ANTIOXIDANTS & REDOX SIGNALING

Volume 10, Number 6, 2008

© Mary Ann Liebert, Inc.
DOI: 10.1089/ars.2007.1989

Forum Review

Nitric Oxide, Tetrahydrobiopterin, Oxidative Stress, and

Endothelial Dysfunction in Hypertension

EBERHARD SCHULZ, THOMAS JANSEN, PHILIP WENZEL, ANDREAS DAIBER,

and THOMAS MÜNZEL

ABSTRACT

Endothelial dysfunction in the setting of cardiovascular risk factors such as hypercholesterolemia, diabetes
mellitus, chronic smoking, as well hypertension, is, at least in part, dependent of the production of reactive
oxygen species (ROS) and the subsequent decrease in vascular bioavailability of nitric oxide (NO). ROS-pro-
ducing enzymes involved in increased oxidative stress within vascular tissue include NADPH oxidase, xan-
thine oxidase, and mitochondrial superoxide producing enzymes. Superoxide produced by the NADPH oxi-
dase may react with NO, thereby stimulating the production of the NO/superoxide reaction product
peroxynitrite. Peroxynitrite in turn has been shown to uncouple eNOS, therefore switching an antiathero-
sclerotic NO producing enzyme to an enzyme that may accelerate the atherosclerotic process by producing
superoxide. Increased oxidative stress in the vasculature, however, is not restricted to the endothelium and
also occurs within the smooth muscle cell layer. Increased superoxide production has important consequences
with respect to signaling by the soluble guanylate cyclase and the cGMP-dependent kinase I, which activity
and expression is regulated in a redox-sensitive fashion. The present review will summarize current concepts
concerning eNOS uncoupling, with special focus on the role of tetrahydrobiopterin in mediating eNOS un-
coupling. Antioxid. Redox Signal. 10, 1115–1126.

INTRODUCTION has been demonstrated to occur in the endothelial cell layer, but
also within the media and adventitia, all of which may impair

T RADITIONALLY, THE ROLE OF THE ENDOTHELIUM was thought

primarily to be that of a selective barrier to the diffusion
of macromolecules from the blood lumen to the interstitial
NO signaling within vascular tissue to endothelium-dependent,
but also endothelium-independent vasodilators. More recent
experimental, but also clinical studies point to a crucial role of
space. During the past 20 years, numerous additional roles for eNOS as a ROS producing enzyme in the setting of arterial hy-
the endothelium have been defined, such as regulation of vas- pertension. This review will briefly address mechanisms under-
cular tone, modulation of inflammation, promotion as well as lying eNOS uncoupling and will focus primarily on the conse-
inhibition of vascular growth, and modulation of platelet ag- quences of intracellular absolute or relative deficiency of the
gregation and coagulation. Endothelial dysfunction is a char- eNOS cofactor tetrahydrobiopterin in causing eNOS uncoupling.
acteristic feature of patients with coronary atherosclerosis, and
more recent studies indicate that it may predict long-term ath-
erosclerotic disease progression as well as cardiovascular event OXIDATIVE STRESS CAUSES
rate (77). Although the mechanisms underlying endothelial dys- ENDOTHELIAL DYSFUNCTION
function are multifactorial, there is a growing body of evidence
that increased reactive oxygen species (ROS) production may The endothelium-derived relaxing factor, previously identi-
contribute considerably to this phenomenon. ROS production fied as nitric oxide (NO) (56) or a closely related compound

II. Medizinische Klinik, Mainz, Kardiologie, Angiologie und Internistische Intensivmedizin, Mainz, Germany.

1115
1116 SCHULZ ET AL.

(50), has potent antiatherosclerotic properties. Nitric oxide re- diovascular events defined as a composite endpoint including car-
leased from endothelial cells works in concert with prostacy- diovascular death, unstable angina, myocardial infarction, coro-
clin to inhibit platelet aggregation (62), it inhibits the attach- nary revascularization, ischemic stroke, and peripheral artery
ment of neutrophils to endothelial cells, and the expression of revascularization (66), as compared to patients who responded to
adhesion molecules. Nitric oxide in high concentrations inhibits intracoronary ACh with vasoconstriction. More recently, we were
the proliferation of smooth muscle cells (16). Therefore, under able to show that peripheral endothelial function also has prog-
all conditions where an absolute or relative nitric oxide deficit nostic meanings (27). Patients with cardiovascular events had a
is encountered, the process of atherosclerosis is being initiated clearly attenuated response to intrabrachial infusion in forearm
or accelerated. The half-life of nitric oxide and therefore its bi- blood flow established with the forearm plethysmography method.
ological activity is decisively determined by oxygen-derived Even more exciting, we could demonstrate that patients who re-
free radicals such as superoxide (21). Superoxide rapidly reacts sponded well to intraarterial infusion with vitamin C with an im-
with nitric oxide to form the highly reactive intermediate per- provement of endothelial function had a worse prognosis as com-
oxynitrite (4). The rapid bimolecular reaction between nitric pared to patients with a low or no vitamin C effects (27) (Fig. 1).
oxide and superoxide yielding peroxynitrite (ONOO, rate con- This finding not only strengthens the concept that oxidative stress
stant: 5–10  109 M1s1) is 3–4 times faster than the dis- indeed is the key player in determining the degree of endothelial
mutation of superoxide by the superoxide dismutase. Therefore, dysfunction but also the prognosis in patients with established
peroxynitrite formation represents a major potential pathway of coronary artery disease. Further studies revealed a clear-cut asso-
nitric oxide reactivity, pending on the rates of tissue superox- ciation between endothelial dysfunction and prognosis in patients
ide production. Peroxynitrite in high concentrations is cytotoxic with chronic congestive heart failure (24), essential hypertension
and may cause oxidative damage to proteins, lipids, and DNA (57), and peripheral artery disease (18). For example, in an excit-
(3). Recent studies also indicate that ONOO may have dele- ing study, Gokce et al. quantified endothelial function in patients
terious effects on activity and function of prostacyclin synthase undergoing peripheral or coronary bypass surgery (17). These pa-
(92) and the endothelial NOS (91). Other reactive oxygen species tients have a high perioperative event rate within the first 30 days
(ROS) such as the dismutation product of superoxide, hydro- after surgery. In all patients, flow mediated dilation (FMD) of the
gen peroxide, and hypochlorous acid cannot be considered as brachial artery was measured before surgery and the patients were
free radicals, but have a powerful oxidizing capacity, which will grouped into three tertiles; FMD  8% between 4%–8% and be-
further contribute to oxidative stress within vascular tissue. low 4%. The authors found that patients with a FMD  8% had
almost no cardiovascular events during a follow-up period of 30
days, while patients with an FMD  8% had substantially more
ENDOTHELIAL DYSFUNCTION AND events (17). This study actually implies that measurement of en-
CARDIOVASCULAR RISK FACTORS dothelial function in the brachial artery may provide information
about plaque stability in coronary arteries.
It is well known that, in the presence of cardiovascular risk In patients with essential hypertension, Perticone et al.
factors, endothelial dysfunction is frequently encountered. This showed that in 225 never-treated patients with essential hyper-
has been shown for chronic smokers, patients with increased LDL tension, that those patients with peripheral endothelial dys-
levels, for patients with diabetes Types I and II, for hypertensive function in forearm arterioles had clearly more cardiovascular
patients and for patients with metabolic syndrome. There are sev- event rates as compared to patients with good endothelial func-
eral potential abnormalities, which could account for reductions tion (57). The excess risk in patients with the worst endothe-
in endothelium-dependent vascular relaxation, including changes lial function was still significant after controlling for individ-
in the activity and/or expression of the eNOS, decreased sensi- ual risk markers (Fig. 2A). The same group also showed that
tivity of vascular smooth muscle cells to NO, or increased degra- the co-existence of left ventricular hypertrophy and endothelial
dation of NO via its interaction with ROS such as superoxide. dysfunction in hypertensive patients increases significantly the
The NO-degradation concept is the most attractive one since in risk of subsequent cardiovascular events (69) (Fig. 2B).
the presence of cardiovascular risk factors such as diabetes mel- Taken together, there is no doubt that measurement of en-
litus, hypertension, chronic smoking, a positive family history for dothelial function provides substantial prognostic information
the development of coronary artery disease, as well as hyper- about future cardiovascular events in secondary prevention,
lipidemia, endothelial dysfunction is established and even more whereas its role in primary prevention remains to be established.
importantly it is markedly improved by the acute administration
of the antioxidant vitamin C (13, 25, 41, 80).
MECHANISMS UNDERLYING INCREASED
OXIDATIVE STRESS
ENDOTHELIAL DYSFUNCTION
AND PROGNOSIS eNOS uncoupling contributes to
endothelial dysfunction
Since 2000, a quite large number of clinical trials have dem-
onstrated a quite close association between coronary and periph- In most situations where endothelial dysfunction due to in-
eral endothelial function and the likelihood of cardiovascular creased oxidative stress is encountered, the expression of the
events. For example Volker Schächinger from Andreas Zeiher’s eNOS has been shown to be paradoxically increased rather than
group has shown that patients who respond to intracoronary acetyl- decreased (22, 29, 40, 83). The mechanisms underlying in-
choline (ACh) with vasodilation have subsequently much less car- creased expression of eNOS is likely to be secondary to in-
cGK-I EXPRESSION AND ACTIVITY 1117

Studies with the isolated enzyme

It has become clear from studies with the purified enzyme
that eNOS may become “uncoupled” [e.g., in the absence of
the NOS substrate L-arginine or the cofactor tetrahydrobiopterin
(BH4)]. In such uncoupled state, electrons normally flowing
from the reductase domain of one subunit to the oxygenase do-
main of the other subunit are diverted to molecular oxygen
rather than to L-arginine (81, 89), resulting in production of su-
peroxide rather than nitric oxide (Fig. 3). There are several pos-
sibilities how eNOS uncoupling may occur.

eNOS uncoupling due to increased

peroxynitrite-mediated BH4 oxidation
Tetrahydrobiopterin seems to be essential for the time-criti-
FIG. 1. Kaplan–Meier analysis demonstrating cumulative cal delivery of one electron to an intermediate in the catalytic
proportion of patients without cardiovascular events during cycle of NOS. The resulting species releases NO and L-cit-
follow up. Effects of vitamin C on ACh-induced forearm dila- rulline. The BH3 radical is reduced by the iron, completing the
tion is divided into values below and above the median. The data cycle by formation of FeIII and BH4. In the absence of BH4, the
clearly show that patients with a strong vitamin C response, re- intermediate reacts with molecular oxygen resulting in super-
flecting high oxidative stress in the vasculature, have subsequently oxide formation.
more cardiovascular events, compared to patients with a weaker
The first evidence that eNOS uncoupling may be a patho-
vitamin C response (adapted from ref. 27). (For interpretation of
the references to color in this figure legend, the reader is referred physiologically relevant phenomenon was provided by in vitro
to the web version of this article at www.liebertonline.com/ars). studies where it was demonstrated that native LDL (61), and
even more pronounced oxidized LDL (84), are able to stimu-
late endothelial superoxide production and that this phenome-
creased endothelial levels of hydrogen peroxide, which increases non is inhibited by the NOS inhibitor L-NAME, pointing to a
the expression of eNOS at the transcriptional and translational specific role of eNOS in superoxide production. These in vitro
level (12). The demonstration of endothelial dysfunction in the observations were rapidly extended by in vivo studies in ani-
presence of increased expression of eNOS indicates that the ca- mals with hypercholesterolemia (52), diabetes mellitus (29), an-
pacity of the enzyme to produce NO may be limited. Very in- giotensin II hypertension (47), and chronic congestive heart fail-
triguing are observations that the eNOS itself can be a super- ure (46), where an uncoupled eNOS was consistently identified
oxide source, thereby causing endothelial dysfunction (14). as a significant superoxide source. In the next paragraphs, we

FIG. 2. (A) Kaplan–Meier Analysis. Event free survival curves in patients with essential hypertension were subdivided into
tertiles of forearm blood flow (FBF). Endothelium-dependent increases in forearm blood flow were achieved with intrabrachial
infusion of the endothelium dependent vasodilator acetylcholine. The cumulative cardiovascular events rate in the first tertile
was 52% in 7 years, compared with a cumulative event rate of 14% in the third tertile (adapted from ref. 57). (B) Rate of total
cardiovascular events in relation to tertiles of echocardiographic mass and peak percentage increase in acetylcholine-induced va-
sodilation. The figure clearly shows that the rate of total cardiovascular events significantly increases from the first to the third
tertile of left ventricular mass index (LVMI) (adapted from ref. 69). (For interpretation of the references to color in this figure
legend, the reader is referred to the web version of this article at www.liebertonline.com/ars).
1118 SCHULZ ET AL.

FIG. 3. Electron flow in coupled versus uncoupled

eNOS. Electron flow starts from NADPH to flavins
FAD and FMN in the reductase domain, which de-
livers the electrons to the iron of the heme (oxyge-
nase domain) and to the BH3, radical generated as an
intermediate in the catalytic cycle. BH4 seems to be
essential to donate an electron and proton to versatile
intermediates in the reaction cycle of L-arginine/O2
to citrullin/NO. Calmodulin (CAM) controls electron
flow in eNOS. When BH4 is limiting, electron trans-
fer becomes uncoupled to L-arginine oxidation, the
ferrous dioxygen complex dissociates, and superox-
ide (O2.) is generated from the oxygenase domain
(adapted from ref. 68). (For interpretation of the ref-
erences to color in this figure legend, the reader is re-
ferred to the web version of this article at www.liebert
online.com/ars).

will discuss the potential mechanisms leading to eNOS uncou- radical to BH4, rather than by directly scavenging superoxide
pling in vascular disease. (38).
In addition to peroxynitrite-mediated oxidation of BH4, there
Decreased BH4 levels and decreased expression may be also an intracellular decrease due to decreased synthe-
sis by inhibition of the GTP cyclohydrolase I (GTP-CH 1; de
of the CTP cyclohydrolase I
novo synthetic pathway; Fig. 4) or by inhibition of the so-called
Strategies to improve endothelial function (e.g., in athero- salvage pathway involving enzymes such as sepiapterin syn-
sclerosis) via increasing the expression of eNOS have yielded thase, sepiapterin reductase, or dihydrofolate reductase (DHFR;
mixed results. Ozaki and coworkers reported that targeted over- Fig. 4). Interestingly, recent studies indicate that, for example,
expression of eNOS in ApoE knockout animals accelerated in the setting of angiotensin II-induced hypertension, a down-
rather than decreased atherosclerotic lesion formation (55). The regulation of the DHFR was observed that was accompanied
authors also established a marked decrease in vascular BH4 con- by decreased vascular BH4 levels and an uncoupled eNOS (7).
tent and identified the endothelium as the pivotal superoxide In the case of eNOS uncoupling due to relative intracellular
source compatible with eNOS uncoupling. Treatment with BH4 BH4 deficiency, this phenomenon could be prevented if simul-
reduced lesion formation, reduced vascular superoxide, and in- taneously the expression of the BH4 synthesizing enzyme, GTP-
creased endothelial NO production (55). These findings clearly CH I, were upregulated. This has been demonstrated in isolated
indicate that the stimulation of increases in eNOS expression cells and in atherosclerotic animal models. More recent clini-
without simultaneously increasing vascular levels of BH4 may cal data also indicate that variants of the GTP-CH I are gov-
lead to eNOS uncoupling due to the stoichiometric relationships erning nitric oxide production, autonomic activity, and in ad-
between endothelial BH4 and NOS activity (5). dition, cardiovascular risk (90).
One very attractive concept to explain intracellular BH4 de- Another proof of the concept that BH4-mediated eNOS un-
pletion is the oxidative modification of BH4 (38). In the setting coupling contributes to endothelial dysfunction was delivered by
of an activation of a vascular superoxide source, superoxide experiments demonstrating that supplementation with BH4 and
will react with NO to form the highly reactive intermediate per- by the BH4 precursor sepiapterin was able to improve endothe-
.
oxynitrite. Peroxynitrite in turn will oxidize BH4 to the BH3 lial dysfunction. BH4 has been shown to improve endothelial dys-
radical, and the resulting decrease in endothelial BH4 triggers function in smokers (26), diabetic subjects (58), hypertensive pa-
eNOS uncoupling (Fig. 3). tients (28), patients with hypercholesterolemia (75), and those
This concept, however, also implies that the uncoupling of with coronary artery disease (70). It is important to note that BH4
eNOS is mediated by ONOO and would invariably require a per se has antioxidant properties. Therefore, it may be difficult
priming event such as superoxide produced by NADPH oxi- to differentiate whether the BH4-induced improvements in en-
dase, xanthine oxidase, or mitochondria. These so-called “kin- dothelial dysfunction are due to recoupling of eNOS or due to
dling radicals” would lead to eNOS uncoupling via increased the antioxidant properties of BH4 itself. To differentiate these
formation of peroxynitrite and subsequent eNOS-mediated su- nonspecific versus specific effects on endothelial dysfunction,
peroxide production (bonfire radical). one has to use the pteridine analogue tetrahydroneopterin (NH4),
Oxidation of BH4 not only reduces BH4 bioavailability, but which has comparable antioxidant properties, but no effect at all
the oxidation products such as BH2 may compete with BH4 for on an uncoupled eNOS (26) (Fig. 5).
binding to eNOS (82), thereby leading to eNOS uncoupling. In- Endothelial dysfunction in the setting of coronary artery dis-
.
terestingly, vitamin C was able to recycle the BH3 radical to ease was also improved by treatment with folic acid (86). Recent
BH4 but not BH2 to BH4. This observation may indicate that experimental studies revealed that folate triggers these beneficial
the beneficial effects of vitamin C on endothelial function in effects by an enhancement of binding affinity of BH4 to NOS by
.
patients may be explained in part by a recycling of the BH3 a pteridine-binding domain serving as a locus through which the
cGK-I EXPRESSION AND ACTIVITY 1119

FIG. 4. Pathways of BH4 synthesis and degradation. BH4 biosynthesis proceeds from GTP via 7,8-dyhydroneopterin triphos-
phate and 6-pyruvoyl-5,6,7,8-tetrahydropterin. The first and rate-limiting step in the pathway is GTP cyclohydrolase (GTP-CH).
Subsequent steps are catalyzed by the enzymes 6-pyruvoyl tetrahydropterin synthase and sepiapterin reductase. An alternative
pathway for BH4 synthesis whereby 6-pyruvoyl-5,6,7,8-tetrahydropterin is converted to sepiapterin by an enzyme termed “sepi-
apterin synthase.” There is limited evidence for a sepiapterin synthesis pathway in mammals. However, exogenous sepiapterin
can be reduced in all cells by sepiapterin reductase to BH2, and further by dihydrofolate reductase to form BH4 (the so-called
salvage pathway). ONOO oxidizes BH4 to the intermediate BH3. radical, which can decay to BH2 or can be converted back to
BH4 by ascorbate (38). (For interpretation of the references to color in this figure legend, the reader is referred to the web ver-
sion of this article at www.liebertonline.com/ars).
1120 SCHULZ ET AL.

eNOS uncoupling and increased production of

Forearm blood flow [mL/100mL tissue/min]

16
BH4 asymmetric dimethyl arginine (ADMA)
12 NH4 Several recently published studies demonstrate that increased
concentrations of ADMA in cultured endothelial cells or in pa-
8 Saline tients with endothelial dysfunction are associated with increased
ROS production (6, 42, 79). The question is whether increased
ROS production is the reason for increased ADMA levels or
4
whether increased production of ADMA actually contributes to
the oxidative stress burden of the vasculature via uncoupling of
0 eNOS. Interestingly, the activity of methylating enzymes such
B 0.75 1.5 3.0 as the S-adenosylmethionine-dependent protein arginine
ACh [mg/100mL/min] methyltransferase (PRMT; Type I) (6) is responsible for the
ADMA synthesis or the activity of ADMA hydrolyzing en-
FIG. 5. Effect of tetrahydrobiopterin (BH4) and tetrahy- zymes such as dimethylarginine dimethylaminohydrolase
droneopterin (NH4) on the ACh dose-response relationship
(DDAH) (42) is redox-sensitive. Thus, oxidative stress in the
in chronic smokers. BH4 significantly improved ACh dose-
response relationship, whereas NH4 was ineffective. Adapted vasculature should always stimulate ADMA production and/or
from ref. 26. (For interpretation of the references to color in inhibit ADMA degradation, in concentrations that significantly
this figure legend, the reader is referred to the web version of inhibit eNOS activity or even uncouple the enzyme which
this article at www.liebertonline.com/ars). would further increase superoxide production in a positive feed-
back fashion (78).
active form 5-methyl tetrahydrofolate (5-MTHF) facilitates the
electron transfer by BH4 from the NOS reductase domain to heme
(76). Folate also enhances regeneration of BH4 from inactive BH2 DOES NADPH OXIDASE PRODUCE THE
by stimulating DHFR and it chemically stabilizes BH4 (85). KINDLING RADICAL FOR INCREASED
PEROXYNITRITE FORMATION
eNOS uncoupling due to peroxynitrite-mediated ULTIMATELY LEADING TO
oxidation of the zinc–thiolate complex ENOS UNCOUPLING?
Another interesting concept concerning eNOS uncoupling
was provided by Zou et al. (91). The authors showed that the The NADPH-oxidase is a superoxide producing enzyme that
exposure of the isolated enzyme to the oxidant peroxynitrite was first characterized in neutrophils (1). Meanwhile, we know
leads to a disruption of the zinc–thiolate cluster, resulting in an that a similar enzyme exists also in endothelial and smooth mus-
uncoupling of the enzyme (91). The authors also demonstrated cle cells, as well as in the adventitia. The activity of the en-
that a similar phenomenon occurs when endothelial cells were zyme in endothelial and smooth muscle cells is increased upon
exposed to high concentrations of glucose. Additional experi- stimulation with angiotensin II (20). The stimulatory effects of
ments revealed that BH4 was oxidized at concentrations being angiotensin II on the activity of this enzyme would suggest that
10- to 100-fold higher than those needed to disrupt the zinc–thi- in the presence of an activated renin angiotensin system (local
olate complex. Based on the findings, the authors suggested that or circulating), vascular dysfunction due to increased vascular
the principal mechanism of uncoupling is rather the oxidation superoxide production is likely to be expected. Experimental
of the zinc–thiolate center and the subsequent release of zinc hypercholesterolemia has been shown to be associated with an
rather than the BH4 oxidation process (91). activation of NADPH oxidase (87); and there is a close asso-
ciation between endothelial dysfunction and clinical risk fac-
tors on one hand and the activity of this enzyme in human saphe-
Potential role of L-arginine in eNOS uncoupling
nous veins in patients with coronary artery disease on the other
Beneficial effects of L-arginine supplementation have been doc- hand (23). In atherosclerotic arteries, there is evidence for in-
umented in both animal studies and humans under pathophysio- creased expression of the NADPH oxidase subunit gp91phox
logical conditions such as hypercholesterolemia and hypertension and nox-4, all of which may contribute to increased oxidative
(11, 30, 32, 64). However, based on in vitro studies, it appears stress within vascular tissue (74).
unlikely that L-arginine concentrations will ever become critical Interestingly, there is a growing body of evidence that the
as a substrate in vivo: the KM of eNOS for L-arginine is 3 mol/L local renin angiotensin system is activated in hypercholes-
(59), L-arginine plasma concentrations are 100 mol/L, and terolemia. In patients, ACE-activity and therefore local an-
there is a 10-fold accumulation of L-arginine within cells (9). giotensin II concentrations are increased in atherosclerotic
Thus, nonsubstrate effects of L-arginine are likely to explain the plaques (10, 53) and inflammatory cells are capable of pro-
beneficial effects quoted above. These include potential direct rad- ducing large amounts of angiotensin II. Increased angiotensin
ical scavenging properties of the guanidino nitrogen group, the II concentrations along with increased levels of superoxide have
cooperativity between L-arginine and BH4 binding sites of NOS been shown in the shoulder region of atherosclerotic plaques
(19, 45), or the competition of L-arginine with the derivative asym- (67). In vessels from hypercholesterolemic animals (87), as well
metric dimethyl-L-arginine (ADMA), which is an endogenous in- as in platelets from hypercholesterolemic patients (51), there is
hibitor of eNOS activity (see below) (78). an increase in the expression of the angiotensin II receptor sub-
cGK-I EXPRESSION AND ACTIVITY 1121

FIG. 6. Effects of NADPH oxidase deficiency (p47phox/) and tetrahydrobiopterin (BH4) treatment on NO production
as detect by paramagnetic resonance. In animals with DOCA-salt hypertension, vascular NO production was clearly dimin-
ished, compatible with eNOS uncoupling. Knockout of an NADPH oxidase subunit as well as tetrahydrobiopterin (BH4) treat-
ment normalized or clearly improved vascular NO production, strongly suggesting that an activation of the vascular NADPH ox-
idase in this hypertension model represents the “priming event” leading to eNOS uncoupling (adapted from ref. 39). (For
interpretation of the references to color in this figure legend, the reader is referred to the web version of this article at www.liebert
online.com/ars).

type AT1. Thus, both experimental and clinical studies have stress and abolished superoxide reducing effects of NOS inhi-
provided evidence for stimulation of the renin angiotensin sys- bition compatible with a prevention of eNOS uncoupling (39)
tem in atherosclerosis and simultaneously for an activation of (Fig. 6).
NADPH oxidase in the arterial wall. Similar evidence for an
activation of this enzyme in the vasculature has been provided
from experimental animal models of different forms of hyper-
tension such as angiotensin II infusion (15, 63) and in sponta- ASSESSMENT OF ENOS UNCOUPLING
neously hypertensive rats (SHR) (48), as well as in different IN VASCULAR TISSUE
forms of diabetes mellitus (29). In animal models of angiotensin
II-induced hypertension (47), streptozotocin- induced diabetes, It is important to note that eNOS-mediated superoxide pro-
as well as in patients with diabetes mellitus, increased ROS pro- duction—by the isolated enzyme or in vascular tissue—is in-
duction by NADPH oxidase was also associated with eNOS un- hibited by NG-nitro-L-arginine (L-NNA) or its methylester NG-
coupling. nitro-L-arginine methylester (L-NAME), since both substances
The proof of the concept that superoxide produced by the antagonize the transfer of electrons to either L-arginine or oxy-
NADPH oxidase may indeed trigger eNOS uncoupling was pro- gen. In contrast, L-NMMA has been previously shown to stim-
vided by David Harrison’s group in the experimental animal ulate rather than to inhibit superoxide production by the iso-
model of DOCA-salt hypertension (39). With these studies, the lated enzyme, due to causing partial uncoupling of NADPH
authors showed that superoxide induced by DOCA-salt treat- oxidation (electron transfer to oxygen). This has been shown
ment caused increased vascular H2O2 production, which was for inducible NOS (iNOS) (54) and for neuronal NOS (nNOS)
significantly reduced by the eNOS inhibitor NG-nitro-L-argi- (60). Similar phenomena may have to be expected when iso-
nine methylester (L-NAME). Treatment of p47phox knockout lated enzymes are exposed to the structurally similar asym-
animals with DOCA-salt markedly reduced levels of oxidative metric dimethyl-L-arginine (ADMA), which will prevent the
1122 SCHULZ ET AL.

FIG. 7. Schematic representation of

the role of reactive oxygen species in
causing endothelial dysfunction. Un-
der normal conditions, nitric oxide (NO),
synthesized by nitric oxide synthase
(eNOS), stimulates soluble guanylate cy-
clase (sGC), increasing cGMP and thus
stimulating cGMP-dependent protein ki-
nase I (cGK-I) and vasorelaxation. cGK-I
also phosphorylates the vasodilator-stim-
ulated phosphoprotein (VASP), which can
be taken as a biochemical parameter of
cGK-I activity. This pathway can, how-
ever, be inhibited at several sites. An-
giotensin II, hypertension, hypercholes-
terolemia, chronic smoking, and diabetes
mellitus will stimulate superoxide produc-
tion within endothelial and smooth mus-
cle cells mainly via activation of the vas-
cular NADPH oxidase. The NADPH
oxidase-derived superoxide may inacti-
vate NO and inhibit sGC directly, thereby
diminishing cGK-I activity. Peroxynitrite
(ONOO) may also inhibit sGC directly,
and it uncouples eNOS by oxidizing the eNOS cofactor to the BH3. radical and BH2, respectively. This concept, however, also means
that uncoupling of eNOS would always require a “priming event” such as superoxide produced by the NADPH oxidase, mitochon-
dria, or the xanthine oxidase (kindling radical). Angiotensin II also increases the catabolism of cGMP by the phosphodiesterase 1A1
(PDE1A1), which is usually accomplished by the phosphodiesterase 5 (adapted from ref. 4). (For interpretation of the references to
color in this figure legend, the reader is referred to the web version of this article at www.liebertonline.com/ars).

oxidation of L-arginine and therefore, reduce NO production, muscle cells (72, 73), and as shown more recently, also in in-
or as mentioned above, will stimulate superoxide production by tact vascular tissues (31, 52). The P-VASP assay appears to be
competing for the same binding site of the enzyme. Up to now, sufficiently sensitive to monitor basal NO release in endothe-
however, evidence that ADMA can trigger eNOS uncoupling lium-intact arteries. For example, incubation of vessels with the
is lacking. NOS inhibitors L-NNA or L-NAME strongly decreased P-VASP
The L-NNA sensitive and L-NAME sensitive inhibition of su- levels (31, 52). Likewise, NO donors such as sodium nitro-
peroxide formation is a convenient method to detect eNOS un- prusside (SNP) and nitroglycerin (NTG) markedly increased P-
coupling in vascular tissues. Depending on the detection sys- VASP in vessels in the absence and presence of an intact en-
tem used, addition of NOS inhibitors to vascular cells will dothelium (31, 52). Mechanical removal of the endothelium in
decrease the superoxide derived signal, such as lucigenin- rat and rabbit aorta almost completely abolished “basal” VASP
enhanced chemiluminescence (5 mol/L) (71) or the dihy- phosphorylation, whereas total VASP levels (phosphorylated
droethidine fluorescence (29, 46). The group of David Harri- and non-phosphorylated) were reduced by 40–50% (31, 52).
son showed that depletion of endogenous BH4 in mouse aorta These findings indicate that VASP Serine239 phosphorylation
by peroxynitrite increased superoxide production and that the clearly depends on the presence of a functional endothelium,
addition of L-NAME as well as endothelial removal or genetic and that the endothelial cell layer in aortic vessels contains a
knockout of eNOS prevented this radical response (39). considerable amount of VASP, which is lost upon removal of
the endothelium. P-VASP analysis also appears suitable to mon-
itor oxidative stress within vascular tissue (Fig. 7). Incubation
of isolated endothelium-intact vessels with diethylthiocarba-
ENOS UNCOUPLING AND DOWNSTREAM mate (DETC), an inhibitor of Cu/Zn superoxide dismutase
SIGNALING BY THE SOLUBLE (SOD), markedly reduced P-VASP levels (52). Concomitantly,
GUANYLYL CYCLASE (SGC) AND CGMP- basal endothelial NO formation was decreased and formation
DEPENDENT PROTEIN KINASE (CGK-I) of superoxide was increased (52).
IN HYPERTENSION

P-VASP, a novel marker suited to assess the ENOS UNCOUPLING AND

activity of the cGK-I CONSEQUENCES FOR THE ACTIVITY
cGK-I activity can be assessed by immunodetection of phos- AND EXPRESSION OF THE SGC
phorylation at Serine239 of the cGK-I substrate vasodilator- IN HYPERTENSION
stimulated phosphoprotein (P-VASP). This approach is a reli-
able surrogate parameter of the integrity of the NO/cGMP Endothelial dysfunction in different animal models of hyper-
signaling pathway in platelets, cultured endothelial and smooth tension has been shown to be associated with increased expres-
cGK-I EXPRESSION AND ACTIVITY 1123

sion of eNOS (44, 47) but decreased vascular NO bioavailability SUMMARY AND CONCLUSIONS
(47). Uncoupled eNOS (47) and NADPH oxidase (72) were iden-
tified as significant superoxide sources by the observation that Taken together, there is mounting evidence that endothelial
NOS inhibition decreased superoxide production in the tissue from dysfunction of the coronary or peripheral circulation has im-
hypertensive animals (47) and by the observed increases in ex- portant prognostic implications for future cardiovascular events
pression and enzyme activity of NADPH oxidase subunits nox1, in patients with coronary artery disease, peripheral vascular dis-
p22phox, p67phox, and gp91phox (8, 47, 63). Increased super- ease, essential hypertension, or congestive heart failure. Al-
oxide production involved all vessel layers, namely the endothe- though the mechanisms underlying endothelial dysfunction are
lium, media, and adventitia (47), and may also increase vasocon- likely multifactorial, it is important to note that increased pro-
strictor tone via stimulation of the expression of endothelin-1 in duction of oxygen-derived free radicals by an uncoupled eNOS
the smooth muscle and endothelial cell layers (34, 35). Acute or markedly contributes to this phenomenon. One of the key mech-
chronic treatment with antioxidants or superoxide dismutase not anisms causing eNOS uncoupling is attributed to a decrease in
only improved endothelial dysfunction but also markedly reduced intracellular BH4 levels caused either by peroxynitrite-induced
blood pressure indicating the potential role of ROS in the initia- BH4 oxidation or by decreased activity and/or expression of the
tion and maintenance of hypertension. GTP CH-I and the DHFR.
Hypertensive animals displayed not only reduced vasodila- Increased superoxide production is not restricted to the en-
tion to endothelium-dependent vasodilators but also to endo- dothelium and also involves the smooth muscle cell layer, and
thelium-independent nitro-vasodilators SNP and NTG (63). may lead to an alteration of the expression of the sGC and to
This finding may be explained at least in part by reduced ex- an inhibition of the activity of sGC and subsequently cGK-I.
pression of sGC since in different hypertensive animal models The inhibition and/or activation of cGK-I in vascular tissue can
the expression of one or both sGC subunits (1 and 1) as well be perfectly monitored by quantifying the degree of phospho-
as NO-dependent sGC activity was significantly decreased. This rylation of VASP, a new tool which reliably reflects vascular
was observed in genetic hypertension, such as aged SHR (37, NO bioavailability.
65), stroke-prone SHR (43), type 2 diabetic, mildly hyperten-
sive Goto–Kakizaki rats (88), and TGR(mREN2)27 renin trans-
genic rats (33), as well as in several models of drug-induced
hypertension.
ABBREVIATIONS
Reduction of hypertension normalized or even enhanced sGC
expression, but had differing effects on vascular superoxide pro- ACE, angiotensin converting enzyme; Ach, acetylcholine;
duction. For example, in the angiotensin II-infusion model, in- ADMA, asymmetric dimethyl-L-arginine; BH4, tetrahydro-
hibition of protein kinase C (PKC) in vivo reduced blood pres- biopterin; cGKI, cGMP dependent proteinkinase; cGMP, cyclic
sure and vascular superoxide formation by inhibiting the guanosine monophosphate; CTP CH-I, GTP cyclohydrolase I;
activity and expression of NADPH oxidase and by preventing DDAH, dimethylarginine dimethylaminohydrolase; DETC, di-
eNOS uncoupling (47) and sGC downregulation (unpublished ethylthiocarbamate; DHFR, dihydrofolate reductase; DOCA,
observation). In contrast, other forms of antihypertensive treat- deoxycorticosterone acetate; ED, endothelial dysfunction;
ment (e.g., with hydralazine), significantly lowered blood pres- eNOS, endothelial nitric oxide synthase; FBF, forearm blood
sure but failed to normalize vascular superoxide formation in flow; FMD, flow-mediated dilation; iNOS, inducible nitric ox-
chronically NOS-inhibited rats (2). Thus, it appears that the de- ide synthase; LDL, low density lipoprotein; 5MTHF, 5-methyl
crease in sGC expression elicited by high blood pressure is not tetrahydrofolate; L-NAME, NG-nitro-L-arginine methylester; L-
exclusively linked to increased superoxide formation and/or to NNA, NG-nitro-L-arginine; LVMI, left ventricular mass index;
increased levels of vasoconstrictor peptides. In contrary, vaso- nNOS, neuronal nitric oxide synthase; NO, nitric oxide; NTG,
constrictor peptides such as angiotensin II or endothelin-1, nitroglycerin; ONOO, peroxynitrite; PDE1A1, phosphodi-
which are increased in hypertension, seem to trigger an upreg- esterase 1A1; PKC, protein kinase C, PRMT, protein arginine
ulation of sGC in vascular smooth muscle cells. This sGC up- N-methyltransferase, ROS, reactive oxygen species; sGC, sol-
regulation is apparently overwhelmed by effects of yet unde- uble guanylyl cyclase; SHR, spontaneously hypertensive rat;
termined mediators of hypertension, which decrease sGC SNP, sodium nitroprusside; SOD, superoxide dismutase; STZ,
expression and NO/cGMP signaling, thereby promoting endo- streptozotocin; VASP, vasodilator-stimulated phosphoproteins.
thelial and smooth muscle dysfunction.
cGK I expression in aortic tissue was not changed in two dif-
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