Nitric oxide deficiency in pulmonary hypertension: Pathobiology and implications for therapy.

2013·1.1 MB·English

by  Tonelli, Adriano R.

Review Article
Nitric oxide deficiency in pulmonary hypertension:
Pathobiology and implications for therapy
Adriano R. Tonelli1, Sarah Haserodt2, Metin Aytekin2,3, and Raed A. Dweik1,2
1Department of Pulmonary, Allergy and Critical Care Medicine, Respiratory Institute, 2Department of Pathobiology,
Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, USA; and 3Department of Medical Biology, Faculty of Medicine,
Erciyes University, Kayseri, Turkey
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.
Key Words: pulmonary hypertension, nitric oxide, physiopathology and therapeutics
NITRIC OXIDE PHYSIOLOGY
Pulmonary arterial hypertension (PAH) is a progressive
disease that has poor prognosis since it may lead to right
[1]
ventricular failure and death. The disease is characterized NO is an endogenously synthesized, diffusible, lipophilic
by excessive pulmonary vasoconstriction and abnormal gas that is produced by a group of enzymes known as nitric
vascular remodeling that result in loss of vascular oxide synthases (NOS). Their role is[ 6t]o convert the amino acid
L-arginine to L-citrulline and NO. For their activity, NOS
cross-sectional area and increase in right ventricular
[2]
require oxygen, reduced nicotinamide-adenine dinucleotide
afterload. One of the proposed mechanisms involved in the
phosphate (NADPH), and other cofactors such as flavin
pathogenesis of the disease is an imbalance in vasoactive
adenine dinucleotide (FAD), flavin monon[u11c-l1e3]otide (FMN),
mediators with reduced levels of the vasodilatory and
[3] calmodulin, and tetrahydrobiopterin (BH4). NOS is active
antiproliferative nitric oxide (NO).
as a homodimer and contains a reductase and an oxygenase
domain. The oxygenase domain is the active site of NO[14 s]ynthesis
Since the breakthrough discovery in 1987 that the
[4,5] with binding sites for heme, L-arginine, and BH4 (Fig. 1).
endothelium-derived relaxing factor was nitric oxide (NO), Three NOS isoforms (Types I, II, and III) have been
this colorless and odorless free-radical gas became identified. These NOS isoforms have important differe[n15c,e16s]
increasingly recognized as a key factor in human physiology in expression and regulation as shown in Table 1.
[6,7]
and disease. NO is an autocrine and paracrine signaling In general, NOS I is expressed in neuronal cells and skeletal
molecule whose functions are diverse and involve smooth muscle; NOS II is found in epithelial, and smooth muscles
muscle relaxation, platelet inhibition, central and autonomic cells as well as in neutrophils, macrophages, and fibroblasts;
neurotransmission, tumor cell lysis, bacterial killing, and and N[1O7S] III is present in endothelial cells throughout the
[7-10]
body. NOS I and III are continuously expressed and
stimulation of hormonal release. This review will focus
regulated by Calcium/Calmodulin; meanwhile, NOS II is
on the role of NO in physiology and pathobiology of lung
Access this article online
diseases, particularly pulmonary hypertension (PH), and the Quick Response Code: Website: www.pulmonarycirculation.org
current and emerging pulmonary hypertension (PH)-specific DOI: 10.4103/2045-8932.109911
treatments based on NO signaling.
Address correspondence to: How to cite this article: Tonelli AR, Haserodt
Dr. Raed A. Dweik S, Aytekin M, Dweik RA. Nitric oxide deficiency
Cleveland Clinic in pulmonary hypertension: Pathobiology
9500 Euclid Ave. A-90 and implications for therapy. Pulm Circ
Cleveland, OH 44195, USA 2013;3:20-30.
Email: [email protected]
20 Pulmonary Circulation | January-March 2013 | Vol 3 | No 1
Tonelli et al.: NO deficiency in PH
Table 1: Characteristics of nitric oxide synthases isoforms
NOS Designation Size Expression Main cellular sources in the Calcium Nitric oxide Chromosome
isoforms (KDa) lung regulation output
I Neural NOS 155 Constitutive Inhibitory non‑adrenergic Dependent Picomolar 12
(nNOS) non‑cholinergic neurons
II Inducible 125 Inducible by cytokines, Airway epithelial cells Independent Nanomolar 17
NOS (iNOS) endotoxin and oxidants
III Endothelial 135 Constitutive Endothelial cells and brush Dependent Picomolar 7
NOS (eNOS) border of ciliated epithelial cells
NOS: nitric oxide synthase
α γ β
regulated at the transcription level. NOS II transcription is
increased by cytokines (e.g., TNF- , int[e10r,1fe8]ron- , and IL-1 ),
endotoxins, oxidants, and shear stress, and is decreased
by corticosteroids, retinoids, transforming growth factor beta,
platelet-derive[d9, 1g0,1r3o,1w9]th factor, insulin-like growth factor 1,
and thrombin. The initial clear distinction between
the constitutive and inducible isoforms has been recently
distorted a[2n0,2d1 ]constitutive isoforms may be induced, and
vice versa.
NOS I acts as a functional antagonist of acetylcholine and
mediates inhibito[2r2y] nonadrenergic non-cholinergic neural
bronchodilation. NOS II is involved in inflammation as it Figure 1: NO synthesis and signaling pathways. BH4, tetrahydrobiopterin;
mediates the cytotoxic activity of activated macrophages cGMP: Cyclic guanosine monophosphate; GMP: Guanosine monophosphate;
and may be a contributin[23g] factor in the vasodilation GTP: Guanosine triphosphate; NO: Nitric oxide; NOS: Nitric oxide synthase;
ONOO-, peroxynitrite; PK: Protein kinases; sGC: Soluble guanylate cyclase;
observed in septic shock. [ 2N4]OS III plays a role in the
SNO‑Hb: S-nitrosothiol – hemoglobin. L-arginine can be regenerated from
regulation of vascular flo[2w5] and may reduce plasma L-citrulline by two enzymes (argininosuccinate synthase and argininosuccinate
exudation in the airways [2 6]and regulate ciliary beating lyase).
and mucociliary clearance. NOS III is the predomin[1a4,n20t]
source of NO production in the pulmonary circulation.
Studies in transgenic mice and humans to assess the
relative contribution of the three NOS isoforms showed cells (e.g., vascular smooth mus[9c,3l4e] cells), acting as an
that NOS II and III are the key regulators of the pulmonary intra- or intercellular messenger. The NO intracellular
circulation tone. NOS III is the key mediator of resting tone diffusion may be limited because NO is readily oxidized
through endo[2t7h] elium-dependent pulmonary vasculature to the more sta[b11l]e metabolic products nitrite (NO2¯) and
vasodilation, while NOS II may [m28]ediate the pulmonary nitrate (NO3¯) and is scavenged predominantly by
circulation’s response to oxygen. Targeted disruption hemoglobin. Upon entering the cells, NO activates the
of the NOS III gene in mice was associated with mild P[2H9] intracellular soluble guanylate cyclase (sGC) to produce
without evidence of pulmonary vascular remodeling, 3’,5’-cyclic guanosine monophosphate (cGMP), which
which likely reflects compensation by the other NOS mediates most of th[1e0 ,3p5]hysiological and pathological
isoforms. effects of NO (Fig. 1).
Vascular smooth muscle and endothelial cells have the ability Two main types of guanylate cyclase (GC) are known: the
to regenerate the NOS substrate L-arginine by synthesizing particulate-associated enzymes, which are transmembrane
argininosuccinate from citrulline and aspartate, a process receptors that contain GC that is activated by atrial and
that requires two enzym[3e0s-3, 2a]rgininosuccinate synthase and brain natriuretic peptide; an[3d6 ,3t7h]e cytosolic or soluble
argininosuccinate lyase. The first enzyme is co-induced type which is activated by NO. NO appears to exert its
with NOS II an[3d3 ]the second is constitutively expressed effect by binding to the heme iron (ferrous state) of the
in these cells. Exogenous citrulline administration sGC, stimulati[n13g,3 4t,h38e] enzyme basal cyclase activity several
effectively stimulated NO production in va[3s1c]ular endothelial hundred-fold. Once cGMP is produced, it mediates
cells by means of regenerating arginine. physiologic responses through its effects in cGMP-gated
ion channels, cGMP-regulated phos[3p4,h39o]diesterases, or
Once NO is produced, it may act within the cell in cGMP-dependent protein kinases. A preferential
wPhulimcohn airty iCsir cguelantioenr a| tJeadnu aorry- Mfraercehl y2 0d13if f|u Vsoel 3i n| tNoo 1adjacent activation of specific target proteins is believed2 1to
Tonelli et al.: NO deficiency in PH
NITRIC OXIDE PHYSIOLOGY IN THE
[40] PULMONARY VASCULATURE
underlie the differential effects of cGMP in various cells.
cGMP-induced vasorelaxation of vascular smooth muscle
is through several mechanisms including activation of
K-channels which hyperpolarizes the cell membrane,
The specific role of NO produced in the pulmonary
inhibition of calcium[14 ,i3n9]flux, and reduction of myofilament
vasculature is still a matter of intense investigation. Data
calcium sensitivity.
suggest that NO[4 i8n-5h0]ibits smooth muscle tone, proliferation,
and migration. NO causes relaxation of th[4e8, 4v9]ascular
The cGMP signal is chiefly limited by phosphodiesterases smooth muscle tone via the activation of cGMP. In fact,
(PDE) which degrade the cyclic nucleotide of GMP. PDE endogenous NO plays a key role in decreasing the pulmonary
degrade both cyclic adenosine monophosphate (cAMP) artery resistance at the time of birth a[n39d,4 9i,n51 ]maintaining the
and cGMP; however, PDE-5 is specific to cGMP, and dilation of the pulmonary vasculature. The production
indeed the[ 4e1]nzyme requires the binding of cGMP for full of NO by NOS II in injured vascular smooth muscle cells may
activation. prevent vasospasm a[5n2-d55 i]nhibit cell proliferation by possibly
inducing apoptosis. In addition, NO in this setting may
NOS inhibitors and NO generators have allowed for a regulate the metabolism of va[5s2]cular smooth muscle cells,
better understanding of the NO signaling pathways. NOS favoring anaerobic glycolys[5i6s], and lead to toxic effects on
inhibitors are analogs of L-arginine that act as a false adjacent endothelial cells.
substrate for this enzyme and inhibit both the constitutive [57]
and inducible forms of NOS. Of these, the most widely NO inhibits vas[5c8u] lar smooth muscle cell proliferation,
used include N-monomethyl-L-arginine (L-NMMA), DNA s[y48n,5t0h,5e8,s59i]s, and collagen production via activation of
N-nitro-L-arginine (L[4-2N] NA), and N-nitro-L-arginine cGMP. Furthermore, NO inhibits vascular smooth
methyl ester (L-NAME). Another commonly utilized NOS muscle cell [m50]igration independent of the effects on
inhibitor [i8s] L-aminoguanidine, a more selective inhibitor proliferation. Higher levels of NO are required to inhibit
of NOS II. NO generators are compounds that release NO proliferation rather than to produce vasodilation, suggesting
such as diethylamine, sodium nitroprusside, and isosorbide a potential concomitant activation by cGMP of the [c60A]MP
dinitrite, among others. kinase pathway which inhibits cellular proliferation. NO
generators can also inhibit proliferation in cells that lack sGC,
The most important endogenous NOS inhibitor suggesting that cGMP-independent mechanisms play a role.
among the methylated argi[n43i,n44e]s is the asymmetric One of these potential mechanisms is the up regulation of
dimethylarginine (ADMA). ADMA is produced Fas, a membrane protein tha[5t4 b]elongs to the TNF receptor
as a result of proteolysis of methylated proteins. The family and induces apoptosis. NO-induced apoptosis could
methylation of arginine is a post-translational modification be the result of a feedback control on calcium responses to
via protein arginine methyltransferaes (PRMT). ADMA growth factors, or deamination of purine and pyrimidine
acts as a false substrate and competitively inhibits [N44O,45S] bases in DNA t[h61a,6t2 ]leads to increased mutagenesis and DNA
activity, blocking the formation of endogenous NO. strand breaks. Although NO has b[6e3e]n reported to inhibit
ADMA undergoes clearance by the dimethylarginine cell proliferation in endothelial cells, o[6t4h]er investigations
dimethylaminohydrolase [(46D]DAH) and it is also partially have shown than either exogenous NO or NO produced
cleared by the kidneys. Methylated arginines, like by NOS II in vascular smooth[6 5m] uscle cells may stimulate
ADMA, may be responsible for the “L-arginine paradox.” endothelial cell proliferation.
At physiological state, NOS is saturated with arginine,
thus an increase in arginine concentration in[3 1p]lasma or Alternative NO pathways include the oxidation of NO
cytosol should have no effect in NO production. However, to form nitrite or reaction with protein thiols to form
elevating plasma arginine or citrulline levels enhance NO S-nitrosothiols, molecules that can lead to vasodilation
production, suggesting some form of competitive inhibition or can regula[6t6e,67 ]protein function by post-translational
of NOS, such as ADMA, is present and can [4b5e,4 7o]vercome modification. NO is oxidiz[1e3d] in the blood and tissues
by increasing the arginine concentration. Another to form nitrite and nitrate. Nitrate is produced by
explanation for the arginine paradox is the potential the reaction of NO with oxy[h13e]moglobin, while nitrite is
existence of a separated cellular pool of arginine allocated to formed by oxidation of NO. These molecules can be
NO synthesis, i.e., caveolar-localized arginine regen[e32r]ation recycled to form NO by an allosterically controlled nitrite
system, in which citrulline is recycled to arginine. This reductase reaction predominantly dur[6i8n-7g0 ]hypoxia, thereby
hypothesis is supported by a study that revealed the complemen−ting the NOS path−way. Deoxygenated
colocalization in discrete cellular domains (caveolae) hemoglobin has nitrite reductase a[6c9t,7i1v]ity, forming NO from
of enzymes[3 1i,n32v]olved in arginine regeneration and NO nitrate (NO3 ) and nitrite (NO2 ), a reaction that can
pr2o2duction. explain tPhulem hoynaproy xCiiarc-uslpateiocnif i|c J vaansuoardyi-Mlaatrocrhy 2 e0f1fe3 c|t Voobl s3e r| vNeod 1 in
Tonelli et al.: NO deficiency in PH
some organs. The NOS pathway is oxygen dependent[ 6w9,7h0]ile Endogenous NO plays an important role in the regulation
the nitrate-nitrite-NO pathway is hypoxia activated. of airwa[2y0] function, having both beneficial and detrimental
β effects. It leads to bronchial smooth muscle r[8e]laxation,
In addition, NO can be responsible for nitrosylation of a potentially modulating the basal airway tone. Inhaled
cysteine residue of the -subunit of hemoglobin, resulting NO decreased pulmonary airway resistance in pigs and
in S-nitrosylated-hem[3o9g]lobin, a protein that exerts NO-like NO gene[r8a]tors relaxed human airway smooth muscle
vasodilator effects. This reaction is favored in the in vitro. Exhaled NO is increased in inflammatory
oxygenated state of hemoglobin, and, once desaturation of airway diseases such as asthma and bronchiectasis likely
hemoglobin occurs, there is release of SNO to acceptor thio[7l2s] due to an increase in NOS II expression in the epithelial
potentially delivering NO to the systemic circulation. cells of these patients with[ 8s,2o0,m77]e contribution from the
S-nitrosylation and nitration enable the systemic transport constitutive NOS isoforms. In asthma the fraction
of the NO signal, a process that w[1o4u] ld not be possible for of exhaled NO is considered a surrogate for eosino[2p0,h85i-l8i7c]
NO due to its very short half-life and strong affinity to airway inflammation and steroid responsiveness.
bind hemoglobin. A reduction in exhaled NO levels is observed in smokers
NITRIC OXIDE AND NITRIC OXIDE possibly as a result of a dysregulation of N[77O,8S8] activity as
cigarette smoke contains high levels of NO.
SYNTHASES IN THE LUNG
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
NO is produced endogenously in the human lung in the
load of antioxidant is low, NO will have oxidant properties;
upper and lower respirato[2r8y,7 3t]ract and it is detectable in
however, when the oxidant load is high, NO plays an
exhaled breath (6-8 ppb). The origin of exhaled NO
antioxidant role[1 6b,7y4] scavenging free radicals and reactive
in the human lung likely depends upon all three isoforms
oxygen species. NO rapidly consumes superoxide (O2¯)
of NOS (I-III) but predominantly derives from the a−irway
by forming peroxynitrite (ONOO¯) which is a less reactive
epithelial expression of NOS II, the high-producer of NO.
NO metabolites like nitrosothiol and nitrite (NO2[)8] are onxitirdaatnet (tNhOat3 ¯c).a[7n9 ] bSeo mfuert hofe rt hmeseeta rbeoalcizteivde toox pyrgoednu scptse cliikees
found in the bronchoalveolar lavage of human lungs. NO
could be responsible for oxidative modification of cellular
is formed in high concentrations in the upper respiratory
proteins such as oxidation of sGC, a reaction that can impair
tract (nasopharynx and paranasal sinu[s28e,7s3)] and in lower the specific activ[8i9t]y of sGC and reduce the ability of NO to
quantities in the lower respiratory tract. It is produced
stimulate cGMP. On the same lines, recombinant human
in a variety of cells including epithelial, endothelial, and
superoxide dismutase decreases oxidative stress and
smooth muscle cells, and also in inhibitory non-adrenergic
increases eNOS activity and expression, stimulating[9 0N-9O2]
non-cholinergic neurons, mastocytes, f[6i,b7,2r8o] blasts, production, and ultimately pulmonary vasodilatation.
macrophages, lymphocytes, and neutrophils. NITRIC OXIDE IN PULMONARY
HYPERTENSION
Immunohistochemical studies have identified the presence
of all three isoforms of NOS[,6 e,1x0,p12r,2e8,s7s4-e76d] in different cells of
the human lung (Table 1). Specifically, NOS I
is located in inhibitory non-adrenergic non-cholinergic
NO is a potent pulmonary vasodilator that is produced locally
neurons; NOS II is expressed in the[ 8a,2i1r,7w6,7a7y] epithelium; and in the lung and has effects on smooth muscle relaxation and
NOS III is found in endothelial cells. Contrary to what proliferation. The close proximity of the airways and vesse[9l3s]
occurs in other organs where NOS II needs to be induced, in the lung[ 2a8l]lows NO produced in high levels in the upper
in the lungs this enzyme is continuous[2l1y] expressed in the and lower airways by NOS II to affect pulmonary vascular
airway epithelium at basal conditions. tone, in concert with the low NO level[3s5 t]hat are produced by
NOS III in the vascular endothelium. NO is considered to
NO is involved in pulmonary neurotransmission, be a selective pulmonary vasodilator because after exerting
host defense, airway and vascular smooth muscle its vasodilator action, NO is scavenged by hemog[3l5o]bin
relaxation, mucociliary clearan[8c,7e7], airway mucus secretion, having minimal effects on systemic hemodynamics.
inflammation, and cytotoxicity. NO plays key roles in lung
biology and has been implicated in the pathophysiology of Disruption of the NO pathway is a major contributor to
several lung diseases such as asthma, cystic fibrosis, the pathobiology of PH. NO in exhaled breath and NO
bronchopulmonary dysplasia, lymphangioleiomyomatosis, biochemical reaction products in bronchoalveolar lavage
and adult respiratory dis[8t,1r2e,2s0,s28 ,s35y,7n8-d84r]ome in addition to are lower in lungs of patients with PAH than contr[4o5l,9s4 ]and
pPuullmmoonnaaryr yC irhcyuplaetiornte |n Jsainouna.ry-March 2013 | Vol 3 | No 1 their level is inversely related to the degree of PH. 2N3O
Tonelli et al.: NO deficiency in PH
in exhaled breath of individuals with idiopathic PAH is levels and decrease in activity of dimethylarginine
significantly lower than subject with PH assoc[i9a5t]ed with dimethylaminohydrolase[1 0(8D]DAH), the endothelial enzyme
other causes or healthy nonsmoking controls. In fact, that metabolizes ADMA. Higher serum levels[1 0o9f,1 1A0]DMA
patients with PH associated with other cau[s95e]s had similar are increased in patients with idiopathic PAH [111] and
levels of exhaled NO than healthy controls. chronic thromboembolic pulmonary hypertension, and
correlate with disease severity and survival. ADMA levels
Early data demonstrated a reduced expression of NOS III, increase predominan[1t1l0y] due to a reduction in the expression
measured by immunostaining, in the vascular endothelium and function DDAH.
of pulmonary arteries in patients with PAH. This reduced
expression inversely correlated[9 6w] ith the severity of the Arginase II, an enzyme that is part of the urea cycle and
morphological arterial changes. More recently, however, breaks down arginine to ornithine, can decrease the
other investigators show[9e7d,98 i]ncreased or unaltered NOS III substrate available to NOS for NO synthesis. Idiopathic and
immunostaining in PH. Moreover, there is eviden[c99e] PAH associated with sickle cell disease patients have been
of high NOS III expression in plexiform lesions in PAH. found to have higher levels of Argina[1s1e2 ]II and lower levels
A unifying hypothesis suggested that the act[i1v00i]ty of NOS III of L-arginine than healthy controls. In the absence [1o4f]
may be reduced rather that its expression. Other NOS L-arginine or BH4, NOS III may become “uncoupled,”
isoforms besides NOS III may con[94t]ribute to the low exhaled resulting in the generation of the free radical superoxide.
NO observed in idiopathic PAH.
CLINICAL IMPLICATIONS IN PH:
DIAGNOSIS AND PROGNOSIS
NO has also been implicated in the important role that
bone morphogenetic protein[1 0r1e]ceptor II (BMPRII) has
in the pathogenesis of PAH. In the lung, BMPRII is
highly expressed in endothelial cells and its activation
prom[1o0t2e]s proliferation, migration, and survival of these Although the vascular endothelium produces large amounts
cells. BMPRII levels are markedly reduced in patients of NO, very little is exhaled as a res ult of the marked af[f2i8n]ity
with heritable or idiopathic[ 1P02A]H, promoting endothelial of NO to hemoglobin in the pulmonary circulation. In
dysfunction and apoptosis. These effects have been spite of this potential limitation, NO can be measured in
recently attributed to a decrease in NOS III activity, since exhaled breath and its concentration is inversely related
BMPRII ligands failed to stimulate NOS III dependent to the exhalation flow. For this reason, the fraction of
protein kinase activation in pulmonary artery endoth[e1l0i3a]l exhaled NO is measured at a constant flow (usually
cells from patients with mutation in the BMPR2 gene. 50 mL/s). Using this approach, the fraction of exhaled
NO is a reliable surrogate of the[1 1m3]aximal flux of NO from
PAH patients treated with epoprostenol had a three-fold the large airway compartment. This method does not
higher exhaled NO than PH patients not receiving this measure the steady-state mean distal airway/alveolar
treatment and two-fold higher than healthy controls. concentration of NO. There is no simple surrogate for
Interestingly, exhaled NO increased at 24 hours[ 9i5n] those measuring this parameter since its determination requires
patients treated with this prostacyclin analog. These the measurement of the fraction of exhaled NO at multiple
data suggest that prostacyclin analogs may in part improve expiratory flow rates and t[h20e,1 1a4]pplication of a modified
PH through effects on NO. In support of this, previous “slope-intercept” algorithm.
work has shown that nebulized epoprostenol increased
the exhaled NO in pati[e10n4]ts with PAH associated with Authors have investigated whether exhaled NO could
congenital heart disease. Similarly, inhaled iloprost also serve as a noninvasive marker of severity of disease and
led to an increase in NO concomitant with a decrease in response to therapy in PH. The value of repetitive exhaled
pulmonary artery p[r1e05s]sure in a patient with PAH associated NO measurements was studied in 17 PAH patients over
with scleroderma. Other PAH-specific therapies that two years. NO levels at entry were inversely correlated
do not directly target the NO pathway may also improve with the number of months from the PAH diagnosis,
the fraction of exhaled nitric oxide sug[9g5,e10s6t,1i0n7g] a crosstalk suggesting a glo[b10a6]l decrease in NO over longer periods
between different signaling pathways. with the disease. Lower exhaled NO levels at entry were
associated with higher pulmonary artery pressures and less
The NO signaling pathways have been found to be affected decrease over time. NO at the beginning of the study was not
in PH at different levels. Endogenous NOS III inhibitors associated with survival; nevertheless, its level increased
may be involved in the pathogenesis of PH. These include over time in the PAH individuals who survived to complete
the symmetric and asymmetric dimethylarginines (ADMA). the study when compared to those who died, and c[1o0r6r]elated
An2i4mal models of hypoxic PH showed increased ADMA with chPaunlmgoensa riyn C pircuullmatioonn a| rJyan auarrtye-Mrya rcphr e2s0s1u3 r|e Vso.l 3 | TNho u1s,
Tonelli et al.: NO deficiency in PH
exhaled NO could be a u[9s4e,9f5u,1l0 6m] arker of disease severity
and response to therapy.
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 po[1t1e5-n11t8i]ally
benefit from long-term calcium-channel blockers. NO
vasodilator challenge in several types of PH can also provide
prognostic information, as responders may have b[1e19t-t1e22r]
outcome, independent of the treatment administered.
CLINICAL IMPLICATIONS IN PH: Figure 2: Therapeutic strategies to increase NO effect. BH4, tetrahydrobiopterin;
cGMP: Cyclic guanosine monophosphate; EPC: Endothelial progenitor cells;
THERAPY
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;
Inhibiting phosphodiesterases-5
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
Therapies that manipulate the downstream NO signaling synthase and argininosuccinate lyase).
have revolutionized the treatment of PH (Fig. 2). Sildenafil
and tadalafil are FDA appr[o12v3-e1d25 ]PDE-5 inhibitors for the
treatment of PAH in adults. The FDA recently placed
difficulties for its delivery and avoidance of the toxic effects
a safety warning on the prescription of sildenafil, not
recommending its use in pediatric patients due to i[n12c5r]ease of N[O13 3o]xidative products have prevented its widespread
use. During continuous administration of NO, NO
mortality with increasing doses in this age group. This
oxidative products (NO2) can build u[p13 5a]nd cause airway
modification was based on the results of the Sildenafil
hyperactivity at low concentra[t13io6]ns and pulmonary
Citrate in Treatment-Naive Children[ 1w26]ith Pulmonary edema at higher concentrations. Methemoglobin is also
Arterial Hypertension (STARTS-2) trial. This extension
formed as NO reacts with oxyhemoglobin. Furthermore,
study, designed to assess the safety and tolerability of
mechanisms should be in place to avoid abrupt cessation of
long-term treatment with oral sildenafil monotherapy in
inhaled[ 1N37O,1 3a8s] this may lead to rebound PAH with deleterious
children (aged 1-17 years) with PAH, showed a higher
effects. Nonetheless, two studies that evaluated the
mortality[1 2r6i]sk in patients randomized to high-dose long-term use (one and three months, respectively) of
sildenafil.
inhaled NO in PAH, chronic thromboembolic PH, and PH due
to COPD reported no significant increase in methemoglobin,
PDE-5 inhibitors prevent the normal hydrolysis of cGMP,
withdrawal syndrome, change in oxyg[1e32n,1a3t4i]on, pulmonary
prolonging the NO effects on tissues. PDE-[512 7i]s mainly function, or systemic hemodynamics.
expressed in the pulmonary vascular bed, thu[10s0 ,1i2t8s]
inhibition has primarily pulmonary-specific ef[1fe27c]ts. > 
Furthermore, PDE5 is upregulated in the lun[12g9] and the Currently, inhaled NO is FDA approved for the treatment of term
hypertrophied right ventricular myocardium of patients and near-term ( 34 week gestation) neonates with hypoxic
with PAH. Therefore, PDE-5 inhibitors are an ideal treatment respiratory failure with clinical and ec[8h1,o82c,1a3r9]diographic
evidence of pulmonary hypertension. Extended
for PAH because they decrease the right ventricular
administration (weeks) of inhaled NO has been studied in
afterload and improve right ventricular [i1n29o]tropy, without
relevant systemic hemodynamic effects. premature infants and does not appear to imp[8r1o-8v3e,1 4s0u,1r4v1]ival
Using nitric oxide as an inhaled gas or prevent bronchopulmonary dysplasia. At
present, inhaled NO is undergoing clinical investigations
to evaluate its utility as a treatment for bronchopulmonary
Inhaled NO was first shown to selectively redu[1c30e] dysplasia (NCT01503801). There are two ongoing studies
pulmonary vascular resistance in a lamb model; using extended administration of inhaled NO in adults
subsequently, mul[1t1i8p,1l3e1,1 3s2t]udies have confirmed this with PH. The PHiano study (NCT01265888, Geno LLC) is
finding in humans. Continuous inhaled NO had an open label, dose-escalation, Phase II study using a NO
beneficial eff[e13c2t-s13 i4n] patients with PAH or PH associated delivery system (NITROsyl) in patients with PAH or PH
wPiutlmho nCaOryP CDirc;ulation | Jhaonwuaeryv-Mear,r chh i2g0h1 3c |o Vsotl 3a n| dN ot 1echnical secondary to idiopathic pulmonary fibrosis. The seco2n5d
Tonelli et al.: NO deficiency in PH
study (NCT01457781, INO Therapeutics) is a randomized, L-arginine replacement aims at providing excess substrate
double blind, placebo control, Phase II study of inhaled NO for the NOS enzyme and stimulating the NO production.
versus placebo as add-on therapy in subjects with PAH. NO L-arginine attenuated PAH in different animal models
[153-155]
iNs idterliicv eorxedid bey da ospneocirasl device (INOpulse DS). of PAH and in patients with PAH associated
with sickle cell disease, idiopathic PAH, and chronic
[156,157]
thromboembolic PH. L-citrulline, a urea cycle
Plasma levels of nitrate and nitrite are low [1in42 ]diseases intermediate, is metabolized to L-arginine in pulmonary
that have endothelial dysfunction like PAH. Inhaled vascular endothelial cells. Oral supplementation increased
nitrite could be converted [t1o43 ]NO and selectively dilate NO synthesis and ameliorated chronic hypoxia-induced
[158]
the pulmonary circulation. The vasodilatory effects
PH in newborn piglets. In children undergoing
of these inorganic anions (nitrite and nitrate) are less
cardiopulmonary bypass, the oral supplementation
potent than the org[6a9n] ic nitrates (nitroglycerin) and of L-citrulline safely increased plasma citrulline and
nitrites (amyl-nitrite); however, the inorganic compounds
arginine concentrations, and more importantly PH did
[159]
do not induce significant systemic vasodilat[6o9r] effects and not occur in those with elevated citrulline levels. The
tachyphylaxis as the organic molecules. Nebulized
safety and effectiveness of intravenous L-citrulline, in
sodium nitrite has been shown to ameliorate PH induced
children undergoing cardiopulmonary bypass for surgical
by hypoxia in animals and humans, [w143i,t1h44 ]a longer duration
repair of a congenital heart defect, is currently being
of action than that of inhaled NO, supporting that
tested (NCT01120964).
nitrites are NO donors particularly during hypoxia as r[e69s,1u4l3t]
of the nitrite reductase action of deoxyhemogobin.
NO production by NOS depends on the de novo biosynthesis
Ethyl nitrate is an organic nitrate given by inhalation that [160,161]
of the enzyme cofactor tetrahydrobiopterin (BH4).
forms S-nitrosothiols and has shown to improve pulmonary
hemodynamics in a model of hypoxia-induced PH without BH4 stabilizes the NOS dimmer assembly and the favorable
altering systemic va[s14c5u] lar resistance or increasing spin state of the Fe (II)-O2 heme intermediate preventing
methemoglobin levels. Its low potency when compared “uncoupli[n14g,1”6 o2,1f6 t3h]e enzyme and formation of reactive oxygen
with inhaled NO can be markedly enhanced both [i1n45 v]ivo and products. The augmentation of tetrahydrobiopterin
in vitro by the addition of the thiol glutathione. showed promising results in a pilot study[ 1i6n4] patients
with PAH or chronic thromboembolic PAH. Another
Efforts in trying to find better stability and prolonged promising therapy is the recombinant human superoxide
half-life in NO delivery led to the discovery of dismutase (rhSOD) that scavenges superoxide anion
[165]
diazeniumdiolates (diethylenetriamine/NO) that can and increases the bioavailability of NO. In a lamb
form a group of adducts called NONOates that are model of persistent PH, rhSOD administered through
complexes of NO with nucleophiles that spontaneously and the endotracheal tube as a bolus enhanced the effects of
[165]
nonenzymatically release NO when dissolved in aqueous inhaled NO on pulmonary vasculature.
neutral PH solutions. This pr[o14c6e,1s47s] prolongs the half-life
of NO release up to 20 hours. Nebulized NO donors
Attractive new potential therapies for PAH include
have been [s1h48o]wn to reduce PVR in hypoxi[a1-4i7n]duced PAH the NOS III enhancers, the delivery of autologous
[166]
in piglet[s14,9] monocrotaline rat model, and ARDS endothelial progenitor cells, or the enzyme NOS II
patients with no systemic adverse effects or toxic
and III using adenoviral-mediated transfer or adult stem
[167]
Oreathcteior nth perroadpucietss obfa NsOe.d on the nitric oxide pathway
cell-based ex vivo gene therapy. Bone marrow-derived
endothelial-like progenitor cells prevented the development
or progression of PH in a monocrotaline rat model of
Several promising therapies which affect the signaling [168]
PH. The engraftment of these progenitor cells in the
pathway of NO are under active investigation (Fig. 1).
pulmonary vasculature may restore the microvascular
Soluble guanylate cyclase stimulators increase cGMP [168]
structure and function. Meanwhile, animals receiving
independently of NO. One of these stimulators, Riociguat,
endothelial-like progenitor cells or mesenchymal stem
increased the activity of soluble guanylate cyclase 73-fold
cells transduced with NOS III had reversal of established
in vivo with partial reduction in pulmonary pressures, [168,169]
PH and improved survival. Adenoviral gene transfer
RV hypertrophy, and pulm[1o5n0]ary artery muscularization
in animal models of PH. This medication showed of NOS III produced an increase in the NOS III expression
promising results in a Phase II stu[1d5y1] in patients with PAH and activity with attenuation in the [h17y0]poxia-induced
and chronic thromboembolic PH. We are awaiting the increase in pulmonary artery pressure. Similarly, NOS
results of two Phase III studies evaluating safety and clinical II gene transfer increased pulmonary NO production with
effectiveness of riociguat in PAH (NCT0081[10526]93) and reducti[o17n1] in hypoxia-induced PH and vascular remodeling
ch2r6onic thromboembolic PH (NCT00855465). in rats. Pulmonary Circulation | January-March 2013 | Vol 3 | No 1
Tonelli et al.: NO deficiency in PH
CONCLUSIONS 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 S, Palmer RM, Higgs EA. Nitric oxide: Physiology, pathophysiology,
and pharmacology. Pharmacol Rev 1991;43:109‑42.
24. Shaul PW. Regulation of endothelial nitric oxide synthase: Location, location,
Nitric oxide mediates diverse key signaling functions in
location. Annu Rev Physiol 2002;64:749‑74.
human physiology and disease. Major progress in the 25. Bernareggi M, Mitchell JA, Barnes PJ, Belvisi MG. Dual action of nitric oxide
understanding of NO signaling pathway has led to the on airway plasma leakage. Am J Respir Crit Care Med 1997;155:869‑74.
approval of PAH-specific treatments and the ongoing 26. Ricciardolo FL, Sterk PJ, Gaston B, Folkerts G. Nitric oxide in health and
disease of the respiratory system. Physiol Rev 2004;84:731‑65.
discovery and development of promising new therapies.
27. Fagan KA, Tyler RC, Sato K, Fouty BW, Morris KG Jr, Huang PL, et al.
Molecules in the NO pathway also have the potential to Relative contributions of endothelial, inducible, and neuronal NOS to tone
in the murine pulmonary circulation. Am J Physiol 1999;277:L472‑8.
be used as biomarkers of disease severity, outcomes, or
28. Dweik RA, Laskowski D, Abu‑Soud HM, Kaneko F, Hutte R, Stuehr DJ,
response to therapy in pulmonary hypertension. et al. Nitric oxide synthesis in the lung. Regulation by oxygen through a
REFERENCES kinetic mechanism. J Clin Invest 1998;101:660‑6.
29. Steudel W, Ichinose F, Huang PL, Hurford WE, Jones RC, Bevan JA,
et al. Pulmonary vasoconstriction and hypertension in mice with targeted
1. Rich S, Dantzker DR, Ayres SM, Bergofsky EH, Brundage BH, Detre KM,
disruption of the endothelial nitric oxide synthase (NOS 3) gene. Circ Res
et al. Primary pulmonary hypertension. A national prospective study. Ann
1997;81:34‑41.
Intern Med 1987;107:216‑23.
30. Hecker M, Sessa WC, Harris HJ, Anggard EE, Vane JR. The metabolism of
2. Tuder RM, Abman SH, Braun T, Capron F, Stevens T, Thistlethwaite PA,
L‑arginine and its significance for the biosynthesis of endothelium‑derived
et al. Development and pathology of pulmonary hypertension. J Am Coll
relaxing factor: Cultured endothelial cells recycle L‑citrulline to L‑arginine.
Cardiol 2009;54:S3‑9.
Proc Natl Acad Sci U S A 1990;87:8612‑6.
3. Schermuly RT, Ghofrani HA, Wilkins MR, Grimminger F. Mechanisms of
31. Flam BR, Hartmann PJ, Harrell‑Booth M, Solomonson LP, Eichler DC. Caveolar
disease: Pulmonary arterial hypertension. Nat Rev Cardiol 2011;8:443‑55.
localization of arginine regeneration enzymes, argininosuccinate synthase,
4. Ignarro LJ, Buga GM, Wood KS, Byrns RE, Chaudhuri G. Endothelium‑derived and lyase, with endothelial nitric oxide synthase. Nitric Oxide 2001;5:187‑97.
relaxing factor produced and released from artery and vein is nitric oxide.
32. Solomonson LP, Flam BR, Pendleton LC, Goodwin BL, Eichler DC. The
Proc Natl Acad Sci U S A 1987;84:9265‑9.
caveolar nitric oxide synthase/arginine regeneration system for NO
5. Palmer RM, Ferrige AG, Moncada S. Nitric oxide release accounts for production in endothelial cells. J Exp Biol 2003;206:2083‑7.
the biological activity of endothelium‑derived relaxing factor. Nature
33. Hattori Y, Campbell EB, Gross SS. Argininosuccinate synthetase mRNA
1987;327:524‑6.
and activity are induced by immunostimulants in vascular smooth muscle.
6. Nathan C, Xie QW. Nitric oxide synthases: Roles, tolls, and controls. Cell Role in the regeneration or arginine for nitric oxide synthesis. J Biol Chem
1994;78:915‑8. 1994;269:9405‑8.
7. Schmidt HH, Walter U. NO at work. Cell 1994;78:919‑25. 34. Schmidt HH, Lohmann SM, Walter U. The nitric oxide and cGMP signal
8. Gaston B, Drazen JM, Loscalzo J, Stamler JS. The biology of nitrogen oxides transduction system: Regulation and mechanism of action. Biochim Biophys
in the airways. Am J Respir Crit Care Med 1994;149:538‑51. Acta 1993;1178:153‑75.
9. Nathan C. Nitric oxide as a secretory product of mammalian cells. FASEB 35. Dweik RA. Pulmonary hypertension and the search for the selective
J 1992;6:3051‑64. pulmonary vasodilator. Lancet 2002;360:886‑7.
10. Dweik RA, Erzurum SC. Effects of Nitric Oxide and Cyclic GMP on 36. Garbers DL. Guanylyl cyclase receptors and their endocrine, paracrine, and
Smooth Muscle Proliferation. In: Moss J, editors. LAM and Other Diseases autocrine ligands. Cell 1992;71:1‑4.
Characterized by Smooth Muscle Proliferation. New York: Marcel Dekker 37. McDonald LJ, Murad F. Nitric oxide and cyclic GMP signaling. Proc Soc
Inc; 1999. p. 333‑49. Exp Biol Med 1996;211:1‑6.
11. Stuehr DJ, Griffith OW. Mammalian nitric oxide synthases. Adv Enzymol 38. Murad F. Regulation of cytosolic guanylyl cyclase by nitric oxide: The
Relat Areas Mol Biol 1992;65:287‑346. NO‑cyclic GMP signal transduction system. Adv Pharmacol 1994;26:19‑33.
12. Dweik RA, Laskowski D, Ozkan M, Farver C, Erzurum SC. High levels 39. Archer SL, Huang JM, Hampl V, Nelson DP, Shultz PJ, Weir EK. Nitric oxide
of exhaled nitric oxide (NO) and NO synthase III expression in lesional and cGMP cause vasorelaxation by activation of a charybdotoxin‑sensitive
smooth muscle in lymphangioleiomyomatosis. Am J Respir Cell Mol Biol K channel by cGMP‑dependent protein kinase. Proc Natl Acad Sci U S A
2001;24:414‑8. 1994;91:7583‑7.
13. Moncada S, Higgs A. The L‑arginine‑nitric oxide pathway. N Engl J Med 40. Lincoln TM, Cornwell TL. Intracellular cyclic GMP receptor proteins. FASEB
1993;329:2002‑12. J 1993;7:328‑38.
14. Coggins MP, Bloch KD. Nitric oxide in the pulmonary vasculature. 41. Rybalkin SD, Yan C, Bornfeldt KE, Beavo JA. Cyclic GMP phosphodiesterases
Arterioscler Thromb Vasc Biol 2007;27:1877‑85. and regulation of smooth muscle function. Circ Res 2003;93:280‑91.
15. Stuehr DJ. Mammalian nitric oxide synthases. Biochim Biophys Acta 42. Alderton WK, Cooper CE, Knowles RG. Nitric oxide synthases: Structure,
1999;1411:217‑30. function and inhibition. Biochem J 2001;357:593‑615.
16. Dweik RA. Nitric oxide, hypoxia, and superoxide: The good, the bad, and 43. Bulau P, Zakrzewicz D, Kitowska K, Leiper J, Gunther A, Grimminger F,
the ugly! Thorax 2005;60:265‑7. et al. Analysis of methylarginine metabolism in the cardiovascular system
17. Dudzinski DM, Igarashi J, Greif D, Michel T. The regulation and identifies the lung as a major source of ADMA. Am J Physiol Lung Cell Mol
pharmacology of endothelial nitric oxide synthase. Annu Rev Pharmacol Physiol 2007;292:L18‑24.
Toxicol 2006;46:235‑76. 44. Tran CT, Leiper JM, Vallance P. The DDAH/ADMA/NOS pathway.
18. Newby AC, Southgate KM, Assender JW. Inhibition of vascular smooth Atheroscler 2003;4:33‑40.
muscle cell proliferation by endothelium‑dependent vasodilators. Herz 45. Dweik RA. The lung in the balance: Arginine, methylated arginines, and
1992;17:291‑9. nitric oxide. Am J Physiol Lung Cell Mol Physiol 2007;292:L15‑7.
19. Nathan C, Xie QW. Regulation of biosynthesis of nitric oxide. J Biol Chem 46. Ogawa T, Kimoto M, Sasaoka K. Purification and properties of a new
1994;269:13725‑8. enzyme, NG, NG‑dimethylarginine dimethylaminohydrolase, from rat
20. Barnes PJ, Dweik RA, Gelb AF, Gibson PG, George SC, Grasemann H, et al. kidney. J Biol Chem 1989;264:10205‑9.
Exhaled nitric oxide in pulmonary diseases: A comprehensive review. Chest 47. Sy BMC, Dweik EE, Dweik RA. Arginine and nitric oxide. In: Shils ME,
2010;138:682‑92. Shike M, Ross AC, Caballero B, Cousins RJ, editors. Modern nutrition
21. Guo FH, De Raeve HR, Rice TW, Stuehr DJ, Thunnissen FB, Erzurum SC. in health and disease. 10th ed. Philadelphia, PA: Lippincott Williams and
Continuous nitric oxide synthesis by inducible nitric oxide synthase in normal Wilkins; 2005. p. 571‑81.
human airway epithelium in vivo. Proc Natl Acad Sci U S A 1995;92:7809‑13. 48. Garg UC, Hassid A. Nitric oxide‑generating vasodilators and 8‑bromo‑cyclic
22. Ward JK, Belvisi MG, Fox AJ, Miura M, Tadjkarimi S, Yacoub MH, et al. guanosine monophosphate inhibit mitogenesis and proliferation of cultured
Pulmonary Circulation | January-March 2013 | Vol 3 | No 1 27
Tonelli et al.: NO deficiency in PH
rat vascular smooth muscle cells. J Clin Invest 1989;83:1774‑7. 74. Dweik RA, Erzurum SC. Regulation of nitric oxide (NO) synthases and gas
49. Thomae KR, Nakayama DK, Billiar TR, Simmons RL, Pitt BR, Davies P. phase NO by oxygen. In: Marczin N, Kharitonov SA, Yacoub MH, Barnes PJ,
The effect of nitric oxide on fetal pulmonary artery smooth muscle growth. editors. Disease markers in exhaled breath. New York: Marcel Dekker Inc;
J Surg Res 1995;59:337‑43. 2003. p. 235‑46.
50. Sarkar R, Meinberg EG, Stanley JC, Gordon D, Webb RC. Nitric oxide 75. Dweik RA, Guo FH, Uetani K, Erzurum SC. Nitric oxide synthase in the
reversibly inhibits the migration of cultured vascular smooth muscle cells. human airway epithelium. Zhongguo Yao Li Xue Bao 1997;18:550‑2.
Circ Res 1996;78:225‑30. 76. Kobzik L, Bredt DS, Lowenstein CJ, Drazen J, Gaston B, Sugarbaker D,
51. Abman SH, Chatfield BA, Hall SL, McMurtry IF. Role of endothelium‑derived et al. Nitric oxide synthase in human and rat lung: Immunocytochemical
relaxing factor during transition of pulmonary circulation at birth. Am J and histochemical localization. Am J Respir Cell Mol Biol 1993;9:371‑7.
Physiol 1990;259:H1921‑7. 77. Barnes PJ, Belvisi MG. Nitric oxide and lung disease. Thorax 1993;48:1034‑43.
52. Geng Y, Hansson GK, Holme E. Interferon‑gamma and tumor necrosis 78. Dweik RA. The promise and reality of nitric oxide in the diagnosis and
factor synergize to induce nitric oxide production and inhibit mitochondrial treatment of lung disease. Cleve Clin J Med 2001;68:486, 8, 90, 93.
respiration in vascular smooth muscle cells. Circ Res 1992;71:1268‑76. 79. Dweik RA, Comhair SA, Gaston B, Thunnissen FB, Farver C, Thomassen MJ,
53. Stein CS, Fabry Z, Murphy S, Hart MN. Involvement of nitric oxide et al. NO chemical events in the human airway during the immediate
in IFN‑gamma‑mediated reduction of microvessel smooth muscle cell and late antigen‑induced asthmatic response. Proc Natl Acad Sci U S A
proliferation. Mol Immunol 1995;32:965‑73. 2001;98:2622‑7.
54. Fukuo K, Hata S, Suhara T, Nakahashi T, Shinto Y, Tsujimoto Y, et al. Nitric 80. Khatri SB, Hammel J, Kavuru MS, Erzurum SC, Dweik RA. Temporal
oxide induces upregulation of Fas and apoptosis in vascular smooth muscle. association of nitric oxide levels and airflow in asthma after whole lung
Hypertension 1996;27:823‑6. allergen challenge. J Appl Physiol 2003;95:436‑40.
55. Schini VB, Vanhoutte PM. Role of the L‑arginine‑nitric oxide pathway in 81. Kinsella JP, Cutter GR, Walsh WF, Gerstmann DR, Bose CL, Hart C, et al.
vascular smooth muscle. Eur Heart J 1993;14:16‑21. Early inhaled nitric oxide therapy in premature newborns with respiratory
56. Thomae KR, Joshi PC, Davies P, Pitt BR, Billiar TR, Simmons RL, et al. Nitric failure. N Engl J Med 2006;355:354‑64.
oxide produced by cytokine‑activated pulmonary artery smooth muscle 82. Ballard RA, Truog WE, Cnaan A, Martin RJ, Ballard PL, Merrill JD, et al.
cells is cytotoxic to cocultured endothelium. Surgery 1996;119:61‑6. Inhaled nitric oxide in preterm infants undergoing mechanical ventilation.
57. Nunokawa Y, Tanaka S. Interferon‑gamma inhibits proliferation of rat N Engl J Med 2006;355:343‑53.
vascular smooth muscle cells by nitric oxide generation. Biochem Biophys
83. Stark AR. Inhaled NO for preterm infants‑getting to yes? N Engl J Med
Res Commun 1992;188:409‑15.
2006;355:404‑6.
58. Nakaki T, Nakayama M, Kato R. Inhibition by nitric oxide and nitric
84. McCurnin DC, Pierce RA, Chang LY, Gibson LL, Osborne‑Lawrence S,
oxide‑producing vasodilators of DNA synthesis in vascular smooth muscle
Yoder BA, et al. Inhaled NO improves early pulmonary function and
cells. Eur J Pharmacol 1990;189:347‑53.
modifies lung growth and elastin deposition in a baboon model of neonatal
59. Kolpakov V, Gordon D, Kulik TJ. Nitric oxide‑generating compounds inhibit chronic lung disease. Am J Physiol Lung Cell Mol Physiol 2005;288:L450‑9.
total protein and collagen synthesis in cultured vascular smooth muscle
85. Zacharasiewicz A, Wilson N, Lex C, Erin EM, Li AM, Hansel T, et al.
cells. Circ Res 1995;76:305‑9.
Clinical use of noninvasive measurements of airway inflammation in steroid
60. Cornwell TL, Arnold E, Boerth NJ, Lincoln TM. Inhibition of smooth muscle reduction in children. Am J Respir Crit Care Med 2005;171:1077‑82.
cell growth by nitric oxide and activation of cAMP‑dependent protein kinase
86. Berry MA, Shaw DE, Green RH, Brightling CE, Wardlaw AJ, Pavord ID.
by cGMP. Am J Physiol 1994;267:C1405‑13.
The use of exhaled nitric oxide concentration to identify eosinophilic airway
61. Clementi E, Sciorati C, Nistico G. Growth factor‑induced Ca2+ responses
inflammation: An observational study in adults with asthma. Clin Exp
are differentially modulated by nitric oxide via activation of a cyclic
Allergy 2005;35:1175‑9.
GMP‑dependent pathway. Mol Pharmacol 1995;48:1068‑77.
87. Smith AD, Cowan JO, Brassett KP, Filsell S, McLachlan C, Monti‑Sheehan G,
62. Nguyen T, Brunson D, Crespi CL, Penman BW, Wishnok JS, Tannenbaum SR.
et al. Exhaled nitric oxide: A predictor of steroid response. Am J Respir Crit
DNA damage and mutation in human cells exposed to nitric oxide in vitro.
Care Med 2005;172:453‑9.
Proc Natl Acad Sci U S A 1992;89:3030‑4.
88. Ischiropoulos H, Mendiguren I, Fisher D, Fisher AB, Thom SR. Role of
63. Yang W, Ando J, Korenaga R, Toyo‑oka T, Kamiya A. Exogenous nitric
neutrophils and nitric oxide in lung alveolar injury from smoke inhalation.
oxide inhibits proliferation of cultured vascular endothelial cells. Biochem
Am J Respir Crit Care Med 1994;150:337‑41.
Biophys Res Commun 1994;203:1160‑7.
89. Weber M, Lauer N, Mulsch A, Kojda G. The effect of peroxynitrite on
64. Ziche M, Morbidelli L, Masini E, Amerini S, Granger HJ, Maggi CA, et al.
the catalytic activity of soluble guanylyl cyclase. Free Radic Biol Med
Nitric oxide mediates angiogenesis in vivo and endothelial cell growth and
2001;31:1360‑7.
migration in vitro promoted by substance P. J Clin Invest 1994;94:2036‑44.
90. Firth AL, Yuan JX. Bringing down the ROS: A new therapeutic approach
65. Fukuo K, Inoue T, Morimoto S, Nakahashi T, Yasuda O, Kitano S, et al.
for PPHN. Am J Physiol Lung Cell Mol Physiol 2008;295:L976‑8.
Nitric oxide mediates cytotoxicity and basic fibroblast growth factor
release in cultured vascular smooth muscle cells. A possible mechanism of 91. Farrow KN, Lakshminrusimha S, Reda WJ, Wedgwood S, Czech L,
neovascularization in atherosclerotic plaques. J Clin Invest 1995;95:669‑76. Gugino SF, et al. Superoxide dismutase restores eNOS expression and
66. Kim‑Shapiro DB, Schechter AN, Gladwin MT. Unraveling the reactions function in resistance pulmonary arteries from neonatal lambs with
of nitric oxide, nitrite, and hemoglobin in physiology and therapeutics. persistent pulmonary hypertension. Am J Physiol Lung Cell Mol Physiol
Arterioscler Thromb Vasc Biol 2006;26:697‑705. 2008;295:L979‑87.
67. Hess DT, Matsumoto A, Kim SO, Marshall HE, Stamler JS. Protein 92. Robbins CG, Horowitz S, Merritt TA, Kheiter A, Tierney J, Narula P, et al.
S‑nitrosylation: Purview and parameters. Nat Rev Mol Cell Biol Recombinant human superoxide dismutase reduces lung injury caused by
2005;6:150‑66. inhaled nitric oxide and hyperoxia. Am J Physiol 1997;272:L903‑7.
68. Zweier JL, Wang P, Samouilov A, Kuppusamy P. Enzyme‑independent 93. Lundberg JO, Farkas‑Szallasi T, Weitzberg E, Rinder J, Lidholm J, Anggåard
formation of nitric oxide in biological tissues. Nat Med 1995;1:804‑9. A, et al. High nitric oxide production in human paranasal sinuses. Nat Med
1995;1:370‑3.
69. Lundberg JO, Weitzberg E, Gladwin MT. The nitrate‑nitrite‑nitric
oxide pathway in physiology and therapeutics. Nat Rev Drug Discov 94. Kaneko FT, Arroliga AC, Dweik RA, Comhair SA, Laskowski D,
2008;7:156‑67. Oppedisano R, et al. Biochemical reaction products of nitric oxide as
70. Duranski MR, Greer JJ, Dejam A, Jaganmohan S, Hogg N, Langston W, et al. quantitative markers of primary pulmonary hypertension. Am J Respir
Cytoprotective effects of nitrite during in vivo ischemia‑reperfusion of the Crit Care Med 1998;158:917‑23.
heart and liver. J Clin Invest 2005;115:1232‑40. 95. Ozkan M, Dweik RA, Laskowski D, Arroliga AC, Erzurum SC. High levels
71. Gladwin MT, Kim‑Shapiro DB. The functional nitrite reductase activity of of nitric oxide in individuals with pulmonary hypertension receiving
the heme‑globins. Blood 2008;112:2636‑47. epoprostenol therapy. Lung 2001;179:233‑43.
72. Singel DJ, Stamler JS. Chemical physiology of blood flow regulation by red 96. Giaid A, Saleh D. Reduced expression of endothelial nitric oxide synthase
blood cells: The role of nitric oxide and S‑nitrosohemoglobin. Annu Rev in the lungs of patients with pulmonary hypertension. N Engl J Med
Physiol 2005;67:99‑145. 1995;333:214‑21.
73. Tsujino I, Miyamoto K, Nishimura M, Shinano H, Makita H, Saito S, et al. 97. Xue C, Johns RA. Endothelial nitric oxide synthase in the lungs of patients
Production of nitric oxide (NO) in intrathoracic airways of normal humans. with pulmonary hypertension. N Engl J Med 1995;333:1642‑4.
Am J Respir Crit Care Med 1996;154:1370‑4. 98. Tuder RM, Cool CD, Geraci MW, Wang J, Abman SH, Wright L, et al.
28 Pulmonary Circulation | January-March 2013 | Vol 3 | No 1
Tonelli et al.: NO deficiency in PH
Prostacyclin synthase expression is decreased in lungs from patients with congenital heart defects and pulmonary arterial hypertension. Eur Heart J
severe pulmonary hypertension. Am J Respir Crit Care Med 1999;159:1925‑32. 2004;25:1651‑6.
99. Mason NA, Springall DR, Burke M, Pollock J, Mikhail G, Yacoub MH, et al. 121. Skoro‑Sajer N, Hack N, Sadushi‑Kolici R, Bonderman D, Jakowitsch J,
High expression of endothelial nitric oxide synthase in plexiform lesions Klepetko W, et al. Pulmonary vascular reactivity and prognosis in patients
of pulmonary hypertension. J Pathol 1998;185:313‑8. with chronic thromboembolic pulmonary hypertension: A pilot study.
100. Hagan G, Pepke‑Zaba J. Pulmonary hypertension, nitric oxide and nitric Circulation 2009;119:298‑305.
oxide‑releasing compounds. Expert Rev Respir Med 2011;5:163‑71. 122. Krasuski RA, Devendra GP, Hart SA, Wang A, Harrison JK, Bashore TM.
101. Thomson JR, Machado RD, Pauciulo MW, Morgan NV, Humbert M, Response to inhaled nitric oxide predicts survival in patients with
Elliott GC, et al. Sporadic primary pulmonary hypertension is associated pulmonary hypertension. J Card Fail 2011;17:265‑71.
with germline mutations of the gene encoding BMPR‑II, a receptor member 123. Galie N, Ghofrani HA, Torbicki A, Barst RJ, Rubin LJ, Badesch D, et al.
of the TGF‑beta family. J Med Genet 2000;37:741‑5. Sildenafil citrate therapy for pulmonary arterial hypertension. N Engl J
102. Atkinson C, Stewart S, Upton PD, Machado R, Thomson JR, Trembath RC, Med 2005;353:2148‑57.
et al. Primary pulmonary hypertension is associated with reduced 124. Galie N, Brundage BH, Ghofrani HA, Oudiz RJ, Simonneau G, Safdar Z,
pulmonary vascular expression of type II bone morphogenetic protein et al. Tadalafil therapy for pulmonary arterial hypertension. Circulation
receptor. Circulation 2002;105:1672‑8. 2009;119:2894‑903.
103. Gangopahyay A, Oran M, Bauer EM, Wertz JW, Comhair SA, Erzurum SC, 125. Label approved for REVATIO, NDA # 021845. FDA, 2012. Available from:
et al. Bone morphogenetic protein receptor II is a novel mediator of http://www.accessdata.fda.gov/scripts/cder/drugsatfda.[Last accessed on
endothelial nitric‑oxide synthase activation. J Biol Chem 2011;286:33134‑40. 2012 April 10].
104. Forrest IA, Small T, Corris PA. Effect of nebulized epoprostenol (prostacyclin) 126. Barst RJ, Ivy DD, Gaitan G, Szatmari A, Rudzinski A, Garcia AE, et al.
on exhaled nitric oxide in patients with pulmonary hypertension due A randomized, double‑blind, placebo‑controlled, dose‑ranging study of
to congenital heart disease and in normal controls. Clin Sci (Lond) oral sildenafil citrate in treatment‑naive children with pulmonary arterial
1999;97:99‑102. hypertension. Circulation 2012;125:324‑34.
105. Rolla G, Colagrande P, Brussino L, Bucca C, Bertero MT, Caligaris‑Cappio F. 127. Wharton J, Strange JW, Moller GM, Growcott EJ, Ren X, Franklyn AP, et al.
Exhaled nitric oxide and pulmonary response to iloprost in systemic Antiproliferative effects of phosphodiesterase type 5 inhibition in human
sclerosis with pulmonary hypertension. Lancet 1998;351:1491‑2. pulmonary artery cells. Am J Respir Crit Care Med 2005;172:105‑13.
106. Machado RF, Londhe Nerkar MV, Dweik RA, Hammel J, Janocha A, 128. Michelakis E, Tymchak W, Lien D, Webster L, Hashimoto K, Archer S. Oral
Pyle J, et al. Nitric oxide and pulmonary arterial pressures in pulmonary sildenafil is an effective and specific pulmonary vasodilator in patients with
hypertension. Free Radic Biol Med 2004;37:1010‑7. pulmonary arterial hypertension: Comparison with inhaled nitric oxide.
Circulation 2002;105:2398‑403.
107. Hirata Y, Emori T, Eguchi S, Kanno K, Imai T, Ohta K, et al. Endothelin
receptor subtype B mediates synthesis of nitric oxide by cultured bovine 129. Nagendran J, Archer SL, Soliman D, Gurtu V, Moudgil R, Haromy A, et al.
endothelial cells. J Clin Invest 1993;91:1367‑73. Phosphodiesterase type 5 is highly expressed in the hypertrophied human
right ventricle, and acute inhibition of phosphodiesterase type 5 improves
108. Millatt LJ, Whitley GS, Li D, Leiper JM, Siragy HM, Carey RM, et al. Evidence
contractility. Circulation 2007;116:238‑48.
for dysregulation of dimethylarginine dimethylaminohydrolase I in chronic
hypoxia‑induced pulmonary hypertension. Circulation 2003;108:1493‑8. 130. Frostell C, Fratacci MD, Wain JC, Jones R, Zapol WM. Inhaled nitric
oxide. A selective pulmonary vasodilator reversing hypoxic pulmonary
109. Kielstein JT, Bode‑Boger SM, Hesse G, Martens‑Lobenhoffer J, Takacs A,
vasoconstriction. Circulation 1991;83:2038‑47.
Fliser D, et al. Asymmetrical dimethylarginine in idiopathic pulmonary
131. Pepke‑Zaba J, Higenbottam TW, Dinh‑Xuan AT, Stone D, Wallwork J. Inhaled
arterial hypertension. Arterioscler Thromb Vasc Biol 2005;25:1414‑8.
nitric oxide as a cause of selective pulmonary vasodilatation in pulmonary
110. Pullamsetti S, Kiss L, Ghofrani HA, Voswinckel R, Haredza P, Klepetko W,
hypertension. Lancet 1991;338:1173‑4.
et al. Increased levels and reduced catabolism of asymmetric and symmetric
132. Vonbank K, Ziesche R, Higenbottam TW, Stiebellehner L, Petkov V, Schenk P,
dimethylarginines in pulmonary hypertension. FASEB J 2005;19:1175‑7.
et al. Controlled prospective randomised trial on the effects on pulmonary
111. Skoro‑Sajer N, Mittermayer F, Panzenboeck A, Bonderman D, Sadushi R, haemodynamics of the ambulatory long term use of nitric oxide and oxygen
Hitsch R, et al. Asymmetric dimethylarginine is increased in chronic in patients with severe COPD. Thorax 2003;58:289‑93.
thromboembolic pulmonary hypertension. Am J Respir Crit Care Med
133. Channick RN, Newhart JW, Johnson FW, Williams PJ, Auger WR, Fedullo PF,
2007;176:1154‑60.
et al. Pulsed delivery of inhaled nitric oxide to patients with primary
112. Xu W, Kaneko FT, Zheng S, Comhair SA, Janocha AJ, Goggans T, et al. pulmonary hypertension: An ambulatory delivery system and initial clinical
Increased arginase II and decreased NO synthesis in endothelial cells of tests. Chest 1996;109:1545‑9.
patients with pulmonary arterial hypertension. FASEB J 2004;18:1746‑8.
134. Perez‑Penate GM, Julia‑Serda G, Ojeda‑Betancort N, García‑Quintana A,
113. American Thoracic Society; European Respiratory Society. ATS/ERS Pulido‑Duque J, Rodríguez‑Pérez A, et al. Long‑term inhaled nitric oxide
recommendations for standardized procedures for the online and offline plus phosphodiesterase 5 inhibitors for severe pulmonary hypertension.
measurement of exhaled lower respiratory nitric oxide and nasal nitric J Heart Lung Transplant 2008;27:1326‑32.
oxide, 2005. Am J Respir Crit Care Med 2005;171:912‑30.
135. Frampton MW, Morrow PE, Cox C, Gibb FR, Speers DM, Utell MJ. Effects
114. Condorelli P, Shin HW, Aledia AS, Silkoff PE, George SC. A simple technique of nitrogen dioxide exposure on pulmonary function and airway reactivity
to characterize proximal and peripheral nitric oxide exchange using in normal humans. Am Rev Respir Dis 1991;143:522‑7.
constant flow exhalations and an axial diffusion model. J Appl Physiol 136. Clutton‑Brock J. Two cases of poisoning by contamination of nitrous
2007;102:417‑25. oxide with higher oxides of nitrogen during anaesthesia. Br J Anaesth
115. Rich S, Kaufmann E, Levy PS. The effect of high doses of calcium‑channel 1967;39:388‑92.
blockers on survival in primary pulmonary hypertension. N Engl J Med 137. Miller OI, Tang SF, Keech A, Celermajer DS. Rebound pulmonary
1992;327:76‑81. hypertension on withdrawal from inhaled nitric oxide. Lancet 1995;346:51‑2.
116. Sitbon O, Humbert M, Jais X, Ioos V, Hamid AM, Provencher S, et al. 138. Christenson J, Lavoie A, O’Connor M, Bhorade S, Pohlman A, Hall JB.
Long‑term response to calcium channel blockers in idiopathic pulmonary The incidence and pathogenesis of cardiopulmonary deterioration after
arterial hypertension. Circulation 2005;111:3105‑11. abrupt withdrawal of inhaled nitric oxide. Am J Respir Crit Care Med
117. Barst RJ, Gibbs JS, Ghofrani HA, Hoeper MM, McLaughlin VV, Rubin LJ, 2000;161:1443‑9.
et al. Updated evidence‑based treatment algorithm in pulmonary arterial 139. Label for INOMAX, NDA # 020845. U.S. Food and Drug Administration.
hypertension. J Am Coll Cardiol 2009;54:S78‑84. 2010. Accessed from: http://www.accessdata.fda.gov/scripts/cder/
118. Tonelli AR, Alnuaimat H, Mubarak K. Pulmonary vasodilator testing and drugsatfda/. [Last accessed on 2012, April 10].
use of calcium channel blockers in pulmonary arterial hypertension. Respir 140. Mercier JC, Hummler H, Durrmeyer X, Sanchez‑Luna M, Carnielli V,
Med 2010;104:481‑96. Field D, et al. Inhaled nitric oxide for prevention of bronchopulmonary
119. Weir EK, Rubin LJ, Ayres SM, Bergofsky EH, Brundage BH, Detre KM, dysplasia in premature babies (EUNO): A randomised controlled trial.
et al. The acute administration of vasodilators in primary pulmonary Lancet 2010;376:346‑54.
hypertension. Experience from the National Institutes of Health Registry 141. Van Meurs KP, Wright LL, Ehrenkranz RA, Lemons JA, Ball MB, Poole WK,
on Primary Pulmonary Hypertension. Am Rev Respir Dis 1989;140:1623‑30. et al. Inhaled nitric oxide for premature infants with severe respiratory
120. Post MC, Janssens S, Van de Werf F, Budts W. Responsiveness to inhaled failure. N Engl J Med 2005;353:13‑22.
nitric oxide is a predictor for mid‑term survival in adult patients with 142. Kleinbongard P, Dejam A, Lauer T, Jax T, Kerber S, Gharini P, et al. Plasma
Pulmonary Circulation | January-March 2013 | Vol 3 | No 1 29