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JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY VOL. 70, NO.

19, 2017

ª 2017 BY THE AMERICAN COLLEGE OF CARDIOLOGY FOUNDATION ISSN 0735-1097/$36.00

PUBLISHED BY ELSEVIER https://doi.org/10.1016/j.jacc.2017.09.1064

THE PRESENT AND FUTURE

STATE-OF-THE-ART REVIEW

The Role of Nitroglycerin and
Other Nitrogen Oxides in
Cardiovascular Therapeutics
Sanjay Divakaran, MD, Joseph Loscalzo, MD, PHD

ABSTRACT

The use of nitroglycerin in the treatment of angina pectoris began not long after its original synthesis in 1847. Since then,
the discovery of nitric oxide as a biological effector and better understanding of its roles in vasodilation, cell permeability,
platelet function, inflammation, and other vascular processes have advanced our knowledge of the hemodynamic
(mostly mediated through vasodilation of capacitance and conductance arteries) and nonhemodynamic effects of organic
nitrate therapy, via both nitric oxide–dependent and –independent mechanisms. Nitrates are rapidly absorbed from
mucous membranes, the gastrointestinal tract, and the skin; thus, nitroglycerin is available in a number of preparations for
delivery via several routes: oral tablets, sublingual tablets, buccal tablets, sublingual spray, transdermal ointment,
and transdermal patch, as well as intravenous formulations. Organic nitrates are commonly used in the treatment of
cardiovascular disease, but clinical data limit their use mostly to the treatment of angina. They are also used in the
treatment of subsets of patients with heart failure and pulmonary hypertension. One major limitation of the use of nitrates
is the development of tolerance. Although several agents have been studied for use in the prevention of nitrate tolerance,
none are currently recommended owing to a paucity of supportive clinical data. Only 1 method of preventing nitrate
tolerance remains widely accepted: the use of a dosing strategy that provides an interval of no or low nitrate exposure
during each 24-h period. Nitric oxide’s important role in several cardiovascular disease mechanisms continues to drive
research toward finding novel ways to affect both endogenous and exogenous sources of this key molecular mediator.
(J Am Coll Cardiol 2017;70:2393–410) © 2017 by the American College of Cardiology Foundation.

I n this review, we detail the discovery of nitro-
glycerin and its early use in the treatment of
angina; the history of the discovery of nitric
oxide (NO), its sources, and its roles; the mechanism
new therapeutic options in the treatment of cardio-
vascular disease.

EARLY HISTORY OF THE USE OF NITRATES IN
of action, preparations, and hemodynamic and non- CORONARY ARTERY DISEASE
hemodynamic effects of the organic nitrates, as well
as their biotransformation; and the clinical uses and William Heberden is credited with coining the term
adverse effects of nitrate therapy, including toler- “angina pectoris” in 1772 (1). Joseph Priestley, an
ance. We conclude by outlining current and future English theologian and chemist, discovered NO 3
work investigating novel modulators of the NO– years later (2). Not until the next century, however,
soluble guanylyl cyclase (sGC)–cyclic guanosine was NO and its congeners, organic nitrates, linked to
Listen to this manuscript’s
monophosphate (cGMP) pathway that may result in the treatment of angina (3). Glyceryl trinitrate, or
audio summary by
JACC Editor-in-Chief
Dr. Valentin Fuster.

From the Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts. This work
was supported in part by National Institutions of Health grants HL61795 and GM107618 (to Dr. Loscalzo). Both authors have re-
ported that they have no relationships relevant to the contents of this paper to disclose.

Manuscript received September 7, 2017; accepted September 19, 2017.
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Nitroglycerin and Nitric Oxide in Cardiovascular Therapeutics NOVEMBER 7, 2017:2393–410

ABBREVIATIONS nitroglycerin, was first synthesized by NO IN THE CARDIOVASCULAR SYSTEM
AND ACRONYMS Ascanio Sombrero in Turin, Italy. In 1847, he
noted that “a very minute quantity put on HISTORY OF NO. In 1975, Diamond and Holmes
ALDH = aldehyde
dehydrogenase
the tongue produced a violent headache for showed that tissue levels of cyclic adenosine mono-
several hours” (4,5). Three year earlier, the phosphate (cAMP) were increased in rat myometrial
BH4 = tetrahydro-L-biopterin
French chemist Antoine Balard synthesized strips maintained in a state of sustained contracture.
cAMP = cyclic adenosine
monophosphate amyl nitrite (4). Frederick Guthrie, an En- They demonstrated that nitroglycerin could relax the
CAV1 = caveolin-1
glish chemist, explored the actions of amyl depolarized muscles without significantly increasing
nitrite and published in 1859 that when it cAMP levels and, therefore, concluded that changes
cGMP = cyclic guanosine
monophosphate was held near the nostrils, “after a lapse of in total tissue levels of cAMP were not responsible for
EDRF = endothelium-derived about 50 seconds, a sudden throbbing of the the uterine relaxation caused by nitroglycerin (10).
relaxing factor arteries of the neck is felt, immediately These investigators went on to show that nitroglyc-
eNOS = endothelial nitric oxide followed by a flushing of neck, temples, erin increased cyclic guanosine monophosphate
synthase and forehead and an acceleration action of (cGMP) levels in depolarized muscle (10). The
iNOS = cytokine-inducible the heart” (6). following year, Diamond and Blisard showed that 200
nitric oxide synthase
T. Lauder Brunton, a Scottish physician mmol/l nitroglycerin increased levels of cGMP by more
nNOS = neuronal nitric oxide
and medical scientist, first described the than 15-fold during relaxation of isolated strips of
synthase
clinical effectiveness of amyl nitrite (4,5,7). phenylephrine-contracted canine femoral arteries
NO = nitric oxide
Brunton began caring for patients with while having no significant effect on cAMP levels (11).
NOS = nitric oxide synthase
angina pectoris as a house physician at the In 1977, pharmacologist Ferid Murad and his col-
P450 = cytochrome P450
Edinburgh Royal Infirmary (7). At the time, leagues published their seminal work on modulating
enzyme(s)
many treatments, including therapeutic contractility in bovine tracheal smooth muscle
sGC = soluble guanylyl cyclase
bleeding, were being used to treat angina, showing that the guanylyl cyclase activators, sodium
VEGF = vascular endothelial
largely unsuccessfully. In 1867, Brunton nitrite, nitroglycerin, and sodium nitroprusside,
growth factor
published the first report of the use of amyl increased cGMP levels and relaxed tracheal smooth
nitrite in the treatment of angina pectoris (7,8). He muscle (12). They went on to demonstrate that solu-
believed “the relief produced by [therapeutic] tions of NO gas increased cGMP activity in soluble and
bleeding to be due to the diminution it occasioned in particulate preparations from various tissues in a
the arterial tension” and “that a substance which dose-dependent fashion, and that NO alone and
possesses the power of lessening it in such an in combination with sodium azide, sodium nitrite,
eminent degree as nitrite of amyl would probably hydroxylamine, and sodium nitroprusside increased
produce the same effect, and might be repeated cGMP levels to approximately the same degree;
as often as necessary without detriment to the they concluded that the 2 methods activate
patient’s health” (7,8). In 1903, Charles-Émile guanylyl cyclase through a “similar but undefined
François-Franck, a French physiologist, first mechanism” (13). Later, the same group postulated
suggested that amyl nitrite was a coronary vasodi- that “while the precise mechanism of guanylate
lator (7). cyclase activation by these agents is not known,
In 1879, William Murrell described the symptom- activation may be due to the formation of NO or
atic effects of placing drops of 1% solution of another reactive material since NO also increased
nitroglycerin in alcohol on the tongue (9). In addi- guanylate cyclase activity” (14).
tion to reporting that it relieved angina and pre- Working independently, Robert Furchgott and
vented subsequent attacks, he also reported the John Zawadzki published their observations on the
symptoms he felt when he “[tried] its action on importance of the endothelium in blood vessel
[himself]” (9). He described a “violent pulsation in relaxation in 1980 (4,15). They reported that acetyl-
[his] head” and noticed that his pulse was “much choline did not always produce blood vessel relaxa-
fuller than natural” (9). In 1914, Brunton, who tion in vitro, even though it was a potent vasodilator
originally thought angina was caused by hyperten- in vivo. They found that loss of relaxation in vitro
sion, acknowledged that the “dilating action of amyl was due to “unintentional rubbing of [the rabbit
nitrite and nitroglycerine upon the coronary vessels thoracic aorta’s] intimal surface against foreign
would readily explain the relief they offer in angina surfaces during its preparation.” When this mechan-
pectoris, even in cases where the blood-pressure is ical injury was avoided during preparation of the
normal” (7). tissue, it always relaxed in response to acetylcholine.
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Therefore, they concluded that acetylcholine-
F I G U R E 1 Sources of NO
mediated relaxation of vascular smooth muscle
required the presence of endothelial cells (15).
L-Arginine
Furchgott and his colleagues eventually proposed
that bradykinin and other molecules acting on the
endothelium caused relaxation of vascular smooth L-Citrulline NOS

muscle via a substance they termed endothelium-
derived relaxing factor (EDRF) (16). The link to NO, NO

however, was not yet appreciated (4).
Acidic environment
Louis Ignarro and Salvador Moncada indepen- (stomach, hypoxic/ischemic tissue)
dently discovered that EDRF is NO in 1987 (4,17).
After a series of experiments, Ignarro et al. (18,19) NO2–
stated that “EDRF released from artery and vein
Nitrate reductase
possesses identical biological and chemical properties
(oral flora)
as NO.” Moncada and colleagues suggested that EDRF
and NO were “identical” as “NO released from endo- NO3–
thelial cells is indistinguishable from EDRF in terms
of biological activity, stability, and susceptibility to
NO is a free radical that is synthesized by the family of NO synthases from L-arginine and
an inhibitor and to a potentiator” (20). Based on their
oxygen, yielding L-citrulline as a coproduct. In the blood vessel wall, NO is mainly
collective contributions to the field, the Nobel Prize in produced by endothelial nitric oxide synthase (NOS). There are 2 other isoforms of NOS
Physiology or Medicine was awarded to Furchgott, that also produce NO from L-arginine: neuronal NOS and cytokine-inducible NOS.
Ignarro, and Murad in 1998 “for their discoveries Additionally, bacterial flora present in the mammalian oral cavity is rich in nitrate
reductases and can convert dietary sources of nitrate to nitrite. In the extremely acidic
concerning NO as a signaling molecule in the cardio-
environment (pH #3) of the gastric lumen, protonation of nitrite can, in turn, yield
vascular system” (4).
nitrous acid, which can spontaneously decompose to NO. This pathway of NO generation
SOURCES OF NO. NO is a free radical that is synthe- is referred to as the enterosalivary nitrate circulation (nitrate-nitrite-NO pathway).
sized by the family of NO synthases from L-arginine NO ¼ nitric oxide; NO2 ¼ inorganic nitrite; NO3 ¼ inorganic nitrate.
and oxygen, yielding L-citrulline as a coproduct
(Figure 1) (21–23). In the blood vessel wall, NO is
mainly produced by endothelial nitric oxide synthase
(eNOS) (23,24). However, there are 2 other isoforms of Hemoglobin has enzymatic behavior as a nitrite
nitric oxide synthase (NOS) that also produce NO from reductase under hypoxic conditions and is a
L-arginine: neuronal nitric oxide synthase (nNOS) sensor and effector of hypoxic vasodilation (34). He-
and cytokine-inducible nitric oxide synthase (iNOS) moglobin’s maximal nitrite reduction rate occurs
(25–27). Although eNOS is the major NOS isoform that when hemoglobin is 40% to 60% saturated with
regulates vascular function, both nNOS and iNOS are oxygen (23,34). Cytochrome P 450 reductase causes NO
implicated as sources in certain tissues and environ- release by reducing nitrate, and can also facilitate
ments (23). For example, vascular injury induces generation of S-nitrosothiols (35).
expression of nNOS in the neointima and medial Bacterial flora present in the mammalian oral
smooth muscle cells, and the production of iNOS cavity is rich in nitrate reductases and can convert
within vascular smooth muscle cells after exposure dietary sources of nitrate to nitrite. In the extremely
to proinflammatory cytokines is a major cause of acidic environment (pH #3) of the gastric lumen,
vasodilation in sepsis (28,29). protonation of nitrite can, in turn, produce nitrous
S-nitrosothiols, such as S-nitrosoglutathione and acid, which can spontaneously decompose to NO
S-nitrosohemoglobin, are also sources of NO (23,24). (36,37). This pathway of NO generation is referred
Under certain conditions, such as in the presence to as the enterosalivary nitrate circulation (nitrate–
of trace transition metal ions or photolysis, nitrite–NO pathway) (Figure 1) and is a major
S-nitrosothiols decompose to liberate NO (30). In source of NO (38,39).
addition, S-nitrosothiols can undergo trans-S- NO’s ROLES. NO is a powerful vasodilator that in-
nitrosation with other thiol groups and thereby duces formation of cGMP by activating sGC in
modify cell or protein function (31–33). vascular smooth muscle cells (Figure 2) (13). cGMP can
Several proteins, such as hemoglobin, cytochrome bind to and enhance protein kinase G activity, cGMP-
P450 reductase, and cytochrome P 450 , can catalyze the gated ion channels, and cGMP-sensitive phosphodi-
reduction of nitrite or nitrate to generate NO (23). esterases (40). Protein kinase G promotes reuptake of
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F I G U R E 2 Regulation of Vascular Tone by NO

Endothelial Cell

Nitrovasodilators
L-Arginine

eNOS

Bioactivation
NO Ca2+

GTP

Vascular Smooth
NO sGC cGMP cGMP-dep Kinase I
Muscle Cell

PDE
GMP ↓Ca2+

MLCK
Actin-Myosin
↓ MLC MLC-Pi
ATPase
MLCP

Vascular Smooth Muscle
Relaxation

NO is a powerful vasodilator that induces formation of cGMP by activating soluble sGC in vascular smooth muscle cells. cGMP can bind to and enhance
protein kinase G activity, cGMP-gated ion channels, and cGMP-sensitive phosphodiesterases. Protein kinase G promotes reuptake of cytosolic calcium into
the sarcoplasmic reticulum, the movement of calcium from the intracellular to the extracellular environment, and the opening of calcium-activated
potassium channels. These changes result in reduction of vascular tone as the reduction in intracellular calcium impairs myosin light chain kinase’s ability
to phosphorylate myosin, resulting in smooth muscle cell relaxation. ATPase ¼ adenosine triphosphatase; Ca2þ ¼ calcium ion; cGMP ¼ cyclic guanosine
monophosphate; cGMP-dep Kinase I ¼ cyclic guanosine monophosphate-dependent protein kinase I; eNOS ¼ endothelial nitric oxide synthase;
GMP ¼ guanosine monophosphate; GTP ¼ guanosine triphosphate; MLC ¼ myosin light chain; MLCK ¼ myosin light chain kinase; MLCP ¼ myosin light
chain phosphatase; MLC-Pi ¼ phosphorylated myosin light chain; NO ¼ nitric oxide; PDE ¼ phosphodiesterase; sGC ¼ soluble guanylyl cyclase.

cytosolic calcium into the sarcoplasmic reticulum, the activating platelet guanylyl cyclase and increasing
movement of calcium from the intracellular to the intraplatelet cGMP (46–49).
extracellular environment, and the opening of Finally, a greater understanding of the conse-
calcium-activated potassium channels (23). These quences of impaired NO synthesis can yield more
changes result in reduction of vascular tone as the information regarding the importance of NO’s role in a
reduction in intracellular calcium impairs myosin well-functioning cardiovascular system. For example,
light chain kinase’s ability to phosphorylate myosin, when low levels of its essential cofactor (6R)-5,6,7,8-
resulting in smooth muscle cell relaxation (41). tetrahydrobiopterin are present, eNOS produces
NO has several other important roles in the superoxide anion instead of NO in a process denoted
vasculature. For example, when vascular endothelial enzymatic uncoupling (50). eNOS uncoupling has been
growth factor (VEGF) binds to VEGF receptor-2, eNOS observed in animal models of cardiovascular disease
is activated and NO is formed (42–44). This NO is and in patients with cardiovascular risk factors, such
essential for VEGF function, as eNOS-deficient mice as hypertension and diabetes mellitus (51,52).
have markedly reduced vascular permeability and
angiogenesis (45). S-nitrosylation of b -catenin by NO MECHANISM OF ACTION OF NITRATES
modulates intercellular contacts between endothelial
cells, likely accounting for NO-dependent changes in Organic nitrates, such as nitroglycerin, isosorbide
endothelial permeability (42). NO and organic nitrates dinitrate, and isosorbide mononitrate, are rapidly
also impair platelet activation and aggregation by absorbed from several sites, such as the
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F I G U R E 3 Chemical Structures of Key Nitrovasodilators

ONO2 OH
H H

O O
O2NO ONO2

ONO2 O O ONO
H H
O2NO O2NO

Isosorbide Isosorbide
Nitroglycerin Amyl nitrite
dinitrate mononitrate

O 2–

N N
O2NO ONO2 N N
C C
2Na+ Fe H
C C
N N N
C ONO2
O2NO ONO2 O
N

Pentaerythrityl Sodium
Nicorandil
tetranitrate nitroprusside

Chemical structures of 7 key nitrovasodilators are shown.

gastrointestinal tract, mucous membranes, and skin, used in hospitalized patients with angina, hyperten-
depending on the preparation (53). These compounds sion, or heart failure (Figure 3, Table 1) (53,59).
are prodrugs with a nitrooxy (O-NO 2) moiety and are Nitroglycerin has a plasma half-life of approximately
metabolized to produce bioactive metabolites (54,55). 1 to 4 min; following hepatic and intravascular
The bioactivation of organic nitrates causes liberation metabolism, its biologically active metabolites have a
of NO, allowing them to serve as NO donors (55,56). half-life of approximately 40 min (53,60).
NO then causes vasodilation via its effect on vascular Isosorbide dinitrate is rapidly absorbed and
smooth muscle cells, and impairs platelet activation undergoes extensive first-pass metabolism by the
as previously discussed (13). liver, which results in low bioavailability (53,55).
Nitrates are also implicated in epigenetic regula- Sustained-release formulations have a slower rate of
tion of vasodilation, including specifically smooth absorption and can provide therapeutic plasma
muscle cell relaxation (57,58). Additionally, nitro- concentrations of the drug for up to 12 h (53).
glycerin has been shown to increase the activity of Oral isosorbide mononitrate is completely absorbed
histone acetylases with nitroglycerin-dependent and has 100% bioavailability as it avoids first-pass
vascular responses influenced by N 3-lysine acetyla- metabolism. This leads to a more predictable dose
tion of contractile proteins (58). response and plasma levels with less variation when
compared with other nitrates (55,61). Pentaerythrityl
PREPARATIONS tetranitrate is a long-acting organic nitrate available
in an oral formulation that acts within 10 to 20 min
Nitrates are rapidly absorbed from mucous mem- and has a duration of action of 8 to 12 h (57).
branes, the gastrointestinal tract, and the skin (55). Preclinical data suggested that pentaerythrityl
Thus, nitroglycerin is available in several prepara- tetranitrate may not cause nitrate tolerance (see the
tions for delivery via several routes: oral tablets, following text) or endothelial dysfunction; however,
sublingual tablets, buccal tablets, sublingual spray, the drug is not currently commonly used, as clinical
transdermal ointment, and transdermal patch; it is studies have not shown any clear benefit in patients
also available in intravenous formulations, which are with chronic, stable angina (62).
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the alveolar-capillary membrane and activates sGC in
T A B L E 1 Properties of Key Nitrovasodilators
the subjacent smooth muscle cells in the pulmonary
Time Until Duration of vasculature (71). N-diazeniumdiolates (NONOates) are
Drug Route/Formulation Dose Onset of Action Action
NO donors that are synthesized by reacting primary or
Nitroglycerin SL spray 0.4 mg 1–3 min 25 min
secondary amines with NO gas at high pressure and
SL tablet 0.3–0.8 mg 1–3 min 25 min
TD patch 0.2–0.8 mg/h 30 min 10–12 h low temperatures; hydrolysis causes spontaneous
IV infusion 5–400 mg/min Within 3–5 min decomposition under physiological conditions and
seconds releases NO (72–74). NO (as N 2O 3 or ONOO) can react
Isosorbide dinitrate PO IR tablet 5–80 mg 60 min 8h
PO SR tablet 40–160 mg 60 min 12 h
with thiols in vivo to form S-nitrosothiols, such as
Isosorbide mononitrate PO IR tablet 5–20 mg 30–45 min 6h S-nitrosocysteine and S-nitrosoglutathione (75).
PO SR tablet 30–240 mg 30–45 min 12–24 h S-nitrosothiol compounds can also be synthesized
Amyl nitrite Inhalation of 0.3 ml 30 s 3–15 min chemically by reacting thiols with nitrous acid. When
ampule
Pentaerythrityl PO tablet 10–80 mg 10–20 min 8–12 h
exposed to physiological fluids, S-nitrosothiols
tetranitrate decompose to release NO (72). Other newer classes of
Sodium nitroprusside IV infusion 0.3–10.0 mg/kg/min 1 min 6–12 min direct NO donors that are actively being studied
Nicorandil PO tablet 5–20 mg 30–60 min 12 h
include furoxans, benzofuroxans, and zeolites
(76,77).
IR ¼ immediate release; IV ¼ intravenous; PO ¼ oral; SL ¼ sublingual; SR ¼ sustained release; TD ¼ transdermal.

BIOTRANSFORMATION OF
Nicorandil is a nicotinamide-nitrate ester whose ORGANIC NITRATES
chemical structure consists of a nicotinamide deriv-
ative combined with a nitrate moiety (55). Nicorandil Organic nitrates must undergo biotransformation
acts as both an NO donor and a K þATP channel to release a vasoactive molecule (78). The detailed
opener to provide antianginal effects (63). The bio- study of the biotransformation of nitroglycerin
activation of nicorandil occurs via the nicotinamide/ has suggested a high-potency pathway mediated
nicotinic acid pathway and involves NO generation by aldehyde dehydrogenase (ALDH)-2 and a low-
via denitration (64). Nicorandil also dilates the potency pathway mediated by other enzymes or
coronary microvasculature and peripheral resistance low-molecular-weight reductants (Figure 4) (78).
arterioles by causing vascular smooth cell hyperpo- The high-potency pathway is important at clinically
larization and closure of L-type voltage-gated relevant nitroglycerin concentrations (<1 m mol/l) and
calcium channels via its action on K þATP channels; is localized to the mitochondria. The mitochondrial
it is rapidly absorbed via the gastrointestinal tract, isoform of aldehyde dehydrogenase, ALDH-2, was
does not undergo first-pass metabolism, reaches found to be a key enzyme in the biotransformation of
maximal plasma concentration after 30 to 60 min, nitroglycerin via this pathway as it generates inor-
and has a half-life of approximately 52 min ganic nitrite and 1,2-glyceryl dinitrate from nitro-
(55,65,66). Nicorandil has an oral bioavailability glycerin (79). There are 3 proposed mechanisms for
of >75%, and its antianginal effects last approxi- the release of the vasoactive molecule via this
mately 12 h (55,64,67). pathway: nitrogen oxides are formed via reduction of
Sodium nitroprusside, which is 44% cyanide by inorganic nitrite; NO is formed directly in response to
weight, comprises a ferrous ion center complexed interaction with ALDH-2; and inorganic nitrite
with 5 cyanic moieties and a nitrosyl group (68). It is released from mitochondria may be reduced by
available intravenously and interacts with oxyhemo- xanthine oxidase in the cytoplasm to form NO (78).
globin to produce methemoglobin and spontaneously The low-potency pathway is important at supra-
release NO and cyanide (69,70). Sodium nitroprusside pharmacological nitroglycerin concentrations (>1 mmol/l),
causes direct venous and arterial vasodilation, is a is found in the smooth endoplasmic reticulum, and
potent pulmonary vasodilator, and is an inhibitor of leads to formation of measurable amounts of NO in
hypoxia-induced pulmonary vasoconstriction (68). It vascular tissues (80,81). In this pathway, nitroglyc-
has an almost immediate onset of action and a very erin is biotransformed by proteins such as deoxy-
short half-life: its effects dissipate within 1 to 2 min hemoglobin, deoxymyoglobin, cytochrome P 450 ,
(68). xanthine oxidase, glutathione-S-transferase, glycer-
Finally, there are several direct NO donors that aldehyde-3-phosphate dehydrogenase, or other
are already being used clinically, or have been ALDH isoforms; and by low-molecular-weight re-
synthesized and studied for use particularly in ductants such as cysteine, N-acetyl-cysteine, thio-
biomaterials. Inhaled NO gas diffuses rapidly across salicylic acid, and ascorbate (57,82–84).
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F I G U R E 4 Bioactivation of Organic Nitrates

High potency nitrates Low potency nitrates
GTN, PETN high dose ISDN, ISDM

low dose
PETriN

GMN
GDN, PEDN P450
PEMN

ALDH2 .
NO
H+
NO2– NOx
?
. Smooth
sGC NO
Mitochondrion Cyt Ox Endoplasmic Reticulum

.
NO cGMP cGK-I Relaxation
XO

Organic nitrates must undergo biotransformation to release a vasoactive molecule via 1 of 2 pathways: a high-potency pathway mediated by ALDH-2, or a low-potency
pathway mediated by other enzymes or low-molecular-weight reductants. The high-potency pathway is important at clinically relevant nitroglycerin concentrations
(<1 mmol/l) and is localized to the mitochondria. The mitochondrial isoform of aldehyde dehydrogenase, ALDH-2, was found to be a key enzyme in the
biotransformation of nitroglycerin via this pathway as it generates inorganic nitrite (NO2) and 1,2-glyceryl dinitrate from nitroglycerin. There are 3 proposed
mechanisms for the release of the vasoactive molecule via this pathway: nitrogen oxides are formed via reduction of inorganic nitrite; nitric oxide is formed directly in
response to interaction with ALDH-2; and inorganic nitrite released from mitochondria may be reduced by xanthine oxidase in the cytoplasm to form NO. The
low-potency pathway is important at suprapharmacological nitroglycerin concentrations (> 1 mmol/l), is found in the smooth endoplasmic reticulum, and leads to
formation of measurable amounts of NO in vascular tissues. In this pathway, nitroglycerin is biotransformed by proteins such as deoxyhemoglobin, deoxymyoglobin,
cytochrome P450, xanthine oxidase, glutathione-S-transferase, glyceraldehyde-3-phosphate dehydrogenase, or other ALDH isoforms; and by low-molecular-weight
reductants such as cysteine, N-acetyl-cysteine, thiosalicylic acid, and ascorbate. Reproduced with permission from Munzel and Daiber (78). ALDH2 ¼ aldehyde
dehydrogenase-2; cGK-I ¼ cyclic guanosine monophosphate-dependent protein kinase I; Cyt Ox ¼ cytochrome c oxidase; GDN ¼ 1,2-glyceryl dinitrate;
GMN ¼ 1,2-glyceryl mononitrate; GTN ¼ glyceryl trinitrate (nitroglycerin); Hþ ¼ hydrogen ion; ISDM ¼ isosorbide mononitrate; ISDN ¼ isosorbide dinitrate;
NO2- ¼ inorganic nitrite; NOx ¼ nitrogen oxides; P450 ¼ cytochrome P450 enzyme(s); PEDN ¼ pentaerythrityl dinitrate; PEMN ¼ pentaerythrityl mononitrate;
PETN ¼ pentaerythrityl tetranitrate; PETriN ¼ pentaerythrityl trinitrate; XO ¼ xanthine oxidase; other abbreviations as in Figure 2.

HEMODYNAMIC EFFECTS OF NITRATES “suggesting the possibility that diastolic coronary
blood flow may be augmented by diminished extra-
The organic nitrates have several hemodynamic ef- vascular resistance to flow” (86).
fects that are mostly mediated through vasodilation Nitrates also dilate large- and medium-sized coro-
of capacitance veins and conductance arteries nary arteries and arterioles >100 m m in diameter. This
(Central Illustration) (53). In 1971, Parker et al. (85) effect reduces left ventricular systolic wall tension via
published the results of a study of hemodynamic decreasing afterload and, therefore, also decreases
indices and coronary blood flow at rest and during myocardial oxygen demand (53,87). The hemody-
exercise before and after administration of nitro- namic effects of nitrates on the coronary vasculature of
glycerin in 15 patients with coronary artery disease atherosclerotic patients also relieve angina. In 1972,
and concluded that nitroglycerin acted primarily by Horwitz, Herman, and Gorlin published a study of 70
reducing left ventricular oxygen requirements patients (49 with coronary artery disease and 21
through a reduction in left ventricular volume (85). without) with anginal chest pain and found that 76% of
NO-mediated dilation of capacitance veins decreases patients with coronary artery disease were regularly
ventricular pre-load, which results in reduction in relieved of their pain within 3 min, while only 19%
myocardial oxygen demand. In 1975, Greenberg et al. of those without coronary artery disease were
(86), after angiographic and hemodynamic assess- regularly relieved of their pain within 3 min (88).
ment of 10 patients, concluded that the mechanism of Nitrates dilate the epicardial coronary arteries,
action of nitroglycerin “seems to relate best to the including stenotic segments, and also improve blood
decrease in systolic wall tension.” They also found flow in coronary collaterals via decreasing resistance
that the end-diastolic wall tension decreased by 57%, to collateral flow (87,89). In 1964, Fam and McGregor
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C E NT R AL IL L U STR AT IO N Hemodynamic Effects of Nitroglycerin

↓ Myocardial O2 Consumption

↓ Preload ↓ Left ventricular
wall tension

↑ Subendocardial
blood flow

Vasodilation of large- and
Vasodilation of Vasodilation of conductance
medium-sized coronary
capacitance veins arteries
arteries and arterioles

Vasodilation of systemic and
Nitroglycerin → NO
pulmonary arterial beds

Dilation of stenotic
Improvement in coronary Epicardial coronary artery
epicardial coronary artery
collateral blood flow dilation
segments

Divakaran, S. et al. J Am Coll Cardiol. 2017;70(19):2393–410.

The organic nitrates have several hemodynamic effects that are largely mediated through vasodilation of capacitance veins and conductance arteries. Seminal work in
the 1970s, as detailed in the main text, showed that nitroglycerin acts primarily by reducing left ventricular oxygen requirements through a reduction in left
ventricular volume. Nitric oxide (NO)–mediated dilation of capacitance veins decreases ventricular pre-load, which results in reduction in myocardial oxygen demand
and left ventricular wall tension. This results in an increase in subendocardial myocardial blood flow. Nitrates also dilate large- and medium-sized coronary arteries
and arterioles >100 mm in diameter. This effect reduces left ventricular systolic wall tension via decreasing afterload and, therefore, also decreases myocardial
oxygen demand. The hemodynamic effects of nitrates on the coronary vasculature also relieve angina. Nitrates dilate the epicardial coronary arteries, including
stenotic segments, and also improve blood flow in coronary collaterals via decreasing resistance to collateral flow. Nitrates, particularly sodium nitroprusside, can also
dilate the systemic arterial bed, and NO gas and sodium nitroprusside dilate the pulmonary vascular bed and inhibit hypoxia-induced pulmonary vasoconstriction.
O2 ¼ oxygen.

studied the effects of nitroglycerin and dipyridamole diameter, which reduces the risk of ischemia from
on coronary collateral blood flow in normal dogs coronary steal (91). Nitrates, particularly sodium
and dogs with chronic myocardial ischemia produced nitroprusside, can also dilate the systemic arterial bed,
by the placement of ameroid constrictors on the left and NO gas and sodium nitroprusside dilate the pul-
anterior descending and left circumflex arteries. monary vascular bed and inhibit hypoxia-induced
Unlike dipyridamole, they found that nitroglycerin pulmonary vasoconstriction (68).
“did not cause a significant reduction in either
retrograde flow or peripheral coronary pressure in NONHEMODYNAMIC EFFECTS OF NITRATES
spite of a greater fall in mean arterial pressure.”
Additionally, when arterial pressure was held The organic nitrates have also been found to have
constant, nitroglycerin caused a large increase of several important, nonhemodynamic effects. Inhibi-
retrograde flow in the dogs with chronic myocardial tion of platelet function by organic nitrates was first
ischemia (90). Importantly, the organic nitrates do not reported in 1967 by Hampton et al. (92), who showed
dilate coronary microvessels that are <100 m m in that nitroglycerin impairs platelet aggregation
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NOVEMBER 7, 2017:2393–410 Nitroglycerin and Nitric Oxide in Cardiovascular Therapeutics

in vitro. Subsequently, the organic nitrates have been difference in mortality in patients who received ni-
shown to inhibit platelet aggregation and function trate therapy (105). The ISIS-4 (Fourth International
by increasing intracellular cGMP and forming Study of Infarct Survival) randomized 58,050 pa-
S-nitrosothiols, which are potent activators of sGC tients presenting up to 24 h after the onset of a
and inhibitors of platelet aggregation (46,93). Organic suspected acute myocardial infarction in a 2-by-2
nitrates also have other antithrombotic effects, as factorial design that involved treatment with capto-
activation of sGC is accompanied by inhibition of pril, isosorbide mononitrate, magnesium sulfate,
agonist-mediated calcium flux, which results in a and/or placebo. There was no significant reduction in
reduction of fibrinogen binding to the glycoprotein mortality attributed to nitrate therapy (106). Of note,
Ilb/IIIa receptor of platelets (94). both the GISSI-3 and ISIS-4 trials were conducted in
Nitrates have also been found to have anti- the fibrinolytic era (i.e., prior to the percutaneous
inflammatory effects via NO’s role in the inflamma- coronary intervention era). By contrast, however, a
tory process (95). NO inhibits neutrophil adhesion meta-analysis of 10 trials, performed in the pre-
and chemotaxis in acute inflammation and modu- fibrinolytic era (and pre-dating the GISSI-3 and
lates microvascular permeability (96–99). However, ISIS-4 trials), of patients randomized to intravenous
increased expression of iNOS has been implicated in nitroglycerin or sodium nitroprusside in acute
chronic inflammatory conditions, as high-flux NO from myocardial infarction showed a reduction in mortal-
iNOS or its derivates in the inflammatory milieu (e.g., ity of 35% associated with nitrate therapy, with the
peroxynitrite) promotes adhesion molecule expres- greatest reduction in mortality occurring during the
sion and leukocyte–endothelial cell interactions, first week of follow-up (107).
among other pro-inflammatory mechanisms (99). Nitroglycerin is the drug most frequently used to
Finally, nitroglycerin has been shown to induce a treat acute episodes of angina. It is usually given as a
protective phenotype that limits damage after sublingual tablet, but is also available as a sublingual
ischemia and reperfusion. Nitroglycerin particularly spray. Sublingual nitroglycerin or nitroglycerin oral
protects against post-ischemic endothelial dysfunc- spray can also be used prior to angina-inducing ac-
tion, in part by impairing the opening of the mito- tivities to prevent the occurrence of acute angina
chondrial permeability transition pore (100,101). (53,108,109).
Winsor and Berger (110) studied 53 patients with
CLINICAL USES OF NITRATES documented angina, and concluded in 1975 that
controlled-release oral nitroglycerin provided a sta-
Organic nitrates, in intravenous, sublingual, and oral tistically significant clinical improvement of angina.
formulations, are often used in the management of In 1974, Reichek et al. (111) published their work on 14
acute coronary syndrome (102,103). Treatment with patients with angina pectoris and concluded that
nitrates causes vasodilation of the capacitance veins nitroglycerin ointment produced a significant in-
and results in reduced ventricular filling pressure, wall crease in exercise capacity, which persisted for at
tension, and myocardial oxygen demand (53). As pre- least 3 h. Several studies have also shown an
viously discussed, nitrates also dilate the epicardial improvement in exercise capacity without angina via
coronary arteries, which improves coronary blood use of transdermal preparations (112–116). Taken
flow, particularly in ischemic zones (87,89). together, all of these studies support the use of oral
These well-demonstrated physiological effects organic nitrates to increase exercise tolerance, pre-
notwithstanding, randomized controlled trial data vent angina, and improve chronic stable angina. In
supporting a clinical outcome benefit for the use of current practice, isosorbide dinitrate and isosorbide
nitrates in acute coronary syndrome are lacking. The mononitrate are often used for these indications (53).
GISSI-3 (Gruppo Italiano per lo Studio della Soprav- Although not available in the United States, nic-
vivenza nell’Infarto Miocardico 3) trial randomly orandil is used in the treatment of chronic, stable
assigned 19,394 patients with acute myocardial angina in other countries. In the IONA (Impact of
infarction in a 2-by-2 factorial design to intravenous Nicorandil in Angina) trial, treatment with nicorandil
nitroglycerin followed by a nitrate patch or placebo, in patients with stable angina statistically signifi-
as well as to lisinopril or placebo. The primary study cantly reduced the primary endpoint of coronary
endpoint of mortality at 6 weeks demonstrated no death, nonfatal myocardial infarction, or unplanned
significant benefit of nitrate therapy. The subset of hospitalization for angina (117).
patients receiving combination therapy with lisino- The established treatment for Prinzmetal variant
pril and nitrates, however, had the lowest mortality angina, or coronary artery spasm, is therapy with
in the trial (104). At 6 months, there was no calcium-channel blockers. Long-acting oral nitrates
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have also been found to be helpful, and it is thought Cochrane review does not support its use for respi-
that their vasodilatory effects are additive to calcium- ratory failure in pre-term infants (129).
channel blockade in this setting (118,119).
Intravenous nitroglycerin and sodium nitroprus- NITRATE TOLERANCE
side are used to treat patients hospitalized with hy-
pertensive urgency and emergency with preference to One major problem with the use of nitrates is the
sodium nitroprusside, as it induces more arterial development of tolerance, which is defined as “the
vasodilation than nitroglycerin. Although nitrates loss of hemodynamic and anti-anginal effects during
have generally not been proven useful in the man- sustained therapy” (53). Tolerance occurs following
agement of hypertension, they are considered one of chronic exposure to all nitrates and results in
the drug classes-of-choice for patients with hyper- “complete or markedly diminished anti-anginal and
tension and stable angina (120). They should ideally anti-ischemic effects throughout the 24-h period of
be used in combination with beta-blockers, or alone if long-term therapy” (130). In 1980, Thadani et al. (131)
beta-blockers are contraindicated or cause unaccept- reported their studies of tolerance to oral isosorbide
able side effects (120). dinitrate and showed that both partial circulatory
Nitrates were previously considered first-line ther- tolerance to isosorbide dinitrate and cross-tolerance
apy for patients with acute heart failure with normal or to nitroglycerin developed rapidly during treatment
high blood pressures owing to their previously with isosorbide dinitrate given every 6 h. They went
described hemodynamic effects on the venous and on to report similar findings regarding its antianginal
arterial systems, pre-load, and afterload (121). A recent effect 2 years later (132). Subsequent studies reported
Cochrane review, however, found no significant dif- similar findings with different formulations of nitrate
ference between nitrate vasodilator therapy and therapy (61,115,130,133–138).
alternative interventions for the treatment of acute The cause of nitrate tolerance is still incompletely
heart failure syndromes with regard to symptom relief understood, but several hypotheses exist. One hy-
and hemodynamic variables (122). Given the striking pothesis argues that chronic treatment with organic
results of the African-American Heart Failure Trial nitrates triggers supersensitivity to vasoconstrictors,
however, the combination of hydralazine and iso- which attenuates the vasodilator effects of nitrates.
sorbide dinitrate, together with angiotensin- This action is mediated by increased autocrine levels
converting enzyme inhibitors, beta-blockers, and of endothelin within the vasculature, with the sub-
aldosterone antagonists, is recommended by current sequent activation of phospholipase C and protein
guidelines for African-Americans who require further kinase C. These pathways lead to increased actomy-
blood pressure control and relief of symptoms from osin activity and myocyte contractility. Additionally,
New York Heart Association functional class III or IV agonist-driven calcium-dependent activation of the
chronic heart failure (120,123,124). This combination RhoA/Rho kinase pathway contributes to vasocon-
can also be useful clinically in patients with chronic striction via inhibition of myosin light chain phos-
systolic heart failure who cannot be given angiotensin- phatase (139–141).
converting enzyme inhibitors or angiotensin receptor Another hypothesis for the mechanism of
blockers (124). nitrate tolerance is that chronic therapy with organic
Inhaled NO gas has several clinical uses in adults. It nitrates desensitizes sGC (142,143). S-nitrosylation of
has a well-established role in vasoreactivity testing in sGC is a means by which “memory” of NO exposure
patients with pulmonary arterial hypertension. This is retained in smooth muscle cells, resulting in
testing is helpful to identify patients who may decreased responsiveness to NO, and could be a
respond to therapy with calcium-channel blockers mechanism of NO tolerance (139–144). Nitroglycerin
(71,125). Although robust data are lacking, inhaled NO metabolism also promotes the production of reactive
gas is also used in patients both with and without a oxygen species. Oxidation of thiol groups in the
prior diagnosis of pulmonary hypertension with acute active site of ALDH-2 by these reactive derivatives has
hypoxemic respiratory failure to improve ventilation- been observed during chronic nitroglycerin treat-
perfusion matching (126). Inhaled NO has an estab- ment. This post-translational modification may cause
lished role in the treatment of severe persistent inhibition of ALDH-2 enzyme activity, which can lead
pulmonary hypertension of the newborn, and a to both reduced nitroglycerin biotransformation and
recently published Cochrane review also supports its efficacy (139,145–147).
use in the treatment of term and near-term infants Additionally, continuous treatment with nitro-
with hypoxic respiratory failure who do not have a glycerin has been shown to cause NO synthase
diaphragmatic hernia (127,128). By contrast, a recent dysfunction, likely through the reduced
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NOVEMBER 7, 2017:2393–410 Nitroglycerin and Nitric Oxide in Cardiovascular Therapeutics

bioavailability of tetrahydrobiopterin (148). NO syn- including sodium thiosulfate, sodium nitrite, amyl
thase dysfunction can cause superoxide anion for- nitrite, and hydroxocobalamin, as well as potential
mation. If not effectively mitigated by superoxide antidotes that are undergoing development, such as
dismutases, increased superoxide anion formation sulfanegen sodium. Sodium thiosulfate is available
can lead to the formation of peroxynitrite anion intravenously, and acts as a sulfur donor to plasma
(through reaction with NO), which is a highly reactive rhodanase, an enzyme that transforms cyanide to
intermediate that promotes oxidant stress and re- thiocyanate, a nontoxic derivative (168). Intravenous
duces NO bioavailability (139,149,150). sodium nitrite and inhaled amyl nitrite are methe-
Nitrate therapy–induced increases in phosphodi- moglobin generators, and methemoglobin’s strong
esterase activity have also been implicated in the affinity for cyanide binding is the rationale for their
development of tolerance (151). As previously dis- use as antidotes (169). Hydroxocobalamin is available
cussed, nitroglycerin is metabolized to NO, which intravenously and contains a cobalt moiety, which
activates sGC to increase cGMP. Phosphodiesterase avidly binds to intracellular cyanide forming cyano-
decreases cGMP levels, and, therefore, NO-induced cobalamin (169–171). Sulfanegen sodium is a prodrug
vasodilation is attenuated by its activity. of 3-mercaptopyruvate that converts cyanide to
Finally, pseudotolerance also complicates chronic thiocyanate (168,172). It was shown to be effective in
treatment with organic nitrates. This adaptive phe- reversing cyanide toxicity in a juvenile pig model, but
nomenon is not considered true vascular tolerance work is ongoing to develop it into an antidote that is
because it occurs in response to every form of vaso- safe for use in humans (173).
dilator therapy. Pseudotolerance is marked by Additionally, there are certain clinical situations
neurohormonal activation, increased catecholamine in which nitrates should not be used, or should be
release rates and circulating catecholamine levels, used with extreme caution. Nitrates should not
sodium retention, and intravascular volume expan- be given to patients who have used a phosphodies-
sion (139,152). terase inhibitor, such as sildenafil or tadalafil, within
Although several agents have been studied for use 24 to 48 h due to the risk of severe hypotension
in the prevention of nitrate tolerance, none are (174,175). Nitrates should also be avoided in
currently recommended due to a paucity of supportive suspected causes of right ventricular infarction, as
clinical data (130,136,153–160). Only 1 method nitrate-induced dilation of the venous capacitance
of preventing nitrate tolerance remains widely beds could cause hypotension given the need for
accepted: the use of a dosing strategy that provides an high filling pressures in this setting (176). Patients
interval of no or low nitrate exposure during each 24-h with hypertrophic cardiomyopathy should not be
period (116,130,137,161–163). Nitrate tolerance is placed on nitrate therapy, as it can increase outflow
rapidly reversed during a nitrate-free interval (53,164). tract obstruction by decreasing pre-load and
ventricular volumes (177).
ADVERSE EFFECTS OF NITRATES
NOVEL MODULATORS OF THE
In addition to the development of tolerance, there are NO-sGC-cGMP PATHWAY
other important considerations to treatment with
organic nitrates. Common side effects of nitrate The organic nitrates have been and continue to be
therapy include headache, flushing, lightheadedness, mainstays in the treatment of several acute and
and postural hypotension. When sodium nitroprus- chronic cardiovascular conditions. Although many of
side is infused, it interacts with oxyhemoglobin to the organic nitrates have been used clinically for de-
form methemoglobin and releases NO and cyanide. cades, there are several new drugs and drug classes
Therefore, patients receiving sodium nitroprusside that have been approved recently or are currently
must be monitored for manifestations of both cyanide being studied that have the potential to add to the list
toxicity, such as altered mental status, seizure, and of modulators of the NO-sGC-cGMP pathway (178).
metabolic acidosis; and methemoglobinemia, such Their methods of modulation of this pathway are
as cyanosis, headache, fatigue, and lethargy summarized in Figure 5.
(68,69,165,166). Cyanide toxicity from sodium nitro- Caveolin-1 (CAV1) is the main coat protein of cav-
prusside therapy is rare and is unlikely if the cumu- eolae, which are invaginations in the plasma mem-
lative dose of nitroprusside does not exceed brane implicated in several biological processes,
0.5 mg/kg/h. Large-infusion doses of nitroglycerin including signaling, in endothelial cells (178–182).
can also cause methemoglobinemia (167). There are eNOS activity is decreased when bound to CAV1
several antidotes available to treat cyanide toxicity, (183,184). Cavnoxin, a peptide that activates eNOS,
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Nitroglycerin and Nitric Oxide in Cardiovascular Therapeutics NOVEMBER 7, 2017:2393–410

F I G U R E 5 Novel Ways of Modulating the NOsGC-cGMP Pathway

Increase Introduce Enhance Modulate the Inhibit NADH- Disrupt hemoglobin
Reverse eNOS
Endogenous Cavnoxin-like transcription of enterosalivary cytochrome b5 α’s binding site for
uncoupling
Cavnoxin peptides eNOS nitrate circulation reductase 3 eNOS

eNOS Fe2+-hemoglobin α
activation to
Fe3+-hemoglobin α

Increase endogenous NO production Increase NO bioavailability

Inhibit
Stimulate sGC
phosphodiesterases

Increase cGMP Decrease cGMP
production breakdown

Several novel modulators of the NO-sGC-cGMP pathway are undergoing active investigation, and the details are summarized in the main text. Methods of increasing
endogenous cavnoxin production or introducing synthesized cavnoxin-like peptides could increase endogenous NO production. Transcriptional enhances of eNOS, the
small molecules AVE3085 and AVE9488, have increased endogenous NO production, reversed eNOS uncoupling, and decreased eNOS production of superoxide anion in
mice. The enterosalivary nitrate circulation (nitrate-nitrite-NO pathway) is a source of NO that is derived from dietary inorganic nitrate intake, and production of NO
from this pathway is enhanced by hypoxia and acidosis. Research dedicated to exploring therapeutic options, such as prebiotics, probiotics, and antimicrobial agents,
that can modulate the microbiome and the nitrate-nitrite-NO pathway in heart failure, pulmonary hypertension, hypertension, obesity, and other cardiovascular
disease states is ongoing. Increasing the bioavailability of endogenous NO is another potential approach to modulating the NO-sGC-cGMP pathway. The oxidized form of
hemoglobin-a has a much lower affinity for NO than the reduced form, and, therefore, allows eNOS-generated NO to diffuse to underlying vascular smooth muscle
cells. Because NADH-cytochrome b5 reductase 3 reduces Fe3þ–hemoglobin-a to Fe2þ–hemoglobin-a, a potential way to increase NO bioavailability would be to inhibit
NADH-cytochrome b5 reductase 3. Another possible strategy would be to disrupt the binding site of hemoglobin-a for eNOS, and a small peptide, hemoglobin-aX, has
been developed as just such an inhibitor. Finally, modulating the NO-sGC-cGMP pathway in an NO-independent fashion is also being studied. The ciguats modulate sGC
activity to increase cGMP production independently of NO. The phosphodiesterases hydrolyze the phosphodiester bond of cGMP. Inhibition of these enzymes de-
creases cGMP breakdown. NADH ¼ nicotinamide adenine dinucleotide; Fe ¼ iron; other abbreviations as in Figure 2.

was identified by studying the key residues in CAV1 oxidized form of hemoglobin-a has a much lower af-
responsible for inhibition of eNOS function (185). In finity for NO than the reduced form, and, therefore,
wild-type mice, cavnoxin increases NO levels, causes a allows eNOS-generated NO to diffuse to underlying
reduction in vascular tone, and lowers systemic blood vascular smooth muscle cells (178,187). Nicotinamide
pressure (185). Therefore, novel methods of increasing adenine dinucleotide (NADH)–cytochrome b5
endogenous cavnoxin production or introducing reductase 3 reduces Fe3þ–hemoglobin-a to Fe 2þ–
synthesized cavnoxin-like peptides could be a prom- hemoglobin-a . Therefore, yet another potential
ising way to increase endogenous NO production. way to increase NO bioavailability would be
Another approach to increasing endogenous NO is to inhibit NADH-cytochrome b5 reductase 3. Still
to increase NO bioavailability by affecting hemoglo- another possible strategy would be to disrupt the
bin- a ’s ability to complex with eNOS. In the micro- binding site of hemoglobin- a for eNOS; a small pep-
circulation, hemoglobin-a forms a macromolecular tide, hemoglobin-a X, has been developed for this
complex with eNOS and controls the flux of purpose. Hemoglobin- a X has been found to decrease
bioavailable NO (178,186). The reduced Fe 2þ–O2 – blood pressure in mice and dilate constricted arteri-
hemoglobin-a reacts with NO rapidly and generates oles isolated from patients with hypertension
nitrate and the oxidized Fe3þ–hemoglobin- a . This (186,188).
JACC VOL. 70, NO. 19, 2017 Divakaran and Loscalzo 2405
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Modulating the transcription, and therefore, NOsGC stimulator that is approved for clinical
translation of NOS is another focus of research and use (196). Riociguat is currently approved for the
development efforts to augment levels of endoge- treatment of pulmonary arterial hypertension and
nous NO. Two small molecules, AVE3085 and inoperable chronic thromboembolic pulmonary hy-
AVE9488, have been identified as transcriptional pertension (178,197,198). There are ongoing trials of
enhancers of eNOS that bind its promoter (178,189). In riociguat’s potential role in the treatment of pulmo-
apolipoprotein E knockout mice, 12 weeks of treat- nary hypertension and heart failure. Other NOsGC-
ment with AVE9488 or AVE3085 reduced atheroscle- stimulating ciguats that are being studied include
rotic plaque formation. Treatment of these mice with vericiguat, which was shown to decreased circulating
AVE9488 also reversed eNOS uncoupling, increased levels of N-terminal pro–B-type natriuretic peptide
vascular content of the essential eNOS cofactor in patients with worsening chronic systolic heart
tetrahydro-L-biopterin (BH4 ), and reduced vascular failure, as well as nelociguat, IW-1973, and IW-1701
cuff-induced neointima formation (189). Four weeks (178,199,200).
of treatment with AVE3085 was shown to attenuate Cinaciguat was identified as an NOsGC activator
cardiac remodeling in an experimental mouse model via high-throughput screening (201,202). Its clinical
involving aortic banding to induce remodeling (190). use has been limited due to significant hypotension.
Finally, 9 weeks of treatment with AVE9488 was In a placebo-controlled trial of 139 patients admitted
shown to improve left ventricular remodeling and with acute, decompensated, systolic heart failure,
contractile dysfunction in an experimental myocar- cinaciguat significantly decreased pulmonary capil-
dial infarction rat model (191). lary wedge pressure, right atrial pressure, pulmo-
As referred to in the previous text with regard to nary vascular resistance, and systemic vascular
treatment with AVE9488, reversing eNOS uncoupling resistance, and also significantly increased cardiac
is desired as it decreases eNOS production of super- index. However, the trial was stopped prematurely
oxide anion (50). Tetrahydrobiopterin has been due to an increased occurrence of hypotension at
shown to improve endothelial dysfunction in patients cinaciguat doses >200 m g/h (203). At lower doses,
with type 2 diabetes mellitus and in patients on cinaciguat has been shown to decrease systemic
cyclosporine A after cardiac transplantation (52,192). blood pressure without improving dyspnea or
However, a study of 49 patients randomized to cardiac index in patients with acute heart failure,
receive 400 mg/day of BH4 , 700 mg/day of BH 4, or therefore limiting its use in this population (204).
placebo for 2 to 6 weeks before coronary artery bypass Another NOsGC activator that is under development
graft surgery found no effect of BH 4 treatment on is ataciguat (178).
vascular function or superoxide production (193). Another class of NOsGC-cGMP pathway modula-
More research is needed to explore alternative ways tors is the phosphodiesterases. These enzymes
to reverse eNOS uncoupling and, thereby, increase inhibit the pathway by hydrolyzing the phospho-
NO production and decrease superoxide anion diester bond of cGMP (178,205). There are 4 phos-
production. phodiesterase 5 inhibitors in current clinical use that
Ignarro et al. (194) showed that sGC activity could inhibit cGMP breakdown: sildenafil, vardenafil,
be modulated in an NO-independent fashion by pro- tadalafil, and avanafil. All 4 are approved for use in
toporphyrin IX, an sGC activator. Protoporphyrin IX erectile dysfunction. Sildenafil and tadalafil are also
was never used clinically due to its severe photo- used for pulmonary arterial hypertension, and
sensitizing effect (178). However, a recently devel- tadalafil is approved for use in benign prostatic
oped class of drugs called the ciguats also modulate hyperplasia (178). Work is ongoing to develop addi-
the NO–sGC–cGMP pathway similarly in an NO- tional phosphodiesterase inhibitors, including in-
independent fashion (178). There are 2 subgroups of hibitors of nonselective phosphodiesterases and
ciguats: NOsGC stimulators (which bind NOsGC and cGMP-selective phosphodiesterases (phosphodies-
act through allosteric regulation) and NOsGC activa- terases 5, 6, and 9) (178).
tors (which occupy the heme binding site of NOsGC Finally, as discussed above, the enterosalivary ni-
and work additively with NO) (178). Lificiguat was the trate circulation (nitrate–nitrite–NO pathway) is a
first NOsGC stimulator that was identified when it source of NO that is derived from dietary inorganic
was found to stimulate sGC in rabbit platelets during nitrate intake (38,39,206). Production of NO from this
a small-molecule screen designed to identify novel pathway is enhanced by hypoxia and acidosis
platelet aggregation inhibitors (195). Further work to (207,208). There is, therefore, interest in studying the
improve the solubility and efficacy of lificiguat led to use of inorganic nitrates to improve exercise capacity,
the development of riociguat, which is the only particularly in patients with heart failure with
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Nitroglycerin and Nitric Oxide in Cardiovascular Therapeutics NOVEMBER 7, 2017:2393–410

preserved left ventricular ejection fraction (206). In symptoms despite the paucity of evidence of benefit
addition, there is ongoing research dedicated to on hard clinical endpoints. As research and develop-
exploring therapeutic options, such as prebiotics, ment of new ways to modulate the NO-sGC-cGMP
probiotics, and antimicrobial agents, that can modu- pathway continue, it may be time to revisit the
late the microbiome and the nitrate–nitrite–NO study of nitrates in large, prospective clinical trials in
pathway in heart failure, pulmonary hypertension, the percutaneous coronary intervention era.
hypertension, obesity, and other cardiovascular dis-
ACKNOWLEDGMENT The authors thank Ms.
ease states (39).
Stephanie Tribuna for expert technical assistance.
CONCLUSIONS
ADDRESS FOR CORRESPONDENCE: Dr. Joseph Loscalzo,
The use of nitrates in cardiovascular disease has a Department of Medicine, Brigham and Women’s
long and storied history. They continue to play a Hospital, 75 Francis Street, Boston, Massachusetts 02115.
major role in current clinical practice to improve E-mail: jloscalzo@rics.bwh.harvard.edu.

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