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THE PRESENT AND FUTURE

STATE-OF-THE-ART REVIEW

The Role of Nitroglycerin andOther Nitrogen Oxides inCardiovascular Therapeutics
Sanjay Divakaran, MD, Joseph Loscalzo, MD, PHD
ABSTRACT

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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 ofangina; the history of the discovery of nitric

oxide (NO), its sources, and its roles; the mechanismof action, preparations, and hemodynamic and non-hemodynamic effects of the organic nitrates, as wellas their biotransformation; and the clinical uses andadverse effects of nitrate therapy, including toler-ance. We conclude by outlining current and futurework investigating novel modulators of the NO–

soluble guanylyl cyclase (sGC)–cyclic guanosinemonophosphate (cGMP) pathway that may result in

m the Department of Medicine, Brigham and Women’s Hospital, Harvar

s supported in part by National Institutions of Health grants HL61795 an

rted that they have no relationships relevant to the contents of this pape

nuscript received September 7, 2017; accepted September 19, 2017.

new therapeutic options in the treatment of cardio-vascular disease.

EARLY HISTORY OF THE USE OF NITRATES IN

CORONARY ARTERY DISEASE

William Heberden is credited with coining the term“angina pectoris” in 1772 (1). Joseph Priestley, anEnglish theologian and chemist, discovered NO 3years later (2). Not until the next century, however,was NO and its congeners, organic nitrates, linked tothe treatment of angina (3). Glyceryl trinitrate, or

d Medical School, Boston, Massachusetts. This work

d GM107618 (to Dr. Loscalzo). Both authors have re-

r to disclose.

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https://doi.org/10.1016/j.jacc.2017.09.1064

ABBR EV I A T I ON S

AND ACRONYMS

ALDH = aldehyde

dehydrogenase

BH4 = tetrahydro-L-biopterin

cAMP = cyclic adenosine

monophosphate

CAV1 = caveolin-1

cGMP = cyclic guanosine

monophosphate

EDRF = endothelium-derived

relaxing factor

eNOS = endothelial nitric oxide

synthase

iNOS = cytokine-inducible

nitric oxide synthase

nNOS = neuronal nitric oxide

synthase

NO = nitric oxide

NOS = nitric oxide synthase

P450 = cytochrome P450

enzyme(s)

sGC = soluble guanylyl cyclase

VEGF = vascular endothelial

growth factor

Divakaran and Loscalzo J A C C V O L . 7 0 , N O . 1 9 , 2 0 1 7

Nitroglycerin and Nitric Oxide in Cardiovascular Therapeutics N O V E M B E R 7 , 2 0 1 7 : 2 3 9 3 – 4 1 0

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nitroglycerin, was first synthesized byAscanio Sombrero in Turin, Italy. In 1847, henoted that “a very minute quantity put onthe tongue produced a violent headache forseveral hours” (4,5). Three year earlier, theFrench chemist Antoine Balard synthesizedamyl nitrite (4). Frederick Guthrie, an En-glish chemist, explored the actions of amylnitrite and published in 1859 that when itwas held near the nostrils, “after a lapse ofabout 50 seconds, a sudden throbbing of thearteries of the neck is felt, immediatelyfollowed by a flushing of neck, temples,and forehead and an acceleration action ofthe heart” (6).

T. Lauder Brunton, a Scottish physicianand medical scientist, first described theclinical effectiveness of amyl nitrite (4,5,7).Brunton began caring for patients withangina pectoris as a house physician at theEdinburgh Royal Infirmary (7). At the time,many treatments, including therapeuticbleeding, were being used to treat angina,largely unsuccessfully. In 1867, Bruntonpublished the first report of the use of amyl

nitrite in the treatment of angina pectoris (7,8). Hebelieved “the relief produced by [therapeutic]bleeding to be due to the diminution it occasioned inthe arterial tension” and “that a substance whichpossesses the power of lessening it in such aneminent degree as nitrite of amyl would probablyproduce the same effect, and might be repeatedas often as necessary without detriment to thepatient’s health” (7,8). In 1903, Charles-ÉmileFrançois-Franck, a French physiologist, firstsuggested that amyl nitrite was a coronary vasodi-lator (7).

In 1879, William Murrell described the symptom-atic effects of placing drops of 1% solution ofnitroglycerin in alcohol on the tongue (9). In addi-tion to reporting that it relieved angina and pre-vented subsequent attacks, he also reported thesymptoms he felt when he “[tried] its action on[himself]” (9). He described a “violent pulsation in[his] head” and noticed that his pulse was “muchfuller than natural” (9). In 1914, Brunton, whooriginally thought angina was caused by hyperten-sion, acknowledged that the “dilating action of amylnitrite and nitroglycerine upon the coronary vesselswould readily explain the relief they offer in anginapectoris, even in cases where the blood-pressure isnormal” (7).

NO IN THE CARDIOVASCULAR SYSTEM

HISTORY OF NO. In 1975, Diamond and Holmesshowed that tissue levels of cyclic adenosine mono-phosphate (cAMP) were increased in rat myometrialstrips maintained in a state of sustained contracture.They demonstrated that nitroglycerin could relax thedepolarized muscles without significantly increasingcAMP levels and, therefore, concluded that changesin total tissue levels of cAMP were not responsible forthe uterine relaxation caused by nitroglycerin (10).These investigators went on to show that nitroglyc-erin increased cyclic guanosine monophosphate(cGMP) levels in depolarized muscle (10). Thefollowing year, Diamond and Blisard showed that 200mmol/l nitroglycerin increased levels of cGMP by morethan 15-fold during relaxation of isolated strips ofphenylephrine-contracted canine femoral arterieswhile having no significant effect on cAMP levels (11).In 1977, pharmacologist Ferid Murad and his col-leagues published their seminal work on modulatingcontractility in bovine tracheal smooth muscleshowing that the guanylyl cyclase activators, sodiumnitrite, nitroglycerin, and sodium nitroprusside,increased cGMP levels and relaxed tracheal smoothmuscle (12). They went on to demonstrate that solu-tions of NO gas increased cGMP activity in soluble andparticulate preparations from various tissues in adose-dependent fashion, and that NO alone andin combination with sodium azide, sodium nitrite,hydroxylamine, and sodium nitroprusside increasedcGMP levels to approximately the same degree;they concluded that the 2 methods activateguanylyl cyclase through a “similar but undefinedmechanism” (13). Later, the same group postulatedthat “while the precise mechanism of guanylatecyclase activation by these agents is not known,activation may be due to the formation of NO oranother reactive material since NO also increasedguanylate cyclase activity” (14).

Working independently, Robert Furchgott andJohn Zawadzki published their observations on theimportance of the endothelium in blood vesselrelaxation in 1980 (4,15). They reported that acetyl-choline did not always produce blood vessel relaxa-tion in vitro, even though it was a potent vasodilatorin vivo. They found that loss of relaxation in vitrowas due to “unintentional rubbing of [the rabbitthoracic aorta’s] intimal surface against foreignsurfaces during its preparation.” When this mechan-ical injury was avoided during preparation of thetissue, it always relaxed in response to acetylcholine.

FIGURE 1 Sources of NO

L-Arginine

Acidic environment(stomach, hypoxic/ischemic tissue)

Nitrate reductase(oral flora)

L-Citrulline NOS

NO

NO2–

NO3–

NO is a free radical that is synthesized by the family of NO synthases from L-arginine and

oxygen, yielding L-citrulline as a coproduct. In the blood vessel wall, NO is mainly

produced by endothelial nitric oxide synthase (NOS). There are 2 other isoforms of NOS

that also produce NO from L-arginine: neuronal NOS and cytokine-inducible NOS.

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

environment (pH #3) of the gastric lumen, protonation of nitrite can, in turn, yield

nitrous acid, which can spontaneously decompose to NO. This pathway of NO generation

is referred to as the enterosalivary nitrate circulation (nitrate-nitrite-NO pathway).

NO ¼ nitric oxide; NO2� ¼ inorganic nitrite; NO3

� ¼ inorganic nitrate.

J A C C V O L . 7 0 , N O . 1 9 , 2 0 1 7 Divakaran and LoscalzoN O V E M B E R 7 , 2 0 1 7 : 2 3 9 3 – 4 1 0 Nitroglycerin and Nitric Oxide in Cardiovascular Therapeutics

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Therefore, they concluded that acetylcholine-mediated relaxation of vascular smooth musclerequired the presence of endothelial cells (15).Furchgott and his colleagues eventually proposedthat bradykinin and other molecules acting on theendothelium caused relaxation of vascular smoothmuscle via a substance they termed endothelium-derived relaxing factor (EDRF) (16). The link to NO,however, was not yet appreciated (4).

Louis Ignarro and Salvador Moncada indepen-dently discovered that EDRF is NO in 1987 (4,17).After a series of experiments, Ignarro et al. (18,19)stated that “EDRF released from artery and veinpossesses identical biological and chemical propertiesas NO.” Moncada and colleagues suggested that EDRFand NO were “identical” as “NO released from endo-thelial cells is indistinguishable from EDRF in termsof biological activity, stability, and susceptibility toan inhibitor and to a potentiator” (20). Based on theircollective contributions to the field, the Nobel Prize inPhysiology or Medicine was awarded to Furchgott,Ignarro, and Murad in 1998 “for their discoveriesconcerning NO as a signaling molecule in the cardio-vascular system” (4).SOURCES OF NO. NO is a free radical that is synthe-sized by the family of NO synthases from L-arginineand oxygen, yielding L-citrulline as a coproduct(Figure 1) (21–23). In the blood vessel wall, NO ismainly produced by endothelial nitric oxide synthase(eNOS) (23,24). However, there are 2 other isoforms ofnitric oxide synthase (NOS) that also produce NO fromL-arginine: neuronal nitric oxide synthase (nNOS)and cytokine-inducible nitric oxide synthase (iNOS)(25–27). Although eNOS is the major NOS isoform thatregulates vascular function, both nNOS and iNOS areimplicated as sources in certain tissues and environ-ments (23). For example, vascular injury inducesexpression of nNOS in the neointima and medialsmooth muscle cells, and the production of iNOSwithin vascular smooth muscle cells after exposureto proinflammatory cytokines is a major cause ofvasodilation in sepsis (28,29).

S-nitrosothiols, such as S-nitrosoglutathione andS-nitrosohemoglobin, are also sources of NO (23,24).Under certain conditions, such as in the presenceof trace transition metal ions or photolysis,S-nitrosothiols decompose to liberate NO (30). Inaddition, S-nitrosothiols can undergo trans-S-nitrosation with other thiol groups and therebymodify cell or protein function (31–33).

Several proteins, such as hemoglobin, cytochromeP450 reductase, and cytochrome P450, can catalyze thereduction of nitrite or nitrate to generate NO (23).

Hemoglobin has enzymatic behavior as a nitritereductase under hypoxic conditions and is asensor and effector of hypoxic vasodilation (34). He-moglobin’s maximal nitrite reduction rate occurswhen hemoglobin is 40% to 60% saturated withoxygen (23,34). Cytochrome P450 reductase causes NOrelease by reducing nitrate, and can also facilitategeneration of S-nitrosothiols (35).

Bacterial flora present in the mammalian oralcavity is rich in nitrate reductases and can convertdietary sources of nitrate to nitrite. In the extremelyacidic environment (pH #3) of the gastric lumen,protonation of nitrite can, in turn, produce nitrousacid, which can spontaneously decompose to NO(36,37). This pathway of NO generation is referredto as the enterosalivary nitrate circulation (nitrate–nitrite–NO pathway) (Figure 1) and is a majorsource of NO (38,39).

NO’s ROLES. NO is a powerful vasodilator that in-duces formation of cGMP by activating sGC invascular smooth muscle cells (Figure 2) (13). cGMP canbind to and enhance protein kinase G activity, cGMP-gated ion channels, and cGMP-sensitive phosphodi-esterases (40). Protein kinase G promotes reuptake of

FIGURE 2 Regulation of Vascular Tone by NO

L-Arginine

Endothelial Cell

Vascular SmoothMuscle Cell

Vascular Smooth MuscleRelaxation

Actin-MyosinATPase↓

Nitrovasodilators

Bioactivation

GTP

cGMPsGC

eNOS

cGMP-dep Kinase I

Ca2+

↓Ca2+

MLC-Pi

GMPPDE

MLCMLCK

MLCP

NO

NO

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.



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cytosolic calcium into the sarcoplasmic reticulum, themovement of calcium from the intracellular to theextracellular environment, and the opening ofcalcium-activated potassium channels (23). Thesechanges result in reduction of vascular tone as thereduction in intracellular calcium impairs myosinlight chain kinase’s ability to phosphorylate myosin,resulting in smooth muscle cell relaxation (41).

NO has several other important roles in thevasculature. For example, when vascular endothelialgrowth factor (VEGF) binds to VEGF receptor-2, eNOSis activated and NO is formed (42–44). This NO isessential for VEGF function, as eNOS-deficient micehave markedly reduced vascular permeability andangiogenesis (45). S-nitrosylation of b-catenin by NOmodulates intercellular contacts between endothelialcells, likely accounting for NO-dependent changes inendothelial permeability (42). NO and organic nitratesalso impair platelet activation and aggregation by

activating platelet guanylyl cyclase and increasingintraplatelet cGMP (46–49).

Finally, a greater understanding of the conse-quences of impaired NO synthesis can yield moreinformation regarding the importance of NO’s role in awell-functioning cardiovascular system. For example,when low levels of its essential cofactor (6R)-5,6,7,8-tetrahydrobiopterin are present, eNOS producessuperoxide anion instead of NO in a process denotedenzymatic uncoupling (50). eNOS uncoupling has beenobserved in animal models of cardiovascular diseaseand in patients with cardiovascular risk factors, suchas hypertension and diabetes mellitus (51,52).

MECHANISM OF ACTION OF NITRATES

Organic nitrates, such as nitroglycerin, isosorbidedinitrate, and isosorbide mononitrate, are rapidlyabsorbed from several sites, such as the

FIGURE 3 Chemical Structures of Key Nitrovasodilators

Isosorbidedinitrate

Isosorbidemononitrate Amyl nitriteNitroglycerin

Pentaerythrityltetranitrate

Sodiumnitroprusside Nicorandil

ONO2

ONO2

O2NO

O2NO

ONO2

ONO2

O2NO

ONO

O

O

ONO2

O2NO

H

H

ONO2

O

HN

N

O

O

OH

O2NO

H

H

2–

2Na+ CCN

N CC N

NN

O

N

C

Fe

Chemical structures of 7 key nitrovasodilators are shown.


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gastrointestinal tract, mucous membranes, and skin,depending on the preparation (53). These compoundsare prodrugs with a nitrooxy (�O-NO2) moiety and aremetabolized to produce bioactive metabolites (54,55).The bioactivation of organic nitrates causes liberationof NO, allowing them to serve as NO donors (55,56).NO then causes vasodilation via its effect on vascularsmooth muscle cells, and impairs platelet activationas previously discussed (13).

Nitrates are also implicated in epigenetic regula-tion of vasodilation, including specifically smoothmuscle cell relaxation (57,58). Additionally, nitro-glycerin has been shown to increase the activity ofhistone acetylases with nitroglycerin-dependentvascular responses influenced by N 3-lysine acetyla-tion of contractile proteins (58).

PREPARATIONS

Nitrates are rapidly absorbed from mucous mem-branes, the gastrointestinal tract, and the skin (55).Thus, nitroglycerin is available in several prepara-tions for delivery via several routes: oral tablets,sublingual tablets, buccal tablets, sublingual spray,transdermal ointment, and transdermal patch; it isalso available in intravenous formulations, which are

used in hospitalized patients with angina, hyperten-sion, or heart failure (Figure 3, Table 1) (53,59).Nitroglycerin has a plasma half-life of approximately1 to 4 min; following hepatic and intravascularmetabolism, its biologically active metabolites have ahalf-life of approximately 40 min (53,60).

Isosorbide dinitrate is rapidly absorbed andundergoes extensive first-pass metabolism by theliver, which results in low bioavailability (53,55).Sustained-release formulations have a slower rate ofabsorption and can provide therapeutic plasmaconcentrations of the drug for up to 12 h (53).Oral isosorbide mononitrate is completely absorbedand has 100% bioavailability as it avoids first-passmetabolism. This leads to a more predictable doseresponse and plasma levels with less variation whencompared with other nitrates (55,61). Pentaerythrityltetranitrate is a long-acting organic nitrate availablein an oral formulation that acts within 10 to 20 minand has a duration of action of 8 to 12 h (57).Preclinical data suggested that pentaerythrityltetranitrate may not cause nitrate tolerance (see thefollowing text) or endothelial dysfunction; however,the drug is not currently commonly used, as clinicalstudies have not shown any clear benefit in patientswith chronic, stable angina (62).

TABLE 1 Properties of Key Nitrovasodilators

Drug Route/Formulation DoseTime Until

Onset of ActionDuration of

Action

Nitroglycerin SL spray 0.4 mg 1–3 min 25 min

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

IV infusion 5–400 mg/min Withinseconds

3–5 min

Isosorbide dinitrate PO IR tabletPO SR tablet

5–80 mg40–160 mg

60 min60 min

8 h12 h

Isosorbide mononitrate PO IR tabletPO SR tablet

5–20 mg30–240 mg

30–45 min30–45 min

6 h12–24 h

Amyl nitrite Inhalation ofampule

0.3 ml 30 s 3–15 min

Pentaerythrityltetranitrate

PO tablet 10–80 mg 10–20 min 8–12 h

Sodium nitroprusside IV infusion 0.3–10.0 mg/kg/min 1 min 6–12 min

Nicorandil PO tablet 5–20 mg 30–60 min 12 h

IR ¼ immediate release; IV ¼ intravenous; PO ¼ oral; SL ¼ sublingual; SR ¼ sustained release; TD ¼ transdermal.



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Nicorandil is a nicotinamide-nitrate ester whosechemical structure consists of a nicotinamide deriv-ative combined with a nitrate moiety (55). Nicorandilacts as both an NO donor and a Kþ

ATP channelopener to provide antianginal effects (63). The bio-activation of nicorandil occurs via the nicotinamide/nicotinic acid pathway and involves NO generationvia denitration (64). Nicorandil also dilates thecoronary microvasculature and peripheral resistancearterioles by causing vascular smooth cell hyperpo-larization and closure of L-type voltage-gatedcalcium channels via its action on Kþ

ATP channels;it is rapidly absorbed via the gastrointestinal tract,does not undergo first-pass metabolism, reachesmaximal plasma concentration after 30 to 60 min,and has a half-life of approximately 52 min(55,65,66). Nicorandil has an oral bioavailabilityof >75%, and its antianginal effects last approxi-mately 12 h (55,64,67).

Sodium nitroprusside, which is 44% cyanide byweight, comprises a ferrous ion center complexedwith 5 cyanic moieties and a nitrosyl group (68). It isavailable intravenously and interacts with oxyhemo-globin to produce methemoglobin and spontaneouslyrelease NO and cyanide (69,70). Sodium nitroprussidecauses direct venous and arterial vasodilation, is apotent pulmonary vasodilator, and is an inhibitor ofhypoxia-induced pulmonary vasoconstriction (68). Ithas an almost immediate onset of action and a veryshort half-life: its effects dissipate within 1 to 2 min(68).

Finally, there are several direct NO donors thatare already being used clinically, or have beensynthesized and studied for use particularly inbiomaterials. Inhaled NO gas diffuses rapidly across

the alveolar-capillary membrane and activates sGC inthe subjacent smooth muscle cells in the pulmonaryvasculature (71). N-diazeniumdiolates (NONOates) areNO donors that are synthesized by reacting primary orsecondary amines with NO gas at high pressure andlow temperatures; hydrolysis causes spontaneousdecomposition under physiological conditions andreleases NO (72–74). NO (as N2O3 or �ONOO) can reactwith thiols in vivo to form S-nitrosothiols, such asS-nitrosocysteine and S-nitrosoglutathione (75).S-nitrosothiol compounds can also be synthesizedchemically by reacting thiols with nitrous acid. Whenexposed to physiological fluids, S-nitrosothiolsdecompose to release NO (72). Other newer classes ofdirect NO donors that are actively being studiedinclude furoxans, benzofuroxans, and zeolites(76,77).

BIOTRANSFORMATION OF

ORGANIC NITRATES

Organic nitrates must undergo biotransformationto release a vasoactive molecule (78). The detailedstudy of the biotransformation of nitroglycerinhas suggested a high-potency pathway mediatedby aldehyde dehydrogenase (ALDH)-2 and a low-potency pathway mediated by other enzymes orlow-molecular-weight reductants (Figure 4) (78).

The high-potency pathway is important at clinicallyrelevant nitroglycerin concentrations (<1 mmol/l) andis localized to the mitochondria. The mitochondrialisoform of aldehyde dehydrogenase, ALDH-2, wasfound to be a key enzyme in the biotransformation ofnitroglycerin via this pathway as it generates inor-ganic nitrite and 1,2-glyceryl dinitrate from nitro-glycerin (79). There are 3 proposed mechanisms forthe release of the vasoactive molecule via thispathway: nitrogen oxides are formed via reduction ofinorganic nitrite; NO is formed directly in response tointeraction with ALDH-2; and inorganic nitritereleased from mitochondria may be reduced byxanthine oxidase in the cytoplasm to form NO (78).

The low-potency pathway is important at supra-pharmacological nitroglycerin concentrations (>1 mmol/l),is found in the smooth endoplasmic reticulum, andleads to formation of measurable amounts of NO invascular tissues (80,81). In this pathway, nitroglyc-erin is biotransformed by proteins such as deoxy-hemoglobin, deoxymyoglobin, cytochrome P450,xanthine oxidase, glutathione-S-transferase, glycer-aldehyde-3-phosphate dehydrogenase, or otherALDH isoforms; and by low-molecular-weight re-ductants such as cysteine, N-acetyl-cysteine, thio-salicylic acid, and ascorbate (57,82–84).

FIGURE 4 Bioactivation of Organic Nitrates

Cyt Ox

XO

?

H+

ALDH2

low dose

high dose

sGC

cGMP cGK-I Relaxation

Mitochondrion

High potency nitratesGTN, PETN

PETriN

GDN, PEDN

NO2– NOx

.NO

.NO

.NO

P450 GMNPEMN

SmoothEndoplasmic Reticulum

Low potency nitratesISDN, ISDM

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.


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HEMODYNAMIC EFFECTS OF NITRATES

The organic nitrates have several hemodynamic ef-fects that are mostly mediated through vasodilationof capacitance veins and conductance arteries(Central Illustration) (53). In 1971, Parker et al. (85)published the results of a study of hemodynamicindices and coronary blood flow at rest and duringexercise before and after administration of nitro-glycerin in 15 patients with coronary artery diseaseand concluded that nitroglycerin acted primarily byreducing left ventricular oxygen requirementsthrough a reduction in left ventricular volume (85).NO-mediated dilation of capacitance veins decreasesventricular pre-load, which results in reduction inmyocardial oxygen demand. In 1975, Greenberg et al.(86), after angiographic and hemodynamic assess-ment of 10 patients, concluded that the mechanism ofaction of nitroglycerin “seems to relate best to thedecrease in systolic wall tension.” They also foundthat the end-diastolic wall tension decreased by 57%,

“suggesting the possibility that diastolic coronaryblood flow may be augmented by diminished extra-vascular resistance to flow” (86).

Nitrates also dilate large- and medium-sized coro-nary arteries and arterioles >100 mm in diameter. Thiseffect reduces left ventricular systolic wall tension viadecreasing afterload and, therefore, also decreasesmyocardial oxygen demand (53,87). The hemody-namic effects of nitrates on the coronary vasculature ofatherosclerotic patients also relieve angina. In 1972,Horwitz, Herman, and Gorlin published a study of 70patients (49 with coronary artery disease and 21without) with anginal chest pain and found that 76% ofpatients with coronary artery disease were regularlyrelieved of their pain within 3 min, while only 19%of those without coronary artery disease wereregularly relieved of their pain within 3 min (88).Nitrates dilate the epicardial coronary arteries,including stenotic segments, and also improve bloodflow in coronary collaterals via decreasing resistanceto collateral flow (87,89). In 1964, Fam and McGregor

CENTRAL ILLUSTRATION Hemodynamic Effects of Nitroglycerin

Vasodilation of large- andmedium-sized coronaryarteries and arterioles

↓ Myocardial O2 Consumption

↓ Preload ↓ Left ventricularwall tension

↑ Subendocardialblood flow

Vasodilation ofcapacitance veins

Vasodilation of systemic andpulmonary arterial beds

Improvement in coronarycollateral blood flow

Dilation of stenoticepicardial coronary artery

segments

Vasodilation of conductancearteries

Epicardial coronary arterydilation

Nitroglycerin → NO

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.



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studied the effects of nitroglycerin and dipyridamoleon coronary collateral blood flow in normal dogsand dogs with chronic myocardial ischemia producedby the placement of ameroid constrictors on the leftanterior descending and left circumflex arteries.Unlike dipyridamole, they found that nitroglycerin“did not cause a significant reduction in eitherretrograde flow or peripheral coronary pressure inspite of a greater fall in mean arterial pressure.”Additionally, when arterial pressure was heldconstant, nitroglycerin caused a large increase ofretrograde flow in the dogs with chronic myocardialischemia (90). Importantly, the organic nitrates do notdilate coronary microvessels that are <100 mm in

diameter, which reduces the risk of ischemia fromcoronary steal (91). Nitrates, particularly sodiumnitroprusside, can also dilate the systemic arterial bed,and NO gas and sodium nitroprusside dilate the pul-monary vascular bed and inhibit hypoxia-inducedpulmonary vasoconstriction (68).

NONHEMODYNAMIC EFFECTS OF NITRATES

The organic nitrates have also been found to haveseveral important, nonhemodynamic effects. Inhibi-tion of platelet function by organic nitrates was firstreported in 1967 by Hampton et al. (92), who showedthat nitroglycerin impairs platelet aggregation


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in vitro. Subsequently, the organic nitrates have beenshown to inhibit platelet aggregation and functionby increasing intracellular cGMP and formingS-nitrosothiols, which are potent activators of sGCand inhibitors of platelet aggregation (46,93). Organicnitrates also have other antithrombotic effects, asactivation of sGC is accompanied by inhibition ofagonist-mediated calcium flux, which results in areduction of fibrinogen binding to the glycoproteinIlb/IIIa receptor of platelets (94).

Nitrates have also been found to have anti-inflammatory effects via NO’s role in the inflamma-tory process (95). NO inhibits neutrophil adhesionand chemotaxis in acute inflammation and modu-lates microvascular permeability (96–99). However,increased expression of iNOS has been implicated inchronic inflammatory conditions, as high-fluxNO fromiNOS or its derivates in the inflammatory milieu (e.g.,peroxynitrite) promotes adhesion molecule expres-sion and leukocyte–endothelial cell interactions,among other pro-inflammatory mechanisms (99).

Finally, nitroglycerin has been shown to induce aprotective phenotype that limits damage afterischemia and reperfusion. Nitroglycerin particularlyprotects against post-ischemic endothelial dysfunc-tion, in part by impairing the opening of the mito-chondrial permeability transition pore (100,101).

CLINICAL USES OF NITRATES

Organic nitrates, in intravenous, sublingual, and oralformulations, are often used in the management ofacute coronary syndrome (102,103). Treatment withnitrates causes vasodilation of the capacitance veinsand results in reduced ventricular filling pressure, walltension, and myocardial oxygen demand (53). As pre-viously discussed, nitrates also dilate the epicardialcoronary arteries, which improves coronary bloodflow, particularly in ischemic zones (87,89).

These well-demonstrated physiological effectsnotwithstanding, randomized controlled trial datasupporting a clinical outcome benefit for the use ofnitrates in acute coronary syndrome are lacking. TheGISSI-3 (Gruppo Italiano per lo Studio della Soprav-vivenza nell’Infarto Miocardico 3) trial randomlyassigned 19,394 patients with acute myocardialinfarction in a 2-by-2 factorial design to intravenousnitroglycerin followed by a nitrate patch or placebo,as well as to lisinopril or placebo. The primary studyendpoint of mortality at 6 weeks demonstrated nosignificant benefit of nitrate therapy. The subset ofpatients receiving combination therapy with lisino-pril and nitrates, however, had the lowest mortalityin the trial (104). At 6 months, there was no

difference in mortality in patients who received ni-trate therapy (105). The ISIS-4 (Fourth InternationalStudy of Infarct Survival) randomized 58,050 pa-tients presenting up to 24 h after the onset of asuspected acute myocardial infarction in a 2-by-2factorial design that involved treatment with capto-pril, isosorbide mononitrate, magnesium sulfate,and/or placebo. There was no significant reduction inmortality attributed to nitrate therapy (106). Of note,both the GISSI-3 and ISIS-4 trials were conducted inthe fibrinolytic era (i.e., prior to the percutaneouscoronary intervention era). By contrast, however, ameta-analysis of 10 trials, performed in the pre-fibrinolytic era (and pre-dating the GISSI-3 andISIS-4 trials), of patients randomized to intravenousnitroglycerin or sodium nitroprusside in acutemyocardial infarction showed a reduction in mortal-ity of 35% associated with nitrate therapy, with thegreatest reduction in mortality occurring during thefirst week of follow-up (107).

Nitroglycerin is the drug most frequently used totreat acute episodes of angina. It is usually given as asublingual tablet, but is also available as a sublingualspray. Sublingual nitroglycerin or nitroglycerin oralspray can also be used prior to angina-inducing ac-tivities to prevent the occurrence of acute angina(53,108,109).

Winsor and Berger (110) studied 53 patients withdocumented angina, and concluded in 1975 thatcontrolled-release oral nitroglycerin provided a sta-tistically significant clinical improvement of angina.In 1974, Reichek et al. (111) published their work on 14patients with angina pectoris and concluded thatnitroglycerin ointment produced a significant in-crease in exercise capacity, which persisted for atleast 3 h. Several studies have also shown animprovement in exercise capacity without angina viause of transdermal preparations (112–116). Takentogether, all of these studies support the use of oralorganic nitrates to increase exercise tolerance, pre-vent angina, and improve chronic stable angina. Incurrent practice, isosorbide dinitrate and isosorbidemononitrate are often used for these indications (53).

Although not available in the United States, nic-orandil is used in the treatment of chronic, stableangina in other countries. In the IONA (Impact ofNicorandil in Angina) trial, treatment with nicorandilin patients with stable angina statistically signifi-cantly reduced the primary endpoint of coronarydeath, nonfatal myocardial infarction, or unplannedhospitalization for angina (117).

The established treatment for Prinzmetal variantangina, or coronary artery spasm, is therapy withcalcium-channel blockers. Long-acting oral nitrates



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have also been found to be helpful, and it is thoughtthat their vasodilatory effects are additive to calcium-channel blockade in this setting (118,119).

Intravenous nitroglycerin and sodium nitroprus-side are used to treat patients hospitalized with hy-pertensive urgency and emergency with preference tosodium nitroprusside, as it induces more arterialvasodilation than nitroglycerin. Although nitrateshave generally not been proven useful in the man-agement of hypertension, they are considered one ofthe drug classes-of-choice for patients with hyper-tension and stable angina (120). They should ideallybe used in combination with beta-blockers, or alone ifbeta-blockers are contraindicated or cause unaccept-able side effects (120).

Nitrates were previously considered first-line ther-apy for patients with acute heart failure with normal orhigh blood pressures owing to their previouslydescribed hemodynamic effects on the venous andarterial systems, pre-load, and afterload (121). A recentCochrane review, however, found no significant dif-ference between nitrate vasodilator therapy andalternative interventions for the treatment of acuteheart failure syndromes with regard to symptom reliefand hemodynamic variables (122). Given the strikingresults of the African-American Heart Failure Trialhowever, the combination of hydralazine and iso-sorbide dinitrate, together with angiotensin-converting enzyme inhibitors, beta-blockers, andaldosterone antagonists, is recommended by currentguidelines for African-Americans who require furtherblood pressure control and relief of symptoms fromNew York Heart Association functional class III or IVchronic heart failure (120,123,124). This combinationcan also be useful clinically in patients with chronicsystolic heart failure who cannot be given angiotensin-converting enzyme inhibitors or angiotensin receptorblockers (124).

Inhaled NO gas has several clinical uses in adults. Ithas a well-established role in vasoreactivity testing inpatients with pulmonary arterial hypertension. Thistesting is helpful to identify patients who mayrespond to therapy with calcium-channel blockers(71,125). Although robust data are lacking, inhaled NOgas is also used in patients both with and without aprior diagnosis of pulmonary hypertension with acutehypoxemic respiratory failure to improve ventilation-perfusion matching (126). Inhaled NO has an estab-lished role in the treatment of severe persistentpulmonary hypertension of the newborn, and arecently published Cochrane review also supports itsuse in the treatment of term and near-term infantswith hypoxic respiratory failure who do not have adiaphragmatic hernia (127,128). By contrast, a recent

Cochrane review does not support its use for respi-ratory failure in pre-term infants (129).

NITRATE TOLERANCE

One major problem with the use of nitrates is thedevelopment of tolerance, which is defined as “theloss of hemodynamic and anti-anginal effects duringsustained therapy” (53). Tolerance occurs followingchronic exposure to all nitrates and results in“complete or markedly diminished anti-anginal andanti-ischemic effects throughout the 24-h period oflong-term therapy” (130). In 1980, Thadani et al. (131)reported their studies of tolerance to oral isosorbidedinitrate and showed that both partial circulatorytolerance to isosorbide dinitrate and cross-toleranceto nitroglycerin developed rapidly during treatmentwith isosorbide dinitrate given every 6 h. They wenton to report similar findings regarding its antianginaleffect 2 years later (132). Subsequent studies reportedsimilar findings with different formulations of nitratetherapy (61,115,130,133–138).

The cause of nitrate tolerance is still incompletelyunderstood, but several hypotheses exist. One hy-pothesis argues that chronic treatment with organicnitrates triggers supersensitivity to vasoconstrictors,which attenuates the vasodilator effects of nitrates.This action is mediated by increased autocrine levelsof endothelin within the vasculature, with the sub-sequent activation of phospholipase C and proteinkinase C. These pathways lead to increased actomy-osin activity and myocyte contractility. Additionally,agonist-driven calcium-dependent activation of theRhoA/Rho kinase pathway contributes to vasocon-striction via inhibition of myosin light chain phos-phatase (139–141).

Another hypothesis for the mechanism ofnitrate tolerance is that chronic therapy with organicnitrates desensitizes sGC (142,143). S-nitrosylation ofsGC is a means by which “memory” of NO exposureis retained in smooth muscle cells, resulting indecreased responsiveness to NO, and could be amechanism of NO tolerance (139–144). Nitroglycerinmetabolism also promotes the production of reactiveoxygen species. Oxidation of thiol groups in theactive site of ALDH-2 by these reactive derivatives hasbeen observed during chronic nitroglycerin treat-ment. This post-translational modification may causeinhibition of ALDH-2 enzyme activity, which can leadto both reduced nitroglycerin biotransformation andefficacy (139,145–147).

Additionally, continuous treatment with nitro-glycerin has been shown to cause NO synthasedysfunction, likely through the reduced


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bioavailability of tetrahydrobiopterin (148). NO syn-thase dysfunction can cause superoxide anion for-mation. If not effectively mitigated by superoxidedismutases, increased superoxide anion formationcan lead to the formation of peroxynitrite anion(through reaction with NO), which is a highly reactiveintermediate that promotes oxidant stress and re-duces NO bioavailability (139,149,150).

Nitrate therapy–induced increases in phosphodi-esterase activity have also been implicated in thedevelopment of tolerance (151). As previously dis-cussed, nitroglycerin is metabolized to NO, whichactivates sGC to increase cGMP. Phosphodiesterasedecreases cGMP levels, and, therefore, NO-inducedvasodilation is attenuated by its activity.

Finally, pseudotolerance also complicates chronictreatment with organic nitrates. This adaptive phe-nomenon is not considered true vascular tolerancebecause it occurs in response to every form of vaso-dilator therapy. Pseudotolerance is marked byneurohormonal activation, increased catecholaminerelease rates and circulating catecholamine levels,sodium retention, and intravascular volume expan-sion (139,152).

Although several agents have been studied for usein the prevention of nitrate tolerance, none arecurrently recommended due to a paucity of supportiveclinical data (130,136,153–160). Only 1 methodof preventing nitrate tolerance remains widelyaccepted: the use of a dosing strategy that provides aninterval of no or low nitrate exposure during each 24-hperiod (116,130,137,161–163). Nitrate tolerance israpidly reversed during a nitrate-free interval (53,164).

ADVERSE EFFECTS OF NITRATES

In addition to the development of tolerance, there areother important considerations to treatment withorganic nitrates. Common side effects of nitratetherapy include headache, flushing, lightheadedness,and postural hypotension. When sodium nitroprus-side is infused, it interacts with oxyhemoglobin toform methemoglobin and releases NO and cyanide.Therefore, patients receiving sodium nitroprussidemust be monitored for manifestations of both cyanidetoxicity, such as altered mental status, seizure, andmetabolic acidosis; and methemoglobinemia, suchas cyanosis, headache, fatigue, and lethargy(68,69,165,166). Cyanide toxicity from sodium nitro-prusside therapy is rare and is unlikely if the cumu-lative dose of nitroprusside does not exceed0.5 mg/kg/h. Large-infusion doses of nitroglycerincan also cause methemoglobinemia (167). There areseveral antidotes available to treat cyanide toxicity,

including sodium thiosulfate, sodium nitrite, amylnitrite, and hydroxocobalamin, as well as potentialantidotes that are undergoing development, such assulfanegen sodium. Sodium thiosulfate is availableintravenously, and acts as a sulfur donor to plasmarhodanase, an enzyme that transforms cyanide tothiocyanate, a nontoxic derivative (168). Intravenoussodium nitrite and inhaled amyl nitrite are methe-moglobin generators, and methemoglobin’s strongaffinity for cyanide binding is the rationale for theiruse as antidotes (169). Hydroxocobalamin is availableintravenously and contains a cobalt moiety, whichavidly binds to intracellular cyanide forming cyano-cobalamin (169–171). Sulfanegen sodium is a prodrugof 3-mercaptopyruvate that converts cyanide tothiocyanate (168,172). It was shown to be effective inreversing cyanide toxicity in a juvenile pig model, butwork is ongoing to develop it into an antidote that issafe for use in humans (173).

Additionally, there are certain clinical situationsin which nitrates should not be used, or should beused with extreme caution. Nitrates should notbe given to patients who have used a phosphodies-terase inhibitor, such as sildenafil or tadalafil, within24 to 48 h due to the risk of severe hypotension(174,175). Nitrates should also be avoided insuspected causes of right ventricular infarction, asnitrate-induced dilation of the venous capacitancebeds could cause hypotension given the need forhigh filling pressures in this setting (176). Patientswith hypertrophic cardiomyopathy should not beplaced on nitrate therapy, as it can increase outflowtract obstruction by decreasing pre-load andventricular volumes (177).

NOVEL MODULATORS OF THE

NO-sGC-cGMP PATHWAY

The organic nitrates have been and continue to bemainstays in the treatment of several acute andchronic cardiovascular conditions. Although many ofthe organic nitrates have been used clinically for de-cades, there are several new drugs and drug classesthat have been approved recently or are currentlybeing studied that have the potential to add to the listof modulators of the NO-sGC-cGMP pathway (178).Their methods of modulation of this pathway aresummarized in Figure 5.

Caveolin-1 (CAV1) is the main coat protein of cav-eolae, which are invaginations in the plasma mem-brane implicated in several biological processes,including signaling, in endothelial cells (178–182).eNOS activity is decreased when bound to CAV1(183,184). Cavnoxin, a peptide that activates eNOS,

FIGURE 5 Novel Ways of Modulating the NOsGC-cGMP Pathway

IncreaseEndogenous

Cavnoxin

IntroduceCavnoxin-like

peptides

Enhancetranscription of

eNOS

Reverse eNOSuncoupling

Modulate theenterosalivary

nitrate circulation

Inhibit NADH-cytochrome b5

reductase 3

Disrupt hemoglobinα’s binding site for

eNOS

Fe2+-hemoglobin αto

Fe3+-hemoglobin α

Increase NO bioavailabilityIncrease endogenous NO production

eNOSactivation

Stimulate sGC

Increase cGMPproduction

Inhibitphosphodiesterases

Decrease cGMPbreakdown

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.



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was identified by studying the key residues in CAV1responsible for inhibition of eNOS function (185). Inwild-type mice, cavnoxin increases NO levels, causes areduction in vascular tone, and lowers systemic bloodpressure (185). Therefore, novel methods of increasingendogenous cavnoxin production or introducingsynthesized cavnoxin-like peptides could be a prom-ising way to increase endogenous NO production.

Another approach to increasing endogenous NO isto increase NO bioavailability by affecting hemoglo-bin-a’s ability to complex with eNOS. In the micro-circulation, hemoglobin-a forms a macromolecularcomplex with eNOS and controls the flux ofbioavailable NO (178,186). The reduced Fe2þ–O2–

hemoglobin-a reacts with NO rapidly and generatesnitrate and the oxidized Fe3þ–hemoglobin-a. This

oxidized form of hemoglobin-a has a much lower af-finity for NO than the reduced form, and, therefore,allows eNOS-generated NO to diffuse to underlyingvascular smooth muscle cells (178,187). Nicotinamideadenine dinucleotide (NADH)–cytochrome b5

reductase 3 reduces Fe3þ–hemoglobin-a to Fe2þ–hemoglobin-a. Therefore, yet another potentialway to increase NO bioavailability would beto inhibit NADH-cytochrome b5 reductase 3. Stillanother possible strategy would be to disrupt thebinding site of hemoglobin-a for eNOS; a small pep-tide, hemoglobin-aX, has been developed for thispurpose. Hemoglobin-aX has been found to decreaseblood pressure in mice and dilate constricted arteri-oles isolated from patients with hypertension(186,188).


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Modulating the transcription, and therefore,translation of NOS is another focus of research anddevelopment efforts to augment levels of endoge-nous NO. Two small molecules, AVE3085 andAVE9488, have been identified as transcriptionalenhancers of eNOS that bind its promoter (178,189). Inapolipoprotein E knockout mice, 12 weeks of treat-ment with AVE9488 or AVE3085 reduced atheroscle-rotic plaque formation. Treatment of these mice withAVE9488 also reversed eNOS uncoupling, increasedvascular content of the essential eNOS cofactortetrahydro-L-biopterin (BH4), and reduced vascularcuff-induced neointima formation (189). Four weeksof treatment with AVE3085 was shown to attenuatecardiac remodeling in an experimental mouse modelinvolving aortic banding to induce remodeling (190).Finally, 9 weeks of treatment with AVE9488 wasshown to improve left ventricular remodeling andcontractile dysfunction in an experimental myocar-dial infarction rat model (191).

As referred to in the previous text with regard totreatment with AVE9488, reversing eNOS uncouplingis desired as it decreases eNOS production of super-oxide anion (50). Tetrahydrobiopterin has beenshown to improve endothelial dysfunction in patientswith type 2 diabetes mellitus and in patients oncyclosporine A after cardiac transplantation (52,192).However, a study of 49 patients randomized toreceive 400 mg/day of BH4, 700 mg/day of BH4, orplacebo for 2 to 6 weeks before coronary artery bypassgraft surgery found no effect of BH4 treatment onvascular function or superoxide production (193).More research is needed to explore alternative waysto reverse eNOS uncoupling and, thereby, increaseNO production and decrease superoxide anionproduction.

Ignarro et al. (194) showed that sGC activity couldbe modulated in an NO-independent fashion by pro-toporphyrin IX, an sGC activator. Protoporphyrin IXwas never used clinically due to its severe photo-sensitizing effect (178). However, a recently devel-oped class of drugs called the ciguats also modulatethe NO–sGC–cGMP pathway similarly in an NO-independent fashion (178). There are 2 subgroups ofciguats: NOsGC stimulators (which bind NOsGC andact through allosteric regulation) and NOsGC activa-tors (which occupy the heme binding site of NOsGCand work additively with NO) (178). Lificiguat was thefirst NOsGC stimulator that was identified when itwas found to stimulate sGC in rabbit platelets duringa small-molecule screen designed to identify novelplatelet aggregation inhibitors (195). Further work toimprove the solubility and efficacy of lificiguat led tothe development of riociguat, which is the only

NOsGC stimulator that is approved for clinicaluse (196). Riociguat is currently approved for thetreatment of pulmonary arterial hypertension andinoperable chronic thromboembolic pulmonary hy-pertension (178,197,198). There are ongoing trials ofriociguat’s potential role in the treatment of pulmo-nary hypertension and heart failure. Other NOsGC-stimulating ciguats that are being studied includevericiguat, which was shown to decreased circulatinglevels of N-terminal pro–B-type natriuretic peptidein patients with worsening chronic systolic heartfailure, as well as nelociguat, IW-1973, and IW-1701(178,199,200).

Cinaciguat was identified as an NOsGC activatorvia high-throughput screening (201,202). Its clinicaluse has been limited due to significant hypotension.In a placebo-controlled trial of 139 patients admittedwith acute, decompensated, systolic heart failure,cinaciguat significantly decreased pulmonary capil-lary wedge pressure, right atrial pressure, pulmo-nary vascular resistance, and systemic vascularresistance, and also significantly increased cardiacindex. However, the trial was stopped prematurelydue to an increased occurrence of hypotension atcinaciguat doses >200 mg/h (203). At lower doses,cinaciguat has been shown to decrease systemicblood pressure without improving dyspnea orcardiac index in patients with acute heart failure,therefore limiting its use in this population (204).Another NOsGC activator that is under developmentis ataciguat (178).

Another class of NOsGC-cGMP pathway modula-tors is the phosphodiesterases. These enzymesinhibit the pathway by hydrolyzing the phospho-diester bond of cGMP (178,205). There are 4 phos-phodiesterase 5 inhibitors in current clinical use thatinhibit cGMP breakdown: sildenafil, vardenafil,tadalafil, and avanafil. All 4 are approved for use inerectile dysfunction. Sildenafil and tadalafil are alsoused for pulmonary arterial hypertension, andtadalafil is approved for use in benign prostatichyperplasia (178). Work is ongoing to develop addi-tional phosphodiesterase inhibitors, including in-hibitors of nonselective phosphodiesterases andcGMP-selective phosphodiesterases (phosphodies-terases 5, 6, and 9) (178).

Finally, as discussed above, the enterosalivary ni-trate circulation (nitrate–nitrite–NO pathway) is asource of NO that is derived from dietary inorganicnitrate intake (38,39,206). Production of NO from thispathway is enhanced by hypoxia and acidosis(207,208). There is, therefore, interest in studying theuse of inorganic nitrates to improve exercise capacity,particularly in patients with heart failure with



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preserved left ventricular ejection fraction (206). Inaddition, there is ongoing research dedicated toexploring therapeutic options, such as prebiotics,probiotics, and antimicrobial agents, that can modu-late the microbiome and the nitrate–nitrite–NOpathway in heart failure, pulmonary hypertension,hypertension, obesity, and other cardiovascular dis-ease states (39).

CONCLUSIONS

The use of nitrates in cardiovascular disease has along and storied history. They continue to play amajor role in current clinical practice to improve

symptoms despite the paucity of evidence of benefiton hard clinical endpoints. As research and develop-ment of new ways to modulate the NO-sGC-cGMPpathway continue, it may be time to revisit thestudy of nitrates in large, prospective clinical trials inthe percutaneous coronary intervention era.

ACKNOWLEDGMENT The authors thank Ms.Stephanie Tribuna for expert technical assistance.

ADDRESS FOR CORRESPONDENCE: Dr. Joseph Loscalzo,Department of Medicine, Brigham and Women’sHospital, 75 Francis Street, Boston, Massachusetts 02115.E-mail: [email protected].

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KEY WORDS angina, nitrate,nitrate-nitrite-NO pathway, nitric oxide,nitroglycerin, soluble guanylyl cyclase








































































































































The Role of Nitroglycerin and Other Nitrogen Oxides in ... · monophosphate (cGMP) pathway that may...

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