Nitric Oxide Deficiency in Pulmonary Hypertension: Pathobiology and Implications for Therapy

Abstract

Nitric oxide (NO) is a diffusible gas with diverse roles in human physiology and disease. Significant progress in the understanding of its biological effects has taken place in recent years. This has led to a better understanding of the pathobiology of pulmonary hypertension (PH) and the development of new therapies. This article provides an overview of the NO physiology and its role in the pathobiology of lung diseases, particularly PH. We also discuss current and emerging specific treatments that target NO signaling pathways in PH.

Keywords

pulmonary hypertension nitric oxide physiopathology and therapeutics

Pulmonary arterial hypertension (PAH) is a progressive disease that has poor prognosis since it may lead to right ventricular failure and death.^[1] The disease is characterized by excessive pulmonary vasoconstriction and abnormal vascular remodeling that result in loss of vascular cross-sectional area and increase in right ventricular afterload.^[2] One of the proposed mechanisms involved in the pathogenesis of the disease is an imbalance in vasoactive mediators with reduced levels of the vasodilatory and antiproliferative nitric oxide (NO).^[3]

Since the breakthrough discovery in 1987 that the endothelium-derived relaxing factor was nitric oxide (NO),^[4,5] this colorless and odorless free-radical gas became increasingly recognized as a key factor in human physiology and disease.^[6,7] NO is an autocrine and paracrine signaling molecule whose functions are diverse and involve smooth muscle relaxation, platelet inhibition, central and autonomic neurotransmission, tumor cell lysis, bacterial killing, and stimulation of hormonal release.^[7–10] This review will focus on the role of NO in physiology and pathobiology of lung diseases, particularly pulmonary hypertension (PH), and the current and emerging pulmonary hypertension (PH)-specific treatments based on NO signaling.

NITRIC OXIDE PHYSIOLOGY

NO is an endogenously synthesized, diffusible, lipophilic gas that is produced by a group of enzymes known as nitric oxide synthases (NOS). Their role is to convert the amino acid L-arginine to L-citrulline and NO.^[6] For their activity, NOS require oxygen, reduced nicotinamide-adenine dinucleotide phosphate (NADPH), and other cofactors such as flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), calmodulin, and tetrahydrobiopterin (BH4).^[11–13] NOS is active as a homodimer and contains a reductase and an oxygenase domain. The oxygenase domain is the active site of NO synthesis with binding sites for heme, L-arginine, and BH4^[14] (Fig. 1). Three NOS isoforms (Types I, II, and III) have been identified. These NOS isoforms have important differences in expression and regulation as shown in Table 1.^[15,16] In general, NOS I is expressed in neuronal cells and skeletal muscle; NOS II is found in epithelial, and smooth muscles cells as well as in neutrophils, macrophages, and fibroblasts; and NOS III is present in endothelial cells throughout the body.^[17] NOS I and III are continuously expressed and regulated by Calcium/Calmodulin; meanwhile, NOS II is regulated at the transcription level. NOS II transcription is increased by cytokines (e.g., TNF-α, interferon-γ, and IL-1β), endotoxins, oxidants, and shear stress,^[10,18] and is decreased by corticosteroids, retinoids, transforming growth factor beta, platelet-derived growth factor, insulin-like growth factor 1, and thrombin.^[9,10,13,19] The initial clear distinction between the constitutive and inducible isoforms has been recently distorted and constitutive isoforms may be induced, and vice versa.^[20,21]

Table 1:

Characteristics of nitric oxide synthases isoforms

NOS isoforms	Designation	Size (KDa)	Expression	Main cellular sources in the lung	Calcium regulation	Nitric oxide output	Chromosome
I	Neural NOS (nNOS)	155	Constitutive	Inhibitory non-adrenergic non-cholinergic neurons	Dependent	Picomolar	12
II	Inducible NOS (iNOS)	125	Inducible by cytokines, endotoxin and oxidants	Airway epithelial cells	Independent	Nanomolar	17
III	Endothelial NOS (eNOS)	135	Constitutive	Endothelial cells and brush border of ciliated epithelial cells	Dependent	Picomolar	7

NOS: nitric oxide synthase

Figure 1:

NO synthesis and signaling pathways. BH4, tetrahydrobiopterin; cGMP: Cyclic guanosine monophosphate; GMP: Guanosine monophosphate; GTP: Guanosine triphosphate; NO: Nitric oxide; NOS: Nitric oxide synthase; ONOO⁻, peroxynitrite; PK: Protein kinases; sGC: Soluble guanylate cyclase; SNO-Hb: S-nitrosothiol — hemoglobin. L-arginine can be regenerated from L-citrulline by two enzymes (argininosuccinate synthase and argininosuccinate lyase).

NOS I acts as a functional antagonist of acetylcholine and mediates inhibitory nonadrenergic non-cholinergic neural bronchodilation.^[22] NOS II is involved in inflammation as it mediates the cytotoxic activity of activated macrophages and may be a contributing factor in the vasodilation observed in septic shock.^[23] NOS III plays a role in the regulation of vascular flow^[24] and may reduce plasma exudation in the airways^[25] and regulate ciliary beating and mucociliary clearance.^[26] NOS III is the predominant source of NO production in the pulmonary circulation.^[14,20] Studies in transgenic mice and humans to assess the relative contribution of the three NOS isoforms showed that NOS II and III are the key regulators of the pulmonary circulation tone. NOS III is the key mediator of resting tone through endothelium-dependent pulmonary vasculature vasodilation,^[27] while NOS II may mediate the pulmonary circulation's response to oxygen.^[28] Targeted disruption of the NOS III gene in mice was associated with mild PH without evidence of pulmonary vascular remodeling,^[29] which likely reflects compensation by the other NOS isoforms.

Vascular smooth muscle and endothelial cells have the ability to regenerate the NOS substrate L-arginine by synthesizing argininosuccinate from citrulline and aspartate, a process that requires two enzymes, argininosuccinate synthase and argininosuccinate lyase.^[30–32] The first enzyme is co-induced with NOS II and the second is constitutively expressed in these cells.^[33] Exogenous citrulline administration effectively stimulated NO production in vascular endothelial cells by means of regenerating arginine.^[31]

Once NO is produced, it may act within the cell in which it is generated or freely diffuse into adjacent cells (e.g., vascular smooth muscle cells), acting as an intra- or intercellular messenger.^[9,34] The NO intracellular diffusion may be limited because NO is readily oxidized to the more stable metabolic products nitrite (NO₂⁻) and nitrate (NO₃⁻)^[11] and is scavenged predominantly by hemoglobin. Upon entering the cells, NO activates the intracellular soluble guanylate cyclase (sGC) to produce 3′,5′-cyclic guanosine monophosphate (cGMP), which mediates most of the physiological and pathological effects of NO (Fig. 1).^[10,35]

Two main types of guanylate cyclase (GC) are known: the particulate-associated enzymes, which are transmembrane receptors that contain GC that is activated by atrial and brain natriuretic peptide; and the cytosolic or soluble type which is activated by NO.^[36,37] NO appears to exert its effect by binding to the heme iron (ferrous state) of the sGC, stimulating the enzyme basal cyclase activity several hundred-fold.^[13,34,38] Once cGMP is produced, it mediates physiologic responses through its effects in cGMP-gated ion channels, cGMP-regulated phosphodiesterases, or cGMP-dependent protein kinases.^[34,39] A preferential activation of specific target proteins is believed to underlie the differential effects of cGMP in various cells.^[40] cGMP-induced vasorelaxation of vascular smooth muscle is through several mechanisms including activation of K-channels which hyperpolarizes the cell membrane, inhibition of calcium influx, and reduction of myofilament calcium sensitivity.^[14,39]

The cGMP signal is chiefly limited by phosphodiesterases (PDE) which degrade the cyclic nucleotide of GMP. PDE degrade both cyclic adenosine monophosphate (cAMP) and cGMP; however, PDE-5 is specific to cGMP, and indeed the enzyme requires the binding of cGMP for full activation.^[41]

NOS inhibitors and NO generators have allowed for a better understanding of the NO signaling pathways. NOS inhibitors are analogs of L-arginine that act as a false substrate for this enzyme and inhibit both the constitutive and inducible forms of NOS. Of these, the most widely used include N-monomethyl-L-arginine (L-NMMA), N-nitro-L-arginine (L-NNA), and N-nitro-L-arginine methyl ester (L-NAME).^[42] Another commonly utilized NOS inhibitor is L-aminoguanidine, a more selective inhibitor of NOS II.^[8] NO generators are compounds that release NO such as diethylamine, sodium nitroprusside, and isosorbide dinitrite, among others.

The most important endogenous NOS inhibitor among the methylated arginines is the asymmetric dimethylarginine (ADMA).^[43,44] ADMA is produced as a result of proteolysis of methylated proteins. The methylation of arginine is a post-translational modification via protein arginine methyltransferaes (PRMT). ADMA acts as a false substrate and competitively inhibits NOS activity, blocking the formation of endogenous NO.^[44,45] ADMA undergoes clearance by the dimethylarginine dimethylaminohydrolase (DDAH) and it is also partially cleared by the kidneys.^[46] Methylated arginines, like ADMA, may be responsible for the “L-arginine paradox.” At physiological state, NOS is saturated with arginine, thus an increase in arginine concentration in plasma or cytosol should have no effect in NO production.^[31] However, elevating plasma arginine or citrulline levels enhance NO production, suggesting some form of competitive inhibition of NOS, such as ADMA, is present and can be overcome by increasing the arginine concentration.^[45,47] Another explanation for the arginine paradox is the potential existence of a separated cellular pool of arginine allocated to NO synthesis, i.e., caveolar-localized arginine regeneration system, in which citrulline is recycled to arginine.^[32] This hypothesis is supported by a study that revealed the colocalization in discrete cellular domains (caveolae) of enzymes involved in arginine regeneration and NO production.^[31,32]

NITRIC OXIDE PHYSIOLOGY IN THE PULMONARY VASCULATURE

The specific role of NO produced in the pulmonary vasculature is still a matter of intense investigation. Data suggest that NO inhibits smooth muscle tone, proliferation, and migration.^[48–50] NO causes relaxation of the vascular smooth muscle tone via the activation of cGMP.^[48,49] In fact, endogenous NO plays a key role in decreasing the pulmonary artery resistance at the time of birth and in maintaining the dilation of the pulmonary vasculature.^[39,49,51] The production of NO by NOS II in injured vascular smooth muscle cells may prevent vasospasm and inhibit cell proliferation by possibly inducing apoptosis.^[52–55] In addition, NO in this setting may regulate the metabolism of vascular smooth muscle cells, favoring anaerobic glycolysis,^[52] and lead to toxic effects on adjacent endothelial cells.^[56]

NO inhibits vascular smooth muscle cell proliferation,^[57] DNA synthesis,^[58] and collagen production via activation of cGMP.^{[48,50,58,59]} Furthermore, NO inhibits vascular smooth muscle cell migration independent of the effects on proliferation.^[50] Higher levels of NO are required to inhibit proliferation rather than to produce vasodilation, suggesting a potential concomitant activation by cGMP of the cAMP kinase pathway which inhibits cellular proliferation.^[60] NO generators can also inhibit proliferation in cells that lack sGC, suggesting that cGMP-independent mechanisms play a role. One of these potential mechanisms is the up regulation of Fas, a membrane protein that belongs to the TNF receptor family and induces apoptosis.^[54] NO-induced apoptosis could be the result of a feedback control on calcium responses to growth factors, or deamination of purine and pyrimidine bases in DNA that leads to increased mutagenesis and DNA strand breaks.^[61,62] Although NO has been reported to inhibit cell proliferation in endothelial cells,^[63] other investigations have shown than either exogenous NO^[64] or NO produced by NOS II in vascular smooth muscle cells may stimulate endothelial cell proliferation.^[65]

Alternative NO pathways include the oxidation of NO to form nitrite or reaction with protein thiols to form S-nitrosothiols, molecules that can lead to vasodilation or can regulate protein function by post-translational modification.^[66,67] NO is oxidized in the blood and tissues to form nitrite and nitrate.^[13] Nitrate is produced by the reaction of NO with oxyhemoglobin, while nitrite is formed by oxidation of NO.^[13] These molecules can be recycled to form NO by an allosterically controlled nitrite reductase reaction predominantly during hypoxia, thereby complementing the NOS pathway.^[68–70] Deoxygenated hemoglobin has nitrite reductase activity, forming NO from nitrate (NO₃⁻) and nitrite (NO₂⁻),^[69,71] a reaction that can explain the hypoxia-specific vasodilatory effect observed in some organs. The NOS pathway is oxygen dependent while the nitrate-nitrite-NO pathway is hypoxia activated.^[69,70]

In addition, NO can be responsible for nitrosylation of a cysteine residue of the β-subunit of hemoglobin, resulting in S-nitrosylated-hemoglobin, a protein that exerts NO-like vasodilator effects.^[39] This reaction is favored in the oxygenated state of hemoglobin, and, once desaturation of hemoglobin occurs, there is release of SNO to acceptor thiols potentially delivering NO to the systemic circulation.^[72] S-nitrosylation and nitration enable the systemic transport of the NO signal, a process that would not be possible for NO due to its very short half-life^[14] and strong affinity to bind hemoglobin.

NITRIC OXIDE AND NITRIC OXIDE SYNTHASES IN THE LUNG

NO is produced endogenously in the human lung in the upper and lower respiratory tract and it is detectable in exhaled breath (6-8 ppb).^[28,73] The origin of exhaled NO in the human lung likely depends upon all three isoforms of NOS (I-III) but predominantly derives from the airway epithelial expression of NOS II, the high-producer of NO. NO metabolites like nitrosothiol and nitrite (NO₂⁻) are found in the bronchoalveolar lavage of human lungs.^[8] NO is formed in high concentrations in the upper respiratory tract (nasopharynx and paranasal sinuses) and in lower quantities in the lower respiratory tract.^[28,73] It is produced in a variety of cells including epithelial, endothelial, and smooth muscle cells, and also in inhibitory non-adrenergic non-cholinergic neurons, mastocytes, fibroblasts, macrophages, lymphocytes, and neutrophils.^[6,7,28]

Immunohistochemical studies have identified the presence of all three isoforms of NOS, expressed in different cells of the human lung (Table 1).^{[6,10,12,28,74–76]} Specifically, NOS I is located in inhibitory non-adrenergic non-cholinergic neurons; NOS II is expressed in the airway epithelium; and NOS III is found in endothelial cells.^[8,21,76,77] Contrary to what occurs in other organs where NOS II needs to be induced, in the lungs this enzyme is continuously expressed in the airway epithelium at basal conditions.^[21]

NO is involved in pulmonary neurotransmission, host defense, airway and vascular smooth muscle relaxation, mucociliary clearance, airway mucus secretion, inflammation, and cytotoxicity.^[8,77] NO plays key roles in lung biology and has been implicated in the pathophysiology of several lung diseases such as asthma, cystic fibrosis, bronchopulmonary dysplasia, lymphangioleiomyomatosis, and adult respiratory distress syndrome in addition to pulmonary hypertension.^{[8,12,20,28,35,78–84]}

Endogenous NO plays an important role in the regulation of airway function, having both beneficial and detrimental effects.^[20] It leads to bronchial smooth muscle relaxation, potentially modulating the basal airway tone.^[8] Inhaled NO decreased pulmonary airway resistance in pigs and NO generators relaxed human airway smooth muscle in vitro.^[8] Exhaled NO is increased in inflammatory airway diseases such as asthma and bronchiectasis likely due to an increase in NOS II expression in the epithelial cells of these patients with some contribution from the constitutive NOS isoforms.^[8,20,77] In asthma the fraction of exhaled NO is considered a surrogate for eosinophilic airway inflammation and steroid responsiveness.^[20,85–87] A reduction in exhaled NO levels is observed in smokers possibly as a result of a dysregulation of NOS activity as cigarette smoke contains high levels of NO.^[77,88]

NO is a key molecule in the oxidative metabolism since it can exert oxidant or antioxidant effects depending on the local tissue milieu. Hence, in an environment where the load of antioxidant is low, NO will have oxidant properties; however, when the oxidant load is high, NO plays an antioxidant role by scavenging free radicals and reactive oxygen species.^[16,74] NO rapidly consumes superoxide (O₂⁻) by forming peroxynitrite (ONOO⁻) which is a less reactive oxidant that can be further metabolized to products like nitrate (NO₃⁻).^[79] Some of these reactive oxygen species could be responsible for oxidative modification of cellular proteins such as oxidation of sGC, a reaction that can impair the specific activity of sGC and reduce the ability of NO to stimulate cGMP.^[89] On the same lines, recombinant human superoxide dismutase decreases oxidative stress and increases eNOS activity and expression, stimulating NO production, and ultimately pulmonary vasodilatation.^[90–92]

NITRIC OXIDE IN PULMONARY HYPERTENSION

NO is a potent pulmonary vasodilator that is produced locally in the lung and has effects on smooth muscle relaxation and proliferation. The close proximity of the airways and vessels in the lung allows NO produced in high levels in the upper^[93] and lower^[28] airways by NOS II to affect pulmonary vascular tone, in concert with the low NO levels that are produced by NOS III in the vascular endothelium.^[35] NO is considered to be a selective pulmonary vasodilator because after exerting its vasodilator action, NO is scavenged by hemoglobin having minimal effects on systemic hemodynamics.^[35]

Disruption of the NO pathway is a major contributor to the pathobiology of PH. NO in exhaled breath and NO biochemical reaction products in bronchoalveolar lavage are lower in lungs of patients with PAH than controls and their level is inversely related to the degree of PH.^[45,94] NO in exhaled breath of individuals with idiopathic PAH is significantly lower than subject with PH associated with other causes or healthy nonsmoking controls.^[95] In fact, patients with PH associated with other causes had similar levels of exhaled NO than healthy controls.^[95]

Early data demonstrated a reduced expression of NOS III, measured by immunostaining, in the vascular endothelium of pulmonary arteries in patients with PAH. This reduced expression inversely correlated with the severity of the morphological arterial changes.^[96] More recently, however, other investigators showed increased or unaltered NOS III immunostaining in PH.^[97,98] Moreover, there is evidence of high NOS III expression in plexiform lesions in PAH.^[99] A unifying hypothesis suggested that the activity of NOS III may be reduced rather that its expression.^[100] Other NOS isoforms besides NOS III may contribute to the low exhaled NO observed in idiopathic PAH.^[94]

NO has also been implicated in the important role that bone morphogenetic protein receptor II (BMPRII) has in the pathogenesis of PAH.^[101] In the lung, BMPRII is highly expressed in endothelial cells and its activation promotes proliferation, migration, and survival of these cells.^[102] BMPRII levels are markedly reduced in patients with heritable or idiopathic PAH, promoting endothelial dysfunction and apoptosis.^[102] These effects have been recently attributed to a decrease in NOS III activity, since BMPRII ligands failed to stimulate NOS III dependent protein kinase activation in pulmonary artery endothelial cells from patients with mutation in the BMPR2 gene.^[103]

PAH patients treated with epoprostenol had a three-fold higher exhaled NO than PH patients not receiving this treatment and two-fold higher than healthy controls. Interestingly, exhaled NO increased at 24 hours in those patients treated with this prostacyclin analog.^[95] These data suggest that prostacyclin analogs may in part improve PH through effects on NO. In support of this, previous work has shown that nebulized epoprostenol increased the exhaled NO in patients with PAH associated with congenital heart disease.^[104] Similarly, inhaled iloprost also led to an increase in NO concomitant with a decrease in pulmonary artery pressure in a patient with PAH associated with scleroderma.^[105] Other PAH-specific therapies that do not directly target the NO pathway may also improve the fraction of exhaled nitric oxide suggesting a crosstalk between different signaling pathways.^[95,106,107]

The NO signaling pathways have been found to be affected in PH at different levels. Endogenous NOS III inhibitors may be involved in the pathogenesis of PH. These include the symmetric and asymmetric dimethylarginines (ADMA). Animal models of hypoxic PH showed increased ADMA levels and decrease in activity of dimethylarginine dimethylaminohydrolase (DDAH), the endothelial enzyme that metabolizes ADMA.^[108] Higher serum levels of ADMA are increased in patients with idiopathic PAH^[109,110] and chronic thromboembolic pulmonary hypertension,^[111] and correlate with disease severity and survival. ADMA levels increase predominantly due to a reduction in the expression and function DDAH.^[110]

Arginase II, an enzyme that is part of the urea cycle and breaks down arginine to ornithine, can decrease the substrate available to NOS for NO synthesis. Idiopathic and PAH associated with sickle cell disease patients have been found to have higher levels of Arginase II and lower levels of L-arginine than healthy controls.^[112] In the absence of L-arginine or BH4, NOS III may become “uncoupled,”^[14] resulting in the generation of the free radical superoxide.

CLINICAL IMPLICATIONS IN PH: DIAGNOSIS AND PROGNOSIS

Although the vascular endothelium produces large amounts of NO, very little is exhaled as a result of the marked affinity of NO to hemoglobin in the pulmonary circulation.^[28] In spite of this potential limitation, NO can be measured in exhaled breath and its concentration is inversely related to the exhalation flow. For this reason, the fraction of exhaled NO is measured at a constant flow (usually 50 mL/s). Using this approach, the fraction of exhaled NO is a reliable surrogate of the maximal flux of NO from the large airway compartment.^[113] This method does not measure the steady-state mean distal airway/alveolar concentration of NO. There is no simple surrogate for measuring this parameter since its determination requires the measurement of the fraction of exhaled NO at multiple expiratory flow rates and the application of a modified “slope-intercept” algorithm.^[20,114]

Authors have investigated whether exhaled NO could serve as a noninvasive marker of severity of disease and response to therapy in PH. The value of repetitive exhaled NO measurements was studied in 17 PAH patients over two years. NO levels at entry were inversely correlated with the number of months from the PAH diagnosis, suggesting a global decrease in NO over longer periods with the disease.^[106] Lower exhaled NO levels at entry were associated with higher pulmonary artery pressures and less decrease over time. NO at the beginning of the study was not associated with survival; nevertheless, its level increased over time in the PAH individuals who survived to complete the study when compared to those who died, and correlated with changes in pulmonary artery pressures.^[106] Thus, exhaled NO could be a useful marker of disease severity and response to therapy.^[94,95,106]

Inhaled NO is frequently used for acute vasodilator challenge during right heart catheterization in patients with PAH. A positive pulmonary vasodilator test (decrease in mean pulmonary artery pressure of at least 10 mmHg to an absolute value less than 40 mmHg without decrease in cardiac output) indicates the patient that can potentially benefit from long-term calcium-channel blockers.^[115–118] NO vasodilator challenge in several types of PH can also provide prognostic information, as responders may have better outcome, independent of the treatment administered.^[119–122]

CLINICAL IMPLICATIONS IN PH: THERAPY

Inhibiting phosphodiesterases-5

Therapies that manipulate the downstream NO signaling have revolutionized the treatment of PH (Fig. 2). Sildenafil and tadalafil are FDA approved PDE-5 inhibitors for the treatment of PAH in adults.^[123–125] The FDA recently placed a safety warning on the prescription of sildenafil, not recommending its use in pediatric patients due to increase mortality with increasing doses in this age group.^[125] This modification was based on the results of the Sildenafil Citrate in Treatment-Naive Children with Pulmonary Arterial Hypertension (STARTS-2) trial.^[126] This extension study, designed to assess the safety and tolerability of long-term treatment with oral sildenafil monotherapy in children (aged 1–17 years) with PAH, showed a higher mortality risk in patients randomized to high-dose sildenafil.^[126]

Figure 2:

Therapeutic strategies to increase NO effect. BH4, tetrahydrobiopterin; cGMP: Cyclic guanosine monophosphate; EPC: Endothelial progenitor cells; GMP: Guanosine monophosphate; GTP: Guanosine triphosphate; HMG coA red, hydroxyl-methylglutaryl-CoA reductase; Inh, inhaled; NO: Nitric oxide; NOS: Nitric oxide synthase; ONOO”, peroxynitrite; PDE: Phosphodiesterases; PK: Protein kinases; rhSOD: Recombinant human superoxide dismutase; sGC: Soluble guanylate cyclase; SNO-Hb: S-nitrosothiol — hemoglobin. L-arginine can be regenerated from L-citrulline by two enzymes (argininosuccinate synthase and argininosuccinate lyase).

PDE-5 inhibitors prevent the normal hydrolysis of cGMP, prolonging the NO effects on tissues. PDE-5 is mainly expressed in the pulmonary vascular bed,^[127] thus its inhibition has primarily pulmonary-specific effects.^[100,128] Furthermore, PDE5 is upregulated in the lung^[127] and the hypertrophied right ventricular myocardium^[129] of patients with PAH. Therefore, PDE-5 inhibitors are an ideal treatment for PAH because they decrease the right ventricular afterload and improve right ventricular inotropy, without relevant systemic hemodynamic effects.^[129]

Using nitric oxide as an inhaled gas

Inhaled NO was first shown to selectively reduce pulmonary vascular resistance in a lamb model;^[130] subsequently, multiple studies have confirmed this finding in humans.^{[118,131,132]} Continuous inhaled NO had beneficial effects in patients with PAH or PH associated with COPD;^[132–134] however, high cost and technical difficulties for its delivery and avoidance of the toxic effects of NO oxidative products have prevented its widespread use.^[133] During continuous administration of NO, NO oxidative products (NO₂) can build up and cause airway hyperactivity at low concentrations^[135] and pulmonary edema at higher concentrations.^[136] Methemoglobin is also formed as NO reacts with oxyhemoglobin. Furthermore, mechanisms should be in place to avoid abrupt cessation of inhaled NO as this may lead to rebound PAH with deleterious effects.^[137,138] Nonetheless, two studies that evaluated the long-term use (one and three months, respectively) of inhaled NO in PAH, chronic thromboembolic PH, and PH due to COPD reported no significant increase in methemoglobin, withdrawal syndrome, change in oxygenation, pulmonary function, or systemic hemodynamics.^[132,134]

Currently, inhaled NO is FDA approved for the treatmentofterm and near-term (> 34 week gestation) neonates with hypoxic respiratory failure with clinical and echocardiographic evidence of pulmonary hypertension.^[81,82,139] Extended administration (weeks) of inhaled NO has been studied in premature infants and does not appear to improve survival or prevent bronchopulmonary dysplasia.^{[81–83,140,141]} At present, inhaled NO is undergoing clinical investigations to evaluate its utility as a treatment for bronchopulmonary dysplasia (NCT01503801). There are two ongoing studies using extended administration of inhaled NO in adults with PH. The PHiano study (NCT01265888, Geno LLC) is an open label, dose-escalation, Phase II study using a NO delivery system (NITROsyl) in patients with PAH or PH secondary to idiopathic pulmonary fibrosis. The second study (NCT01457781, INO Therapeutics) is a randomized, double blind, placebo control, Phase II study of inhaled NO versus placebo as add-on therapy in subjects with PAH. NO is delivered by a special device (INOpulse DS).

Nitric oxide donors

Plasma levels of nitrate and nitrite are low in diseases that have endothelial dysfunction like PAH.^[142] Inhaled nitrite could be converted to NO and selectively dilate the pulmonary circulation.^[143] The vasodilatory effects of these inorganic anions (nitrite and nitrate) are less potent than the organic nitrates (nitroglycerin) and nitrites (amyl-nitrite);^[69] however, the inorganic compounds do not induce significant systemic vasodilator effects and tachyphylaxis as the organic molecules.^[69] Nebulized sodium nitrite has been shown to ameliorate PH induced by hypoxia in animals and humans, with a longer duration of action than that of inhaled NO,^[143,144] supporting that nitrites are NO donors particularly during hypoxia as result of the nitrite reductase action of deoxyhemogobin.^[69,143] Ethyl nitrate is an organic nitrate given by inhalation that forms S-nitrosothiols and has shown to improve pulmonary hemodynamics in a model of hypoxia-induced PH without altering systemic vascular resistance or increasing methemoglobin levels.^[145] Its low potency when compared with inhaled NO can be markedly enhanced both in vivo and in vitro by the addition of the thiol glutathione.^[145]

Efforts in trying to find better stability and prolonged half-life in NO delivery led to the discovery of diazeniumdiolates (diethylenetriamine/NO) that can form a group of adducts called NONOates that are complexes of NO with nucleophiles that spontaneously and nonenzymatically release NO when dissolved in aqueous neutral PH solutions. This process prolongs the half-life of NO release up to 20 hours.^[146,147] Nebulized NO donors have been shown to reduce PVR in hypoxia-induced PAH in piglets,^[148] monocrotaline rat model,^[147] and ARDS patients^[149] with no systemic adverse effects or toxic reaction products of NO.

Other therapies based on the nitric oxide pathway

Several promising therapies which affect the signaling pathway of NO are under active investigation (Fig. 1). Soluble guanylate cyclase stimulators increase cGMP independently of NO. One of these stimulators, Riociguat, increased the activity of soluble guanylate cyclase 73-fold in vivo with partial reduction in pulmonary pressures, RV hypertrophy, and pulmonary artery muscularization in animal models of PH.^[150] This medication showed promising results in a Phase II study in patients with PAH and chronic thromboembolic PH.^[151] We are awaiting the results of two Phase III studies evaluating safety and clinical effectiveness of riociguat in PAH (NCT00810693) and chronic thromboembolic PH (NCT00855465).^[152]

L-arginine replacement aims at providing excess substrate for the NOS enzyme and stimulating the NO production. L-arginine attenuated PAH in different animal models of PAH^[153–155] and in patients with PAH associated with sickle cell disease, idiopathic PAH, and chronic thromboembolic PH.^[156,157] L-citrulline, a urea cycle intermediate, is metabolized to L-arginine in pulmonary vascular endothelial cells. Oral supplementation increased NO synthesis and ameliorated chronic hypoxia-induced PH in newborn piglets.^[158] In children undergoing cardiopulmonary bypass, the oral supplementation of L-citrulline safely increased plasma citrulline and arginine concentrations, and more importantly PH did not occur in those with elevated citrulline levels.^[159] The safety and effectiveness of intravenous L-citrulline, in children undergoing cardiopulmonary bypass for surgical repair of a congenital heart defect, is currently being tested (NCT01120964).

NO production by NOS depends on the de novo biosynthesis of the enzyme cofactor tetrahydrobiopterin (BH4).^[160,161] BH4 stabilizes the NOS dimmer assembly and the favorable spin state of the Fe (II)-O2 heme intermediate preventing “uncoupling” of the enzyme and formation of reactive oxygen products.^[14,162,163] The augmentation of tetrahydrobiopterin showed promising results in a pilot study in patients with PAH or chronic thromboembolic PAH.^[164] Another promising therapy is the recombinant human superoxide dismutase (rhSOD) that scavenges superoxide anion and increases the bioavailability of NO.^[165] In a lamb model of persistent PH, rhSOD administered through the endotracheal tube as a bolus enhanced the effects of inhaled NO on pulmonary vasculature.^[165]

Attractive new potential therapies for PAH include the NOS III enhancers, the delivery of autologous endothelial progenitor cells,^[166] or the enzyme NOS II and III using adenoviral-mediated transfer or adult stem cell-based ex vivo gene therapy.^[167] Bone marrow-derived endothelial-like progenitor cells prevented the development or progression of PH in a monocrotaline rat model of PH.^[168] The engraftment of these progenitor cells in the pulmonary vasculature may restore the microvascular structure and function.^[168] Meanwhile, animals receiving endothelial-like progenitor cells or mesenchymal stem cells transduced with NOS III had reversal of established PH and improved survival.^[168,169] Adenoviral gene transfer of NOS III produced an increase in the NOS III expression and activity with attenuation in the hypoxia-induced increase in pulmonary artery pressure.^[170] Similarly, NOS II gene transfer increased pulmonary NO production with reduction in hypoxia-induced PH and vascular remodeling in rats.^[171]

CONCLUSIONS

Nitric oxide mediates diverse key signaling functions in human physiology and disease. Major progress in the understanding of NO signaling pathway has led to the approval of PAH-specific treatments and the ongoing discovery and development of promising new therapies. Molecules in the NO pathway also have the potential to be used as biomarkers of disease severity, outcomes, or response to therapy in pulmonary hypertension.

Footnotes

Source of Support: Dr. Adriano R. Tonelli is supported by CTSA KL2 Grant # RR024990 from the National Center for Research Resources (NCRR). Dr. Raed A. Dweik is supported by the following grants: HL081064, HL107147, HL095181, and RR026231 from the National Institutes of Health (NIH), and BRCP 08–049 Third Frontier Program grant from the Ohio Department of Development (ODOD). Dr. Metin Aytekin is supported by 0826095H from the American Heart Association (AHA)

Conflict of Interest: None declared.

References

Rich

Dantzker

Ayres

Bergofsky

Brundage

Detre

. Primary pulmonary hypertension. A national prospective study. Ann Intern Med 1987;107:216–23.

Tuder

Abman

Braun

Capron

Stevens

Thistlethwaite

. Development and pathology of pulmonary hypertension. J Am Coll Cardiol 2009;54:S3–9.

Schermuly

Ghofrani

Wilkins

Grimminger

. Mechanisms of disease: Pulmonary arterial hypertension. Nat Rev Cardiol 2011;8:443–55.

Ignarro

Buga

Wood

Byrns

Chaudhuri

. Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide. Proc Natl Acad Sci U S A 1987;84:9265–9.

Palmer

Ferrige

Moncada

. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 1987;327:524–6.

Nathan

Xie

. Nitric oxide synthases: Roles, tolls, and controls. Cell 1994;78:915–8.

Schmidt

Walter

. NO at work. Cell 1994;78:919–25.

Gaston

Drazen

Loscalzo

Stamler

. The biology of nitrogen oxides in the airways. Am J Respir Crit Care Med 1994;149:538–51.

Nathan

. Nitric oxide as a secretory product of mammalian cells. FASEB J 1992;6:3051–64.

10.

Dweik

Erzurum

. Effects of Nitric Oxide and Cyclic GMP on Smooth Muscle Proliferation. In: Moss

, editors. LAM and Other Diseases Characterized by Smooth Muscle Proliferation. New York: Marcel Dekker Inc; 1999. p. 333–49.

11.

Stuehr

Griffith

. Mammalian nitric oxide synthases. Adv Enzymol Relat Areas Mol Biol 1992;65:287–346.

12.

Dweik

Laskowski

Ozkan

Farver

Erzurum

. High levels of exhaled nitric oxide (NO) and NO synthase III expression in lesional smooth muscle in lymphangioleiomyomatosis. Am J Respir Cell Mol Biol 2001;24:414–8.

13.

Moncada

Higgs

. The L-arginine-nitric oxide pathway. N Engl J Med 1993;329:2002–12.

14.

Coggins

Bloch

. Nitric oxide in the pulmonary vasculature. Arterioscler Thromb Vasc Biol 2007;27:1877–85.

15.

Stuehr

. Mammalian nitric oxide synthases. Biochim Biophys Acta 1999;1411:217–30.

16.

Dweik

. Nitric oxide, hypoxia, and superoxide: The good, the bad, and the ugly! Thorax 2005;60:265–7.

17.

Dudzinski

Igarashi

Greif

Michel

. The regulation and pharmacology of endothelial nitric oxide synthase. Annu Rev Pharmacol Toxicol 2006;46:235–76.

18.

Newby

Southgate

Assender

. Inhibition of vascular smooth muscle cell proliferation by endothelium-dependent vasodilators. Herz 1992;17:291–9.

19.

Nathan

Xie

. Regulation of biosynthesis of nitric oxide. J Biol Chem 1994;269:13725–8.

20.

Barnes

Dweik

Gelb

Gibson

George

Grasemann

. Exhaled nitric oxide in pulmonary diseases: A comprehensive review. Chest 2010;138:682–92.

21.

Guo

De Raeve

Rice

Stuehr

Thunnissen

Erzurum

. Continuous nitric oxide synthesis by inducible nitric oxide synthase in normal human airway epithelium in vivo. Proc Natl Acad Sci U S A 1995;92:7809–13.

22.

Ward

Belvisi

Fox

Miura

Tadjkarimi

Yacoub

. Modulation of cholinergic neural bronchoconstriction by endogenous nitric oxide and vasoactive intestinal peptide in human airways in vitro. J Clin Invest 1993;92:736–42.

23.

Moncada

Palmer

Higgs

. Nitric oxide: Physiology, pathophysiology, and pharmacology. Pharmacol Rev 1991;43:109–42.

24.

Shaul

. Regulation of endothelial nitric oxide synthase: Location, location, location. Annu Rev Physiol 2002;64:749–74.

25.

Bernareggi

Mitchell

Barnes

Belvisi

. Dual action of nitric oxide on airway plasma leakage. Am J Respir Crit Care Med 1997;155:869–74.

26.

Ricciardolo

Sterk

Gaston

Folkerts

. Nitric oxide in health and disease of the respiratory system. Physiol Rev 2004;84:731–65.

27.

Fagan

Tyler

Sato

Fouty

Morris

Jr Huang

. Relative contributions of endothelial, inducible, and neuronal NOS to tone in the murine pulmonary circulation. Am J Physiol 1999;277:L472–8.

28.

Dweik

Laskowski

Abu-Soud

Kaneko

Hutte

Stuehr

. Nitric oxide synthesis in the lung. Regulation by oxygen through a kinetic mechanism. J Clin Invest 1998;101:660–6.

29.

Steudel

Ichinose

Huang

Hurford

Jones

Bevan

. Pulmonary vasoconstriction and hypertension in mice with targeted disruption of the endothelial nitric oxide synthase (NOS 3) gene. Circ Res 1997;81:34–41.

30.

Hecker

Sessa

Harris

Anggard

Vane

. The metabolism of L-arginine and its significance for the biosynthesis of endothelium-derived relaxing factor: Cultured endothelial cells recycle L-citrulline to L-arginine. Proc Natl Acad Sci U S A 1990;87:8612–6.

31.

Flam

Hartmann

Harrell-Booth

Solomonson

Eichler

. Caveolar localization of arginine regeneration enzymes, argininosuccinate synthase, and lyase, with endothelial nitric oxide synthase. Nitric Oxide 2001;5:187–97.

32.

Solomonson

Flam

Pendleton

Goodwin

Eichler

. The caveolar nitric oxide synthase/arginine regeneration system for NO production in endothelial cells. J Exp Biol 2003;206:2083–7.

33.

Hattori

Campbell

Gross

. Argininosuccinate synthetase mRNA and activity are induced by immunostimulants in vascular smooth muscle. Role in the regeneration or arginine for nitric oxide synthesis. J Biol Chem 1994;269:9405–8.

34.

Schmidt

Lohmann

Walter

. The nitric oxide and cGMP signal transduction system: Regulation and mechanism of action. Biochim Biophys Acta 1993;1178:153–75.

35.

Dweik

. Pulmonary hypertension and the search for the selective pulmonary vasodilator. Lancet 2002;360:886–7.

36.

Garbers

. Guanylyl cyclase receptors and their endocrine, paracrine, and autocrine ligands. Cell 1992;71:1–4.

37.

McDonald

Murad

. Nitric oxide and cyclic GMP signaling. Proc Soc Exp Biol Med 1996;211:1–6.

38.

Murad

. Regulation of cytosolic guanylyl cyclase by nitric oxide: The NO-cyclic GMP signal transduction system. Adv Pharmacol 1994;26:19–33.

39.

Archer

Huang

Hampl

Nelson

Shultz

Weir

. Nitric oxide and cGMP cause vasorelaxation by activation of a charybdotoxin-sensitive K channel by cGMP-dependent protein kinase. Proc Natl Acad Sci U S A 1994;91:7583–7.

40.

Lincoln

Cornwell

. Intracellular cyclic GMP receptor proteins. FASEB J 1993;7:328–38.

41.

Rybalkin

Yan

Bornfeldt

Beavo

. Cyclic GMP phosphodiesterases and regulation of smooth muscle function. Circ Res 2003;93:280–91.

42.

Alderton

Cooper

Knowles

. Nitric oxide synthases: Structure, function and inhibition. Biochem J 2001;357:593–615.

43.

Bulau

Zakrzewicz

Kitowska

Leiper

Gunther

Grimminger

. Analysis of methylarginine metabolism in the cardiovascular system identifies the lung as a major source of ADMA. Am J Physiol Lung Cell Mol Physiol 2007;292:L18–24.

44.

Tran

Leiper

Vallance

. The DDAH/ADMA/NOS pathway. Atheroscler 2003;4:33–40.

45.

Dweik

. The lung in the balance: Arginine, methylated arginines, and nitric oxide. Am J Physiol Lung Cell Mol Physiol 2007;292:L15–7.

46.

Ogawa

Kimoto

Sasaoka

. Purification and properties of a new enzyme, NG, NG-dimethylarginine dimethylaminohydrolase, from rat kidney. J Biol Chem 1989;264:10205–9.

47.

BMC

Dweik

. Arginine and nitric oxide. In: Shils

Shike

Ross

Caballero

Cousins

, editors. Modern nutrition in health and disease. 10th ed. Philadelphia, PA: Lippincott Williams and Wilkins; 2005. p. 571–81.

48.

Garg

Hassid

. Nitric oxide-generating vasodilators and 8-bromo-cyclic guanosine monophosphate inhibit mitogenesis and proliferation of cultured rat vascular smooth muscle cells. J Clin Invest 1989;83:1774–7.

49.

Thomae

Nakayama

Billiar

Simmons

Pitt

Davies

. The effect of nitric oxide on fetal pulmonary artery smooth muscle growth. J Surg Res 1995;59:337–43.

50.

Sarkar

Meinberg

Stanley

Gordon

Webb

. Nitric oxide reversibly inhibits the migration of cultured vascular smooth muscle cells. Circ Res 1996;78:225–30.

51.

Abman

Chatfield

Hall

McMurtry

. Role of endothelium-derived relaxing factor during transition of pulmonary circulation at birth. Am J Physiol 1990;259:H1921–7.

52.

Geng

Hansson

Holme

. Interferon-gamma and tumor necrosis factor synergize to induce nitric oxide production and inhibit mitochondrial respiration in vascular smooth muscle cells. Circ Res 1992;71:1268–76.

53.

Stein

Fabry

Murphy

Hart

. Involvement of nitric oxide in IFN-gamma-mediated reduction of microvessel smooth muscle cell proliferation. Mol Immunol 1995;32:965–73.

54.

Fukuo

Hata

Suhara

Nakahashi

Shinto

Tsujimoto

. Nitric oxide induces upregulation of Fas and apoptosis in vascular smooth muscle. Hypertension 1996;27:823–6.

55.

Schini

Vanhoutte

. Role of the L-arginine-nitric oxide pathway in vascular smooth muscle. Eur Heart J 1993;14:16–21.

56.

Thomae

Joshi

Davies

Pitt

Billiar

Simmons

. Nitric oxide produced by cytokine-activated pulmonary artery smooth muscle cells is cytotoxic to cocultured endothelium. Surgery 1996;119:61–6.

57.

Nunokawa

Tanaka

. Interferon-gamma inhibits proliferation of rat vascular smooth muscle cells by nitric oxide generation. Biochem Biophys Res Commun 1992;188:409–15.

58.

Nakaki

Nakayama

Kato

. Inhibition by nitric oxide and nitric oxide-producing vasodilators of DNA synthesis in vascular smooth muscle cells. Eur J Pharmacol 1990;189:347–53.

59.

Kolpakov

Gordon

Kulik

. Nitric oxide-generating compounds inhibit total protein and collagen synthesis in cultured vascular smooth muscle cells. Circ Res 1995;76:305–9.

60.

Cornwell

Arnold

Boerth

Lincoln

. Inhibition of smooth muscle cell growth by nitric oxide and activation of cAMP-dependent protein kinase by cGMP. Am J Physiol 1994;267:C1405–13.

61.

Clementi

Sciorati

Nistico

. Growth factor-induced Ca2⁺ responses are differentially modulated by nitric oxide via activation of a cyclic GMP-dependent pathway. Mol Pharmacol 1995;48:1068–77.

62.

Nguyen

Brunson

Crespi

Penman

Wishnok

Tannenbaum

. DNA damage and mutation in human cells exposed to nitric oxide in vitro. Proc Natl Acad Sci U S A 1992;89:3030–4.

63.

Yang

Ando

Korenaga

Toyo-oka

Kamiya

. Exogenous nitric oxide inhibits proliferation of cultured vascular endothelial cells. Biochem Biophys Res Commun 1994;203:1160–7.

64.

Ziche

Morbidelli

Masini

Amerini

Granger

Maggi

. Nitric oxide mediates angiogenesis in vivo and endothelial cell growth and migration in vitro promoted by substance P. J Clin Invest 1994;94:2036–44.

65.

Fukuo

Inoue

Morimoto

Nakahashi

Yasuda

Kitano

. Nitric oxide mediates cytotoxicity and basic fibroblast growth factor release in cultured vascular smooth muscle cells. A possible mechanism of neovascularization in atherosclerotic plaques. J Clin Invest 1995;95:669–76.

66.

Kim-Shapiro

Schechter

Gladwin

. Unraveling the reactions of nitric oxide, nitrite, and hemoglobin in physiology and therapeutics. Arterioscler Thromb Vasc Biol 2006;26:697–705.

67.

Hess

Matsumoto

Kim

Marshall

Stamler

. Protein S-nitrosylation: Purview and parameters. Nat Rev Mol Cell Biol 2005;6:150–66.

68.

Zweier

Wang

Samouilov

Kuppusamy

. Enzyme-independent formation of nitric oxide in biological tissues. Nat Med 1995;1:804–9.

69.

Lundberg

Weitzberg

Gladwin

. The nitrate-nitrite-nitric oxide pathway in physiology and therapeutics. Nat Rev Drug Discov 2008;7:156–67.

70.

Duranski

Greer

Dejam

Jaganmohan

Hogg

Langston

. Cytoprotective effects of nitrite during in vivo ischemia-reperfusion of the heart and liver. J Clin Invest 2005;115:1232–40.

71.

Gladwin

Kim-Shapiro

. The functional nitrite reductase activity of the heme-globins. Blood 2008;112:2636–47.

72.

Singel

Stamler

. Chemical physiology of blood flow regulation by red blood cells: The role of nitric oxide and S-nitrosohemoglobin. Annu Rev Physiol 2005;67:99–145.

73.

Tsujino

Miyamoto

Nishimura

Shinano

Makita

Saito

. Production of nitric oxide (NO) in intrathoracic airways of normal humans. Am J Respir Crit Care Med 1996;154:1370–4.

74.

Dweik

Erzurum

. Regulation of nitric oxide (NO) synthases and gas phase NO by oxygen. In: Marczin

Kharitonov

Yacoub

Barnes

, editors. Disease markers in exhaled breath. New York: Marcel Dekker Inc; 2003. p. 235–46.

75.

Dweik

Guo

Uetani

Erzurum

. Nitric oxide synthase in the human airway epithelium. Zhongguo Yao Li Xue Bao 1997;18:550–2.

76.

Kobzik

Bredt

Lowenstein

Drazen

Gaston

Sugarbaker

. Nitric oxide synthase in human and rat lung: Immunocytochemical and histochemical localization. Am J Respir Cell Mol Biol 1993;9:371–7.

77.

Barnes

Belvisi

. Nitric oxide and lung disease. Thorax 1993;48:1034–43.

78.

Dweik

. The promise and reality of nitric oxide in the diagnosis and treatment of lung disease. Cleve Clin J Med 2001;68:486, 8, 90, 93.

79.

Dweik

Comhair

Gaston

Thunnissen

Farver

Thomassen

. NO chemical events in the human airway during the immediate and late antigen-induced asthmatic response. Proc Natl Acad Sci U S A 2001;98:2622–7.

80.

Khatri

Hammel

Kavuru

Erzurum

Dweik

. Temporal association of nitric oxide levels and airflow in asthma after whole lung allergen challenge. J Appl Physiol 2003;95:436–40.

81.

Kinsella

Cutter

Walsh

Gerstmann

Bose

Hart

. Early inhaled nitric oxide therapy in premature newborns with respiratory failure. N Engl J Med 2006;355:354–64.

82.

Ballard

Truog

Cnaan

Martin

Ballard

Merrill

. Inhaled nitric oxide in preterm infants undergoing mechanical ventilation. N Engl J Med 2006;355:343–53.

83.

Stark

. Inhaled NO for preterm infants-getting to yes? N Engl J Med 2006;355:404–6.

84.

McCurnin

Pierce

Chang

Gibson

Osborne-Lawrence

Yoder

. Inhaled NO improves early pulmonary function and modifies lung growth and elastin deposition in a baboon model of neonatal chronic lung disease. Am J Physiol Lung Cell Mol Physiol 2005;288:L450–9.

85.

Zacharasiewicz

Wilson

Lex

Erin

Hansel

. Clinical use of noninvasive measurements of airway inflammation in steroid reduction in children. Am J Respir Crit Care Med 2005;171:1077–82.

86.

Berry

Shaw

Green

Brightling

Wardlaw

Pavord

. The use of exhaled nitric oxide concentration to identify eosinophilic airway inflammation: An observational study in adults with asthma. Clin Exp Allergy 2005;35:1175–9.

87.

Smith

Cowan

Brassett

Filsell

McLachlan

Monti-Sheehan

. Exhaled nitric oxide: A predictor of steroid response. Am J Respir Crit Care Med 2005;172:453–9.

88.

Ischiropoulos

Mendiguren

Fisher

Thom

. Role of neutrophils and nitric oxide in lung alveolar injury from smoke inhalation. Am J Respir Crit Care Med 1994;150:337–41.

89.

Weber

Lauer

Mulsch

Kojda

. The effect of peroxynitrite on the catalytic activity of soluble guanylyl cyclase. Free Radic Biol Med 2001;31:1360–7.

90.

Firth

Yuan

. Bringing down the ROS: A new therapeutic approach for PPHN. Am J Physiol Lung Cell Mol Physiol 2008;295:L976–8.

91.

Farrow

Lakshminrusimha

Reda

Wedgwood

Czech

Gugino

. Superoxide dismutase restores eNOS expression and function in resistance pulmonary arteries from neonatal lambs with persistent pulmonary hypertension. Am J Physiol Lung Cell Mol Physiol 2008;295:L979–87.

92.

Robbins

Horowitz

Merritt

Kheiter

Tierney

Narula

. Recombinant human superoxide dismutase reduces lung injury caused by inhaled nitric oxide and hyperoxia. Am J Physiol 1997;272:L903–7.

93.

Lundberg

Farkas-Szallasi

Weitzberg

Rinder

Lidholm

Anggåard

. High nitric oxide production in human paranasal sinuses. Nat Med 1995;1:370–3.

94.

Kaneko

Arroliga

Dweik

Comhair

Laskowski

Oppedisano

. Biochemical reaction products of nitric oxide as quantitative markers of primary pulmonary hypertension. Am J Respir Crit Care Med 1998;158:917–23.

95.

Ozkan

Dweik

Laskowski

Arroliga

Erzurum

. High levels of nitric oxide in individuals with pulmonary hypertension receiving epoprostenol therapy. Lung 2001;179:233–43.

96.

Giaid

Saleh

. Reduced expression of endothelial nitric oxide synthase in the lungs of patients with pulmonary hypertension. N Engl J Med 1995;333:214–21.

97.

Xue

Johns

. Endothelial nitric oxide synthase in the lungs of patients with pulmonary hypertension. N Engl J Med 1995;333:1642–4.

98.

Tuder

Cool

Geraci

Wang

Abman

Wright

. Prostacyclin synthase expression is decreased in lungs from patients with severe pulmonary hypertension. Am J Respir Crit Care Med 1999;159:1925–32.

99.

Mason

Springall

Burke

Pollock

Mikhail

Yacoub

. High expression of endothelial nitric oxide synthase in plexiform lesions of pulmonary hypertension. J Pathol 1998;185:313–8.

100.

Hagan

Pepke-Zaba

. Pulmonary hypertension, nitric oxide and nitric oxide-releasing compounds. Expert Rev Respir Med 2011;5:163–71.

101.

Thomson

Machado

Pauciulo

Morgan

Humbert

Elliott

. Sporadic primary pulmonary hypertension is associated with germline mutations of the gene encoding BMPR-II, a receptor member of the TGF-beta family. J Med Genet 2000;37:741–5.

102.

Atkinson

Stewart

Upton

Machado

Thomson

Trembath

. Primary pulmonary hypertension is associated with reduced pulmonary vascular expression of type II bone morphogenetic protein receptor. Circulation 2002;105:1672–8.

103.

Gangopahyay

Oran

Bauer

Wertz

Comhair

Erzurum

. Bone morphogenetic protein receptor II is a novel mediator of endothelial nitric-oxide synthase activation. J Biol Chem 2011;286:33134–40.

104.

Forrest

Small

Corris

. Effect of nebulized epoprostenol (prostacyclin) on exhaled nitric oxide in patients with pulmonary hypertension due to congenital heart disease and in normal controls. Clin Sci (Lond) 1999;97:99–102.

105.

Rolla

Colagrande

Brussino

Bucca

Bertero

Caligaris-Cappio

. Exhaled nitric oxide and pulmonary response to iloprost in systemic sclerosis with pulmonary hypertension. Lancet 1998;351:1491–2.

106.

Machado

Nerkar

Londhe MV

Dweik

Hammel

Janocha

Pyle

. Nitric oxide and pulmonary arterial pressures in pulmonary hypertension. Free Radic Biol Med 2004;37:1010–7.

107.

Hirata

Emori

Eguchi

Kanno

Imai

Ohta

. Endothelin receptor subtype B mediates synthesis of nitric oxide by cultured bovine endothelial cells. J Clin Invest 1993;91:1367–73.

108.

Millatt

Whitley

Leiper

Siragy

Carey

. Evidence for dysregulation of dimethylarginine dimethylaminohydrolase I in chronic hypoxia-induced pulmonary hypertension. Circulation 2003;108:1493–8.

109.

Kielstein

Bode-Boger

Hesse

Martens-Lobenhoffer

Takacs

Fliser

. Asymmetrical dimethylarginine in idiopathic pulmonary arterial hypertension. Arterioscler Thromb Vasc Biol 2005;25:1414–8.

110.

Pullamsetti

Kiss

Ghofrani

Voswinckel

Haredza

Klepetko

. Increased levels and reduced catabolism of asymmetric and symmetric dimethylarginines in pulmonary hypertension. FASEB J 2005;19:1175–7.

111.

Skoro-Sajer

Mittermayer

Panzenboeck

Bonderman

Sadushi

Hitsch

. Asymmetric dimethylarginine is increased in chronic thromboembolic pulmonary hypertension. Am J Respir Crit Care Med 2007;176:1154–60.

112.

Kaneko

Zheng

Comhair

Janocha

Goggans

. Increased arginase II and decreased NO synthesis in endothelial cells of patients with pulmonary arterial hypertension. FASEB J 2004;18:1746–8.

113.

American Thoracic Society; European Respiratory Society. ATS/ERS recommendations for standardized procedures for the online and offline measurement of exhaled lower respiratory nitric oxide and nasal nitric oxide, 2005. Am J Respir Crit Care Med 2005;171:912–30.

114.

Condorelli

Shin

Aledia

Silkoff

George

. A simple technique to characterize proximal and peripheral nitric oxide exchange using constant flow exhalations and an axial diffusion model. J Appl Physiol 2007;102:417–25.

115.

Rich

Kaufmann

Levy

. The effect of high doses of calcium-channel blockers on survival in primary pulmonary hypertension. N Engl J Med 1992;327:76–81.

116.

Sitbon

Humbert

Jais

Ioos

Hamid

Provencher

. Long-term response to calcium channel blockers in idiopathic pulmonary arterial hypertension. Circulation 2005;111:3105–11.

117.

Barst

Gibbs

Ghofrani

Hoeper

McLaughlin

Rubin

. Updated evidence-based treatment algorithm in pulmonary arterial hypertension. J Am Coll Cardiol 2009;54:S78–84.

118.

Tonelli

Alnuaimat

Mubarak

. Pulmonary vasodilator testing and use of calcium channel blockers in pulmonary arterial hypertension. Respir Med 2010;104:481–96.

119.

Weir

Rubin

Ayres

Bergofsky

Brundage

Detre

. The acute administration of vasodilators in primary pulmonary hypertension. Experience from the National Institutes of Health Registry on Primary Pulmonary Hypertension. Am Rev Respir Dis 1989;140:1623–30.

120.

Post

Janssens

Van de Werf

Budts

. Responsiveness to inhaled nitric oxide is a predictor for mid-term survival in adult patients with congenital heart defects and pulmonary arterial hypertension. Eur Heart J 2004;25:1651–6.

121.

Skoro-Sajer

Hack

Sadushi-Kolici

Bonderman

Jakowitsch

Klepetko

. Pulmonary vascular reactivity and prognosis in patients with chronic thromboembolic pulmonary hypertension: A pilot study. Circulation 2009;119:298–305.

122.

Krasuski

Devendra

Hart

Wang

Harrison

Bashore

. Response to inhaled nitric oxide predicts survival in patients with pulmonary hypertension. J Card Fail 2011;17:265–71.

123.

Galie

Ghofrani

Torbicki

Barst

Rubin

Badesch

. Sildenafil citrate therapy for pulmonary arterial hypertension. N Engl J Med 2005;353:2148–57.

124.

Galie

Brundage

Ghofrani

Oudiz

Simonneau

Safdar

. Tadalafil therapy for pulmonary arterial hypertension. Circulation 2009;119:2894–903.

125.

Label approved for REVATIO, NDA # 021845. FDA, 2012. Available from: http://www.accessdata.fda.gov/scripts/cder/drugsatfda. [Last accessed on 2012 April 10].

126.

Barst

Ivy

Gaitan

Szatmari

Rudzinski

Garcia

. A randomized, double-blind, placebo-controlled, dose-ranging study of oral sildenafil citrate in treatment-naive children with pulmonary arterial hypertension. Circulation 2012;125:324–34.

127.

Wharton

Strange

Moller

Growcott

Ren

Franklyn

. Antiproliferative effects of phosphodiesterase type 5 inhibition in human pulmonary artery cells. Am J Respir Crit Care Med 2005;172:105–13.

128.

Michelakis

Tymchak

Lien

Webster

Hashimoto

Archer

. Oral sildenafil is an effective and specific pulmonary vasodilator in patients with pulmonary arterial hypertension: Comparison with inhaled nitric oxide. Circulation 2002;105:2398–403.

129.

Nagendran

Archer

Soliman

Gurtu

Moudgil

Haromy

. Phosphodiesterase type 5 is highly expressed in the hypertrophied human right ventricle, and acute inhibition of phosphodiesterase type 5 improves contractility. Circulation 2007;116:238–48.

130.

Frostell

Fratacci

Wain

Jones

Zapol

. Inhaled nitric oxide. A selective pulmonary vasodilator reversing hypoxic pulmonary vasoconstriction. Circulation 1991;83:2038–47.

131.

Pepke-Zaba

Higenbottam

Dinh-Xuan

Stone

Wallwork

. Inhaled nitric oxide as a cause of selective pulmonary vasodilatation in pulmonary hypertension. Lancet 1991;338:1173–4.

132.

Vonbank

Ziesche

Higenbottam

Stiebellehner

Petkov

Schenk

. Controlled prospective randomised trial on the effects on pulmonary haemodynamics of the ambulatory long term use of nitric oxide and oxygen in patients with severe COPD. Thorax 2003;58:289–93.

133.

Channick

Newhart

Johnson

Williams

Auger

Fedullo

. Pulsed delivery of inhaled nitric oxide to patients with primary pulmonary hypertension: An ambulatory delivery system and initial clinical tests. Chest 1996;109:1545–9.

134.

Perez-Penate

Julia-Serda

Ojeda-Betancort

García-Quintana

Pulido-Duque

Rodríguez-Pérez

. Long-term inhaled nitric oxide plus phosphodiesterase 5 inhibitors for severe pulmonary hypertension. J Heart Lung Transplant 2008;27:1326–32.

135.

Frampton

Morrow

Cox

Gibb

Speers

Utell

. Effects of nitrogen dioxide exposure on pulmonary function and airway reactivity in normal humans. Am Rev Respir Dis 1991;143:522–7.

136.

Clutton-Brock

. Two cases of poisoning by contamination of nitrous oxide with higher oxides of nitrogen during anaesthesia. Br J Anaesth 1967;39:388–92.

137.

Miller

Tang

Keech

Celermajer

. Rebound pulmonary hypertension on withdrawal from inhaled nitric oxide. Lancet 1995;346:51–2.

138.

Christenson

Lavoie

O'Connor

Bhorade

Pohlman

Hall

. The incidence and pathogenesis of cardiopulmonary deterioration after abrupt withdrawal of inhaled nitric oxide. Am J Respir Crit Care Med 2000;161:1443–9.

139.

Label for INOMAX, NDA # 020845. U.S. Food and Drug Administration. 2010. Accessed from: http://www.accessdata.fda.gov/scripts/cder/drugsatfda/. [Last accessed on 2012, April 10].

140.

Mercier

Hummler

Durrmeyer

Sanchez-Luna

Carnielli

Field

. Inhaled nitric oxide for prevention of bronchopulmonary dysplasia in premature babies (EUNO): A randomised controlled trial. Lancet 2010;376:346–54.

141.

Van Meurs

Wright

Ehrenkranz

Lemons

Ball

Poole

. Inhaled nitric oxide for premature infants with severe respiratory failure. N Engl J Med 2005;353:13–22.

142.

Kleinbongard

Dejam

Lauer

Jax

Kerber

Gharini

. Plasma nitrite concentrations reflect the degree of endothelial dysfunction in humans. Free Radic Biol Med 2006;40:295–302.

143.

Hunter

Dejam

Blood

Shields

Kim-Shapiro

Machado

. Inhaled nebulized nitrite is a hypoxia-sensitive NO-dependent selective pulmonary vasodilator. Nat Med 2004;10:1122–7.

144.

Ingram

Pinder

Bailey

Fraser

James

. Low-dose sodium nitrite vasodilates hypoxic human pulmonary vasculature by a means that is not dependent on a simultaneous elevation in plasma nitrite. Am J Physiol Heart Circ Physiol 2010;298:H331–9.

145.

Brandler

Powell

Craig

Quick

McMahon

Goldberg

. A novel inhaled organic nitrate that affects pulmonary vascular tone in a piglet model of hypoxia-induced pulmonary hypertension. Pediatr Res 2005;58:531–6.

146.

Keefer

Nims

Davies

Wink

. “NONOates” (1-substituted diazen-1-ium-1,2-diolates) as nitric oxide donors: Convenient nitric oxide dosage forms. Methods Enzymol 1996;268:281–93.

147.

Hampl

Tristani-Firouzi

Hutsell

Archer

. Nebulized nitric oxide/nucleophile adduct reduces chronic pulmonary hypertension. Cardiovasc Res 1996;31:55–62.

148.

Young

Ladino

Navarrete

Dabrowska

Hehre

Bancalari

. The effect of a nebulized NO donor, DPTA/NO, on acute hypoxic pulmonary hypertension in newborn piglets. Biol Neonate 2004;85:195–202.

149.

Lam

Van Heerden

Blott

Roberts

Ilett

. The selective pulmonary vasodilatory effect of inhaled DETA/NO, a novel nitric oxide donor, in ARDS-a pilot human trial. J Crit Care 2004;19:48–53.

150.

Schermuly

Stasch

Pullamsetti

Middendorff

Müller

Schlüter

. Expression and function of soluble guanylate cyclase in pulmonary arterial hypertension. Eur Respir J 2008;32:881–91.

151.

Ghofrani

Hoeper

Halank

Meyer

Staehler

Behr

. Riociguat for chronic thromboembolic pulmonary hypertension and pulmonary arterial hypertension: A phase II study. Eur Respir J 2010;36:792–9.

152.

Schermuly

Janssen

Weissmann

Stasch

Grimminger

Ghofrani

. Riociguat for the treatment of pulmonary hypertension. Expert Opin Investig Drugs 2011;20:567–76.

153.

Wei

Huang

Luo

Wang

. L-arginine restores endothelial nitric oxide synthase-coupled activity and attenuates monocrotaline-induced pulmonary artery hypertension in rats. Am J Physiol Endocrinol Metab 2010;298:E1131–9.

154.

Howell

Costello

Sands

Dooley

McLoughlin

. L-Arginine promotes angiogenesis in the chronically hypoxic lung: A novel mechanism ameliorating pulmonary hypertension. Am J Physiol Lung Cell Mol Physiol 2009;296:L1042–50.

155.

Mitani

Maruyama

Sakurai

. Prolonged administration of L-arginine ameliorates chronic pulmonary hypertension and pulmonary vascular remodeling in rats. Circulation 1997;96:689–97.

156.

Nagaya

Uematsu

Oya

Sato

Sakamaki

Kyotani

. Short-term oral administration of L-arginine improves hemodynamics and exercise capacity in patients with precapillary pulmonary hypertension. Am J Respir Crit Care Med 2001;163:887–91.

157.

Morris

Jr. Hagar

Van Warmerdam

Claster

Kepka-Lenhart

. Arginine therapy: A new treatment for pulmonary hypertension in sickle cell disease? Am J Respir Crit Care Med 2003;168:63–9.

158.

Ananthakrishnan

Barr

Summar

Smith

Kaplowitz

Cunningham

. L-Citrulline ameliorates chronic hypoxia-induced pulmonary hypertension in newborn piglets. Am J Physiol Lung Cell Mol Physiol 2009;297:L506–11.

159.

Smith

Canter

Christian

Drinkwater

Scholl

Christman

. Nitric oxide precursors and congenital heart surgery: A randomized controlled trial of oral citrulline. J Thorac Cardiovasc Surg 2006;132:58–65.

160.

Nakayama

Geller

Di Silvio

Bloomgarden

Davies

Pitt

. Tetrahydrobiopterin synthesis and inducible nitric oxide production in pulmonary artery smooth muscle. Am J Physiol 1994;266:L455–60.

161.

Gross

Levi

. Tetrahydrobiopterin synthesis. An absolute requirement for cytokine-induced nitric oxide generation by vascular smooth muscle. J Biol Chem 1992;267:25722–9.

162.

Raman

Martasek

Kral

Masters

Poulos

. Crystal structure of constitutive endothelial nitric oxide synthase: A paradigm for pterin function involving a novel metal center. Cell 1998;95:939–50.

163.

Venema

Zou

Ryan

Venema

. Subunit interactions of endothelial nitric-oxide synthase. Comparisons to the neuronal and inducible nitric-oxide synthase isoforms. J Biol Chem 1997;272:1276–82.

164.

Robbins

Hemnes

Gibbs

Christman

Howard

Meehan

. Safety of sapropterin dihydrochloride (6r-bh4) in patients with pulmonary hypertension. Exp Lung Res 2011;37:26–34.

165.

Steinhorn

Albert

Swartz

Russell

Levine

Davis

. Recombinant human superoxide dismutase enhances the effect of inhaled nitric oxide in persistent pulmonary hypertension. Am J Respir Crit Care Med 2001;164:834–9.

166.

Wang

Zhang

Shang

Zhu

Xie

Tao

. Transplantation of autologous endothelial progenitor cells may be beneficial in patients with idiopathic pulmonary arterial hypertension: A pilot randomized controlled trial. J Am Coll Cardiol 2007;49:1566–71.

167.

Deng

Bivalacqua

Champion

Hellstrom

Murthy

Kadowitz

. Gene therapy techniques for the delivery of endothelial nitric oxide synthase to the lung for pulmonary hypertension. Methods Mol Biol 2010;610:309–21.

168.

Zhao

Courtman

Deng

Kugathasan

Zhang

Stewart

. Rescue of monocrotaline-induced pulmonary arterial hypertension using bone marrow-derived endothelial-like progenitor cells: Efficacy of combined cell and eNOS gene therapy in established disease. Circ Res 2005;96:442–50.

169.

Kanki-Horimoto

Horimoto

Mieno

Kishida

Watanabe

Furuya

. Implantation of mesenchymal stem cells overexpressing endothelial nitric oxide synthase improves right ventricular impairments caused by pulmonary hypertension. Circulation 2006;114:I181–5.

170.

Janssens

Bloch

Nong

Gerard

Zoldhelyi

Collen

. Adenoviral-mediated transfer of the human endothelial nitric oxide synthase gene reduces acute hypoxic pulmonary vasoconstriction in rats. J Clin Invest 1996;98:317–24.

171.

Budts

Pokreisz

Nong

Van Pelt

Gillijns

Gerard

. Aerosol gene transfer with inducible nitric oxide synthase reduces hypoxic pulmonary hypertension and pulmonary vascular remodeling in rats. Circulation 2000;102:2880–5.