J Appl Physiol 116: 463–477, 2014.
First published January 9, 2014; doi:10.1152/japplphysiol.01100.2013.
Synthesis
Inorganic nitrite supplementation for healthy arterial aging
Amy L. Sindler, Allison E. DeVan, Bradley S. Fleenor, and Douglas R. Seals
Department of Integrative Physiology, University of Colorado Boulder, Boulder, Colorado
Submitted 1 October 2013; accepted in final form 3 January 2014
arterial stiffness; vascular endothelial dysfunction; oxidative stress; inflammation;
mitochondrial biogenesis; nitric oxide
AGING, ARTERIAL DYSFUNCTION, AND CARDIOVASCULAR
DISEASES
Cardiovascular diseases (CVD) remain the leading cause of
morbidity and mortality in modern societies, and most forms of
CVD are linked to dysfunction of arteries (59, 111). Aging is
the primary risk factor for CVD, and therefore, most forms of
CVD are diseases of aging (59, 98, 112, 136, 137). Thus there
is something about aging that causes dysfunction of arteries,
which in turn, leads to an increased risk for CVD (100). Given
the changing demographics of aging worldwide, these events
represent a pathophysiological platform for a new baby boomer-driven epidemic of CVD in the near future. Indeed, a recent
projection suggests that without effective intervention, 40% of
U.S. adults will have at least one form of CVD by 2030, and
medical costs for these disorders will triple, primarily as a
result of population aging (68). As such, establishing the
efficacy of treatments that prevent and/or reverse age-associated arterial dysfunction is a high biomedical priority because
it has the potential to prevent age-associated CVD (91, 117,
155).
Address for reprint requests and other correspondence: A.L. Sindler, Univ.
of Colorado Boulder, 354 UCB, Boulder, CO 80309 (e-mail: amy.sindler
@colorado.edu).
http://www.jappl.org
ARTERIAL DYSFUNCTION WITH AGING
Aging causes numerous changes to arteries that increase the
risk for CVD, but two key contributors are stiffening of the
large elastic arteries (aorta and carotid arteries) and the development of vascular endothelial dysfunction (100, 142, 164,
194).
Large Elastic Artery Stiffness
Large elastic artery stiffening with aging is mediated by
changes in both structural and functional factors (99, 136, 195).
Structural factors include changes to the extracellular matrix
involving increases in collagen (fibrosis) and reductions in
elastin (i.e., the major structural proteins in the arterial wall
that confer stiffness and elasticity, respectively) (41). The
cytokine growth factor, transforming growth factor-␤ (TGF-␤),
may play an important role in signaling collagen synthesis with
aging (13, 163). The formation of advanced glycation end
products (AGEs), glucose-derived molecules that cross-link
structural proteins in the arterial wall, also increases with age
and contributes to large elastic artery stiffening (62). Functional influences on arterial stiffness involve factors that modulate vascular smooth muscle tone, including paracrine molecules, of which endothelial cell-synthesized nitric oxide (NO)
is among the most important (42, 52, 123, 173, 191, 195). In
vivo, the gold standard for assessing large elastic artery stiff-
8750-7587/14 Copyright © 2014 the American Physiological Society
463
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Sindler AL, DeVan AE, Fleenor BS, Seals DR. Inorganic nitrite supplementation for healthy arterial aging. J Appl Physiol 116: 463– 477, 2014. First published
January 9, 2014; doi:10.1152/japplphysiol.01100.2013.—Aging is the major risk
factor for cardiovascular diseases (CVD). This is attributable primarily to adverse
changes in arteries, notably, increases in large elastic artery stiffness and endothelial dysfunction mediated by inadequate concentrations of the vascular-protective
molecule, nitric oxide (NO), and higher levels of oxidative stress and inflammation.
Inorganic nitrite is a promising precursor molecule for augmenting circulating and
tissue NO bioavailability because it requires only a one-step reduction to NO.
Nitrite also acts as an independent signaling molecule, exerting many of the effects
previously attributed to NO. Results of recent studies indicate that nitrite may be
effective in the treatment of vascular aging. In old mice, short-term oral sodium
nitrite supplementation reduces aortic pulse wave velocity, the gold-standard
measure of large elastic artery stiffness, and ameliorates endothelial dysfunction, as
indicated by normalization of NO-mediated endothelium-dependent dilation. These
improvements in age-related vascular dysfunction with nitrite are mediated by
reductions in oxidative stress and inflammation, and may be linked to increases in
mitochondrial biogenesis and health. Increasing nitrite levels via dietary intake of
nitrate appears to have similarly beneficial effects in many of the same physiological and clinical settings. Several clinical trials are being performed to determine the
broad therapeutic potential of increasing nitrite bioavailability on human health and
disease, including studies related to vascular aging. In summary, inorganic nitrite,
as well as dietary nitrate supplementation, represents a promising therapy for
treatment of arterial aging and prevention of age-associated CVD in humans.
Synthesis
464
Inorganic Nitrite, and Arterial Aging
ness is aortic pulse wave velocity (PWV) (105, 126); the
greater the PWV, the greater the stiffness. Intrinsic stiffness
of these arteries also can be assessed ex vivo in aortic rings
(1, 53, 73).
Vascular Endothelial Dysfunction
Mechanisms of NO Insufficiency
The key mechanism for reduced NO bioavailability with
aging is oxidative stress (49, 164, 177). Oxidative stress can be
defined as increased bioactivity of reactive oxygen species
(ROS) relative to antioxidant defenses (97). Oxidative stress
reduces NO bioavailability via excessive production of superoxide, which reacts with NO to form peroxynitrite. Peroxynitrite in turn causes nitration of tyrosine residues on proteins
(nitrotyrosine), providing a useful cellular marker of oxidative
stress (107, 159), and also oxidizes tetrahydrobiopterin (BH4),
an essential cofactor for NO synthesis by eNOS, to its inactive
form, BH2 (54, 165). This reduction in bioavailability of BH4
leads to the uncoupling of eNOS, which produces more superoxide and less NO in a vicious cycle that further reduces NO
bioavailability (101, 106). Increased superoxide production by
oxidant enzymes such as NADPH oxidase and reduced expression/activity of endogenous antioxidant enzymes that remove
superoxide (e.g., superoxide dismutases; SOD) are among the
key players contributing to oxidative stress, reduced NO, and
vascular dysfunction with aging (15, 64, 130). Consistent with
the known synergistic interaction between oxidative stress and
inflammation (181), vascular inflammation develops with aging and also contributes to arterial dysfunction (34, 44, 108,
149, 186). Finally, recent evidence suggests that mitochondrial
dysfunction may play an important role in large elastic artery
stiffening and endothelial dysfunction with aging via modulation of oxidative stress (33, 35, 56, 198).
Sindler AL et al.
In summary, on the basis of our present knowledge of the
mechanisms underlying vascular aging, it is reasonable to
postulate that treatments that increase NO bioavailability, reduce oxidative stress and inflammation, and perhaps improve
mitochondrial health, have the potential to improve arterial
dysfunction in middle-aged and older adults.
NO, NITRITE, AND NITRATE
NO is a gaseous signaling molecule that plays an essential
role in regulating systemic physiological function, and maintaining NO homeostasis is essential for optimal function and
health (116, 134, 178). NO has a very short half-life (i.e.,
milliseconds) and is rapidly and sequentially oxidized to nitrite
(NO2⫺) and nitrate (NO3⫺). These molecules have much
longer half-lives (minutes to hours) and can be measured in the
plasma, where in particular, nitrite has been established as a
marker of NO flux/formation (93, 104).
Two major pathways contribute to systemic NO formation.
The most well-known pathway is the production of NO from
L-arginine in the presence of oxygen by isoforms of the enzyme
NO synthase (L-arginine-NO pathway) (Fig. 1, left side) (114).
In the presence of several cofactors, most notably BH4, eNOS
constitutively produces small amounts of NO (inducible NOS
can synthesize large amounts of NO in certain inflammatory
conditions) (165). In healthy young adults, constitutive NO
production by eNOS is sufficient for normal physiological
function in most cases. However, NOS is dysfunctional in most
if not all physiological and pathophysiological states associated
with chronic NO insufficiency, including aging (10, 27, 184).
Indeed, in these states activation of NOS often results in
increased formation of superoxide instead of NO due to
inadequate availability of BH4, further exacerbating oxidative stress (90, 106). Thus activation of NOS may not be an
effective strategy to boost NO bioavailability in such settings (47).
More recently, an alternative pathway of NO formation, the
nitrate-nitrite-NO pathway, has been identified (Fig. 1, right
Fig. 1. Two primary pathways for nitric oxide (NO) production; the L-arginine-NO and nitrate-nitrite-NO pathways. Modified with permission from (114).
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Reduced bioavailability of NO as a result of decreased
production by endothelial NO synthase (eNOS), increased
degradation, or both, also is the key mechanism mediating
vascular endothelial dysfunction with aging (118, 164, 177).
The vascular endothelium is a single cell layer at the interface
between the flow of blood in the lumen of the vessel and the
vessel wall. These cells produce a remarkable variety of
biologically active molecules that act in autocrine and/or paracrine fashion to regulate the function and health of the vasculature. Endothelial dysfunction can be defined as any alteration
in the phenotype of the normal healthy endothelium (36, 48,
133). However, endothelial function is most commonly assessed as the dilation produced by a mechanical or chemical
(e.g., acetylcholine) stimulus that evokes increased production
of NO by the endothelial cells, leading to activation of guanylate cyclase, increases in cyclic GMP, and relaxation of
vascular smooth muscle (55, 75). The greater the endotheliumdependent dilation (EDD) observed, the greater the endothelial
function (health).
Large elastic artery stiffness and vascular endothelial dysfunction are not independent functions. Endothelial dysfunction is believed to contribute to large elastic artery stiffness
(173), and stiffening of arteries likely restricts EDD. Inadequate NO would serve as a critical factor linking these events
(173, 195).
•
Synthesis
Inorganic Nitrite, and Arterial Aging
Sindler AL et al.
465
ciency, and each has relative strengths and weaknesses, conceptually. Sodium nitrite has been advanced as a more direct
strategy for increasing nitrite concentrations in the body because it involves only a one-step reduction to NO. Direct
supplementation with nitrite also evades the need for increased
consumption of green leafy vegetables and/or beets/beetroot
juice, which like other dietary interventions, may not be sustainable for many individuals (although pharmacological supplementation of nitrate also would obviate this potential limitation). Sodium nitrite also may show greater efficacy for
increasing nitrite concentrations in some populations, as has
been reported in older adults (124). Moreover, reductions of
nitrate to nitrite depend entirely on oral commensal bacteria,
and individual differences in microflora may affect nitrate
reduction (61, 86, 174). Indeed, there may be sex differences in
endogenous nitrate processing involving the entero-salivary
circulation (86). In addition, the use of mouthwash and/or
antibiotic medications may disturb the oral bacterial flora
required for nitrate reduction, thereby attenuating the NOdependent benefits of nitrate supplementation (61). These issues may be avoided by supplementing with inorganic nitrite.
Dietary supplementation via increased intake of foods high in
nitrate, on the other hand, may be considered a more natural
approach to boosting nitrite and NO bioavailability (72, 119).
Currently, however, there is an absence of information from
studies comparing the health benefits of nitrite vs. nitrate
supplementation. This is an important area of future investigation.
INORGANIC NITRITE AS AN ORGAN-PROTECTIVE
MOLECULE
Ischemia/Reperfusion Injury in the Heart
Much of the initial work on the cardiovascular effects of
nitrite shows that pretreatment with this compound, delivered
as a salt in the form of sodium nitrite, protects against ischemia/reperfusion (I/R) injury in the heart in preclinical models
(7, 19, 20, 40, 46, 60, 79, 189). In agreement with this, in
healthy young humans, acute venous infusion of sodium nitrite
increases resistance to temporary (reversible) I/R injury in the
peripheral circulation, as assessed noninvasively by measuring
the decrease in brachial artery flow-mediated dilation from
normal baseline levels following cuff inflation-induced blood
flow occlusion to the forearm (76). In adults with inducible
myocardial ischemia, venous infusion of sodium nitrite improves functional cardiac responses during dobutamine stress
echocardiography, suggesting that nitrite acts as a coronary
vasodilator (76). The results of studies using nitrate administration also lend support to the concept that nitrite-boosting
interventions promote resistance to I/R injury (86, 190).
Several ongoing clinical trials are currently investigating the
therapeutic effects of nitrite administration during coronary
angioplasty (80) (clinical trial NCT01584453), coronary
artery bypass surgery (clinical trial NCT01098409), and
acute myocardial infarction (clinical trials NCT01388504,
NCT00924118, and NCT01178359).
Injury in Other Organs/Tissues
Numerous investigations have reported that nitrite is protective against I/R and other forms of injury in multiple tissues
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side) (114, 178). In this pathway, nitrate and nitrite are boosted
by consuming foods rich in these compounds (e.g., green leafy
vegetables, beets, etc.) (71, 160) or administering nitrate or
nitrite salts orally (63, 74, 86); or through inhalation (200),
infusion (121, 151), or topical application (144). Nitrate and
nitrite are then sequentially reduced to the same bioactive form
of NO that is synthesized by NOS. Nitrate is readily converted
to nitrite by commensal bacteria in the mouth (115), and the
reduction of nitrite to NO occurs systemically, mediated by a
variety of endogenous nitrite reductases (17, 26). Therefore,
nitrate and nitrite are physiologically recycled in the blood and
tissues, acting as precursors that can be easily converted to
bioactive NO on demand (178).
By whatever means of delivery, nitrite is an attractive
candidate for restoring physiological NO signaling in states of
chronic NO insufficiency such as aging because it is rapidly
absorbed from the circulation by peripheral tissues and stored
in cells until conversion to NO is needed (21, 51, 138). Nitrite
has an immense capacity to produce NO, in some tissues
synthesizing 10,000 times the amount of constitutive NO
formation from NOS (66, 178). Importantly, at physiological
concentrations, nitrite, but not nitrate, acts as an independent
signaling molecule, performing many of the same actions
previously attributable to NO (21, 160, 178). Thus increasing
nitrite bioavailability likely has dual-acting effects on vascular
and systemic physiological function and health in states of NO
insufficiency.
Growing evidence supports the clinical efficacy and safety
of inorganic nitrite. Nitrite is classified by the Food and Drug
Administration as “Generally Recognized as Safe” for use as a
food additive and for the treatment of cynanide poisoning (9,
23, 183). Over the last decade, it has been shown that earlier
health concerns regarding nitrite intake are not supported by
the data (16, 18, 67, 115, 170), and nitrite is well tolerated with
little or no side effects in humans and nonhuman primates
when administered at nontoxic doses (Table 1; note that wide
ranging doses were used in these studies, in many cases
given acutely to assess dose-response and safety). Inorganic
nitrite and nitrate also have different chemical compositions
and physiological effects than organic nitrite or nitrate
(143). Most health care professionals are familiar with
organic nitrates that have been used for many years in
medicine, such as nitroglycerin for the treatment of angina.
Although highly effective under such acute conditions,
chronic use of organic nitrites and nitrates leads to tolerance
and adverse physiological effects such as endothelial dysfunction (131, 132, 143). Furthermore, organic nitrites and
nitrates also have potent vasodilatory and blood pressure
lowering effects, whereas inorganic nitrites and nitrates are
milder vasodilators and have small or no effects on blood
pressure in nonhypertensive adults (39, 63, 141).
Several recent investigations suggest that administration of
nitrate through consumption of green leafy or root vegetables
(e.g., via beetroot juice) has therapeutic potential to treat a
variety of clinical diseases and improve exercise performance,
likely via nitrate conversion to nitrite within the body (11, 87,
88, 109, 116, 152). Because the cardiovascular effects of
nitrate supplementation have been recently reviewed elsewhere
(109), the focus of this synthesis article will be on nitrite
supplementation. In general, both approaches appear to have
physiological and clinical benefits in settings of NO insuffi-
•
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Inorganic Nitrite, and Arterial Aging
•
Sindler AL et al.
Table 1. Sodium nitrite administration in humans and nonhuman primates
Duration per dose
Sample
size
Administration
Dose
Arterial infusion
75 mg
Acute (30 min)
18
Arterial infusion
0.75 mg
Acute (5 min)
10
Arterial infusion
⬃86 mg
Acute (30 min)
5
Arterial infusion
⬃20 mg
Acute (45 min)
15
Arterial infusion
⬃210 mg/day
Arterial infusion
14 days
3
⬃84 mg
Acute (bolus)
3
Venous infusion
1 mg
Acute (5 min)
3
Venous Infusion
⬃3–29 mg
Acute (3–8 h)
9
Oral
80 mg
Acute
12
⬃76–164 mg
Acute (25–145 min)
5
Venous infusion
290–380 mg
Acute
9
Oral
140–190 mg
Acute
9
Oral
290–380 mg
Acute
9
Young & MA
healthy adults
Young & MA
healthy adults
Young healthy
adults
Young healthy
adults
Cynomolgus
primates
Cynomolgus
primates
Adults in cardiac
arrest
Cynomolgus
primates
MA & older adults
with diabetes
Young healthy
adults
Young healthy
adults
Young healthy
adults
Young healthy
adults
Avg 2
MAP,
mmHg
⬍1.5
⫺7
NR
(32)
NR
NR
NR
(32)
3.2
⫺15
NR
(37)
NR
NR
NR
(37)
NR
⫹3
NR
(37)
NR
⫺18
NR
(37)
1.1
NS
NR
(39)
NR
NS
NR
(50)
NS
⫺4
(63)
NS
NR
Headache (n ⫽ 1–2);
hot flush (n ⫽ 2);
nausea (n ⫽ 1)
NR
ⱕ12.0
⫺14
(74)
ⱕ4.5
NS
ⱕ11.0
⫺11
NR
NS
NS
NS
Mild headache
(n ⫽ 5);
nausea (n ⫽ 1)
Mild headache
(n ⫽ 4)
Mild headache
(n ⫽ 4);
nausea (n ⫽ 2)
NR
NR
NS
NS
NR
(76)
⬍13.0
⫺12
(96)
⬍5.0
⫺8
⬍12.0
⫺11
Mild headache
(n ⫽ 5);
nausea (n ⫽ 1)
Mild headache
(n ⫽ 3)
Mild headache
(n ⫽ 3);
nausea (n ⫽ 2);
vomiting (n ⫽ 1)
Mild dizziness
(n ⫽ 1);
tiredness (n ⫽ 1)
None
Side effects
Reference
(70)
(74)
(74)
Venous infusion
Venous infusion
⬃4 mg
⬃2 mg
Acute (60 min)
Acute (20 min)
12
10
Venous infusion
⬃2 mg
Acute (20 min)
19
Venous infusion
290–380 mg
Acute (30 min)
9
Oral
140–190 mg
Acute
9
Oral
290–380 mg
Acute
9
Venous infusion
90–130 mg
Acute (10 min)
3
Young healthy
adults
⬍4.0
⫺10
Venous infusion
90–130 mg
Acute (30 min)
3
⬍4.0
⫺9
Venous infusion
190–250 mg
Acute (30 min)
3
⬍8.0
⫺5
Venous infusion
290–370 mg
Acute (30 min)
3
Young healthy
adults
Young healthy
adults
Young healthy
adults
⬍12.0
⫺10
Arterial infusion
⬍1 mg
Acute (bolus)
3
NR
NR
Arterial infusion
25 mg
Acute
14
⬍5.0
NS
Transient nausea
(n ⫽ 1)
(120)
Arterial infusion
⬃0.01–25 mg
Acute (40–180 min)
40
NR
NR
NR
(122)
Arterial infusion
⬃25 mg
Acute (120 min)
20
⬍2.0
NS
None
(121)
Arterial infusion
⬃25 mg
Acute (120 min)
21
⬍2.0
NS
None
(121)
Venous infusion
⬃24 mg
Acute (80 min)
15
⬍2.0
NS
None
(121)
Young healthy men
MA & older adults
with inducible
myocardial
ischemia
Young healthy
adults
Young healthy
adults
Young healthy
adults
Young healthy
adults
Young healthy
adults
Young & MA
adults with sickle
cell disease
MA & older
healthy adults
MA & older
healthy adults
MA & older adults
with heart failure
MA & older adults
with heart failure
Mild arm tingling
(n ⫽ 1)
Mild dizziness
(n ⫽ 1); arm
tingling (n ⫽ 1)
NR
(77)
(76)
(96)
(96)
(95)
(95)
(95)
(95)
(104)
Continued
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Arterial infusion
Population
Peak
MetHb,
%
Synthesis
Inorganic Nitrite, and Arterial Aging
•
467
Sindler AL et al.
Table 1.—Continued
Administration
Dose
Venous infusion
⬃223–445 mg/day
Venous infusion
135 mg
Venous infusion
Duration per dose
Sample
size
9
Acute (bolus)
3
180 mg/day
14 days
3
Venous infusion
⬃10–557 mg/day
2 days
9
Venous infusion
⬃364 mg
⬃1 day
1
Venous infusion
⬃1,230 mg/day
2 days
1
Venous infusion
⬃91 mg
180 min
1
Venous infusion
⬃2 mg
Acute (bolus)
3
Oral
Oral
200–300 mg
200–300 mg
Acute (30 min)
Acute (30 min)
Oral
200–300 mg
Acute (30 min)
Oral
120–180 mg
Acute
MA & older adults
with
subarachnoid
hemorrhage
Cynomolgus
primates
Cynomolgus
primates
Young & MA
healthy adults
MA man (subject
8)
Young woman
(subject 11)
Young woman
(subject 12)
Young healthy
adults
Avg 2
MAP,
mmHg
ⱕ3.3
NS
Asymptomatic
(141)
10.8
⫺17
NR
(150)
⬍3.0
NS
NR
(150)
ⱕ1.6
NS
Asymptomatic
(151)
⬍4.0
⫺25
Asymptomatic
(151)
⬍6.0
⫺10
Asymptomatic
(151)
⬍2.5
⫺28
Asymptomatic
(151)
NR
NR
NR
Headache (n ⫽ 3);
faintness (n ⫽ 1);
dizziness (n ⫽ 1)
Drowsy (n ⫽ 1);
fainted (n ⫽ 2);
cyanotic/weak
(n ⫽ 1)
Headache (n ⫽ 10);
cyanotic (n ⫽ 4);
dizziness (n ⫽ 1);
faintness (n ⫽ 2);
palpitation
(n ⫽ 1);
flush (n ⫽ 1);
fainted (n ⫽ 1)
(158)
(192)
Unclear
(193)
10
Young & MA
healthy adults
NR
⫺13
14
Young & MA with
renal disorders or
nephrectomy
NR
⫺12
NR
⫺26
NR
NC
36
7
Young, MA &
older with Stage
1 & Stage 2
hypertension
Young healthy
adults
Side effects
Reference
(192)
(192)
MetHb, methemoglobin; MAP, mean arterial pressure; MA, middle-aged (40 – 60 yr); NR, not reported; NS, no significant change; NC, not clear.
and organs (81, 125, 188), as reviewed extensively elsewhere
(157). Indeed, nitrite administration reduces skeletal muscle
(188), renal (125, 179), and liver (46, 154, 169) injury after
ischemia. Infarct size and hematoma volume decreases and
blood flow enhances in the brain after stroke with nitrite
pretreatment (81, 83), and a nitrate-rich diet has been reported
to increase cerebral perfusion in older adults (152). Within the
lung, nitrite, nitrate, or both limits injury and enhances recovery after ischemia (140, 176) and ameliorates pulmonary hypertension in experimental animal models (8, 22, 145). In
humans, nitrite reduces pulmonary arterial pressure during
hypoxia (77) and improves cell activation and wound healing in human airway epithelial cells (187). Nitrite treatment
lessens renal injury in hypertensive rats (84) and can protect
against brain death-mediated renal injury while also improving posttransplant renal function (89). Nitrite, but not nitrate, therapy is effective in restoring blood flow and stimulating vascular remodeling, thereby preventing tissue necrosis in aged-diabetic mice after unilateral femoral ligation
(12). Recent studies in humans (39, 77) and ongoing clinical trials (NCT01431313, NCT01725256, NCT01725269,
NCT01715883, and NCT01316796) may provide evidence
in humans supporting these preclinical findings.
Cardiovascular Disease and its Risk Factors
The effects of dietary nitrate on cardiovascular outcomes
have been reviewed elsewhere, and generally support the
efficacy of this approach (109). Regarding inorganic nitrite,
multiple studies in preclinical models suggest that nitrite also
may be effective for the prevention and/or treatment of many
cardiovascular disorders or risk factors for CVD including
peripheral artery disease (4, 69, 146), atherosclerosis (2, 3, 5,
127), chronic heart failure (32, 122), stroke (50, 82, 150),
diabetes (138), and hypertension (22, 57, 58, 128). Presently,
there is some evidence that treatment with sodium nitrite may
have beneficial cardiovascular effects in humans by acutely (by
either mildly or moderately) decreasing blood pressure (32, 37,
63) (Table 1); however, many of these studies administered
much higher acute doses of nitrite than are currently being used
in clinical trials. In addition, acute infusions of sodium nitrite
increase forearm blood flow under normoxic (32, 74, 122, 151)
and hypoxic (77, 122) conditions in healthy adults and in
patients with heart failure (121). Recently published studies in
humans provide additional evidence for nitrite or nitrate as a
therapeutic agent for the prevention and treatment of cardiovascular disorders or risk factors for CVD, and ongoing clinical investigations will extend present insight (clinical trials
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14 days
Population
Peak
MetHb,
%
Synthesis
468
Inorganic Nitrite, and Arterial Aging
•
Sindler AL et al.
sodium nitrite was added to the drinking water (50 mg/l) for 3
wk in young (4 – 6 mo) and old (26 –28 mo) male C57/BL6
mice (53, 171). Nitrite concentrations in the plasma and large
elastic arteries (aorta and carotid arteries) were lower in older
animals, but sodium nitrite supplementation increased nitrite
concentrations in the old mice up to or above those of young
control animals (Fig. 2) (171). Thus sodium nitrite supplementation was effective in restoring circulating and tissue nitrite
bioavailability in old mice. Because sodium nitrite had no
effect on arterial function in the young mice in this study (171),
and constitutive NO production by eNOS is sufficient in young
animals/humans in most cases, only the influence of nitrite
therapy on arterial function of old mice will be described in the
sections below.
Large Elastic Artery Stiffness
NCT01401517, NCT01681810, NCT01961427, NCT01919177,
NCT01983826, NCT01430780, NCT01682356, NCT01785524,
NCT02000856, NCT01684930, and NCT02022670).
Despite accumulating evidence suggesting that acute nitrite
administration may be broadly effective in treating cardiovascular disease (4, 24, 114), little preclinical or clinical information is available with regard to chronic nitrite therapy and
arterial function/health per se (128, 175). In the present synthesis article, we summarize the results of recent work aimed at
establishing the efficacy of inorganic nitrite as a treatment for
arterial aging and discuss the potential physiological mechanisms suggested.
INORGANIC NITRITE: AN ANTI-AGING TREATMENT
FOR ARTERIES?
The potential efficacy of sodium nitrite in vascular aging
was initially assessed in a preclinical investigation in which
Fig. 3. Aortic pulse wave velocity (A), aortic advanced glycation end-products (AGEs) assessed by histological cross-sections (B, top) with quantification
(B, bottom), and effects of AGEs (100 ␮g/ml) and sodium nitrite (100 ␮M) on arterial stiffness in cultured aortic segments (C). Values are means ⫾ SE. *P ⱕ
0.05 vs. all. Data were adapted from (53, 171).
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Fig. 2. Plasma, arterial, and heart concentrations of nitrite from young and
old control and old nitrite-supplemented (Old Nitrite). Values are means ⫾
SE. *P ⱕ 0.05 Young Control vs. Old Control; †P ⱕ 0.05 Old Control vs. Old
Nitrite. Data were adapted from (171).
Among the measurements of large elastic artery stiffness,
aortic PWV has emerged as the strongest and most consistent
predictor of cardiovascular events and risk with aging (126,
185). In this initial study (171), aortic PWV was markedly
higher (i.e., the aorta was stiffer) in old compared with young
control mice (Fig. 3A). Sodium nitrite treatment reduced aortic
PWV in old mice to well below old controls and not significantly different from that in young animals. These results
suggested that sodium nitrite supplementation, started late in
life, can substantially reduce large elastic artery stiffness in old
mice.
In a follow-up study (53), the potential mechanisms of this
destiffening effect by sodium nitrite were investigated. Aortic
PWV was higher in old mice and this was associated with
higher adventitial TGF-␤ and collagen levels, and lower medial elastin and higher adventitial and medial abundance of
AGEs in the aorta. Sodium nitrite reduced aortic PWV in old
mice, but did not influence aortic TGF-␤, collagen, or elastin.
However, nitrite supplementation normalized total, adventitial,
and medial AGEs in the aorta of old mice (Fig. 3B). In aortic
rings from young mice studied ex vivo, administration of
AGEs directly induced stiffening, an effect prevented by incubation with sodium nitrite (Fig. 3C).
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469
Vascular Endothelial Dysfunction
In an initial preclinical study (171), vascular endothelial
function as assessed by carotid artery EDD in response to
acetylcholine, was impaired in old compared with young control animals (Fig. 4, top). Inhibition of NO using the NO
synthase inhibitor L-NAME, markedly reduced EDD in young
animals while having less effect in old mice, indicating that the
impaired EDD in old animals was mediated by reduced NO
bioavailability. Sodium nitrite treatment restored EDD in old
mice by increasing NO-dependent dilation (Fig. 4, bottom).
Nitrite did not affect endothelium-independent dilation to sodium nitroprusside (171), indicating that the improvements in
EDD were not induced by increased vascular smooth muscle
sensitivity to NO. Thus nitrite supplementation appears to
ameliorate vascular endothelial dysfunction in old animals by
restoring endothelial NO bioavailability.
These results obtained in a setting of primary aging are in
agreement with previous observations in young hypercholesterolemic mice (175), and also are consistent with the vasodilatory effects and vascular protection against I/R injury (76, 86,
190) afforded by dietary nitrite (32, 37, 120 –122) and nitrate
(14, 69) administration in healthy adults, as well as patients
with risk factors for CVD (156). Together, these findings
suggest that inorganic nitrite or nitrate supplementation may
have broad therapeutic efficacy for treating arterial endothelial
dysfunction in a number of clinical states.
Potential Mechanisms of Action
Oxidative stress. Recent preclinical investigations found that
the staining intensity for nitrotyrosine, a cellular marker of
oxidant modification of proteins, was markedly greater in aorta
of old compared with young control mice (53, 171) (Fig. 5A).
Sodium nitrite treatment normalized aortic nitrotyrosine staining in old mice, indicating amelioration of age-associated
arterial oxidative stress. This is consistent with recent observations that sodium nitrite supplementation reduces markers of
oxidative stress in a model of experimental hypertension (128),
after cerebral or cardiac I/R injury (81, 92), following alcohol-
Fig. 4. Endothelium-dependent dilation to acetylcholine (ACh) (A) and NOdependent dilation from young and old control and old nitrite-supplemented
(Old Nitrite) mice with and without an inhibitor of nitric oxide synthase,
L-NAME. Values are means ⫾ SE. *P ⱕ 0.05 Young Control vs. Old Control;
†P ⱕ 0.05 Old Control vs. Old Nitrite. Data were adapted from (171).
induced liver injury (81, 113), and after femoral ligation in
diabetic mice (12). Nitrate supplementation reduced oxidative
stress in spontaneously hypertensive rats (30), in rats with
unilateral nephrectomy that consumed a high-salt diet (25), and
in a mouse model of cardiomyopathy (196, 199). Collectively,
this work supports the concept that nitrite and nitrate have
strong antioxidant effects in a variety of tissues, including
arteries, under physiological and pathophysiological conditions
associated with oxidative stress.
Superoxide production. To gain insight into the source of
arterial oxidative stress with aging and its mitigation with
nitrite administration, aortic superoxide production was directly assessed using electron paramagnetic resonance spectroscopy in the above referenced studies (53, 171). Superoxide
production was much greater in aorta from old compared with
young control mice, and short-term treatment with sodium
nitrite completely normalized superoxide formation in old
animals (53, 171) (Fig. 5B). These observations indicate that
sodium nitrite treatment reverses arterial oxidative stress at
least in part by reducing superoxide production, and are in
agreement with recent findings in hypertensive rats showing
reduced staining for superoxide in aortic rings following nitrite
supplementation (128, 129).
To link these age- and nitrite-related effects on arterial
superoxide production and oxidative stress to arterial function, the superoxide scavenger TEMPOL (4-hydroxy-2,2,6,6-
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To our knowledge, the effects of inorganic nitrite supplementation on arterial stiffness have not been investigated in
humans. However, 4 wk of sodium nitrate supplementation
was reported to decrease aortic PWV in older adults with risk
factors for CVD (156). In addition, several studies have investigated the effects of acute nitrate administration in human
subjects. An acute oral dose of potassium nitrate was found to
decrease aortic PWV in young healthy adults (6), whereas a
single dose of beetroot juice reduced aortic PWV in patients
with Stage 1 essential hypertension (57). A meal high in nitrate
had no significant effect on aortic PWV in a group of healthy
adults, although an indirect measure of arterial stiffness derived from measurements at the radial artery was found to
decrease (110).
In summary, these findings indicate that short-term supplementation with sodium nitrite reduces aortic stiffness in old
mice, perhaps by reversing AGEs formation. Moreover, there
is some evidence from preliminary studies that suggests that
boosting nitrite bioavailability via dietary nitrate intake may
hold promise for reducing large elastic artery stiffness in
humans.
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•
Sindler AL et al.
tetramethylpiperidine-N-oxyl) was administered to carotid arteries ex vivo and EDD was assessed (171). TEMPOL had no
effect on EDD in old mice treated with sodium nitrite, but
completely restored EDD in old control animals (i.e., the group
with excessive superoxide-associated oxidative stress at baseline). Overall, these results are consistent with the idea that
sodium nitrite treatment restores NO-mediated EDD in old
mice by reducing superoxide production and oxidative stress.
Tetrahydrobiopterin and eNOS uncoupling. To determine
whether age-associated oxidative stress and its reversal with
sodium nitrite might be influencing arterial function via BH4
deficiency-dependent eNOS uncoupling, carotid arteries have
been treated ex vivo with sepiapterin (171), which increases
BH4 bioavailability, recouples eNOS, and boosts NO production in states of BH4 deficiency (38). Sepiapterin restored
acetylcholine-induced EDD selectively in carotid arteries of
old control mice although it had no effect on EDD in old mice
treated with sodium nitrite (171). These data suggest that
superoxide-related oxidative stress suppresses arterial function
in old control mice by causing oxidation of BH4 and uncoupling eNOS, thus reducing NO production. Sodium nitrite
restores NO-mediated vascular endothelial function in part by
reversing these effects of aging on superoxide formation and
the oxidation of BH4, thus preserving eNOS-mediated NO
production. These observations are consistent with recent findings of increases in BH4 and the BH4/BH2 ratio in the liver
after sodium nitrite treatment for 3 wk in mice fed a high-
cholesterol diet (175), and further support the concept that
nitrite has antioxidant actions in vivo.
NADPH oxidase. To determine the source of superoxideassociated oxidative stress with arterial aging and the therapeutic effects of sodium nitrite supplementation, the contribution of the major oxidant-producing enzyme in vascular tissue,
NADPH oxidase has been assessed (171). Protein expression
of NADPH oxidase was threefold greater in aorta of old mice
compared with those of young control mice, but this was
normalized after sodium nitrite treatment (Fig. 5C). To link
NADPH oxidase-associated superoxide production to age- and
sodium nitrite-related effects on arterial function, the influence
of inhibiting the enzyme with apocynin was assessed on carotid
artery EDD ex vivo (171). Inhibition of NADPH oxidase with
apocynin had no effect on EDD in arteries of old animals
treated with sodium nitrite, but restored EDD in old control
mice (171). These observations support the postulate that
sodium nitrite treatment ameliorates superoxide-dependent impairment of NO-mediated vascular endothelial function in old
mice at least in part by reducing superoxide production by
NADPH oxidase.
Taken together, these results are consistent with recent
findings in an experimental model of hypertension in which
sodium nitrite supplementation reduced NADPH oxidase activity and apocynin decreased staining for superoxide in aortic
rings of control and/or hypertensive rats (128). In subsequent
in vitro experiments (liver homogenates) from that study,
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Fig. 5. Nitrotyrosine (NT) abundance (A), mean electron paramagnetic resonance (EPR) signal for superoxide (B), protein expression of p67 subunit of NADPH
oxidase (C), and superoxide dismutase (SOD) enzymatic activity (D) in aorta from young and old control and old nitrite-supplemented (Old Nitrite) mice. Values
are means ⫾ SE. *P ⱕ 0.05 Young Control vs. Old Control; †P ⱕ 0.05 Old Control vs. Old Nitrite. Data were adapted from (171).
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Sindler AL et al.
471
sistent with this finding, sodium nitrite decreases plasma IL-6
concentrations and increases survival rate in a crush injury
model in rats (134), and reduces liver granulocyte infiltrates
after endotoxemic shock (28, 29, 65). Nitrite also lessens
inflammation caused by ischemia in various organs (89, 125,
140, 146, 147, 176, 197) and normalizes inflammation in a
mouse model of inflammatory bowel disease (139). Moreover,
studies conducted in microvascular endothelial cell culture
demonstrate that nitrite prevents TNF-␣-induced upregulation
of intracellular adhesion molecule type 1, providing evidence
of a direct anti-inflammatory effect of nitrite in vascular cells
(78). Taken together, these results support the view that nitrite
therapy exerts a potent systemic and vascular anti-inflammatory effect that is associated with improvements in arterial
function.
Mitochondrial function. Accumulating evidence implicates
mitochondrial dysregulation in impaired arterial function both
in animal models of cardiovascular disease (94, 161, 182) and
human diabetes mellitus (166). Emerging evidence suggests
that NO deficiency is associated with impaired mitochondrial
function and that restoration of NO bioavailability induces
mitochondrial biogenesis (127, 135, 167, 168). A recent study
reported nitrite-induced stimulation of mitochondrial biogenesis is associated with increases in adenosine monophosphateactivated protein kinase and peroxisome proliferator-activated
receptor gamma coactivator 1-␣ signaling in aortic smooth
muscle cells in an experimental model of carotid artery injury
(127). Pretreatment with nitrite under normoxic conditions
protects cardiomyocytes from cell death after a hypoxic challenge and is dependent on activation of protein kinase A, which
inhibits dynamin-related protein-1 leading to enhanced mitochondrial fusion (153). Nitrite also minimizes mitochondrial
damage in a mouse shock model induced by a lethal dose of
TNF-␣ (29), and nitrate normalizes mitochondrial-specific antioxidant proteins in a cardiotoxicity model in mice (196). In
humans, boosting inorganic nitrite either from oxidation of
endogenously produced NO or from dietary supplementation
of nitrate, enhances mitochondrial function and efficiency by
improving oxidative phosphorylation (102, 103). Collectively,
these observations have potentially important implications for
the prevention and treatment of metabolic disorders, agerelated physiological dysfunction, and chronic diseases. We
expect future studies to determine the effects of nitrite on
age-associated mitochondrial dysfunction and its role in vascular function.
Integrative Working Hypothesis
Fig. 6. Expression of proinflammatory cytokines interleukin (IL)-1␤, IL6,
interferon-␥ (IFNg), and tumor necrosis factor-␣ (TNF-alpha) in aorta from
young and old control and old nitrite-supplemented (Old Nitrite) mice. Values
are means ⫾ SE. *P ⱕ 0.05 Young Control vs. Old Control; †P ⱕ 0.05 Old
Control vs. Old Nitrite. Data were adapted from (171).
How might oral inorganic nitrite or nitrate supplementation
produce the beneficial vascular effects observed in preclinical
studies in old mice and, perhaps, in recent investigations in
models of cardiovascular disease (3, 84, 129, 175)? Although
the precise mechanisms of action are incompletely understood,
we offer the following reasoned speculation as illustrated in
Fig. 7. Dietary supplementation of inorganic nitrate that results
in significant increases in circulating and tissue nitrite levels
would presumably have the same actions.
To briefly review, aging leads to increases in vascular
production of superoxide anion, resulting in a state of oxidative
stress and causing synergistic development of vascular inflammation. The excessive vascular superoxide bioactivity induces
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nitrite administration did not exert direct antioxidant effects,
inhibit the oxidant enzyme xanthine oxidase, or suppress mitochondrial superoxide production, but rather appeared to exert
its antioxidant influence via inhibition of NADPH oxidase
protein expression and activity (128).
Antioxidant defenses. Arterial antioxidant enzyme defenses
also have been assessed in response to nitrite treatment in old
mice (171). The activity of the major antioxidant enzyme SOD
was found to be ⬃50% lower in aorta of old compared with
young control mice, and short-term treatment with sodium
nitrite restored SOD activity in aorta of old mice to levels
observed in young controls (Fig. 5D). These data provided the
first evidence for an antioxidant-boosting effect of sodium
nitrite, and suggest that activation of SOD may play a significant role in the ability of nitrite supplementation to reverse
age-associated oxidative stress and improve arterial function.
These findings are supported by recent work demonstrating
that nitrite normalizes SOD activity in a model of renovascular hypertension (129), and in gills and liver of fish (31).
Nitrite supplementation also preserves or increases antioxidant
capacity in the heart after an acute nitrite exposure under basal
conditions (43, 89, 113, 148), after I/R injury (37), and in a rat
model of high altitude-induced hypoxia (172). Finally, nitrite
protects the kidneys after brain death-induced renal injury (74)
and liver after an acute alcohol-induced injury (97) by enhancing antioxidant defenses.
Inflammation. Chronic low-grade inflammation develops in
arteries with aging (44, 180) and is a key mechanism mediating
age-related arterial dysfunction (34, 45, 149, 162). Consistent
with these earlier observations, expression of the proinflammatory cytokines interleukin (IL)-1␤, IL-6, interferon-␥, and
tumor necrosis factor-␣ (TNF-␣) were shown to be greater in
aorta of old mice than young control mice (171) (Fig. 6). Most
importantly, nitrite supplementation completely reversed this
age-associated arterial inflammation (171). These findings are
in agreement with a report showing that sodium nitrite treatment normalizes plasma C-reactive protein concentrations,
leukocyte markers of inflammation, and improves vascular
endothelial function in hypercholesterolemic mice (175). Con-
•
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Inorganic Nitrite, and Arterial Aging
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Sindler AL et al.
Fig. 7. Proposed mechanisms for the detrimental effects
of aging on the vasculature and possible beneficial
effects of nitrite. O2⫺, superoxide; AGEs, advanced
glycation end-products; NO, nitric oxide; SOD, superoxide dismutase.
trite supplementation also may induce mitochondrial biogenesis and improve mitochondrial health in arteries, and exert
broadly beneficial actions in multiple cell stress pathways in
the aging vasculature. Finally, nitrite therapy also holds promise for treating many other age-related clinical disorders associated with increased risk for CVD, including hypercholesterolemia (175), hypertension (58, 128), and chronic kidney
disease (84).
Increasing the bioavailability of NO by boosting concentrations of its precursor, nitrite (either by direct supplementation
of nitrite or via increased dietary nitrate intake), represents a
new paradigm that has great potential for improving NO
homeostasis and vascular function with aging. However, much
of the work to date determining the beneficial effects of nitrite
or nitrate supplementation per se has occurred using preclinical
models or acute administration in humans. As such, more
studies are needed to determine the broad therapeutic potential
and feasibility with chronic nitrite or nitrate administration on
physiological and pathophysiological outcomes. Several ongoing clinical trials assessing the efficacy of nitrite and nitrate
supplementation should provide new insight into the potential
pleiotropic effects of increasing nitrite bioavailability on ageassociated physiological dysfunction and disease. Despite its
potential clinical implications, additional research is needed to
establish the efficacy of chronic nitrite or nitrate supplementation for the promotion of vascular health and the prevention
and treatment of CVD.
GRANTS
This work was supported by National Institutes of Health Grants AG013038, HL-107105, HL-007822, AG-000279, and TR-000154.
CONCLUSIONS AND CLINICAL IMPLICATIONS
DISCLOSURES
In conclusion, nitrite and nitrate are biologically important
precursors of NO that have the potential to restore NO bioavailability in states of NO insufficiency via the nitrate-nitrite-NO pathway. Recent findings provide experimental support that oral supplementation of inorganic nitrite or nitrate
may have the ability to ameliorate key features of arterial aging
that are believed to be clinically important antecedents of
CVD, including large elastic artery stiffening, endothelial dysfunction, and vascular oxidative stress and inflammation. Ni-
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: A.L.S. and D.R.S. conception and design of research;
A.L.S. performed experiments; A.L.S. and B.S.F. analyzed data; A.L.S., B.S.F.,
and D.R.S. interpreted results of experiments; A.L.S., A.E.D., and B.S.F. prepared
figures; A.L.S. and A.E.D. drafted manuscript; A.L.S., A.E.D., B.S.F., and D.R.S.
edited and revised manuscript; A.L.S., A.E.D., B.S.F., and D.R.S. approved final
version of manuscript.
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several changes to arteries including modifications of structural
proteins (greater levels of collagen, fragmentation of elastin,
formation of AGEs, etc.) and greater generation of the ROS
peroxynitrite (via superoxide reaction with NO), with consequent oxidation of BH4 and uncoupling of eNOS. The latter
series of events result in lower NO bioavailability due to the
combination of direct superoxide reaction with NO and less
eNOS production of NO. Reduced bioavailability of NO in
turn results in less circulating nitrite because less NO is being
metabolized into nitrite.
We postulate that nitrite supplementation reverses the vascular effects of aging, possibly by multiple mechanisms of
action. Nitrite, perhaps in part through modulation of gene
transcription (21, 85, 172), reduces superoxide by suppressing
ROS production by the potent vascular oxidant enzyme NADPH
oxidase and, possibly, by mitochondria, while boosting the
activity of the important superoxide scavenging antioxidant
enzyme, SOD. This normalization of vascular superoxide reverses the changes in structural proteins and its reaction with
NO, reduces peroxynitrite formation, and recouples eNOS,
such that NO bioavailability is increased via less destruction
(by superoxide) and more production by eNOS. Endothelial
NOS also produces less superoxide under these conditions
(reversing the vicious cycle) and suppresses inflammation. In
addition to these effects on superoxide, nitrite supplementation
increases NO bioavailability via subsequent reductions in the
circulation and tissues to NO, mediated by both enzymatic and
nonenzymatic pathways (114). Thus, circulating and arterial
concentrations of nitrite are restored in old animals and, collectively, these events lead to improvement, even normalization in some cases, of NO homeostasis and vascular function.
Synthesis
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