Journal of the American College of Cardiology
2013 by the American College of Cardiology Foundation
Published by Elsevier Inc.
Vol. 62, No. 2, 2013
ISSN 0735-1097/$36.00
http://dx.doi.org/10.1016/j.jacc.2013.03.070
STATE-OF-THE-ART PAPER
Effects of Nitric Oxide on Cell Proliferation
Novel Insights
Claudio Napoli, MD, PHD,*y Giuseppe Paolisso, MD,z Amelia Casamassimi, BIOLD,*
Mohammed Al-Omran, MD,x Michelangela Barbieri, MD,z Linda Sommese, BIOLD,*
Teresa Infante, BIOLD,y Louis J. Ignarro, PHDk
Naples, Italy; Riyadh, Kingdom of Saudi Arabia; and Los Angeles, California
Nitric oxide (NO) has been suggested to be a pathophysiological modulator of cell proliferation, cell cycle arrest, and
apoptosis. In this context, NO can exert opposite effects under diverse conditions. Indeed, several studies have
indicated that low relative concentrations of NO seem to favor cell proliferation and antiapoptotic responses and
higher levels of NO favor pathways inducing cell cycle arrest, mitochondria respiration, senescence, or apoptosis.
Here we report the effects of NO on both promotion and inhibition of cell proliferation, in particular in regard to
cardiovascular disease, diabetes, and stem cells. Moreover, we focus on molecular mechanisms of action involved
in the control of cell cycle progression, which include both cyclic guanosine monophosphate–dependent
and –independent pathways. This growing field may lead to broad and novel targeted therapies against
cardiovascular diseases, especially concomitant type 2 diabetes, as well as novel bioimaging NO-based diagnostic
tools. (J Am Coll Cardiol 2013;62:89–95) ª 2013 by the American College of Cardiology Foundation
Nitric oxide (NO) is a signaling molecule that regulates
many functions, such as vascular tone, blood pressure,
neurotransmission, immune response, and oxidation-sensitive
mechanisms (1–4). NO may act as an autocrine or paracrine
messenger, and its production and degradation are cell type
dependent. NO synthesis is catalyzed from L-arginine by
a family of nitric oxide synthases (NOSs), varying from
picomolar/nanomolar range for short periods to micromolar
range for protracted periods (3). NO reacts with molecules
such as oxygen, superoxide or metals, nucleic acids, and
proteins. The major reactions involving NO support its rapid
oxidation into nitrate and nitrite, which are now considered
not inert end products but actors of a reverse pathway that
represents an alternative source of NO when the endogenous
L-arginine/NOS pathway is dysfunctional (5,6).
The effect of NO production on cellular processes is
also dependent on its concentration and on the presence of
other free radicals. Peroxynitrites, generated from reaction
with a superoxide, can interact with several cellular components and are implicated in NO signaling mechanisms
From the *Department of General Pathology, Excellence Research Centre on
Cardiovascular Diseases, U.O.C. Immunohematology, Second University of Naples,
Naples, Italy; zDivision of Geriatrics, 1st School of Medicine, Second University
of Naples, Naples, Italy; yFondazione SDN, IRCCS, Naples, Italy; xCollege of
Medicine, King Saud University, Riyadh, Kingdom of Saudi Arabia; and the
kDepartment of Pharmacology, David Geffen School of Medicine, University of
California Los Angeles, Los Angeles, California. This study was supported in part by
Regione Campania 2008 Research Funds. The authors have reported that they have no
relationships relevant to the content of this paper to disclose.
Manuscript received February 15, 2013; accepted March 19, 2013.
involving protein modifications (6). Lower concentrations of
NO have been suggested to exert a direct effect on processes
such as cell proliferation and survival, whereas higher concentrations have an indirect effect through both oxidative and nitrosative stresses (3,5,6). Free radical interactions
also influence NO signaling. One of the consequences of
reactive oxygen species (ROS) generation is reduced concentrations of NO (1). The resulting reactive nitrogen
species can also have biological effects and increase oxidative
and nitrosative stress responses. Overall, cellular responses
are differentially regulated by specific NO concentrations,
with lower NO concentrations (picomolar to nanomolar)
generally promoting cell survival and proliferation and
higher concentrations (micromolar) favoring cell cycle arrest,
apoptosis, and senescence (6) (Fig. 1A). However, observed divergent effects can be explained by cellular context,
cell cycle point, and oncogenic state (6). The molecular
mechanisms underlying the proliferative action of NO at
low concentrations are not yet clear, but key molecules
and pathways involved in NO-mediated inhibition have
been better studied. Some basic distinct concentrations of
NO have also been proposed for activity, such as for cyclic
guanosine monophosphate (cGMP)-mediated processes
(30 nmol/l) and nitrosative stress (1 mmol/l) (6).
Molecular Mechanisms of NO Involved in
Progression of the Cell Cycle
NO blocks the progression of the cell cycle primarily at the
G1/S transition. Hence, NO-induced G1 arrest has been
observed in vascular smooth muscle cells (VSMCs) and
90
Napoli et al.
NO and Cell Proliferation
other cell lines (7–9). In other
contexts and after treatment with
NO donors of the nonsteroidal
BMC = bone marrow cell(s)
anti-inflammatory drug family, the
cGMP = cyclic guanosine
entry into the G2/M phase was
monophosphate
NO sensitive (8,10,11).
EGFR = epidermal growth
As expected, many cell cycle
factor receptor
proteins (p21Cip1/Waf1, p27, cyeNOS = endothelial nitric
clins, Cdk2, pRb, and the like)
oxide synthase
are good candidates for final moEPC = endothelial progenitor
lecular targets of NO, as revealed
cell(s)
by exogenous NO, endothelial
GC = guanylate cyclase
nitric oxide synthase (eNOS) transNO = nitric oxide
fection, and whole expression studNOS = nitric oxide synthase
ies (7,9,12–18). NO is also able
ROS = reactive oxygen
to regulate cell proliferation by
species
targeting important mitogenic
T2DM = type 2 diabetes
receptors and their downstream
mellitus
pathways, such as epidermal growth
VSMC = vascular smooth
factor receptor (EGFR) signaling
muscle cell(s)
pathway (8,19). Moreover, 26S
proteasome and apoptosis factors represent other important
NO targets (20,21) (Fig. 1B).
cGMP-dependent and -independent pathways. A canonical NO pathway involves the selective activation of soluble
guanylate cyclase (GC), the generation of cGMP, and the activation of specific cGMP-dependent protein kinases (1,22).
However, further mechanisms have emerged that mainly
include the formation of NO-induced post-translational
modifications (8,22,23). Overall, these modifications activate
pathways that are cGMP independent (Fig. 1B).
The involvement of cGMP in growth inhibition has
been described in VSMCs, in which NO activates GC with
a subsequent increase in cGMP leading to the phosphorylation of a vasodilator-stimulated phosphoprotein and subsequent inhibition of the epidermal growth factor signaling
pathway (24,25). Recent findings indicate that other nuclear
effects of NO altering the cell cycle can occur. A mechanism
involved in the regulation of the cell cycle is the direct interaction of NO-sensitive GC with chromosomes during
mitosis (26). More recently, NO has been shown to modulate chromatin folding in human endothelial cells through
class II histone deacetylases (27).
The antiproliferative effect of NO does not necessarily
require cGMP as an intermediate effector because it was
also shown in a fibroblast cell line lacking soluble GC (19)
or with GC inhibitors (16,28,29). Inhibition of the G1/S
transition has been shown to be mostly a cGMP-independent
process (7,9), although dual mechanisms were found to
mediate the antimitogenic effects in VSMCs (30).
A mechanism for cGMP-independent proliferative arrest
is S-nitrosylation, which can reversibly inhibit the catalytic
activity of the 26S proteasome and enhance Ras guanine
nucleotide exchange under nitrosative stress (31,32). A recent study has also demonstrated that NO-mediated Ras
nitrosylation in different subcellular compartments regulates
Abbreviations
and Acronyms
JACC Vol. 62, No. 2, 2013
July 9, 2013:89–95
downstream pathways stimulating cell proliferation (33).
In addition, the EGFR trans(auto)phosphorylation inhibition was found to be independent of cGMP and directly
inhibited by NO (19). However, an increase in NOmediated cGMP enables the disruption of the downstream
mitogenic signal through Raf1 phosphorylation (25).
eNOS S-glutathionylation is a pivotal redox regulator
of endothelial function and vascular tone. Indeed, in
endothelial cells, this modification reversibly decreases
eNOS activity with increased production of superoxide
anions. Moreover, in hypertensive vessels, eNOS S-glutathionylation is increased with impaired vasodilation (34).
The impact of another mechanism, tyrosine nitration, was
demonstrated in a recent study that identified several nitrated
proteins involved in the different checkpoints toward the cell
cycle (35).
Another cGMP-independent mechanism involves mitochondria that influence several pathways modulating cell
proliferation, cell cycle arrest, and apoptosis through oxidative and nitrosative reactions that are mediated by the
NOS mitochondrial isoform (23,36). Moreover, peroxynitrite can also change calcium homeostasis, allowing the
opening of the mitochondrial permeability transition pore
that promotes mitochondrial signaling of cell death (37). In
this context, NO instead exerts a protective action by directly
inhibiting the opening of the mitochondrial permeability
transition pore. Furthermore, cytochrome c oxidase is one of
the most important targets for NO signaling, leading to
inhibition of mitochondrial oxidative phosphorylation, the
control of apoptosis, and ROS generation (37,38).
Cell Cycle NO-Dependent Effects in the
Cardiovascular System
Cell proliferation, inhibition, and apoptosis. NO is able
to inhibit cell growth and proliferation and to induce
apoptosis in a dose-dependent manner (7,12,20,28,29,39,40)
(Table 1). Indeed, an adequate concentration of NO is
required to induce inhibition of cell proliferation. There is a
lack of correlation between increased NOS expression and
inhibition of cell proliferation in some cellular systems,
probably due to the short life of NO that is transformed into
inactive compounds (6,8,28).
NO donors and drugs affecting the NO pathway through
different mechanisms have been used to inhibit cell proliferation of several cell types. Indeed, it is known that
endogenously produced NO can negatively regulate cell
proliferation and/or the proliferation of neighboring cells.
The increase of endogenous NO depends on the availability
of L-arginine and/or inducible NOS expression mediated by
several cytokines (41–43).
A proliferative arrest induced by endogenous NO production has been found in different cell types, including
VSMCs (8). Proliferation of these cells has been accepted
as a common event in the pathophysiology of many vascular diseases. Delivery of L-arginine, pharmacological NO
Napoli et al.
NO and Cell Proliferation
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July 9, 2013:89–95
Figure 1
91
Major Molecular Mechanisms Involved in the Effects of NO on the Cell Cycle
(A) Dual role of different doses of nitric oxide (NO) on the cell cycle and their effects on cardiovascular-related cells. (B) NO synthesis from its precursor L-arginine is catalyzed by
nitric oxide synthases (NOSs). NO is rapidly oxidized into nitrate (NO3) and nitrite (NO2), which can be reduced into bioactive NO in a reverse pathway. NO can also react with
superoxide (O2) to generate peroxynitrite anion (ONOO), which is implicated in NO signaling mechanisms involving post-translational modifications. Molecules involved in both
cyclic guanosine monophosphate–dependent and –independent pathways are shown. NO can influence the epidermal growth factor receptor (EGFR)/Ras/mitogen-activated
protein kinase (MAPK) proliferative pathway by both mechanisms. Examples are EGFR and Ras S-nitrosylation. Mitochondria-related mechanisms, molecules involved in the
apoptosis, insulin/insulin-like growth factor (IGF)-I/Akt, and 26S proteasome pathways are also represented.
donors, NO gas, or overexpression of NOS proteins can
inhibit proliferation of VSMCs and reduce the injury
responses within the blood vessel wall. Preclinical models
have been essentially used to establish the potential of NO in
the inhibition of VSMC proliferation and induction of cell
cycle arrest in models of neointimal hyperplasia and restenosis (11,16,44). A recent study also suggested that NO can
inhibit neointimal hyperplasia in a sex- and hormonedependent manner (44).
Consistent with these observations, VSMCs transfected
with eNOS showed inhibition of cell proliferation and of
key cell cycle regulatory molecules (45). Moreover,
apolipoprotein E is able to increase expression of inducible
NOS in VSMCs, thereby inhibiting their proliferation (46).
NO has been implicated in both apoptotic and necrotic
cell death, depending on several factors including cellular
redox state. Additionally, it can be either an antiapoptotic or
a proapoptotic regulator, depending on the pathway and
mechanism involved. For instance, at high concentrations of
NO, programmed cell death was observed after inhibition of
cell proliferation in cardiomyocytes (47). Caspase activation
and mitochondrial changes are also involved in the apoptosis
initiated after treatment with GEA3162 in murine bone
marrow cells (BMCs) (48). NO can also confer protection
92
Napoli et al.
NO and Cell Proliferation
Table 1
JACC Vol. 62, No. 2, 2013
July 9, 2013:89–95
Effects of Exogenous NO on the Proliferation of Cardiovascular-Related Cells
Action
First Author (Ref. #)
Aortic VSMC
Cell Type
DETA/NO
NO Donors
100 mmol/l to 1 mmol/l
NO Donor
Tanner et al. (7) and
Kapadia et al. (20)
Umbilical arteries VSMC
SNAP
100 mmol/l
Ishida et al. (12,39)
HUVEC/coronary aortic endothelial cells
SNAP, S-nitrosoglutathione, SNP
100 mmol/l
Heller et al. (28)
Peripheral blood mononuclear cells
GEA3162, GEA3175, SNAP
1–30 mmol/l GEA; 100–500 mmol/l SNAP
Kosonen et al. (29)
HUVEC
SNAP, SNP, DETA/NO
100–600 mmol/l
Gooch et al. (40)
Murine bone marrow cells (Jaws II)
GEA3162
30–100 mmol/l
Taylor et al. (48)
Embryonic stem cells
DETA-NO
2–20 mmol/l
þ
Tejedo et al. (21)
Murine bone marrow stromal cells (OP9)
SNAP
10–50 mmol/l
þ
Wong et al. (51)
DETA/NO ¼ N-{4-[1-(3-aminopropyl)-2-hydroxy-2 nitrosohydrazino]butyl}propane-1,3-diamine; GEA3162 ¼ 1,2,3,4-oxatriazolium,5-amino-3-(3,4-dichlorophenyl)-chloride; GEA3175 ¼ 1,2,3,4-oxatriazolium,
3-(3-chloro-2-methylphenyl)-5-[[(4-methylphenyl) sulfonyl]amino], hydroxide inner salt; HUVEC ¼ human umbilical vein endothelial cells; NO, nitric oxide; SNAP ¼ S-nitroso-N-acetylpenicillamine; SNP ¼ sodium
nitroprusside; VSMC ¼ vascular smooth muscle cells.
from apoptosis and promote survival of embryonic stem cells,
thus delaying their differentiation (21). NO donors were
found to cause a dramatic and concentration-dependent
induction of apoptosis in a caspase-dependent manner in
human neutrophils and murine BMCs (8,48).
Effects on cell proliferation. NO can also promote cell
proliferation under certain conditions, although the mechanisms responsible for this effect have remained poorly
understood (2,6). Interestingly, a recent in vivo study (49)
showed that eNOS deficiency may cause collateral vessel
rarefaction, thus impairing the activation of a cell cycle gene
network during arteriogenesis; this finding has suggested a
novel role for eNOS in maintaining native collateral density
during growth to adulthood and in collateral remodeling in
obstructive disease through regulation of cell proliferation
(49). Similarly, NO derived from eNOS has been shown to
play an important role in cardiomyocyte proliferation and
maturation during early neonatal heart development (50).
Still, NO donor treatment determines cell proliferation in
several contexts (Table 1) (8,21,51). Interestingly, an NO
donor at low concentrations (10 to 50 mmol/l) protected
murine bone marrow stromal cells against spontaneous
apoptosis (51). Similarly, exposure of embryonic stem cells
to low concentrations of NO (2 to 20 mmol/l) can regulate
their differentiation by arresting the loss of self-renewal
markers and promoting cell survival through inhibition of
apoptosis (21). Overall, these studies support the concept
that the final effect of NO on the cell cycle is dependent on
both concentration and the surrounding context (Fig. 1A).
The existence of multipotent resident cardiac stem cells
and adult BMCs that participate in homeostasis of the heart
and regeneration of the injured tissue make these cells a
valuable resource for the treatment of cardiovascular disease
(52,53). The involvement of NO signaling in stem cell
cardiovascular biology was demonstrated in the differentiation of embryonic stem cells into myocardial cells (54).
Furthermore, a recent study in a porcine model showed
that activation of the NO pathway directs BMCs to
a preferential cardiomyogenic phenotype and also stimulates
cell proliferation (55). However, the precise role of NO
in cardiac progenitors and stem cells remains poorly
understood.
NO scavenger treatment can increase the proliferation of
bone marrow–derived endothelial progenitor cells (EPCs),
a subpopulation of adult stem cells that are recruited from
bone marrow to the injured vessel to promote endothelial
regeneration and neovascularization (53). Data from several
studies indicate that the NO pathway may improve the
paracrine and angiogenesis efficiency of BMCs in experimental hind limb ischemia (56–58) and in patients with
chronic critical limb ischemia (59,60).
The NO Pathway and Cell Proliferation
in Type 2 Diabetes
Hyperglycemia, associated with endothelial dysfunction and
reduced new blood vessel growth, is a primary cause of
vascular complications in diabetes. Moreover, the ability of
patients with type 2 diabetes mellitus (T2DM) to develop
coronary collateral vessels is diminished due to a diabetesassociated impairment of EPC count and mobilization
(61). The EPC count of patients with T2DM and peripheral arterial disease is substantially lower than that of healthy
subjects, nondiabetic patients with vascular disease, and
patients with T2DM who do not have vascular disease
(53,60,62). Moreover, EPCs isolated from patients with
T2DM displayed impaired proliferation and function (63).
Additionally, proliferation of EPCs was inversely correlated
with plasma glycated hemoglobin levels of these patients,
suggesting a relationship between glycemic control and EPC
number and proliferation (63).
EPCs require NO-cGMP signaling for proper function,
including migration, and decreased bioavailability of NO has
been proposed as one of the determinants of vascular
damage in diabetes (61). Patients with T2DM have a lower
overall systemic fraction of L-arginine that is converted to
NO compared with healthy individuals (64). Interestingly,
eNOS uncoupling in diabetic EPCs resulted in excessive
superoxide anion production and reduced bioavailability of
NO, implying an intimate relationship between oxidative
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July 9, 2013:89–95
stress and EPC damage (65). Prolonged exposure of early
and late EPCs to high glucose concentrations reduces their
number and proliferative ability and the extent of phosphorylation of eNOS and some members of the PI3-kinase/Akt
signaling pathway (66). This study also showed that
impaired NO-related rather than oxidative stress–related
mechanisms are involved in the EPC dysfunction induced
by high levels of glucose (66). In light of such evidence,
pharmacological or genetic interventions affecting the molecular pathway involving eNOS activity have been hypothesized to counteract diabetic EPC dysfunction. Thus, the
stimulation of NO production or its signaling cascades may
increase the number and function of EPCs and attenuate
endothelium damage, independently of the vasodilatory
effects of NO. In this regard, a recent study showed that
treatment of glucose-stressed EPCs with superoxide dismutase attenuated generation of O2, restored production of
NO, and partially restored their ability to form colonies,
whereas treatment with insulin increased production of NO
but did not change generation of O2 and their ability to form
colonies. However, this ability was fully restored after
combined treatment with superoxide dismutase and insulin
(61). Additionally, Chen et al. reported that in vitro treatment of EPCs from patients with T2DM with an NO donor
drug could also reverse impairment of EPCs induced by high
levels of glucose acting on cell proliferation (66).
New Concepts in the Role of NO
in the Cell Cycle and Clinical Insights
The NO pathway is involved in many cardiovascular
conditions, and delivery of specific concentrations of exogenous NO is an attractive therapeutic option for these
disorders, including those that are cell proliferation based. A
well-known example of cell proliferation–based pathology is
neointimal hyperplasia, which has been found to decrease
with NO-based therapies in animal model studies. A recent
study suggested that particular attention should be paid to
the patient’s sex and hormone status when developing these
therapies (44). A significant protective role of NO is also
implicated in therapeutic neoangiogenesis associated with
peripheral ischemia (56–60).
Interestingly, vasorelaxant prostanoids used in clinical
practice, such as prostacyclin or its derivatives, have been
shown to exert protective effects on endothelial cells by
mechanisms that partly involve cyclic adenosine monophosphate–mediated formation of NO (67). Indeed, prostacyclin analogues such as Beraprost or Iloprost can increase
the number and migration of EPCs in humans and in
ischemic tissues of experimental animal models (68,69).
Prostacyclin has been found to exert a direct effect on the
function of EPCs in an autocrine or a paracrine manner
through an NO-dependent mechanism (70). In this regard,
NO-dependent vasoprotective agents such as prostacyclin or
statins could have a significant therapeutic role in cardiovascular diseases under pathological conditions, such as
Napoli et al.
NO and Cell Proliferation
93
diabetes, where EPCs are impaired. Moreover, other drugs
such as angiotensin-converting enzyme inhibitors also prevent endothelial dysfunction by increasing eNOS protein
expression and activity (71–73).
The biology of NO has stimulated the development of
pharmacological agents with different actions on cell proliferation; for example, some agents are able to release NO,
such as NO-donating nonsteroidal anti-inflammatory drugs
and NO-aspirin, and may also be used against vascular
damage. Indeed, NCX-4016, an aspirin-like NO donor, has
been found to reduce experimental restenosis (11) and the
development of atherosclerosis (74), which are linked to
proliferation of VSMCs. Similarly, nebivolol, a beta-blocker
that releases NO, was able to reduce experimental atherosclerosis (75). Consistently, there are numerous NO-based
therapies for pulmonary hypertension (76), another disease
linked to proliferation of VSMCs. Preclinical studies
revealed a potent effect of inhaled nitrite in the inhibition of
experimental pulmonary arterial hypertrophy and proliferation of VSMCs, although further studies are required to
better establish doses, potential toxic effects, and mechanisms of their therapeutic action (77). The chemical versatility of NO has led to the synthesis of a wide range of NO
donors, each with a different mode and rate of release and
action, suitable for different disease targets. However, longterm use of current NO donors is limited by development
of tolerance and toxicity issues, suggesting that novel alternatives should be identified. Nitrosyl-cobinamide has been
found to be an effective NO-releasing compound in several
models, suggesting that it could be a useful drug for treating hypertension and cardiovascular disease (78). Furthermore, a recent study showed the positive effect of infusion
of NO-releasing hydrogel/glass hybrid nanoparticles that
induce a reduction of blood pressure and an increase in
vascular relaxation without any tolerance and immunologic
response (79).
Additionally, in vitro and in vivo studies have shown that
some mitochondrial antioxidants are able to decrease ROS
production, leading to reduced apoptosis and improved
cardiac function. However, the use of these molecules in
humans require the development of specific and sensitive
tools to monitor mitochondrial oxidative stress and the
development of orally available compounds (80).
One of the major weaknesses in studying ROS and reactive nitrogen species is the lack of proper tools to monitor
their production in vivo. Thus, sensitive and specific detection methods can be useful for elucidation of the effects of
NO on cell proliferation in pathophysiological conditions
both in vitro and in vivo. To date, fluorescent probes based
on small organic molecules provide dynamic information
concerning the localization and quantity of biological molecules of interest (81). This approach could be worthwhile to
evaluate their functions in the living body by using less
invasive techniques, without the need for isolating tissues or
cellular constituents. Today, several design strategies for
specific reactive nitrogen species fluorescent probes to use in
94
Napoli et al.
NO and Cell Proliferation
bioimaging technologies, including photo-induced electron
transfer, are well established and have been applied to many
probes. The use of this approach should be suitable for
real-time analysis of NO-dependent mechanisms (82–84).
Reprint requests and correspondence: Prof. Claudio Napoli,
Department of General Pathology, Excellence Research Centre on
Cardiovascular Diseases, U.O.C. Immunohematology, 1st School
of Medicine, Second University of Naples, 80138 Naples, Italy.
E-mail: [email protected] or [email protected].
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Key Words: cardiovascular diseases - cell cycle - cell proliferation
diagnostic and therapeutic tools - imaging - nitric oxide type 2 diabetes.
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