Translating the oxidative stress hypothesis into the clinic: NOX versus NOS.

REVIEW
Translating the oxidative stress hypothesis into the clinic:
NOX versus NOS
Melanie E. Armitage & Kirstin Wingler &
Harald H. H. W. Schmidt & Mylinh La
Received: 19 August 2009 /Revised: 14 September 2009 /Accepted: 17 September 2009 /Published online: 16 October 2009
# Springer-Verlag 2009
Abstract Cardiovascular diseases remain the leading cause
of death in industrialised nations. Since the pathomechanisms
of most cardiovascular diseases are not understood, the
majority of therapeutic approaches are symptom-orientated.
Knowing the molecular mechanism of disease would enable
more targeted therapies. One postulated underlying mecha-
nism of cardiovascular diseases is oxidative stress, i.e. the
increased occurrence of reactive oxygen species such as
superoxide. Oxidative stress leads to a dysfunction of vascular
endothelium-dependent protective mechanisms. There is
growing evidence that this scenario also involves impaired
nitric oxide (NO)-cyclic GMP signalling. Out of a number of
enzyme families that can produce reactive oxygen species,
NADPH oxidases stand out, as they are the only enzymes
whose sole purpose is to produce reactive oxygen species.
This review focuses on the clinically validated targets of
oxidative stress, NO synthase (NOS) and the NO
receptor, soluble guanylate cyclase as well as the
source of ROS, e.g. NADPH oxidases. We place recent
knowledge in the function and regulation of these
enzyme families into clinical perspective. For a compre-
hensive overview of the biology and pharmacology of
oxidative stress and possible other sources and targets,
we refer to other literature overviews.
Keywords Nitric oxide . Reactive oxygen species .
Oxidative stress . sGC activators . sGC stimulators .
NADPH oxidases (NOX) . Soluble guanylate cyclase
In cardiovascular diseases (CVD) such as hypertension,
atherosclerosis and chronic heart failure, endothelial dys-
function correlates with and can even predict long-term
disease progression and outcome [1]. Endothelial dysfunc-
tion is defined as the impairment of endothelium-dependent
relaxation. Whilst a number of factors contribute to
endothelial dysfunction, compromised nitric oxide-cyclic
GMP (NO-cGMP) signalling is a hallmark of this condi-
tion. Indeed, loss of vasodilatory and anti-platelet effects of
NO may result in CVD initiation and progression. There is
increasing evidence that pathophysiological production of
reactive oxygen species (ROS) interferes with NO-cGMP
signalling and may play a significant role in the develop-
ment of endothelial dysfunction. At least three underlying
mechanisms have been proposed (Fig. 1).
Firstly, ROS directly reduce the bioavailability of NO by
chemical scavenging. NO reacts with excess superoxide,
forming peroxynitrite (ONOO−) [2]. Secondly, ROS indi-
rectly affect NO bioavailability by uncoupling endothelial
NO synthase (eNOS). Mechanistically, this involves oxida-
tion of the essential NOS redox-sensitive cofactor tetrahy-
drobiopterin (BH4, see below) by ROS [1], which
subsequently uncouples eNOS, which then produces super-
oxide instead of NO [3]. Thirdly, ROS alter both the
expression and activity of the NO receptor, soluble
guanylate cyclase (sGC). This mechanism involves oxida-
tion of the sGC haem and subsequent haem loss [4, 5],
ubiquitination of the empty haem pocket [6] and proteaso-
mal degradation [7].
Several enzymes are capable of initiating this scenario,
including xanthine oxidase, cyclooxygenase, lipoxygenase,
uncoupled eNOS, cytochrome p450 and the mitochondrial
electron chain. However, NADPH oxidases stand out as the
major source of ROS as they are the only known enzyme
family solely dedicated to ROS production. All other
M. E. Armitage : K. Wingler : H. H. H. W. Schmidt (*) : M. La
Centre for Vascular Health, Department of Pharmacology,
Monash University,
Building 13E, Wellington Rd, Clayton,
Victoria 3800, Australia
e-mail: harald.schmidt@med.monash.edu.au
J Mol Med (2009) 87:1071–1076
DOI 10.1007/s00109-009-0544-2

known enzymes produce ROS as a by-product or as a
consequence of a biochemical “accident”. Importantly, of
all known ROS targets, NOS and sGC show the clearest
clinical relevance, as demonstrated by several ongoing drug
development programmes in different clinical stages or
even existing drugs in clinical practise. This brief review
will therefore focus on the NADPH oxidase-NOS-governed
fine balance of radicals. For further information into other
sources of ROS, we refer the reader to several excellent
reviews [8, 9].
Enhancing endothelial NO synthesis
NO is a ubiquitous signalling molecule with distinctive
roles in diverse tissues and species [10]. It is synthesised
either enzymatically from the amino acid L-arginine by
NOS or generated non-enzymatically from nitrite under
acidic conditions, e.g. in ischaemia/reperfusion [11]. Three
isoforms of NOS exist: neuronal (nNOS/NOS1), inducible
(iNOS/NOS2) and endothelial (eNOS/NOS3). Of these
three isoforms, eNOS is the most relevant in cardiovascular
system. eNOS is primarily present in endothelial cells, is
constitutively expressed and synthesises NO for short time
periods in response to receptor or physical stimulation. NO
released by eNOS mediates several physiological and
vasoprotective functions including the inhibition of vascu-
lar contraction and platelet activation. The activity of eNOS
is dependent on the availability of BH4 and other cofactors.
Three approaches have been pursued to increase sub-
optimal levels of NO: the use of NO donors, stimulating
eNOS activity by substrate supply or more recently by
enhancing eNOS expression. NO donors have problematic
pharmacokinetics, are prone to tolerance phenomena and
lead to enhanced nitrative chemistry in the vascular wall
[9]. The second approach, i.e. to stimulate NOS activity by
co-factor (BH4) or substrate (L-arginine) supplementation is
mechanistically plausible, but the clinical efficacies of these
approaches remain to be shown [12]. The bioavailability of
orally supplied L-arginine may be limited by first-pass
effects [13]. Moreover, L-arginine may act not only by
enhancing substrate supply but also by displacing an
inhibitory methylated L-arginine analogue, asymmetric
dimethylarginine (ADMA), from the enzyme's substrate
binding site [12]. In fact, plasma ADMA levels may
represent one of the better markers of endothelial dysfunc-
tion and cardiovascular disease risk [12]. More recently, an
eNOS enhancer, AVE9488, was introduced as a third
approach to overcome insufficient NO synthesis by
upregulating eNOS expression. AVE9488 protects against
ischaemia/reperfusion injury in animal models, but the
mechanism is not fully understood [14]. However, one
foreseeable risk is the presence of oxidative stress as
additionally expressed eNOS may become uncoupled,
which can exacerbate the problem [3]. Nevertheless, the
potential clinical benefits of eNOS enhancers certainly
provide a new approach to treat CVD.
Interestingly, some pathological vascular conditions may
be linked with excessive rather than reduced NO produc-
tion. Stroke-induced neuronal death appears in part to be
caused by excessive nNOS activation [15]. Similarly,
unwarranted upregulation of iNOS causes refractory hypo-
A. Physiological B. Pathological
Fig. 1 The NO-sGC signalling pathway and potential drug targets
under physiological and pathophysiological conditions. Under
physiological conditions (A), NO, synthesised by NOS from
L-arginine, activates soluble guanylate cyclase (sGC) leading to the
formation of cGMP and downstream effector mechanisms. sGC
stimulators enhance the sensitivity of sGC to low levels of
bioavailable NO. Under pathological condition (B) such as oxidative
stress, reactive oxygen species, e.g. superoxide (O2
−) most likely
derived from NADPH oxidases (NOX), affect the NO-sGC system by
three mechanisms: Superoxide scavenges NO; superoxide induces
eNOS uncoupling, reducing NO production and enhanced superoxide
production; superoxide oxidises the NO receptor, sGC, rendering it
unresponsive to NO activation. Potential therapeutic strategies to
diminish oxidative stress include the application of NADPH oxidase
inhibitors, eNOS recoupler such as BH4 or eNOS enhancer (AVE
9488), and sGC stimulators of reduced (Fe2+) or sGC activators of the
oxidised (Fe3+) and haem-free (apo-) sGC
1072 J Mol Med (2009) 87:1071–1076

tension in septic shock [16]. Early studies showed that
inhibitors of NOS were effective in normalising blood
pressure in different animal models of sepsis, but attempts
to translate these findings into an effective clinical therapy
were disappointing to date. A multi-centre phase III study
of 800 patients with a NOS inhibitor failed to show any
benefit and had to be terminated early due to a high mortality
rate [16]. Given that NO is a mediator of many essential
physiological functions, it is not surprising that global
inhibition of NOS has adverse effects. Therefore, selective
iNOS inhibition reducing local—as opposed to a global—
NO production may have more favourable effects. Recently,
new selective iNOS inhibitors have become available.
Nevertheless, their efficacy and safety in the clinic remain
to be determined [17]. Interestingly, a recent provocative
hypothesis proposed that NO is rather protective in sepsis.
Indeed, enhancing NO formation via exogenous nitrite
enhanced survival in a sepsis model [18]. Thus, we may
see soon a dogma shift on the role of NO in sepsis, whilst
the negative role in stroke seems unchallenged; therefore,
stroke may be an indication for NOS inhibition.
Potentiating NO via sGC stimulators
Many physiological functions of NO are mediated through
its primary receptor, sGC. sGC is a heterodimeric protein
consisting of a homologous α and a haem-containing β
sub-unit. The haem group is located on the axial ligand
histidine105 of the β subunit [5]. sGC activation by NO is
haem-dependent and leads to the conversion of GTP to
cGMP, which modulates the activity of several down-
stream effector molecules including protein kinases, phos-
phodiesterase (PDE) and ion channels [5]. According to the
simplest model for sGC activation by NO, sGC is activated
in two steps: NO first binds to the haem of sGC to form a
six-coordinated NO-Fe2+-Histidine105 intermediate complex,
which is then rapidly converted to a penta-complex [19]. It is
believed that the breakage of the haem–histidine105 bond
on the β subunit leads to the full activation of the enzyme
[5].
Pharmacological activation of sGC has been employed
for more than a century in the form of organic nitrates for
the treatment of angina, acute and chronic heart failure.
More recently, inhaled NO is used in acute respiratory
distress syndrome and congenital heart defects. However,
their clinical application is limited due to the development
of tolerance from chronic administration and the route of
administration, respectively [20]. Recently, it was discov-
ered that sGC has an allosteric binding site where so-called
sGC stimulators bind and potentiate sGC activation by NO.
Given the shortcomings of organic nitrates, the first
report of a direct, NO-independent stimulator of sGC,
YC-1, in 1994 raised much interest [21]. In 2001, Bayer
Healthcare AG reported a new class of sGC stimulators,
with BAY 41-2272 as prototype [22]. sGC stimulators
require an intact and functional prosthetic haem moiety in
sGC and act synergistically with NO. In comparison to
YC-1, BAY 41-2272 is a more potent sGC stimulator, is
orally active and has no or less PDE-inhibiting properties
[22]. Mechanistically, sGC stimulators increase the half-life
of the nitrosyl-haem complex [5]. Despite their effective-
ness in a variety of animal models of CVD, stimulators
such as BAY 41-2272 and BAY 41-8543 showed
unfavourable drug metabolism and pharmacokinetic
(DMPK) properties. More recently another stimulator,
Riociguat (BAY 63-2521), was found to have an improved
DMPK profile. It has beneficial effects on pulmonary
haemodynamics and exercise capacity in patients with
pulmonary hypertension. Riociguat is currently in phase
III clinical development for the oral treatment of pulmo-
nary hypertension [23]. However, both enhanced NO
synthesis, NO donors and sGC stimulators fail when the
sGC haem is oxidatively damaged and lost. Here, a
different class of sGC-modulating drug becomes impor-
tant, sGC activators.
Replacing NO by sGC activators
sGC activators helped to elucidate a previously unrecog-
nised alternative pathway to generate cGMP from sGC
beyond the binding of NO to the Fe2+ haem of the sGC
β subunit. One key observation was the existence of a
haem-oxidised sGC (ox-sGC) and even haem-free sGC
(apo-sGC) [5]. It is now clear that ox-/apo-sGC is
ubiquitously present in cells and tissues [5, 24]. It has
been speculated that the oxidised form is elevated in CVD
associated with increased levels of ROS [5].
In contrast to sGC stimulators which synergize with NO,
BAY 58-2667 showed additive effects when combined with
NO donors [5]. Further investigations revealed that NO and
BAY 58-2667 activate sGC independently. It was proposed
that BAY 58-2667 activates sGC by replacing the prosthetic
haem or—in the case of the haem-free enzyme—by directly
occupying the orphaned haem-pocket. At this year's 4th
International Conference on cGMP, the crystal structure of
a domain similar to that of the N terminus of sGCβ and its
interaction with BAY 58-2667 was reported. It was shown
that BAY 58-2667 can indeed displace the haem by
occupying the pocket and thus mimicking the interaction
of haem with sGC [25]. More recently, BAY 58-2667 has
demonstrated its efficacy in a proof-of-concept phase IIb
clinical study in heart failure patients, reducing pre- and
post-load and increasing cardiac output from baseline [26].
Another sGC activator, HMR1766 (Sanofi-Aventis) is
J Mol Med (2009) 87:1071–1076 1073

currently in a phase II clinical study for treatment of
neuropathic pain.
In summary, the physiological existence of haem-free
apo-sGC can no longer be rejected. This raises questions
about the ratio of haem-free to oxidised (Fe3+) to reduced
(Fe2+) sGC in physiological and pathophysiological con-
ditions, and why apo-sGC seems to be more prevalent
under disease conditions. Given that many pathological
conditions are associated with oxidative stress, the link
between ROS and different redox states of sGC needs
further elucidation.
Having described three validated and clinically relevant
mechanisms by which ROS affect NOS and cGMP
signalling and several pharmacologic approaches to reverse
these, a more rational approach is to prevent oxidative
damage occurring in the first place by identifying and
targeting the major sources of ROS, e.g. NADPH oxidases.
The relevant sources of ROS
ROS include free radicals such as the superoxide (O2
−), the
hydroxyl radical (HO) and non-radical species such as
hydrogen peroxide (H2O2), hyperchlorous acid (HClO) and
peroxynitrite. ROS contribute to the homeostasis of a
number of physiological systems including the respiratory,
immune and neurological system [27]. Under physiological
conditions, antioxidant defence enzymes scavenge and
detoxify excess ROS. Endogenous antioxidants include
superoxide dismutase, which dismutates superoxide into
H2O2, as well as catalase and peroxidases, which convert
H2O2 into water or other metabolites. Oxidative stress
results from an imbalance between ROS generation and
ROS inactivation. This may well be a highly localised sub-
cellular event, as opposed to a systemic phenomenon.
Decreased inactivation of ROS appears to be a less
common cause for oxidative stress [28] when compared to
an increase in ROS generation. Thus, inhibiting enhanced
ROS production is a promising strategy to restore endothe-
lial function and to manage CVD. Of all the potential
sources of ROS, NADPH oxidases are the only known
enzyme family that has ROS as their sole enzymatic
product [29]. NADPH oxidase was first identified in
phagocytic cells as the enzyme responsible for the
respiratory burst [30]. It later became apparent that these
enzymes are also expressed in a variety of non-phagocytic
cells, including all cells in the blood vessel wall [31],
suggesting other functions besides host defence [9].
NADPH oxidases are enzyme complexes primarily distin-
guished by the membrane spanning catalytic NOX sub-unit
that transfers electrons from NADPH to molecular oxygen.
Five mammalian members of the NOX family have been
identified (NOX1-5) [27] with NOX1, 2, 4 and 5 being
expressed in the blood vessel wall. Importantly, NOX5 is
not expressed in rodents, which may turn out to be a
previously unrecognised limitation of mouse and rat
models of oxidative stress. NOX isoforms rely to varying
degrees on the association of regulatory proteins. NOX1-
and NOX2-containing isoforms require the association of
a smaller membrane protein, p22phox, an “organiser”
protein (p47phox or NOXO1), an “activator” protein
(p67phox or NOXA1) and a small GTPase, Rac1 [9].
NOX4 may only require the association of p22phox,
whilst NOX5 appears to function independently of any
associated proteins but is regulated by [Ca2+] [9]. NADPH
oxidase activity in vascular cells is acutely increased by
various pathological stimuli, such as angiotensin II,
endothelin-1, growth factors, cytokines, metabolic factors
(e.g. advanced glycation end products), oxidised LDL,
mechanical forces and ischaemia-related stimuli [32]. For
a more comprehensive overview on the molecular biology
and regulation of NADPH oxidases, we refer to these
reviews [9, 27, 29, 33–36].
Vascular NADPH oxidase-dependent superoxide pro-
duction appears to be a driving force in the development of
endothelial dysfunction in several animal models of CVD
[1, 9]. In coronary arteries of patients with CAD, NADPH
oxidase activity is increased [37, 38], as is expression of
NOX5 [39]. In patients with diabetes, superoxide produced
by NADPH oxidases and uncoupled eNOS significantly
contributes to the endothelial dysfunction of arteries from
these patients [40]. Nevertheless, the exact structure,
regulation, distribution and patho-physiological role of the
individual human NOX isoforms are only being unravelled.
How to prevent or treat oxidative stress?
As ROS are derived from a number of sources, it was
deemed feasible to readjust the balance of ROS production
and detoxification by supplementing antioxidants. Howev-
er, clinical trials testing this hypothesis showed little
benefits in reducing cardiovascular events or mortality;
some of the treatments even caused harm [41]. Indeed, in
vivo detoxification of increased ROS by antioxidant
supplementation may be impossible due to the poor
bioavailability of the antioxidants at the correct sub-
cellular localisation and at the optimal time point. Anti-
oxidants may also promote new radical chain reactions
initiated by their oxidised forms. Importantly, the global
removal of ROS may result in unwanted effects since ROS
also regulate important physiological functions. Therefore,
the current strategy is to identify and target the major
sources of ROS, including NADPH oxidases.
A major constraint in NADPH oxidase research and
translation is the lack of specific inhibitors. All commonly
1074 J Mol Med (2009) 87:1071–1076

used compounds, including apocynin and diphenylene-
iodonium, are unspecific [42] (Wind et al., unpublished
observations), and there are no specific NOX inhibitors
commercially available. The novel NADPH oxidase inhib-
itor, VAS2870, inhibits PDGF-stimulated cell migration and
NADPH activity in vascular smooth muscle cells [43] as
well as oxidised LDL-stimulated ROS formation in endo-
thelial cells [44]. However, its mechanism of action and
selectivity for different NOX isoforms is unknown. In vivo
experiments with a small peptide inhibitor, gp91ds-tat,
which interferes with the interaction between NOX2 and
the regulator p47phox, had promising results, e.g. it
reduced angioplasty-induced superoxide and neointimal
hyperplasia of rat carotid artery [45]. As individual NOX
isoforms are regulated by different subunits, targeting these
protein–protein interactions is one strategy for the devel-
opment of isoform-specific NOX inhibitors. For more
details on NADPH oxidase inhibitors and their limitations,
we refer to other published reviews [32, 46–48]. Once
specific inhibitors become available, their potential to
manage CVD by preventing ROS formation can be fully
explored. This concept may also be applicable to other
chronic diseases associated with oxidative stress.
Conclusions
The dissection of the oxidative stress hypothesis into a
detailed identification of clinically relevant enzymes targeted
by ROS offers for the first time a more mechanistic approach
to CVD therapy. eNOS recouplers/enhancers as well as sGC
stimulators/activators target specific oxidative damage, which
currently can only be detected at a functional level. Never-
theless, clinical efficacy and proof-of-concept animal models
promise a far more positive outcome than for previous
antioxidant approaches. Thinking foward, the NADPH
oxidase field represents an uncharted territory in the manage-
ment of CVD. We believe that the development of NOX
isoform selective inhibitors in the next decade will reveal the
clinical relevance of this enzyme family.
Twenty years after its discovery [49], NO has come of
age. It may be not so much NO synthesis itself but the
signalling and oxidative events surrounding this unusual
molecule that may produce the therapeutic outcomes that
have been postulated for this highly protective vascular
pathway and for CVD management.
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