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Endothelial Nitric Oxide Synthase
Host Defense Enzyme of the Endothelium?
Ton J. Rabelink, Thomas F. Luscher
Abstract—This article explores the physiology of superoxide generation by endothelial nitric oxide synthase (eNOS), the
so-called “uncoupled” state of the enzyme. The fact that this alternative chemistry of the eNOS enzyme is evolutionary
strongly conserved, suggests that it may play a physiological role. It is proposed that this uncoupled state may contribute
to defense against infections. As the switch from NO production to reactive oxygen species by eNOS is also the final
common pathway in atherogenesis, the uncoupling of eNOS further builds on the hypothesis that atherogenesis is driven
by cellular mechanisms that originally serve host defense. The central role of uncoupled eNOS in redox signaling in the
endothelium may open up new avenues for therapy to prevent atherosclerosis. (Arterioscler Thromb Vasc Biol. 2006;
26:267-271.)
Key Words: endothelium 䡲 nitric oxide 䡲 atherosclerosis
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T
he concept that endothelium-derived nitric oxide (NO) is
an important molecule in prevention of (progression of)
atherosclerosis has been well established. Consequently, the
endothelial enzyme that produces NO, endothelial NO synthase (eNOS) (NOSIII), is considered to be a protective
enzyme and loss of NO production to be dysfunctional. In the
eNOS enzyme loss of NO release is usually associated with
production of reactive oxygen by the enzyme. In the current
article the hypothesis is explored that this change in eNOS
function represents normal physiology aimed at host defense.
This concept would imply that eNOS “dysfunction” in the
setting of aging and atherosclerosis is basically about how
cardiovascular risk factors use cellular pathways for defense
against bacteria.
tional modification of vital proteins. As was proposed in a
seminal article by Carl Nathan redox reactive molecules may
play a role in integrating signaling events and determine the
duration and strength of signaling.4 Interestingly, eNOS is not
a prerequisite for life and genetic deletion of eNOS appears to
have little effect on basal vasodilation or basal leukocyte
adhesion to the endothelium, supporting a more permissive
role for NO under basal conditions.5,6 If one reviews the most
important cellular effects of NO they seem to be involved in
conservation of metabolism. Probably one important function
of NO in the (endothelial) cell is its competition with oxygen
as an electron acceptor at complex IV in oxidative phosphorylation.7 This means that with diminishing oxygen availability NO will predominate as an electron acceptor and reduce
oxidative phosphorylation thus limiting energy expenditure.
NO also participates in defense against hypoxia by increasing
hypoxia-inducible factor-1␣ (HIF-1␣) binding activity and
HIF-1␣ protein levels.8 In contrast S-nitrosylation of inflammatory transcription factors including NF-␬B, AP1, and DNA
methyltransferases generally leads to reduced transcriptional
activity.3 In fact also the vasodilator effects of NO serve a state
of metabolic preservation by ensuring energy substrate delivery.
By S-nitrosylation of HDM2, the protein that regulates p53
degradation, cell cycle arrest will be induced, again inducing a
state of quiescence (Figure).
Like for NO, reactive oxygen species may also serve
permissive signaling. In the intracellular milieu, where superoxide dismutase is present, the predominant reactive oxygen
species will be H2O2.9 H2O2 can also react with cysteine
residues leading to formation of sulfenic acid (SOH) and
Endothelial Protection and
Permissive Signaling
NO is a reactive molecule that can rapidly diffuse throughout
the cell. It can react with sulfur groups on cysteine residues in
proteins, particularly when these cysteines are present in an
acid– cysteine– base motif. This results in reversible
S-nitrosylation of these proteins and in the presence of
reducing enzymes, such as GSNO-reductase, may also lead to
disulphide bridges between cysteine residues in proteins.1,2 In
addition, because of its unpaired electron it can associate with
transition metals such as heme leading to nitrosyl modifications.3 The rapid access to different parts of the endothelial
cell and the reactivity with a wide range of biological
molecules makes NO very suitable for a role in coordination
and synchronization of cellular processes via posttransla-
Original received August 15, 2005; final version accepted October 27, 2005.
From the Department of Nephrology and Hypertension (T.J.R.), Leiden University Medical Center, The Netherlands; and the Department of Cardiology
(T.F.L.), University Hospital Zurich, Switzerland.
Correspondence to Ton J. Rabelink, Department of Nephrology and Hypertension, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden,
The Netherlands. E-mail [email protected]
© 2006 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org
267
DOI: 10.1161/01.ATV.0000196554.85799.77
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February 2006
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disulfide modifications in proteins.10 This reaction typically
would occur in proteins with low pKa cysteine residues.11
Like NO, H2O2 can also rapidly diffuse throughout the cell.
However, the pattern of cellular events that emerges is
different from that induced by NO; H2O2 results in phosphorylation of transcription factors such as NF-␬B, AP1, and
CREB1, it induces chromatin remodeling in the nucleus, thus
allowing transactivation of genes by these transcription factors, and it will activate proteases.9 Besides initiating a
coordinated proinflammatory transcriptional response in the
endothelium, NF-␬B activation also protects the endothelium
against cytokine- or infection-induced apoptosis by controlling the intracellular levels and localization of members of the
antiapoptotic Bcl-2 protein family.12 In other words, signaling
by reactive oxygen species seems to serve activation of host
defense.
A very interesting feature of the NOS enzymes is that they
not only generate NO but may also produce reactive oxygen
species themselves.
The Catalytic Conundrum
This capacity to release reactive oxygen species is related to
the basic chemistry that occurs at the enzyme. Basically,
eNOS, as well as the other NOS isoforms, are NADPHconsuming enzymes. As a consequence there will be electrons flowing in the enzyme from the reductase domain
toward the heme containing oxygenase domain. In the eNOS
enzyme, the cofactor calmodulin is an important checkpoint
in this chemistry as it is required for shuttling of electrons
toward the heme group.10 In the presence of oxygen a very
reactive superoxy ferrous–peroxy ferric complex is formed.
The eNOS cofactor tetrahydrobiopterin (BH4), another important checkpoint in the chemistry, facilitates the reaction of
electrons and oxygen with L-arginine leading to the formation
of L-citrulline and NO.13 However, if either BH4 or the
substrate L-arginine are lacking, the superoxy ferrous–peroxy
ferric complex may dissociate to yield superoxide (if BH4 is
lacking) or H2O2 (if L-arginine is lacking).13,14 This latter state
is referred to as the so-called “uncoupled” state of eNOS.
This uncoupled state has been associated with risk factors for
atherosclerosis and consequently has been regarded as an
abnormality of eNOS function. For example, models of
diabetes, hypertension, and atherosclerosis have been associated with reduced tissue BH4 levels, increased superoxide
generation, and impaired endothelial function.15–17
As explained before, uncoupling of eNOS implies that the
endothelial cell switches from a quiescent state (NO) into a
state that is adapted for host defense (H2O2). In this respect
generation of reactive oxygen species by uncoupled eNOS
could be regarded as physiological signaling during injury
and infection, and in fact could even be an essential effector
mechanism in the host defense response. If one considers that
the NO synthases are highly conserved in evolution, and the
same BH4-dependent catalytic chemistry is present in plants,
fish, insects, and mammals,18,19 eNOS uncoupling may represent a widely spread physiological mechanism. The knockout animal experiments have taught us that eNOS-derived
NO does not seem to be critical for normal cell physiology6;
one can raise, however, the question whether uncoupling of
eNOS is an essential physiological mechanism in the setting
of host defense.
Uncoupling and Host Defense
A model to look at host defense testing both innate immunity
as well as adaptive immune responses is organ transplantation. Instead of bacterial antigens, innate immune cells such
as monocytes and dendritic cells in the adventitia present
allo-antigens that with proper costimulation give a T cell and
B cell response leading to rejection of the transplanted
organ.20 In such a MHC class mismatched allograft model21
we found that increasing BH4 availability by administering
the precursor compound sepiapterin leads to reduction of
reactive oxygen species and nitrotyrosine while NO production by the allograft increases at the same time.22 This
strongly suggests that increased coupling of NO synthases
had occurred. This coupling was associated with a profound
immune suppressive effect. Macrophage influx in the transplanted organ went down 60% despite the fact that no
immune suppressive drugs were given.22 These observations
could not be explained by a nonspecific antioxidant effect of
BH4, as sepiapterin had no effect on superoxide production
and inflammation in transplantation of isografts, whereas
ischemia-reperfusion injury was present but no antigen presentation takes place.22 It thus appeared that uncoupling of
NOS is an important proinflammatory mechanism in the
immune response to allografts.
As vascular inflammation involves endothelial cell activation as well as concomitant influx of leukocytes, both
uncoupling of eNOS as well as iNOS induction in leukocytes
could modulate this immune response.22,23 The role of iNOS
induction in host defense is well recognized. Recently,
however, it has been suggested that iNOS induction may in
fact be secondary to eNOS uncoupling. For example, during
endotoxemia a marked reduction in inducible NO synthase
(iNOS) protein was observed in the heart of eNOS knock-out
mice compared with wild type, indicating that for iNOS
induction initial eNOS activation is required.24 Although the
article did not directly address whether such activation of
eNOS implied uncoupling and subsequent redox signaling,
this is a plausible scenario as iNOS induction is dependent on
redox signaling.
If uncoupling of eNOS is involved in host defense one
would also expect increased susceptibility to infection in the
absence of the eNOS enzyme. This issue had not extensively
been addressed, but a recent study on colitis in mice indeed
demonstrated increased bacterial invasion of the colon in
eNOS knock-out mice.25
Another consequence of this hypothesis is that the eNOS
enzyme should have a Janus face, which allows the enzyme to
switch from the coupled to the uncoupled mode when it
encounters infectious agents—from cardiovascular homeostasis to host defense. Indeed, activation and endothelial
deposition of the complement cascade, a first-line bacterial
defense component of the innate immune system, causes
redox signaling in the endothelial cell.26 Also, phagocytic
cells take up bacteria through toll-like receptors or Fcy
receptors and activate NADPH oxidase. Such activation of
NADPH oxidases in phagocytes has been shown to induce
Rabelink and Luscher
eNOS: Host Defense Enzyme of the Endothelium?
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Left, Resting endothelial function. In the
presence of sufficient amount of tetrahydrobiopterin (BH4) and the substrate L-arginine,
the eNOS enzyme produces NO.
S-nitrosylation of transcription factors
involved in endothelial activation such as
NF-␬B and cell-cycle proteins, the endothelial cell maintains a quiescent state. In addition, NO functions in the mitochondria to
limit energy expenditure. Right, Endothelial
activation. In the presence of cytokines or
complement activation the endothelial isoform of NADPH oxidase (NOX4) is activated
leading to oxidative stress and oxidation of
the cofactor tetrahydrobiopterin into BH2. In
addition, arginase is induced shuttling the
eNOS substrate L-arginine toward L-ornitine
and urea production. As a result eNOS produces oxyradical species leading to H2O2
signaling into the cell. As a result there is
transcriptional activation of the cellular
inflammatory response and increased energy
expenditure. Although this system is appropriate in the defense against bacteria, it may
lead to inappropriate recruitment of leukocytes to the vessel wall if activation occurs
by cardiovascular risk factors.
downstream redox signaling in endothelial cells when these
phagocytes adhere to endothelium.27 Many cell types including endothelial cells, furthermore, produce low levels of H2O2
in response to cytokines (transforming growth factor [TGF]␤, tumor necrosis factor [TNF]-␣, and interleukin [IL]),
peptide growth factors (PDGF; EGF, VEGF, bFGF), and
agonists of G protein– coupled receptors (eg, angiotensin II,
thrombin, lysophosphatidicacid, sphingosine 1-phosphate,
histamine, and bradykinin) involved in inflammation.28 An
important feature of BH4 is that it is very susceptible to
oxidation and in fact can autooxidize leading to accelerated
degradation of BH4 under conditions of oxidative stress and
redox signaling.29 In this way activation of the innate immune
system will also induce uncoupling of eNOS and subsequent
proinflammatory transcription in the endothelial cell (Figure).
Cytokines released by phagocytes will not only reduce
BH4 availability. They can also induce arginase in the
endothelium and shuttle L-arginine metabolism toward urea
production. This may reduce L-arginine availability for the
eNOS enzyme and further contribute to eNOS uncoupling.30
L-arginine deficiency at the eNOS enzyme may also result
from increased presence of endogenous inhibitors of NOS
such as ADMA and L-NMMA. Interestingly, the ADMAgenerating enzymes, the type I protein arginine methyltransferases, are upregulated by oxidative stress while activity of
the ADMA metabolizing enzyme DDAH is reduced.31,32
Whereas binding of ADMA itself to the enzyme probably
also decreases the accessibility of O2 to bind to the heme, thus
basically shutting down the enzyme, other methylated arginines such as L-NMMA can induce uncoupling of NOS.33
Atherosclerosis: Hijacking the Host
Defense System
The concept that uncoupling of eNOS is part of the normal
physiology of endothelial cell activation also implies that
aging and atherogenesis may be the price we pay for an
efficient host defense system. Indeed, molecular pathways
activated in these conditions may have originally evolved for
defense against bacteria, at times when this was the primary
threat and long-living organisms were unforeseen. It is quite
interesting that one can indeed explain many of the effects of
cardiovascular risk factors through this mechanism. For
example, cytokines released from adipocytes in the metabolic
syndrome constitute an important cardiovascular risk. This is
reflected in the poor cardiovascular outcome that is associated with elevated levels of the acute phase reactant
C-reactive protein (CRP).34 Such inflammatory cytokines
(TNF-␣, IL-6) and possibly also CRP itself can directly
activate NADPH oxidases in endothelial cells.35 The ensuing
oxidative signaling would then lead to uncoupling of eNOS.
Also, metabolic risk factors may primarily lead to redox
signaling in the endothelial cell. Increased glucose and free
fatty acid uptake by endothelial cells in insulin resistance and
diabetes will cause overload of the mitochondria with substrate for oxidative phosphorylation resulting in incomplete
reduction of O2 and production of H2O2.36 Again the net result
would be uncoupling of eNOS, and in agreement uncoupling
of eNOS has been demonstrated in diabetes models.37 Also
hypercholesterolemia, and in particular oxidized lipoproteins,
can directly through the LOX-1 receptor activate NADPH
oxidases in the endothelial cell and lead to uncoupled
eNOS.38
What Are the Implications?
Rather than blocking one risk factor or one pathophysiological mechanism and be confronted with therapy failure
attributable to redundancy in risk factors and atherogenic
mechanisms, this concept suggests that we should rather aim
at the final common pathways of aging and atherogenesis.
One of the main paradigm shifts in treatment of cardiovascular disease will be to prevent patients with cardiovascular
risk factors from becoming high-risk patients. The endothe-
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February 2006
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lium is a central transducer by which risk factors can mediate
progression to clinically overt cardiovascular disease, and
uncoupled eNOS could be the critical checkpoint here that
leads to endothelial cell activation.
With this concept in mind administration of L-arginine has
been explored in conditions such as hypercholesterolemia,
diabetes, and coronary artery disease. Whereas in acute
studies generally an increase in NO bioavailability was
shown,39,40 chronic L-arginine administration could not demonstrate a benefit.41 It was postulated by Loscalzo that
possible beneficial effects of chronic L-arginine administration may be offset by simultaneous increments in S-adenosylL-homocysteine as a side product of the L-arginine associated
creatinine production.42 Vascular increments in S-adenosylL-homocysteine may lead to accumulation of homocysteine
and ADMA and reduce NO bioavailability at the same time.
An alternative strategy to maintain an NO-dominated state
in the endothelial cell in the presence of risk factors could be
the administration of BH4 or its precursor protein sepiapterin.
This has proven to be a very effective strategy in animal
models of cardiovascular disease.43 Also in humans acute
administration of tetrahydrobiopterin could restore NO activity in hypercholesterolemia,16 diabetes,44 and coronary artery
disease.45 Unfortunately no studies on chronic administration
of biopterins on endothelial function or atherosclerosis progression have been published to date. This may be a particularly interesting option as chronic oral treatment with BH4
has been used successfully for treatment of patients with
hyperphenylalaninemia for more than 10 years with no
side-effects reported.46
There is irony in the idea that we may have to pay the price
of atherosclerosis for an enzyme system that so beautifully
serves adaptation to metabolic as well as immunologic
demands. The appreciation of this irony may, however, help
discover new therapeutic avenues in cardiovascular disease.
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Endothelial Nitric Oxide Synthase: Host Defense Enzyme of the Endothelium?
Ton J. Rabelink and Thomas F. Luscher
Arterioscler Thromb Vasc Biol. 2006;26:267-271; originally published online November 17,
2005;
doi: 10.1161/01.ATV.0000196554.85799.77
Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association, 7272
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Copyright © 2005 American Heart Association, Inc. All rights reserved.
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