FORUM REVIEW ARTICLE

Nitrite Signaling in Pulmonary Hypertension:Mechanisms of Bioactivation, Signaling, and Therapeutics

Marta Bueno,1 Jun Wang,1 Ana L. Mora,1,2 and Mark T. Gladwin1,2

Abstract

Significance: Pulmonary arterial hypertension (PAH) is a disorder characterized by increased pulmonary vas-cular resistance and mean pulmonary artery pressure leading to impaired function of the right ventricle, reducedcardiac output, and death. An imbalance between vasoconstrictors and vasodilators plays an important role inthe pathobiology of PAH. Recent Advances: Nitric oxide (NO) is a potent vasodilator in the lung, whosebioavailability and signaling pathway are impaired in PAH. It is now appreciated that the oxidative product ofNO metabolism, the inorganic anion nitrite (NO2

- ), functions as an intravascular endocrine reservoir of NObioactivity that can be reduced back to NO under physiological and pathological hypoxia. Critical Issues: Theconversion of nitrite to NO is controlled by coupled electron and proton transfer reactions between heme- andmolybdenum-containing proteins, such as hemoglobin and xanthine oxidase, and by simple protonation anddisproportionation, and possibly by catalyzed disproportionation. The two major sources of nitrite (and nitrate)are the endogenous l-arginine–NO pathway, by oxidation of NO, and the diet, with conversion of nitrate fromdiet into nitrite by oral commensal bacteria. In the current article, we review the enzymatic formation of nitriteand the available data regarding its use as a therapy for PAH and other cardiovascular diseases. Future Di-rections: The successful efficacy demonstrated in several animal models and safety in early clinical trials suggestthat nitrite may represent a promising new therapy for PAH. Antioxid. Redox Signal. 18, 1797–1809.

Introduction

Pulmonary hypertension (PH) is a life-threatening pro-gressive disorder characterized by persistent elevation in

pulmonary artery pressure ( > 25 mmHg at rest), and in-creased pulmonary vascular resistance. With increasing pul-monary vascular resistance, there is a progressive increase inafter-load on the right ventricle, leading to concentric hyper-trophy, dilation, and decreased function (Fig. 1). This exertssignificant stress on the right heart that will eventually fail ifleft untreated, leading to a drop in cardiac output. Right heartfailure and a low cardiac output lead to the major symptomsof pulmonary arterial hypertension (PAH), such as dyspneaon exertion and syncope, and increasing risk of death.

PAH is a multifactorial process, but early disease is related toa dysregulation of critical vasodilator pathways (down-regulation of nitric oxide [NO] and prostaglandin signaling)and vasoconstrictor pathways (upregulation of endothelin-1and reactive oxygen species [ROS] signaling) (75). PAH is as-sociated with decreased bioavailability and responsiveness ofNO (14, 32). NO is produced in mammalian cells primarily inthe metabolism of l-arginine. NO synthase (NOS) catalyzes the

oxidation of l-arginine to produce l-citrulline in the presence ofoxygen and NADPH (Fig. 2). Although the expression levels ofendothelial NOS (eNOS) in patients with PAH can vary fromhigh to normal, the formation of NO and eNOS activity havebeen found reduced in patients with idiopathic PAH (75). Re-cent studies suggest that eNOS uncoupling may relate to im-paired NO production in PAH, a process by which the enzymetransfers electrons from the NOS reductase domain to theoxygenase domain and diverted to molecular oxygen formingsuperoxide rather than NO. This dysfunctional state of theeNOS enzyme in the presence of high levels of superoxidegeneration may increase the formation of peroxynitrite for-mation and enhance the vascular disease during PH.

Inhaled NO (iNO) is considered as a potential therapytargeting the NO pathway (33, 39), and a potent and selectivepulmonary vasodilator (65). iNO therapy has been proposedfor treatment of PAH, but also for persistent PH of the new-born (15), and bronchopulmonary dysplasia in prematurelyborn infants (21) and post-cardiac surgery (3). Other possibleapplications of iNO therapy include the treatment of pulmo-nary ischemia-reperfusion injury (9), the acute respiratorydistress syndrome (18), and hypoxemia in the setting of severe

1Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, Pennsylvania.2Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania.

ANTIOXIDANTS & REDOX SIGNALINGVolume 18, Number 14, 2013ª Mary Ann Liebert, Inc.DOI: 10.1089/ars.2012.4833

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chronic obstructive pulmonary disease (26). However, iNOgas has its limitations regarding dose and duration of theexposure, cumbersome and expensive delivery systems, off-target reactions of NO with oxygen to form reactive nitrogenspecies, and possible rebound PH when the intervention isinterrupted (38). These limitations open the door to othermolecules that can be a source of NO and nitrosative signal-ing, including the anion nitrite (NO2

- ) (55). Nitrite is a uniquereactive nitrogen species, as it is relatively stable compared toNO [51.4 min (67) vs. 0.05–1.8 ms (68) half-life in whole blood]and can readily be reduced to NO, oxidized to nitrogen di-oxide, and protonated to form S-nitrosothiol, providing NO-dependent and NO-independent signaling effects that havebeen reported to modulate vascular remodeling (2, 86).

Rediscovering Nitrite in Biology

Although in the ancient Chinese medicine, the sodium saltsof nitrate and nitrite were used as a remedy for heart painsand ischemia (circa 800 AD) (54), only until recently, nitrate(NO3

- ) and nitrite (NO2- ) were mostly considered undesired

residues in the food chain with potential carcinogenic activity.Nitrate salts have also been used by early civilizations to curemeats, which not only improve their storage time and pro-duce their reddish color but also kill pathogenic bacteria (69).The mechanism of the meat preservation by nitrate wascharacterized in the 19th century, when it was discovered tobe converted to NO (bound to myoglobin). During the 1970s,public concern over toxicity increased, when nitrate andnitrite were associated to the endogenous formation of N-nitrosamines, which are carcinogenic. While remaining ex-tremely controversial, the epidemiological links betweennitrate/nitrite exposure, for example, in high-nitrate foodssuch as leafy green vegetables, remain unclear (25, 79).

The first relationship between nitrite formation and NOproduction was found by studying immune responses in ac-tivated macrophages. Analyses of bacterial-infected (74) ortumor-bearing (36) mice showed that macrophages use l-arginine to produce nitrite and l-citrulline. This was the firstdemonstration of the oxidation of a terminal nitrogen atom ofl-arginine to an inorganic nitrogen oxide, an enzymatic ac-tivity later shown to be mediated by inducible NOS (iNOS).Marletta and colleagues define that a terminal guanidino ni-trogen atom was the precursor of nitrite and nitrate synthe-sized by activated macrophages (41), and Tannenbaum et al.(76) confirmed that nitrate and nitrite are formed de novo in theintestine of the human body, reflecting in vivo NO formationand oxidation. These observations were followed by thedemonstration of NO synthesis by mammalian endothelialcells. Moving forward another decade, NO was identified as acritical regulator of vascular homeostasis, neurotransmission,

FIG. 1. Radiological imaging in PH. (A) Contrast en-hanced-CT image showing enlarged pulmonary artery inPAH patient. (B) CT image obtained from patient with se-vere PH. The right-sided chambers are dilated, the rightventricle hypertrophied. (C) Apical four-chamber two-dimensional echocardiography image showing enlargedright-sided chambers and small left ventricle. CT, computedtomography; LA, left atrium; LV, left ventricle; PA, pulmonaryartery; PAH, pulmonary arterial hypertension; PH, pulmonaryhypertension; RA: right atrium; RV, right ventricle.

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1798 BUENO ET AL.

and host defense; and nitrite was considered a biomarker ofNO levels or NO formation in vivo (40, 62).

More recently, increasing evidence suggests that nitrite notonly is a biomarker of NO formation from NO synthesis butalso represents a storage reservoir for NO that can be con-verted back to NO during physiological and pathologicalhypoxia (8, 16, 56). It is now known that nitrate and nitrite canbe recycled to NO (or other bioactive forms of nitrogen) inblood and peripheral tissue (27, 55), representing an alterna-tive to the classical l-arginine-NOS-NO signaling. In situa-tions of hypoxia, when the oxygen-dependent function of theNOS may become compromised, nitrite reduction is en-hanced. Chemically, nitrite shows a unique redox positionbetween oxidative (NO2 radical) and reductive (NO radical)signaling and has a relatively long half-life in blood and tissue(67), representing a storage pool supporting NO signalingduring hypoxic or metabolic stress.

The Nitrate-Nitrite-NO Signaling Pathway

The reductive mammalian nitrate-nitrite-NO pathwaymediates important signaling events and complements thetraditional oxidative l-arginine-NOS-NO pathway. The twomajor sources of nitrite (and nitrate) are the endogenous l-arginine-NO pathway and the diet. Our daily intake of ex-ogenous nitrite mainly comes from food additives, preserva-tives, and drinking water, and nitrate from leafy greens androot vegetables. Upon intake of nitrate (Fig. 3), symbioticbacteria in the oral cavity can reduce nitrate to nitrite pro-ducing a high concentration of nitrite (up to low millimolar) insaliva (8, 56). Salivary glands are able to concentrate and se-crete nitrate (from plasma or diet), increasing the amount ofnitrate available to the oral microbiome to be reduced to ni-trite. When saliva is then swallowed and reaches the low pH

milieu of the stomach, nitrite is converted nonenzymaticallyto NO via protonation and reduction (8) (Table 1). Nitrate andnitrite can be absorbed in the gastrointestinal tract into thecirculation. Excess of nitrate will be excreted, and nitrite canbe further reduced to NO by different enzymes through ourbody (55). NO can be oxidized to nitrate by oxyhemoglobin(29) or to nitrite by reaction with oxygen [hydrophobic NOautoxidation (53)] and by reaction with the copper-containingceruloplasmin (73). This new pool of nitrite and nitrate isavailable again for uptake by the salivary glands, closingwhat is known as the enterosalivary circulation of nitrate (55).

Mechanisms of Nitrite Bioactivation

Nitrite can be reduced to NO in vivo by both enzymatic andnonenzymatic processes (Fig. 4). Biological non-enzymatic NOformation was first reported by Zweier et al. (87) in an ischemicrat tissue where, at low pH, there was NOS-independent NOproduction. Benjamin et al. (8) showed that in the highly acidicenvironment (pH 3) of the stomach, nitrite is converted to NOgas. Nitrite production of NO at acidic pH is also enhanced bythe presence of reducing compounds such as copper, ascorbate,and polyphenols (82) [Table 1, Eqs. (1)–(3)].

Enzymatic NO formation from nitrite, via facilitated protonand electron transfer reactions, has been reported for a widevariety of metal-containing proteins, including hemoglobin (22),myoglobin (70), neuroglobin (77), cytochrome c (4), cytochrome coxidase (12), eNOS (78), xanthine oxidoreductase (XOR) (84),aldehyde oxidase (AO) (50), and carbonic anhydrase (1).

Heme-containing proteins

Deoxyhemoglobin. Deoxyhemoglobin (deoxy-Hb) can cata-lyze nitrite reduction accompanied with NO generation and

FIG. 2. The classical l-arginine-NOS-NO signaling pathway. NO is produced in mammalian cells by an oxygen-dependent oxidation of a guanidine nitrogen of l-arginine (with citrulline as a side product). This multistep reaction is catalyzedby the heme-containing protein NOS, which also requires two flavin molecules and tetrahydrobiopterin as cofactors. In mostendothelial cells, the type III isoform is expressed (or eNOS) as is regulated by calcium-dependent binding of calmodulin and bytyrosine phosphorylation. Target tissue effects of NO depend on its quantity. At higher concentrations, NO rapidly reacts withoxygen (and especially with superoxide), forming the highly reactive peroxynitrite. At lower concentrations, NO serves aregulatory role via the activation of soluble guanylate cyclase, resulting in increased cGMP levels in target cells. In vascularsmooth muscle, cGMP causes relaxation by reducing the intracellular calcium concentration and by downregulating the con-tractile apparatus. These actions are mostly (although not exclusively) mediated by type I cGMP-dependent protein kinases.cGMP, cyclic guanosine monophosphate; eNOS, endothelial NOS; NO, nitric oxide; NOS, nitric oxide synthase. (To see thisillustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars.)

NITRITE SIGNALING IN PULMONARY HYPERTENSION 1799

production of ferric heme (Fe + 3). The formed NO can thenbind to another deoxyheme to form iron-nitrosyl (Fe + 2-NO)(Table 1) (22). As for other heme proteins, the reaction is fasterat low pH, indicating the involvement of the protonated ni-trite (nitrous acid—HNO2) [Table 1, Eq. (6)]. The reaction isallosterically regulated, being slow in the T-state, but muchfaster for the R-state deoxy-Hb (37). There is a balancebetween the faster reaction of nitrite with the R-state oxyhe-moglobin and the availability of deoxyhemes to bind tonitrite, leading to a maximal rate of nitrite reduction at about50% hemoglobin–oxygen saturation. These factors have sug-gested that the reaction can respond to change in pH andoxygen tension in blood and tissues, and potentially mediatehypoxic vasodilation (28, 29, 37).

Deoxymyoglobin. It reduces nitrite to NO at a faster ratethan deoxy-Hb, and has been shown to modulate mitochon-drial respiration via inhibition of cytochrome c oxidase (70, 72)

(Fig. 5). Confirmatory studies in myoglobin knockout micesuggest a role for myoglobin in regulating respiration andcardiac energetics, and new studies suggest a contribution tohypoxic vasodilation (35). As indicated for hemoglobin, thereaction rates increase with low pH, which is usually linked tohypoxia. Therefore, myoglobin can use nitrite efficiently toproduce NO during hypoxic or ischemic conditions (72).

Cytochrome c. It is located at the intermembrane space ofmitochondria, where it shuttles electrons from complex III ofthe mitochondrial respiratory chain to cytochrome c oxidase(complex IV). Under hypoxic conditions and those in whichthe cytochrome c heme becomes pentacoordinate, cytochrome

FIG. 3. The enterosalivary circulation of nitrate in hu-mans. The activity of orally ingested inorganic nitrate (fromdietary sources—entry point as a green arrow) is thought to liein its conversion to nitrite by facultative anaerobic bacteriafound on the dorsal surface of the tongue. By swallowing,saliva enters the acidic stomach (around 1 L per day), wheremuch of the nitrate is rapidly protonated to form nitrous acid,which decomposes further to form NO (absorption arrow inblue) and other bioactive nitrogen species. Nitrate and re-maining nitrite are then absorbed from the intestine into thecirculation and can be converted to bioactive NO in blood(absorption arrow in blue). Later on, NO can again be oxidizedto nitrite and/or nitrate in tissue. Although much of the ni-trate is excreted in the urine (excretion arrow in red), salivaryglands actively concentrate nitrate from plasma that can goback again to the mouth to be reduced to nitrite. (To see thisillustration in color, the reader is referred to the web version ofthis article at www.liebertpub.com/ars.)

Table 1. Biological Reactions

of the Reduction of Nitrite

Agent Reaction

Protons, acidicmilieu

NO2- + H + 4HNO2 Eq. (1)

2HNO24N2O3 + H2O Eq. (2)N2O34NO + NO2 Eq. (3)

Ascorbate NO2- + H + 4HNO2

2HNO2 + Asc/2NO+ dehydroAsc + 2H2O

Eq. (4)

Polyphenols NO2- + H + 4HNO2

Ph-OH + HNO2/PH-O+ NO + H2O

Eq. (5)

Heme-containingproteins

NO2- + Fe2 + + H + /NO

+ Fe3 + + OH-Eq. (6)

Molybdopterin-containing proteins

NO2- + Mo4 + + H + /NO

+ Mo5 + + OH-Eq. (7)

FIG. 4. Nitrite chemistry, physiology, and therapeutics.Nitrite reduction to NO is favored by decreasing physiologicaloxygen tensions and low pH, via nonenzymatic pathways (inthe presence of acid or reducing substrates) or enzymaticpathways catalyzed by metal-containing enzymes. The gen-erated NO modulates critical signal transduction processes,inducing cytoprotection, vasodilation, and inhibition of SMCproliferation (30). SMC, smooth muscle cell. (To see thisillustration in color, the reader is referred to the web version ofthis article at www.liebertpub.com/ars.)

1800 BUENO ET AL.

c can bind nitrite and reduce nitrite to NO (4). The reduction ofnitrite to NO by cytochrome c is vastly increased in the five-coordinate state, which is promoted by cardiolipin liposomes(6). This reductive reaction occurs between ferrous cyto-chrome c (Fe2 + ) and nitrite and represents a typical electron–proton transfer reaction [Table 1, Eq. (6)] (4).

Cytochrome c oxidase (complex IV). Nitrite can reactwith cytochrome c oxidase to form NO (12). This reductive re-action occurs between ferrous cytochrome c oxidase (Fe2 + ) andnitrite and represents a typical electron–proton transfer reaction[Table 1, Eq. (6)]. Note that nitrite can also be reduced by otherproteins, such as myoglobin, to form NO, which in turn can bindto ferrous cytochrome c oxidase and inhibit respiration. Theformation of NO and inhibition of cytochrome c oxidase havebeen proposed as a pathway to reduce oxygen consumption atlow oxygen levels, thus promoting oxygen diffusion deeper intoischemic tissues (35, 70, 71). In summary, cytochrome c oxidasehas been proposed to function as both a nitrite reductase thatgenerates NO, as well as a target for nitrite-NO-dependentregulation of respiration and oxygen consumption.

Neuroglobin. Evolved from a common ancestor shared byhemoglobin and myoglobin, human neuroglobin is found inbrain neurons and also possess nitrite reductase activity.Neuroglobin, like cytochrome c, is a six-coordinate hemeprotein, with two histidines bound to the heme iron. Thebinding and reduction of nitrite to NO are favored when theheme is five coordinated. Tiso et al. (77) reported that deox-yneuroglobins where the sixth ligand (Histidine 64) is mu-tated to a nonheme-binding side chain (H64L and H64Q) can

reduce nitrite at a very high rate. The wild-type neuroglobinsix-to-five coordination is also regulated in a redox-dependentfashion, with oxidation of two surface redox-sensitive thiolsto disulfides increasing the open probability of the heme andincreasing the rate of nitrite binding and reduction to NO.Tiso et al. showed in these studies that the nitrite reductasereaction of neuroglobin could modulate mitochondrial respi-ration, similar to that shown for myoglobin (Table 2) (77). Thispathway suggests a possible redox sensor mechanism forneuroglobin-mediated nitrite reduction. (Fig. 6)

Nitric oxide synthase. While though NOS is responsiblefor normoxic NO generation from arginine oxidation, it hasbeen recently reported that NOS also catalyzes nitrite reduc-tion to NO under anoxic conditions. Vanin et al. reported thateNOS catalyzes anoxic NO formation in murine microvascularbrain endothelial cells, which is abolished by the eNOS

FIG. 5. Sites of mitochon-drial nitrite reduction. Upperpanel: In normoxia, electronsenter the respiratory chain atcomplex I or II and are shut-tled through the Q cycle tocomplex III. Electrons are thenshuttled to cytochrome c andthen to complex IV, whereoxygen acts as the terminalelectron acceptor. Protons arepumped from the matrix tothe intermembrane spacethrough the complexes to setup a proton gradient for ATPgeneration. Lower panel: Dur-ing hypoxia, nitrite can be re-duced at complex III (7) orcytochrome c oxidase (com-plex IV) (12). If cytochrome c isconverted to its pentacoordi-nate form (through oxidation,nitration, or association withanionic lipid), it can reducenitrite to NO (6, 72). (To seethis illustration in color, thereader is referred to the webversion of this article at www.liebertpub.com/ars.)

Table 2. Summary of Bimolecular Reaction Rates

of Heme-Containing Proteins with Nitrite

Protein k (M - 1 s - 1)

Hemoglobin (T-state)a 0.12Hemoglobin (R-state)a 6Myoglobinb 5.6Neuroglobin ( - SS - )c 0.12Neuroglobin ( - SH)c 0.062

aHuman, at 37�C, pH 7.4 (37).bSperm whale, at 25�C, pH 7.4 (77).cHuman, at 25�C, pH 7.4 (77).

NITRITE SIGNALING IN PULMONARY HYPERTENSION 1801

inhibitors Nx-nitro-l-arginine or Nx-nitroarginine methyl ester(L-NAME) (78). This pathway may represent an alternative NOsource during severe hypoxia in tissues. Webb et al. have shownthat eNOS exists on whole red blood cells (81). When nitritereacted with red blood cells in the presence of L-NAME or l-arginine at low oxygen tensions, NO production was inhibitedby about 60%, while d-arginine, the inactive isomer, showed nosignificant effects, leading them to propose that this pathwaymay have some relevance to vasodilation. Nevertheless, furtherstudies are needed to reconcile how this pathway may functionin vivo, because it requires extremely low oxygen tensions.

Molybdopterin-containing proteins

XOR and AO are two dimeric molybdopterin enzymesubiquitously expressed in human tissues, containing one mo-lybdenum (Mo) center, two nonidentical Fe2S2 clusters, and aflavin adenine dinucleotide cofactor. XOR catalyzes the ter-minal two steps in purine degradation and exists in cells pri-marily as a dehydrogenase (xanthine dehydrogenase [XDH])where substrate-derived electrons reduce NAD + to NADH.During inflammatory conditions, oxidation of critical cysteineresidues or limited proteolysis converts XDH to xanthine oxi-dase (XO) (80). XO transfers substrate-derived electrons to O2,generating O2

� - and H2O2. However, conversion to XO is not arequisite for ROS production, as XDH displays partial oxidaseactivity under conditions in which the NADH/NAD + ratio isincreased such as the hypoxemia (34). It is also under hypoxic(0–1% O2) conditions that both XO- and XDH-mediated nitritereductase activities have been reported (47, 51). This is evi-denced by inhibition of XO- and XDH-mediated reduction ofnitrite to NO by the XOR-specific inhibitor, oxypurinol, and/orhigh concentrations of xanthine. This oxypurinol- and xan-thine-induced inhibition of NO generation rates results fromtheir binding to the Mo site, which is also the catalytic center fornitrite binding and reduction. A similar effect on nitrite re-

duction was observed for AO, which is inhibited by raloxifene.As the inhibitor for the flavin site, diphenylene iodoniumchloride does not affect nitrite reduction when xanthine or al-dehyde served as the electron donor, but considerably reducesrates of NO formation rates when NADH is the reducingsubstrate. These experiments indicate that nitrite is reduced atthe Mo center of XO or AO, but electrons can be provided eitherat the Mo or the flavin site by various substrates (Fig. 7). It alsosuggests that electron withdrawal from XOR or AO by nitritemay serve not only to produce NO but also to reduce ROSformation and as such elicit salutary actions regarding NOderived from alternative sources.

Nitrite disproportionation

Another mechanism for nitrite bioactivation is dispropor-tionation, with nitrous acid or NO + reacting with nitrite toform N2O3 [Table 1, Eqs. (2) and (3)]. This reaction has beenproposed to be catalyzed by ferric hemoglobin (5) as well ascarbonic anhydrase. Aamand et al. (1) recently demonstrated

FIG. 6. Neuroglobin acts as a nitrite reductase under ox-idative stress conditions. The thiol state of the glutathione isa good indicator of the oxidative stress in vivo conditions andcan be modulated in vitro. In normal conditions, cells keep ahigh concentration of reduced glutathione (GSH) and lowoxidized glutathione (GSSG). In these circumstances, the dis-ulfide bond of Ngb is not formed, and the protein has a lownitrite reductase activity (left). As oxidative stress conditionsdevelop (right), reduced glutathione is consumed, and thenumber of neuroglobin molecules with formed disulfidebonds increases. This leads to increased production of NOfrom nitrite, causing the inhibition of respiratory enzymes andlimiting oxygen consumption and reactive oxygen species-producing reactions [reproduced with permission from (77)].(To see this illustration in color, the reader is referred to theweb version of this article at www.liebertpub.com/ars.)

FIG. 7. The nitrite reductase activity of XOR and AO canbe inhibited at either molybdenum or flavin site. Oxypur-inol or high concentrations of xanthine inhibit nitrite reduc-tion by binding to the molybdenum center of XOR. AO canbe inhibited specifically by raloxifene, which binds to themolybdenum site. Electron can be provided at the flavin site,which is transferred via Fe2S2 clusters to the molybdenumsite. DPI blocks the electron transfer at the flavin site, thusinhibiting the nitrite reductase activity of XOR or AO. Therelative distances of the four redox-active centers were takenfrom the crystal structure of the bovine XOR (PDB code:1fo4). AO, aldehyde oxidase; DPI, diphenylene iodoniumchloride; XOR, xanthine oxidoreductase. (To see this illus-tration in color, the reader is referred to the web version ofthis article at www.liebertpub.com/ars.)

1802 BUENO ET AL.

that NO was generated by nitrite reactions with carbonicanhydrase under both normoxia and hypoxia, which wasstimulated by dorzolamide and acetazolamide, two specificinhibitors of carbonic anhydrase (Table 3). They propose thatnitrite is structurally similar to bicarbonate and may reactwith histidines in the enzyme catalytic site to disproportionateto NO. However, more work is needed to fully explore thisnewly described pathway.

Therapeutic Application of Nitrite for PH

Recent studies have investigated the therapeutic effects ofnitrite in PH models (85). Hunter et al. (38) first reported theeffects of nebulized nitrite as a selective pulmonary vasodi-lator in an ovine hypoxia model. In this study, vasoconstric-tion induced by hypoxia was reduced by inhalation of NO gas(20 ppm) or nebulized nitrite (300 mg in 5 ml of phosphate-buffered saline for 20 min). Nebulized nitrite significantlyreduced the pulmonary arterial pressures with lesser effectson systemic mean arterial blood pressure. The kinetic thera-peutic responses to nitrite and NO were very different: Thenitrite response was slower and lasted longer (over 60 min afterinhalation), whereas the effect of NO response was faster, butreturns rapidly to the hypoxic baseline when the treatment wasdiscontinued (Fig. 8). The nitrite effect appears to be mediatedby its slow conversion to NO, since measurements of exhaledNO gas increased, with values above baseline maintained afterthe end of the treatment. In addition, consistent with a possiblerole of deoxyhemoglobin as the nitrite reductase protein, for-mation of iron-nitrosyl-hemoglobin was also reported (38).More recently, using an ovine model of hemolysis-inducedpulmonary vasoconstriction, the effect of nebulized nitrite (0.87M), iNO (20 ppm), and intravenous sodium nitrite was com-pared. Both iNO and inhaled nitrite were able to reduce PH,whereas intravenous nitrite was ineffective at the concentra-tions studied. Nebulized sodium nitrite formulations were alsostudied in a rabbit model by Egemnazarov et al. (24). In thisstudy, nitrite solutions acidified with ascorbic and citric acid(pH 5.3–5.4) showed a longer-lasting vasodilatory effect, al-though all formulations were effective in reducing hypoxicpulmonary vasoconstriction.

Repeated treatment with nebulized inhaled nitrite has beenstudied by Zuckerbraun et al. (86) in two animal models ofPH: a rat, monocrotaline-induced PH model and a mousehypoxia-induced PH model. They demonstrated that inhalednebulized nitrite can prevent and reverse establish PH. In thehypoxic mouse model, intervention after 2 weeks of hypoxia,after the establishment of the PAH, halted its progression and

reversed high right ventricular pressures. In the monocrotaline-induced PAH rat model, nebulized nitrite was able to diminishmonocrotaline-induced muscularization and hyperplasia of thesmall pulmonary arteries. Nitrite effects in both models wereblocked by the XO inhibitor allopurinol (in vitro) and bytungsten-enriched diet (in vivo), which replaces Mo in the activesites of enzymes such as XO and AO, inhibiting their activity.These data suggest that the protective effect of nitrite againstthe development and progression of hypoxia-induced PAH ismediated by XOR (86). Additional work suggested that thenitrite effect was mediated by NO–cyclic guanosine mono-phosphate signaling and downstream induction of the cellcycle checkpoint inhibitor p21, which inhibits smooth musclecell proliferation (Fig. 9).

Intravenous administration of sodium nitrite has been alsoexplored in the context of PH. Casey et al. (11) investigated theeffect of intravenous sodium nitrite (10–100 lmol/kg) as a pul-monary vasodilator in rats under hypoxic conditions. A vaso-dilatory effect in both pulmonary and systemic arterial pressurewas observed, and these effects were inhibited by allopurinol,consistent with other rodent PAH models (86). While thesestudies suggest a role for XO as a nitrite reductase in rodentmodels, human studies have been performed testing a role forXO in nitrite-dependent vasodilation. Infusions of oxypurinolfor 30 min, followed by coinfusions of nitrite, did not blocknitrite-dependent vasodilation in humans (17). Paradoxically,oxypurinol increased nitrite-dependent vasodilation by about10%. Future studies are required to reconcile findings in rodentdisease models compared with human physiological studies.

Other Therapeutic Opportunities for Nitritein Vascular Disease

In any pathological condition where NO bioavailability iscompromised, nitrite administration may provide a thera-peutic approach to restore NO levels through the nitrate-nitrite-NO signaling pathway. This approach has a directrelevance for cardiovascular disease, given the well-knownvasodilatory effect of NO, and now appreciated vasodilatoryeffects of nitrite. We will summarize here some of the currentevaluations of nitrite in vascular disease.

High blood pressure

As discussed earlier, plasma nitrite levels can be modifiedby dietary nitrate intake. Larsen et al. (49) studied the effect ofdietary nitrate supplementation on healthy volunteers andfound a significant reduction in blood pressure and increases inplasma nitrate and nitrite. Other studies have confirmed that

Table 3. Kinetic Parameters for Other Enzymes That Catalyze Nitrite Reduction

Enzyme Active center Km for NO2- Anaerobic NO generation rate

Cytochrome c oxidasea Heme-Fe, Cu — 0.001 lM min - 1

Xanthine oxidoreductaseb Mo 35.7 mM 0.3 nmol mg - 1 s - 1

Aldehyde oxidasec Mo 2.7 mM 0.34 nmol U - 1 s - 1

Carbonic anhydrased Zn 63 mM —

aReducing agent: TMPD = N,N,N¢,N¢-tetramethyl-p-phenylene diamine; pH 7.0 (12).bReducing agent: NADH = Nicotinamide adenine dinucleotide; pH 7.4 (52).cReducing agent: NADH = Nicotinamide adenine dinucleotide; pH 7.4 (50).dAs reported in Ref. (1).Mo, molybdenum; NO, nitric oxide.

NITRITE SIGNALING IN PULMONARY HYPERTENSION 1803

dietary nitrate can decrease arterial blood pressure and en-hance other parameters linked to NO metabolism, such asplatelet aggregation and endothelial function during ischemia.Interestingly, these effects may be greater in men than that inwomen (46). In general, the dietary effects of nitrate are com-parable to effects of vegetable-rich diet, suggesting that the highnitrate content of vegetables can be a part of their observedeffects on blood pressure (59). Low doses of intraperitoneal,

inhaled, or oral sodium nitrite have been shown to generate NOin blood vessels and to inhibit proliferative responses of smoothmuscle cells in murine models of carotid injury.

Angiogenesis

Vascular endothelial growth factor-induced proliferationand organization of human endothelial cells have been shown

FIG. 8. Comparison of the physiological effects of nebulized nitrite versus NO. Duration of effect of NO gas inhalation(A) or nitrite nebulization (B) on hemodynamic and metabolic measurements during hypoxic-induced PH. Treatment withnitrite aerosol resulted in a rapid sustained reduction in hypoxic-induced pulmonary vasoconstriction and a graded increasein exhaled NO gas concentration with no change in mean arterial blood pressure. These results are contrasted to the rapidreturn of pulmonary artery pressure to hypoxic baseline after termination of inhaled NO gas. Methemoglobin concentrationsincreased after nitrite nebulization from 2.1 – 0.1% to 2.8 – 0.8%. Note that exhalated NO concentrations reach the limit ofdetection during NO inhalation. [reproduced with permission from (38)]. (To see this illustration in color, the reader isreferred to the web version of this article at www.liebertpub.com/ars.)

1804 BUENO ET AL.

to be dependent on eNOS and NO production (63), suggest-ing that nitrite-based therapies could be used to promoteangiogenesis. Consistently, chronic intravenous administra-tion of sodium nitrite has been shown to induce angiogenesisin a mouse model of hindlimb ischemia (48), and sodium ni-trate therapy stimulated ischemic vasodilation and angio-genic activity after permanent femoral artery ligation (64).

Sickle cell disease

The pathology of sickle cell disease relates to vaso-occlusionby sickled erythrocytes and vasoconstriction due to increaseNO scavenging associated with hemolytic anemia (83). Patientswith sickle cell disease develop PH as they age, suggesting apotential role for nitrite therapy. A phase Ib clinical study byMack et al. has shown that infused sodium nitrite was safe andincreased forearm blood flow in patients with sickle cell disease(57), although sickle cell patients show a somewhat reducedresponse to nitrite as compared to healthy volunteers (16, 58).

Myocardial ischemia

Nitrite has protective effects in different ischemia reperfu-sion injury models (19). The effects are at least partly due to theability of nitrite to reversibly inhibit mitochondrial metabolism,

via S-nitrosation of complex I, and reduce reperfusion ROSformation (20, 60, 72). This reduction in reperfusion ROS isassociated with a prevention of opening of the mitochondrialpermeability transition pore and release of cytochrome c (72)(Fig. 10). Several studies have focused on the effects of nitritetreatment on myocardial ischemia. Duranski et al. showed alarge effect in decrease of the infarct size when nitrite was ap-plied during ischemia (23). The beneficial effect of nitrite incardiac infarct has been found be independent of the time ofadministration during the ischemic phase and can be obtainedby dietary intervention (31). The use of intravenous sodiumnitrite to ameliorate the consequences of acute myocardial in-farction is currently being evaluated in phase I–II clinical trials.

Stroke

Several studies have investigated the use of nitrite on stroketherapy in preclinical models. Early administration of nitriteduring reperfusion shows favorable effects in rats (42), althoughresponses depended on the nitrite dosage. High amounts ofnitrite provided no protection, and low doses reduced the in-farction size and enhanced local cerebral blood flow and func-tional recovery (44). Long-term, high-dose (100 lg/kg) nitritetreatment showed improvement in the recovery after stroke in arat ischemia model (45). Some positive effects have been alsoobserved in a rat intracranial hemorrhage model (43).

Nitrite therapy has also been applied in the treatment ofdelayed cerebral vasospasm after subarachnoid hemorrhage,a subtype of hemorrhagic stroke. This condition is charac-terized by decreased middle cerebral artery flow due to thenarrowing of large-capacitance arteries and often developsdays after subarachnoid hemorrhage. Several studies indicatethat the vasospasm is related to decreased bioavailability ofNO, at least in part due to inhibition of eNOS and NO scav-enging by hemoglobin from the subarachnoid clot. Pluta et al.(66) studied the effect of nitrite in a primate model of sub-arachnoid hemorrhage, and showed that nitrite prevented thedevelopment of cerebral vasospasm.

Therapeutic Developments

Nitrite has been proposed and tested successfully in severalanimal models and patients, with a variety of delivery

FIG. 9. Nitrite-induced SMC proliferation is dependenton XOR. Hypoxia-induced PAH is inhibited by NO gener-ation from nitrite, which is catalyzed by XOR. NO produc-tion from nitrite increases expression of p21, which inhibitsSMC proliferation. Allopurinol or tungsten diet treatmentinhibits XOR activity (85, 86).

FIG. 10. Nitrite mediates cyto-protection in ischemia/reperfusioninjury. Nitrite potently mediatescytoprotection after ischemia/reperfusion (I/R) through the tran-sient inhibition of complex I (viaS-nitrosation) and subsequent limi-tation of oxidative damage. Becauseoxidants have been shown to sen-sitize the permeability transitionpore, cytoprotective effects of nitritecould in part be caused by nitrite-dependent protection against poreopening after I/R (72). (To see thisillustration in color, the reader isreferred to the web version of thisarticle at www.liebertpub.com/ars.)

NITRITE SIGNALING IN PULMONARY HYPERTENSION 1805

systems and formulations. A mixture of sodium nitrite andacidifying agents such as ascorbic acid can rapidly releaseNO. This topical administration has been evaluated for itsantimicrobial activity in various skin infections (61) and, withsome modifications, in preventing catheter-associated urinarytract infections (10). It is also well established that nitrite canbe given orally, but its bioavailability can be difficult to assess,because it is extremely dependent on the oral microbiome andits variable metabolism within the gastrointestinal tract.However, the use of organic allylic nitrocompounds as nitritedonors may overcome this issue (13). A third possibility is themuch anticipated use of intravenous infusions of nitrite, butthe dose and the duration of the treatment still need to beadjusted (67). One of the adverse effects of inorganic nitriteis the formation of methemoglobin. Nitrite can oxidize theiron at the heme group, modifying it from a ferrous (Fe2 + )oxygen-binding form to a ferric (Fe3 + ) nonoxygen-bindingstate. If the increase of methemoglobin in circulating blood ishigher than 5%, it can lead to cyanosis. However, studies withnitrite intravenous studies only report undetectable or modestincrease (67).

As described above, inhaled sodium nitrite is a pulmonaryvasodilator that can effectively prevent or reverse PAH inanimal models. Studies suggest that it can be delivered safely,and it is ready for clinical translation for PAH patients. Ani-mal toxicology studies with inhaled nitrite in rodents anddogs have been completed and phase Ia and Ib studies innormal volunteers completed. In a phase Ia study, inhalednebulized sodium nitrite, at doses > 17 mg, increases exhaledNO, whereas methemoglobin levels remained < 3.5% in allsubjects (Bradley et al., unpublished data). A proof-of-conceptphase II trial of inhaled nitrite in patients with PAH is cur-rently enrolling sites in the United States and Europe.

Summary and Conclusions

PAH is associated with decreased bioavailability and re-sponsiveness of NO. Despite the potential therapeutic effect ofiNO as a selective pulmonary vasodilator, its administrationis inconvenient and difficult. The discovery that nitrite is anaturally occurring molecule in the body and may act en-dogenously as a reservoir of NO has suggested the idea thatthis anion may represent an alternative strategy for an effec-tive NO-based therapy. NO can only be administered as a gasand cannot be mixed with oxygen, or it reacts to NO2. Nitrite(as a soluble salt) can be effectively delivered as a nebulizedliquid, dry powder, intravenous solution, or oral formulation.Also, when compared with iNO, nitrite has a longer half-life.However, two caveats arise for either iNO or nitrite therapies:(i) both may have effects in conditions with soluble guanylatecyclase dysfunction (oxidation or downregulation); and (ii)potential harmful side products may be formed by reactionwith ROS (NO2, peroxynitrite, or methemoglobin formation).Nitrite has demonstrated efficacy in multiple animal modelsnot only of cardiovascular disorders but also of inflammatorydiseases and bacterial infections. Clinical trials are ongoingand more planned in the near future to demonstrate thetherapeutic efficacy of nitrite in patients with PAH.

Acknowledgments

We thank Dr. Jesus Tejero, Dr. Courtney Sparacino-Wat-kins, and Dr. Eric Kelley (University of Pittsburgh). Dr.

Gladwin receives research support from the NIH grantsR01HL098032, RO1HL096973, and PO1HL103455, the In-stitute for Transfusion Medicine and the Hemophilia Centerof Western Pennsylvania. Dr. Bueno, Dr. Mora, and Dr. Wangare supported by the Vascular Medicine Institute of the Uni-versity of Pittsburgh.

Author Disclosure Statement

Dr. Gladwin is listed as a coinventor on an NIH govern-ment patent for the use of nitrite salts in cardiovascular dis-eases. Dr. Gladwin consults with Aires Pharmaceuticals onthe development of a phase II proof-of-concept trial usinginhaled nitrite for pulmonary arterial hypertension.

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Address correspondence to:Dr. Mark T. Gladwin

Division of Pulmonary and Critical Care MedicineDepartment of MedicineUniversity of Pittsburgh

NW 628 Montefiore Hospital3459 Fifth Avenue

Pittsburgh, PA 15213

E-mail: gladwinmt@upmc.edu

Date of first submission to ARS Central, July 19, 2012; date ofacceptance, August 7, 2012.

Abbreviations Used

AO¼ aldehyde oxidasecGMP¼ cyclic guanosine monophosphate

CT¼ computed tomographydeoxy-Hb¼deoxyhemoglobin

DPI¼diphenylene iodonium chlorideeNOS¼ endothelial NOS

GSNO¼ S-nitroso glutathioneiNO¼ inhaled NO

iNOS¼ inducible NOSLA¼ left atrium

L-NAME¼Nx-nitroarginine methyl esterLV¼ left ventricleMo¼molybdenumNO¼nitric oxide

NOS¼nitric oxide synthasePA¼pulmonary artery

PAH¼pulmonary arterial hypertensionPH¼pulmonary hypertensionRA¼ right atrium

ROS¼ reactive oxygen speciesRV¼ right ventricle

sGC¼ soluble guanylate cyclaseSMC¼ smooth muscle cellXDH¼ xanthine dehydrogenase

XO¼ xanthine oxidaseXOR¼ xanthine oxidoreductase

NITRITE SIGNALING IN PULMONARY HYPERTENSION 1809