lee AN, Belanger AM, Diz DI, Laurienti P, Caudell DL, Wang J, Gladwin MT, Kim-Shapiro DB. Mechanisms of human erythrocytic bioactivation of nitrite [published online ahead of print December 3, 2014]. J Biol Chem. doi: 10.1074/jbc.M114.609222. http://www.jbc.org/content/ early/2014/12/03/jbc.M114.609222.long. Key Words: Editorials ◼ nitrates ◼ nitric oxide
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Harnessing the Nitrate−Nitrite−Nitric Oxide Pathway for Therapy of Heart Failure With Preserved Ejection Fraction Rebecca Vanderpool and Mark T. Gladwin Circulation. 2015;131:334-336; originally published online December 22, 2014; doi: 10.1161/CIRCULATIONAHA.114.014149 Circulation is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 2014 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7322. Online ISSN: 1524-4539
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N
itrate accumulates in the plasma from oral intake of foods rich in nitrate such as green leafy vegetables and root plants such as beet root or from the intravascular oxidation of nitric oxide (NO), produced by the NO synthase enzymes, to nitrate by oxyhemoglobin. Nitrate is then concentrated in the saliva and reacts with oral commensal bacteria, which contain nitrate reductase enzymes.1 Humans do not possess nitrate reductase enzymes and thus require these bacteria for conversion of nitrate to nitrite. Nitrite is then swallowed and systemically absorbed so that it can be further reduced via single-electron transfer reactions with hemoglobin, myoglobin, neuroglobin, and molybdopterin-containing enzymes (such as xanthine oxidase, aldehyde oxidase, and mARC).2–7 Now referred to as the nitrate-nitrite-NO pathway, this involves a series of oxygen-independent and NO synthase–independent single-electron transfer reactions [Figure (A)].
Articles see p 371 and p 381 Nitrate and nitrite have now been shown to regulate blood pressure, hypoxic vasodilation, mitochondrial efficiency, and exercise performance.2,10–14 Infusions of nitrite have been shown to directly vasodilate the forearm arterial and venous circulation, with significant potentiation during hypoxia.10,14 Systemic infusions of nitrite in dogs exhibit both arteriolar and venous dilation, with more potent effects on the systemic vascular resistance, increasing cardiac output.15 Numerous normal human volunteer studies have now demonstrated that nitrate supplementation can increase exercise efficiency, as ⋅ defined by total oxygen consumption (Vo2) for total work 11 performed (Watts). In preclinical studies, nitrite has been shown to exhibit therapeutic efficacy in mouse, rat, and sheep models of pulmonary hypertension and mouse models of heart failure.16,17 Additionally, a recent study indicates that dietary supplementation of nitrate reverses the features of metabolic syndrome in endothelial NO synthase–deficient mice.18 Considering the known association between features of the The opinions expressed in this article are not necessarily those of the editors or of the American Heart Association. From the Pittsburgh Heart, Lung, Blood and Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, PA (R.V., M.T.G.); and Division of Pulmonary, Allergy and Critical Care Medicine, UPMC and University of Pittsburgh, Pittsburgh, PA (R.V., M.T.G.). Correspondence to Mark T. Gladwin, MD, Pittsburgh Heart, Lung, Blood and Vascular Medicine Institute, Division of Pulmonary, Allergy and Critical Care Medicine, Department of Medicine, University of Pittsburgh, E1244 BST 200 Lothrop St, Pittsburgh, PA 15213. E-mail [email protected] (Circulation. 2015;131:334-336. DOI: 10.1161/CIRCULATIONAHA.114.014149.) © 2014 American Heart Association, Inc. Circulation is available at http://circ.ahajournals.org DOI: 10.1161/CIRCULATIONAHA.114.014149
metabolic syndrome and heart failure with preserved ejection fraction (HFpEF), as well as the secondary development of pulmonary vascular disease and exercise intolerance in this disease, a potential therapeutic role for nitrate and nitrite for HFpEF is being considered and evaluated. In this issue of Circulation, Zamani and colleagues8 enrolled 17 subjects with HFpEF in a double-blind, placebocontrolled, crossover trial of a single dose of 12.9 mmol nitrate in 140 mL beet root juice or nitrate-depleted placebo beet root juice to address this question. The rationale for studying HFpEF was based on a number of pathogenic features that might be responsive to NO therapeutics, including abnormal diastolic left ventricular function, reduced exercise tolerance, reduced vasodilatory reserve during exercise, and abnormal skeletal muscle perfusion and function [Figure (A)]. The diagnosis of HFpEF was defined by clinical signs and symptoms of HFpEF on echocardiography, an increased ratio of the early mitral inflow velocity (E) to septal tissue Doppler velocity (e′) >8, and indirect or direct evidence of elevated left ventricular filling pressures. The primary end point of the trial was exercise efficiency, defined by the ratio of total work performed to total oxygen consumed, measured by supine cycle ergometer 2 hours after consumption of the beet root juice or placebo. A number of additional analyses were performed at maximal effort and constant-intensity cardiopulmonary exercise testing, including the evaluation of cardiac function by echocardiography, skeletal muscle deoxygenation during exercise in the calf using near-infrared spectroscopy, forearm postocclusive oxygen consumption rates and reactive hyperemia measured by Doppler ultrasound, and levels of plasma NO metabolites. Although there was no observed significant change in exercise efficiency, the primary end point of the trial, both exercise oxygen consumption and total work performed significantly increased. In addition, the exercise duration significantly increased by almost 1 minute, which was associated with significant reductions in aortic augmentation index, increases in cardiac output, and decreases in calculated systemic vascular resistance. There were no significant changes in near-infrared spectroscopy and postocclusive oxygen consumption or hyperemic flows, although trends for improvements were observed. These findings suggest that nitrate improves exercise capacity in HFpEF by reducing systemic vascular resistance and left ventricular pulsatile afterload and enhancing cardiac output and oxygen delivery to exercising muscles [Figure (B)]. Although these findings are provocative and the trial was well designed, a major limitation is a reliance on secondary end points, with a primary end point that is not consistent with a number of studies on the published effects of nitrate in normal volunteers.11 A central effect observed with nitrate
Downloaded from http://circ.ahajournals.org/ by guest on April 24, 2015 334
Vanderpool and Gladwin Harnessing the Nitrate-Nitrite-NO Pathway 335
Figure. A, The nitrate–nitrite–nitric oxide (NO) pathway is a series of oxygen-independent and NO synthase–independent single-electron transfer reactions that ultimately facilitate vasodilation. NO therapeutics (nitrate [NO3−] or nitrite [NO2−]) are potentially effective at reversing pathological features of heart failure with preserved ejection fraction (HFpEF). B, Findings from Zamani et al8 suggest that nitrate improves exercise capacity in HFpEF by reducing systemic vascular resistance (SVR) and left ventricular (LV) pulsatile afterload and enhancing cardiac output and oxygen delivery to exercising muscles. C, Webb et al9 found that nitrite significantly dilated both conduit and peripheral arteries. The mechanism by which nitrite dilates conduit arteries is still unknown. ETC indicates electron transport chain; and oxyHb, oxyhemoglobin.
repletion in prior studies was the effect on improved exercise efficiency, characterized by an increase in work performed for any level of oxygen consumption.11 On the other hand, the finding of an increase in exercise duration has been reported, particularly in disease states such as chronic obstructive pulmonary disease.19 The study also uses noninvasive measurements of hemodynamics and a single oral dose of nitrate, with no long-term follow-up to evaluate long-term effects on both exercise capacity or signs and symptoms of heart failure and the metabolic syndrome.
How Does Nitrite Vasodilate? Implications for HFpEF Therapy A second study by Omar and colleagues9 published in this issue of Circulation explored in more detail the pharmacological properties of nitrite in the human circulation, reporting for the first time that nitrite vasodilates not only the arteriolar and venous circulation, as previously described, but also the conduit blood vessels, an effect similar to that observed with nitroglycerin. This effect was associated with a reduction in central systolic blood pressure, augmentation index, and pulsed-wave velocity, which represent hemodynamic effects that might show therapeutic promise in the setting of HFpEF [Figure (C)]. Interestingly, although the effects of nitrite on arteriolar blood flow (measured by venous occlusion strain-gauge plethysmography) were potentiated by hypoxia, as previously described and quite distinct from the effects of nitroglycerin,14 the vasodilatory effects on the conduit arteries were actually inhibited under hypoxia and hyperoxia conditions. The mechanism for this normoxic nitrite bioactivation remains uncertain, but the lack of significant deoxygenation of hemoglobin or myoglobin and the dominant oxidase activities of molybdopterin enzymes under normoxic conditions
suggest that these pathways are unlikely to account for this conduit vasodilatory effect. Omar and colleagues9 infused 2 drugs to test the mechanisms for nitrite bioactivation in conduit arteries. The first was reloxifene, an inhibitor of aldehyde oxidize, classically considered an oxidase that generates superoxide but recently shown also to possess nitrite reductase activity under hypoxic conditions.20,21 Infusion of the aldehyde oxidase inhibitor actually increased nitrite-dependent conduit artery vasodilation, suggesting that this enzyme is in fact generating superoxide, which can scavenge NO. Reduced production of superoxide likely increases NO bioavailability from nitrite and increases vasodilator potency, similar to previous observations with inhibition of xanthine oxidase during nitrite infusions in the human forearm.10 The authors next tested infusions of acetazolamide, an inhibitor of carbonic anhydrase, also proposed to be a nitrite reductase (anhydrase).22 This inhibitor also increased the potency of nitrite. Although Aamand and colleagues22 have proposed that classic inhibitors of carbonic anhydrase can paradoxically increase nitrite reduction by this enzyme, we have recently shown that acetazolamide and dorzolamide reduce nitrite conversion to NO by carbonic anhydrase23, raising questions about this pathway in nitrite reduction. Clearly, carbonic anhydrase and aldehyde oxidase are major enzymes mediating nitrite conversion to NO, and more work is required to explore pathways for the observed conduit artery normoxic vasodilation. Nitrite is an inorganic salt (NO2−) and must be clearly differentiated from the organic nitrates such as nitroglycerin that are characterized by covalently bound nitro (NO2-R) groups adducted to carbon-based small molecules. The latter species require enzymatic metabolism to form NO and nitrite, and this enzymatic process is subject to tolerance. Studies in humans and primates suggest that nitrite is not subject to enzymatic
Downloaded from http://circ.ahajournals.org/ by guest on April 24, 2015
336 Circulation January 27, 2015 tolerance and continues to produce sustained vasodilation with long-term exposure.10 This lack of enzymatic tolerance suggests potential greater therapeutic potential for nitrite as a long-term therapy for HFpEF. These new studies now clarify that nitrite drives pharmacological arteriolar, venous, and conduit artery vasodilation, all effects that may prove beneficial as a long-term therapy for HFpEF. More study is required to understand how nitrite dilates conduit arteries under normoxic conditions and to test therapeutic effects of more long-term treatments with nitrate or nitrite in patients with HFpEF.
Disclosures Dr Gladwin is a coinventor on a National Institutes of Health government patent for the use of nitrite salts in cardiovascular diseases. Dr Gladwin consults with Aires/MAST Pharmaceuticals on the development of a phase II proof-of-concept trial using inhaled nitrite for pulmonary hypertension. Dr Vanderpool reports no conflicts.
References 1. Lundberg JO, Weitzberg E, Gladwin MT. The nitrate-nitrite-nitric oxide pathway in physiology and therapeutics. Nat Rev Drug Discov. 2008;7:156–167. doi: 10.1038/nrd2466. 2. Cosby K, Partovi KS, Crawford JH, Patel RP, Reiter CD, Martyr S, Yang BK, Waclawiw MA, Zalos G, Xu X, Huang KT, Shields H, Kim-Shapiro DB, Schechter AN, Cannon RO 3rd, Gladwin MT. Nitrite reduction to nitric oxide by deoxyhemoglobin vasodilates the human circulation. Nat Med. 2003;9:1498–1505. doi: 10.1038/nm954. 3. Shiva S, Huang Z, Grubina R, Sun J, Ringwood LA, MacArthur PH, Xu X, Murphy E, Darley-Usmar VM, Gladwin MT. Deoxymyoglobin is a nitrite reductase that generates nitric oxide and regulates mitochondrial respiration. Circ Res. 2007;100:654–661. doi: 10.1161/01. RES.0000260171.52224.6b. 4. Tiso M, Tejero J, Basu S, Azarov I, Wang X, Simplaceanu V, Frizzell S, Jayaraman T, Geary L, Shapiro C, Ho C, Shiva S, Kim-Shapiro DB, Gladwin MT. Human neuroglobin functions as a redox-regulated nitrite reductase. J Biol Chem. 2011;286:18277–18289. doi: 10.1074/jbc.M110.159541. 5. Zhang Z, Naughton DP, Blake DR, Benjamin N, Stevens CR, Winyard PG, Symons MC, Harrison R. Human xanthine oxidase converts nitrite ions into nitric oxide (NO). Biochem Soc Trans. 1997;25:524S. 6. Sparacino-Watkins CE, Tejero J, Sun B, Gauthier MC, Thomas J, Ragireddy V, Merchant BA, Wang J, Azarov I, Basu P, Gladwin MT. Nitrite reductase and nitric-oxide synthase activity of the mitochondrial molybdopterin enzymes mARC1 and mARC2. J Biol Chem. 2014;289:10345– 10358. doi: 10.1074/jbc.M114.555177. 7. Kim-Shapiro DB, Gladwin MT. Mechanisms of nitrite bioactivation. Nitric Oxide. 2014;38:58–68. doi: 10.1016/j.niox.2013.11.002. 8. Zamani P, Rawat D, Prithvi S-K, Geraci S, Bhuva R, Konda P, Doulias P-T, Ischiropoulos H, Townsend R, Margulies K, Cappola T, Poole D, Chirinos J. Effect of inorganic nitrate on exercise capacity in heart failure with preserved ejection fraction. Circulation. 2015;131:371–380. 9. Omar S, Fok H, Tilgner K, Nair A, Hunt J, Jiang B, Taylor P, Chowienczyk P, Webb A. Paradoxical normoxia-dependent selective actions of inorganic nitrite in human muscular conduit arteries and related selective actions on central blood pressures. Circulation. 2015;131:381–389. 10. Dejam A, Hunter CJ, Tremonti C, Pluta RM, Hon YY, Grimes G, Partovi K, Pelletier MM, Oldfield EH, Cannon RO 3rd, Schechter AN, Gladwin
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Harnessing the Nitrate−Nitrite−Nitric Oxide Pathway for Therapy of Heart Failure With Preserved Ejection Fraction Rebecca Vanderpool and Mark T. Gladwin Circulation. 2015;131:334-336; originally published online December 22, 2014; doi: 10.1161/CIRCULATIONAHA.114.014149 Circulation is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 2014 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7322. Online ISSN: 1524-4539
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