eNOS Uncoupling in Cardiovascular Diseases - the Role of Oxidative Stress and Inflammation

Abstract

Many cardiovascular diseases and drug-induced complications are associated with - or even based on - an imbalance between the formation of reactive oxygen and nitrogen species (RONS) and antioxidant enzymes catalyzing the break-down of these harmful oxidants. According to the "kindling radical" hypothesis, the formation of RONS may trigger in certain conditions the activation of additional sources of RONS. According to recent reports, vascular dysfunction in general and cardiovascular complications such as hypertension, atherosclerosis and coronary artery diseases may be connected to inflammatory processes. The present review is focusing on the uncoupling of endothelial nitric oxide synthase (eNOS) by different mechanisms involving so-called "redox switches". The oxidative depletion of tetrahydrobiopterin (BH4), oxidative disruption of the dimeric eNOS complex, S-glutathionylation and adverse phosphorylation as well as RONS-triggered increases in levels of asymmetric dimethylarginine (ADMA) will be discussed. But also new concepts of eNOS uncoupling and state of the art detection of this process will be described. Another part of this review article will address pharmaceutical interventions preventing or reversing eNOS uncoupling and thereby normalize vascular function in a given disease setting. We finally turn our attention to the inflammatory mechanisms that are also involved in the development of endothelial dysfunction and cardiovascular disease. Inflammatory cell and cytokine profiles as well as their interactions, which are among the kindling mechanisms for the development of vascular dysfunction will be discussed on the basis of the current literature.

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Current Pharmaceutical Design, 2014, 20, 3579-3594 3579
eNOS Uncoupling in Cardiovascular Diseases - the Role of Oxidative Stress and
Inflammation
Susanne Karbach1, Philip Wenzel1,2, Ari Waisman3, Thomas Münzel1 and Andreas Daiber1¶
12nd Medical Clinic, Department of Cardiology, 2Center of Thrombosis and Hemostasis and 3The Institute for Molecular Medicine,
Medical Center of the Johannes Gutenberg University, Mainz, Germany
Abstract: Many cardiovascular diseases and drug-induced complications are associated with - or even based on - an imbalance between
the formation of reactive oxygen and nitrogen species (RONS) and antioxidant enzymes catalyzing the break-down of these harmful oxi-
dants. According to the “kindling radical” hypothesis, the formation of RONS may trigger in certain conditions the activation of addi-
tional sources of RONS. According to recent reports, vascular dysfunction in general and cardiovascular complications such as hyperten-
sion, atherosclerosis and coronary artery diseases may be connected to inflammatory processes. The present review is focusing on the un-
coupling of endothelial nitric oxide synthase (eNOS) by different mechanisms involving so-called “redox switches”. The oxidative deple-
tion of tetrahydrobiopterin (BH4), oxidative disruption of the dimeric eNOS complex, S-glutathionylation and adverse phosphorylation as
well as RONS-triggered increases in levels of asymmetric dimethylarginine (ADMA) will be discussed. But also new concepts of eNOS
uncoupling and state of the art detection of this process will be described. Another part of this review article will address p harmaceutical
interventions preventing or reversing eNOS uncoupling and thereby normalize vascular function in a given disease setting. We finally
turn our attention to the inflammatory mechanisms that are also involved in the development of endothelial dysfunction and cardiovascu-
lar disease. Inflammatory cell and cytokine profiles as well as their interactions, which are among the kindling mechanisms for the devel-
opment of vascular dysfunction will be discussed on the basis of the current literature.
Keywords: Oxidative stress, nitric oxide synthase uncoupling, redox switches in nitric oxide synthase, inflammatory cells.
INTRODUCTION
Oxidative stress was demonstrated to be a hallmark of most
cardiovascular and neurodegenerative diseases [1, 2]. The term
oxidative stress defines a state with either increased (uncontrolled)
formation of reactive oxygen and nitrogen species (RONS) and/or
impaired cellular antioxidant defense system (e.g. down-regulation
of important antioxidant proteins) with subsequent depletion of low
molecular weight antioxidants and a shift in the cellular redox bal-
ance. The most common RONS include superoxide radicals, hydro-
gen peroxide, hydroxyl radicals, carbon-centered peroxides and
peroxyl radicals, nitric oxide radicals (•NO), nitrogen dioxide radi-
als, peroxynitrite, hypochlorite and others. Some of these species
have a role as cellular messengers and contribute to redox signaling
[3-5] such as •NO which acts as an important vasodilator.
A direct interaction between •NO and superoxide was proven
by the existence of peroxynitrite (ONOO-) that is formed by th e
diffusion-controlled reaction of •NO with superoxide [6]. Peroxyni-
trite is a much more potent oxidant than •NO and superoxide and its
contribution to cardiovascular and neurodegenerative disease is
meanwhile accepted [2, 7]. Therefore, in many aspects superoxide
can be regarded as a direct antagonist of •NO. The superoxide anion
(•O2
-) can be formed from different sources such as xanthine oxi-
dase, NADPH oxidases, uncoupled NO synthases and the mito-
chondrial respiratory chain. The discovery of superoxide dismu-
tases (mitochondrial manganese superoxide dismutase (Mn-SOD)
and cytosolic/extracellular Cu,Zn-SOD) by Fridovich and cowork-
ers in den 1960s [8] suggested that superoxide is formed in the
organism and in living cells. Moreover, the existence of SODs im-
plied that superoxide is a harmful species involved in pathological
processes forcing the organism to express SODs for protection.
¶Address correspondence to this author at the Universitätsmedizin der Jo-
hannes Gutenberg-Universität Mainz, II. MedizinischeKlinik und Poliklinik
– Labor für Molekulare Kadiologie, Geb. 605 – Raum 3.262, Langen-
beckstr. 1, 55131 Mainz, Germany; Tel: +49 (0)6131 176280;
Fax +49 (0)6131 176293, E-mail: andreas.daiber@bioredox.com
The first direct evidence for a role of RONS for the regulation
of the vascular tone was already provided before the commonly
accepted identification of the “endothelium-derived relaxing factor
(EDRF)” to be nitric oxide by the observation that EDRF-mediated
vasodilation was impaired by superoxide anions and that its func-
tion was preserved in the presence of SOD [9]. In these studies
Gryglewski et al. used cultured endothelial cells, stimulated them
with bradykinine, the supernatants (containing all endothelial me-
diators in response to bradykinine) was used to perfuse isolated
aortic smooth muscle strips and the vasodilatory activity of the
endothelial perfusate was tested by isometric tension recordings.
The authors observed that the stability of EDRF, now well known
as nitric oxide, is significantly decreased in the presence of super-
oxide and preserved by addition of SOD. An indirect clinical proof
for endothelial dysfunction triggered by RONS is based on the ob-
servation that patients with pronounced improvement of endothelial
function (measured by acetylcholine-induced forearm vasodilation)
in response to vitamin C infusion were at higher risk for cardiovas-
cular events as compared to those patients displaying only minor
improvement of endothelial function in response to vitamin C infu-
sion [10]. The main conclusion of this study was that patients with
increased vascular oxidative stress (making them more responsive
to vitamin C) displayed a higher incidence of cardiovascular events.
Recent experimental data indicate that inflammatory processes
play an important role for the development of endothelial dysfunc-
tion and hypertension [11-13] and especially the cytokine interleu-
kin 17 plays an essential role in this process [14]. In the present
review, the role of inflammatory cells and the phagocytic NADPH
oxidase for the induction of vascular oxidative stress, eNOS dys-
function (uncoupling) and atherothrombotic events will be dis-
cussed in detail (Fig. 1).
THE DAMAGING ROLE OF OXIDATIVE STRESS IN
CARDIOVASCULAR DISEASES
Harrison and Ohara first described the role of oxidative stress in
the progression and pathophysiology of cardiovascular disease in an
experimental model of hypercholesterolemia [15, 16]. A lot of pre-
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3580 Current Pharmaceutical Design, 2014, Vol. 20, No. 22 Karbach et al.
clinical studies using genetic tools (e.g. knockout mice) followed
and clarified the involvement of ROS producing or degrading en-
zymes in the onset and progression of cardiovascular disease. They
provided the molecular proof of the pathophysiological role of oxi-
dative stress in cardiovascular disease: For example, it was shown
in mice that the genetic deletion of the NADPH oxidase subunit
p47phox almost normalized vascular •NO bioavailability, reduced
ROS formation, and improved heart function as well as the survival
rate by 20% after myocardial infraction (MI) [17]. Besides, the
deletion of the NADPH oxidase subunits p47phox and Nox1 has a
protective effect on blood pressure and endothelial function in an-
giotensin-II (AT-II)-induced hypertension in mice [18, 19]. Over-
expression of Nox1 in these transgenic mice caused a further in-
crease in blood pressure [20]. Vice versa, partial deletion of the
mitochondrial superoxide dismutase (MnSOD+/-) increased age-
dependent mitochondrial oxidative stress and endothelial dysfunc-
tion [21]. Also partial deficiency in the mitochondrial superoxide
dismutase rendered mice more susceptible to nitroglycerin-induced
nitrate tolerance and endothelial dysfunction [22] and deletion of
the glutathione peroxidase-1 resulted in an elevated ath erosclerotic
plaque lesion size in ApoE-/- mice [23]. These data (going in line
with sev eral other ones in literature) are a molecular proof of the
crucial role of oxidative stress in the development of cardiovascular
disease.
THE “KINDLING RADICAL” HYPOTHESIS
According to the concept of “kindling radicals” (or also “bon-
fire” hypothesis), initial formation of ROS (e.g. from NADPH oxi-
dases) triggers further damage such as eNOS uncoupling by differ-
ent mechanisms (see “redox switches” below and Fig. 2). The ROS-
induced ROS production concept can be extended to almost any
kind of source of RONS as almost all of these sources contain “re-
dox switches”. Beyond cytoplasmic enzymes, there is clear evi-
dence of a cross-talk between mitochondrial ROS formation and
NADPH oxidases [24]: mitochondrial ROS can open the mitochon-
drial permeability transition pore (mPTP), which is also subject to
redox regulation [25]. Upon release of the mtROS to the cytosol,
they can activate the redox-sensitive zinc-finger-like complex in the
protein kinase C (PKC) [26] and thereby confer the translocation of
cytosolic NADPH oxidase subunits triggering NADPH oxidase
dependent superoxide release. Vice versa, NADPH oxidase-derived
cytosolic superoxide or peroxynitrite may stimulate mitochondrial
ROS formation via opening of the mitochondrial ATP-sensitive
potassium channel (mtKATP) [27] leading to changes in the mito-
chondrial membrane potential [28, 29]. Examples and the underly-
ing mechanisms for this mtROS-NADPH oxidase crosstalk were
recently summarized and discussed in detail in three different re-
view articles [24, 30, 31].
Other sources of oxidative stress possess similar redox switches
(for review see [30, 31]): The conversion of xanthine dehydro-
genase (XDH) to the oxidase form (XO) needs for example oxida-
tion of critical thiol residues [32, 33] as observed in AT-II induced
hypertension [34]. The uncoupling of eNOS (and also other iso-
forms) is based on increased formation of oxidants. There exist
various reports revealing that eNOS function is improved and un-
coupling is reversed when the sources of oxidative stress are either
inhibited by pharmacological manipulation (e.g. by PKC inhibitors
or NADPH oxidase inhibitors but also AT1-receptor-blockers) [35-
40] or genetic deletion of the p47phox or gp91phox subunit leading to
a dysfunctional phagocytic NADPH oxidase [41-43]. Similar ob-
servations were made when antioxidants were added acutely and at
high concentrations to the system (e.g. infusion of vitamin C) [10,
44]. The alliance and interconnection between the different sources
of oxidative stress may be the reason why the inhibition of only one
source of RONS can be sufficient to completely normalize a car-
diovascular disease state. This could for example be demonstrated
for the inhibition of xanthine oxidase derived ROS by allopurinol in
experimental diabetes, hypertension and pulmonary arterial hyper-
tension [34, 45, 46] and for the prevention of mitochondrial ROS
formation by mitochondria-targeted antioxidants in hypertension
and ischemia/reperfusion [47, 48]. Finally, the exclu sive inhibition
of the NADPH oxidase by apocynin in diabetes, hypertension and
ischemia/reperfusion led to an appreciable normalization of the
vascular complications [49-51].
Fig. (1). Inflammatory cells, vascular dysfunction and atherothrombosis. The scheme illustrates the activation of immune cells and recruitment to vascular
tissues leading to activation of secondary RONS sources such as NADPH oxidase and uncoupled eNOS, all of which contributes to vascular dysfunction.
These processes lead to late-stage cardiovascular complication such as atherosclerosis with plaque formation and thrombosis. The color version of the figure is
available in the electronic copy of the article.

Vascular Dysfunction, eNOS Uncoupling and Inflammation Current Pharmaceutical Design, 2014, Vol. 20, No. 22 3581
REDOX SWITCHES IN ENDOTHELIAL NITRIC OXIDE
SYNTHASE (ENOS)
Next to the classical regulation principles of the enzymatic ac-
tivity of eNOS (e.g. by calcium/calmodulin, caveolin, HSP90,
palmitoylation and myristoylation), there exist other regulatory
pathways as phosphorylation and S-glutathionylation, which are
directly linked to the formation of redox-active species. These „re-
dox switches“ in eNOS confer alterations in enzymatic eNOS activ-
ity and may contribute to uncoupling of eNOS (see Fig. 2). In the
process of eNOS uncoupling, electrons leak from the transport
chain in the reductase domain (from NADPH over FMN and FAD)
or directly from the iron-oxy complex during the catalytic cycle.
They are then transferred to molecular oxygen to yield superoxide
instead of nitric oxide. This development is even more dangerous
than the exclusive inhibition of eNOS because the uncoupling trans-
forms eNOS from a beneficial to a harmful enzyme [52, 53]. Not
only eNOS can be uncoupled and produce superoxide but also the
neuronal NOS (type 1) [54-57] and the inducible NOS (type 2) [58-
61] can be uncoupled and then produce superoxide themselves.
Among the regulatory pathways (“redox switches”) of eNOS,
the concept of the oxidative depletion of tetrahydrobiopterin (BH4)
is the most fundamental one. BH4 is not only important as steric
stabilizer of the NOS dimers. It is also directly involved in the elec-
tron transfer from the co-substrate NADPH via the iron-oxy com-
plex to th e L-arginine substrate. BH4 provides an electron just in
time to avoid the decay of the iron-oxy complex under generation
of superoxide and iron(III) (see Fig. 3). The resulting BH4
+-radicals
are reduced back to BH4 in a subsequent reaction step. As BH4is
highly active in redox processes, oxidants easily react with BH4 in
1-electron reactions. Several independent working groups pointed
at the substantial evidence for a causative role of BH4 depletion in
Fig. (3). Role of tetrahydrobiopterin (BH4) in prevention of the decom-
position of the iron-oxy complex under formation of iron(III) and su-
peroxide. Upon activation of molecular oxygen by its binding to the
iron(III) of the eNOS catalytic (iron-porphyrin) site and transfer of electrons
from the NADPH cofactor via the flavins in the reductase domain, an iron-
oxy complex is formed that may decompose under formation of iron(III)
and superoxide. BH4 stabilizes this iron-oxy complex by transfer of an elec-
tron under formation of a BH4-cation-radical and thereby allows further
activation of the oxygen at the iron and subsequent transfer of a hydroxy-
group to the guanidino-nitrogen of L-arginine under formation of the inter-
mediate NG-hydroxy-L-arginine and reduced BH4. BH4 may participate in a
similar fashion in the subsequent reaction step in which L-citrulline and
nitric oxide is formed. Adapted from Daiber and Münzel, Steinkopff Verlag
Darmstadt 2006 [182]. The color version of the figure is available in the
electronic copy of the article.
Fig. (2). Redox switches in endot helial nitric oxide synthase. X-ray structure of human eNOS with the iron-porphyrin (blue), the substrate L-arginine
(green), the P450-forming axial iron-thiolate ligand from a cysteine residue (yellow), the cofactor BH4 (purple), the zinc-thiolate complex forming cysteines
(red, two from each subunit) and the zinc ion (brown). The red boxes represent the “redox switches” in eNOS triggering regulatory pathways that depend on
oxidants and reductants. From Schulz et al.,Antioxid. Redox Signal. 2012 [31]. With permission of Mary Ann Liebert Inc. Copyright © 2012. The color
version of the figure is available in the electronic copy of the article.

3582 Current Pharmaceutical Design, 2014, Vol. 20, No. 22 Karbach et al.
the process of NOS uncoupling characterized by superoxide forma-
tion in the presence of the electron source NADPH [57, 62, 63]: An
efficient oxidative degradation of BH4 by peroxynitrite to dihydro-
biopterin (BH2) was shown by Milstein and Katusic providing an
explanation of how RONS (especially peroxynitrite) may contribute
to oxidative uncoupling of eNOS [64]. The understanding of the
importance of Vitamin C is important for the recycling or rescue of
the •BH4
+ radicals (once BH2 is formed only energy-consuming
enzymatic reaction confers reduction to BH4) [65-68], and this led
to an attractive explanation for the highly beneficial effects of vita-
min C infusion on improvement of the endothelial function in
smokers [44] and diabetic patients [69].
First direct evidence of th e role of BH4 depletion in eNOS un-
coupling and subsequent endothelial dysfunction in vivo was ob-
tained in hypertensive mice in 2003 [41]. Soon afterwards, the en-
zymatic source for BH4 synthesis in form of the GTP-cyclohy-
drolase-1,was identified as an important regulator of eNOS and
endothelial function [70]. An overexpression of GTP-cyclohydro-
lase-1, improved the endothelial function in atherosclerotic ApoE-/-
mice [71] and vessels from diabetic rats and mice [72, 73]. Besides
the GTP-cyclohydrolase-1 dependent de novo synthesis of BH4,
there exists the “salvage pathway”, which is of high physiological
relevance. It consists of the recycling of oxidized BH2 back to BH4
by dihydrofolate reductase [74, 75]. BH4 but not tetrahydroneop-
terin (NH4), which indeed shares the same antioxidant properties
with BH4without being a cofactor for eNOS led to an improvement
of endothelial function in smokers [76, 77]. In line with this, sup-
plementation with th e BH4 analogue folic acid improved endothe-
lial function in humans [78, 79]. Treatment w ith the BH4 precursor
sepiapterin also led to a re-establishment of endothelial function in
experimental hypertension and in atherosclerosis [80, 81].
Another direct redox-regulatory pathway for eNOS function is
the oxidative disruption of the zinc-sulfur-complex (ZnCys4) in the
binding region of the eNOS dimer resulting in a loss of SDS-
resistant eNOS dimers. This has first been described by Zou and
coworkers for peroxynitrite-mediated oxidation of eNOS [82].
Later they showed that hypochlorous acid also confers disruption of
the zinc-sulfur-complex in eNOS [42]. eNOS dysfunction and al-
tered eNOS dimer stability by hypochlorous acid was also de-
scribed by Keaney Jr. and his coworkers without determination of
the zinc content o f the enzyme [83]. Wenzel and colleagues also
observed this shift in eNOS dimer/monomer ratio in diabetic rats
[84]. Although there is no decisive evidence in favor or against a
relevant role of the oxidative disruption of the zinc-sulfur-complex
for the eNOS uncoupling process, the theory is highly attractive:
Peroxynitrite anion (ONOO-) confers high specificity for the oxida-
tion of “activated” thiols as thiolate groups that are found in the
neighborhood of proton-abstracting amino acids or in zinc-
complexes [5]. The reaction of ONOO- with the ZnCys2His com-
plex at the active site of alcohol dehydrogenase has been reported to
proceed with a kinetic constant of 2.6-5.2*105 M
-1s-1[85] and al-
ready nanomolar flux rates of superoxide, nitric oxide and per-
oxynitrite are able to inactivate the enzyme by oxidative disruption
of the ZnCys2His complex associated with zinc release and disul-
fide formation [86].
S-glutathionylation is an important redox regulatory mechanism
for many enzymes (e.g. mitochondrial aldehyde dehydrogenase
[87], sirtuin-1 [88] or SERCA [89]) and was also reported for
eNOS. eNOS has been recently described to be adversely regulated
and uncoupled (leading to superoxide formation) by S-gluta-
thionylation at one or more cysteine residues of the reductase do-
main [90]. In a subsequent study, Chen et al. demonstrated a super-
oxide-induced thiyl radical formation in eNOS with subsequent
intracellular disulfide formation or S-glutathionylation, which both
lead to an eNOS uncoupling [91]. Based on the observations by
Knorr and colleagues, eNOS S-glutathionylation is largely in-
creased in nitroglycerin-treated endothelial cells and aortic tissue
from nitroglycerin-infused rats, probably contributing to eNOS
uncoupling and endothelial dysfunction in the setting of nitrate
tolerance. This could be prevented by therapy with the AT1-
receptor telmisartan [40]. An eNOS S-glutathionylation could also
be demonstrated in a rat model of streptozotocin-induced type dia-
betes mellitus. Prevention was possible by cotreatment with the
organic nitrate pentaerithrityl tetranitrate (PETN) suggesting plei-
otropic antioxidant effects based on heme oxygenase-1 (HO-1)
induction[92].
Another redox-sensitive regulatory pathway of eNOS is its
phosphorylation. Here, we must discriminate 3 different eNOS
phosphorylation modifications of relevance: On the one hand´s side
the activating phosphorylation at serine1177 is mediated by the Akt
pathway. This process is calcium-independent and increases the
nitric oxide producing activity of eNOS [93]. Second, the inactivat-
ing phosphorylation takes place at tyrosine657, which is arranged
by the protein kinase-2 (PYK-2) inhibiting the enzyme without
evidence for uncoupling of the enzyme [94]. And third, there exists
the inactivating phosphorylation at threonine495 mediated by pro-
tein kinase C (PKC), which can also contribute to uncoupling and
superoxide production by eNOS [95, 96]. The phosphorylation of
eNOS at Thr495 or Tyr657 are activated by oxidative stress. [26,
94, 97]. That means that the phosphorylation at Thr495 and Tyr657
can be regarded as “redox switches” in eNOS.
Asymmetric dimethylarginine (ADMA) has often been de-
scribed to be the most potent endogenous inhibitor of eNOS [98]. It
is still discussed if ADMA itself really evokes uncoupling of eNOS
[99]. What is certainly known is that high ADMA serum/plasma
levels are a reliable risk marker for cardiovascular ev ents and prog-
nosis in patients with cardiovascular disease [100, 101]. Oxidative
stress in the vasculature may significantly contribute to ADMA
production or to inhibition of ADMA degradation, leading to
ADMA concentrations that significantly inhibit eNOS activity
[102] or may even uncouple the enzyme and switch it to a superox-
ide synthase. It has been described in literature that oxidative stress
can increase the expression of PRMTs and thereby lead to increased
ADMA-formation [103, 104]. There exists growing evidence that
the activity of the ADMA demethylating enzyme (DDAH) is ad-
versely regulated by oxidative and nitrosative stress [105]. So the
regulation of eNOS activity (and maybe uncoupling) by ADMA
seems to be redox-sensitive and may significantly contribute to
endothelial dysfunction under oxidative stress conditions (Fig. 4).
A recently published work conferred more importance to the
concept of ADMA-dependent eNOS regulation and provided a new
potential mechanism of endothelial ADMA accumulation despite
moderate increases in plasma ADMA levels, which is based on the
decreased expression/activity of the y+L amino acid transporters
(y+LAT-1) [106]. This concept makes it also easier to understand
the so-called L-arginine paradox which consists in the beneficial
effect of oral L-arginine on vascular function in patient cohorts
despite sufficient L-arginine plasma concentrations for adequate
substrate supply to eNOS. According to the recent findings, the
export of ADMA in endothelial cells is mediated by y+L amino acid
transporters (y+LAT-1 and -2) [107, 108]: These transporters ex-
change intracellular cationic amino acids against extracellular neu-
tral amino acids and sodium cations and thus provide an active
(energy-dependent) efflux pathway for ADMA. Down-regulation of
the y+LAT isoforms in cultured endothelial cells by siRNA, lead to
an intracellular ADMA accumulation [107, 108]. Consequently, an
attractive explanation for the beneficial effects of L-arginine is the
export of ADMA via the cationic amino acid transporter (CAT-1)
using cationic amino acids (e.g. L-arginine) in exchange in the set-
ting of dysfunctional or down-regulated expression of y+LAT [106].
This theory found clinical support by observations in a patient with
multiple coronary artery sp asm and a genetic defect in the y+LAT
expression, causing increased ADMA plasma levels upon admini-
stration of high dose L-arginine, most probably by CAT-1/L-

Vascular Dysfunction, eNOS Uncoupling and Inflammation Current Pharmaceutical Design, 2014, Vol. 20, No. 22 3583
arginine driven export of ADMA from endothelial cells [106].
These results were actually confirmed by cell culture studies using
stable overexpression of CAT-1 [109].
THERAPEUTIC TARGETING OF UNCOUPLED ENOS –
“RECOUPLING ENOS”
One therapeutic option for eNOS “recoupling” is supplementa-
tion with BH4 or its precursors sepiapterin and folic acid, which are
cheaper and more stable. BH4 supplementation has proven highly
beneficial in many studies on endothelial function such as im-
provement in diabetic patients or chronic smokers [110]. Overex-
pression or activity enhancement of GTP-cyclohydrolase represents
another pathway to increase the BH4 levels as demonstrated for
diabetic mice [111]. Under certain condition s the levels of ADMA
are increased in endothelial cell, which may contribute to uncou-
pling of eNOS, and supplementation with high dose L-arginine may
be of great benefit by replacing ADMA or even support its export
from endothelial cells by driving the cationic amino acid transport-
ers. This may be of great clinical importance since ADMA is a
valid predictor of cardiovascular events and increases cardiovascu-
lar mortality [100]. Besides these directly eNOS-targeting therapeu-
tic interventions any cardiovascular therapy that reduces oxidative
stress will contribute to “recoupling” of eNOS as demonstrated for
angiotensin-converting-enzyme (ACE) inhibition, AT1-receptor-
blocker (ARB) therapy, treatment with statins or antihyperglyce-
mics and to a lesser extent treatment with calcium-antagonists or -
blockers. In general, promising strategies to prevent eNOS uncou-
pling or to “recouple” an uncoupled eNOS may be based on the
increase in cellular antioxidant defense mechanisms. A prominent
target for this purpose is the activation of the HO system (most
easily of the inducible isoform HO-1). Figure 5 summarizes ho w
induction of HO-1 or the constitutive isoform HO-2 confer protec-
tion of the eNOS function in the setting of diabetes mellitus (e.g. by
preventing oxidative depletion of BH4) [112, 113]. Importantly,
induction of HO yields low levels of carbon monoxide and biliver-
din, further converted to bilirubin, which have anti-atherosclerotic,
anti-inflammatory, antithrombotic, vasodilato ry and potent antioxi-
dant properties. In addition, these products induce Cu/Zn-
superoxide dismutase expression, which further contributes to the
antioxidant and beneficial profile of HO in diabetic complications.
Interestingly, HO products also induce GTP-cyclohydrolase-1,
providing a direct link for eNOS regulation [92, 114].
Many cardiovascular drugs have pleiotropic antioxidant proper-
ties such as the decrease in NADPH oxidase activity and “recou-
pling” of eNOS by ACE inhibitors, ARBs and statins. An interest-
ing example of a cardiovascular drug with pleiotropic antioxidant
effects is PETN. Once developed in the US, it was abandoned, then
used for many years in the former Eastern part of Germany (DDR)
and after the reunion of Germany seized the nitrate market. During
the last years, it was the best selling nitrate in Germany. PETN is
the only organic nitrate in clinical use that is devoid of nitrate toler-
ance and endothelial dysfunction [115, 116]. Based on previous
observations, PETN is a potent inducer of the intrinsic antioxidant
HO-1 system [81] and of extracellular SOD [117]. Even more sur-
prisingly, a recent gene array study revealed that PETN, in contrast
to GTN, induces a number of cardioprotective genes, whereas GTN
induces cardiotoxic genes [118]. Although both compounds are
organic nitrates and regarded as nitric oxide donors, they show
completely different gene regulation profiles indicating that organic
nitrates are not a homogenous class of drugs [116]. Preliminary data
indicate that PETN also improves vascular complications in an
animal model of type 1 diabetes mellitus.
Figure 6 summarizes the beneficial action of several drugs on
eNOS coupling state and also introduces different techniques to
assess eNOS uncoupling (eNOS-derived superoxide formation).
Fig. (4). Schematic overview of the biochemical pathways related to ADMA. N-methyltransferases utilize S-adenosylmethionine as a methyl group donor
and confer the methylation of arginine residues within proteins or polypeptides. Free ADMA is present in the cytoplasm as a product of proteolytic breakdown
of proteins and its circulating levels can be detected in human blood plasma. ADMA is a competitive inhibitor of all NOS isoforms replacing the physiological
substrate of this enzyme, L-arginine, which contributes to endothelial dysfunction and, as a late consequence, atherosclerosis. The elimination of ADMA from
the body proceeds via urinary excretion and, alternatively, via breakdown by the enzyme dimethylarginine dimethylamino hydrolase (DDAH) to citrulline and
dimethylamine. Adapted from Böger, Cardiovasc. Res.2003 [183]. The color version of the figure is available in the electronic copy of the article.

3584 Current Pharmaceutical Design, 2014, Vol. 20, No. 22 Karbach et al.
Fig. (5). The scheme summarizes the simplified mechanisms underlying oxidative stress-induced endothelial dysfunction (and probably nitrate resis-
tance) in dia betes mellitus. It should be noted that the oxidative stress concept provides an explanation for a part of diabetic complications and probably repre-
sents one important pathological pathway among several. Prevention of diabetic cardiovascular complications by induction of the HO antioxidant system. Key
mediators of these beneficial effects are carbon monoxide (CO) bilirubin, extracellular superoxide dismutase (ecSOD), coupling of endothelial nitric oxide
synthase (eNOS) by normalization of tetrahydrobiopterin (BH4) levels and decrease in superoxide levels. Adopted from Abraham and Kappas, Pharmacol. Rev.
2008 [112] and Oelze et al., Exp. Diabetes Res. 2010 [113]. Copyright © 2010 Matthias Oelze et al. The color version of the figure is available in the electronic
copy of the article.
Fig. (6). Experimental data on eNOS uncoupling and recoupling. (A) Nitric oxide bioavailability was measured indirectly by hemoglobin-nitric oxide com-
plex (electron paramagnetic resonance spectroscopy) levels in whole blood samples and by nitrite concentrations (nitric oxide analyzer, n=6) in plasma samples
from AT-II infused, hypertensive rats with nebivolol therapy. The electron paramagnetic resonance spectra were averaged from 3 independent measurements.
From Oelze et al., Hypertension 2006 [119]. With permission of Wolters Kluwer Health. Copyright © 2006. (B) eNOS uncoupling by oxidative fluorescence
microtopography was detected in aortic cryo section from AT-II infused, hypertensive rats with organic nitrate (PETN versus ISMN) therapy. Densitometric
quantification of DHE staining was performed in the endothelial cell layer which was extracted from the whole microscope image. A fixed area was used for
-30
-10
10
30
50
Change in Aortic Superoxide
Lucigenin Signal by L-NAME [%]
CC+TSS+T
ABC
DE

Vascular Dysfunction, eNOS Uncoupling and Inflammation Current Pharmaceutical Design, 2014, Vol. 20, No. 22 3585
densitometric quantification and the procedure is shown for one representative endothelial cell layer of AT-II treatment group. eNOS uncoupling was previ-
ously assessed by the effects of L-NAME on DHE staining [39, 119]. The method of densitometric quantification of endothelial DHE staining was adopted
from the protocol of Alp et al. [111]. From Schuhmacher et al., Hypertension 2010 [81]. With permission of Wolters Kluwer Health. Copyright © 2010. (C)
Lucigenin-derived chemiluminescence was used to assess vascular ROS formation in intact aortic ring segments from diabetic rats with telmisartan therapy.
eNOS-dependent superoxide formation was assessed by substraction of lucigenin signal of L-NAME-treated aortic rings from lucigenin signal without L-
NAME and expressed as percentage change of signal based on the L-NAME-free group. Data are shown for control (C), control/telm isartan (C+T), STZ (S),
and STZ/telmisartan (S+T) animals. From Wenzel et al., Free Radic. Biol. Med. 2008 [39]. With permission of Elsevier. Copyright © 2008. (D) Levels of
eNOS monomer and dimer were determined in aorta from diabetic and atorvastatin treated using Western blot after SDS-PAGE using a 4°C gel and non-
reducing conditions. From Wenzel et al., Atherosclerosis 2008 [84]. With permission of Elsevier. Copyright © 2008. (E) S-glutathionylation of eNOS was
determined in aorta from nitrate tolerant rats with temisartan therapy by eNOS immunoprecipitation, followed by anti-glutathione staining and normalization
on eNOS. Disappearance of the anti-glutathione staining in the presence of 2-mercaptoethanol served as a control. Representative blots are shown at the bottom
of each densitometric quantification along with the respective loading control. From Knorr et al., Arterioscler. Thromb. Vasc. Biol. 2011 [40]. With permission
of Wolters Kluwer Health. Copyright © 2011. The color version of the figure is available in the electronic copy of the article.
The third generation -blocker nebivolol had highly beneficial ef-
fects on AT-II induced endothelial dysfunction in an experimental
model of hypertension [119]. eNOS dysfunction/uncoupling in AT-
II treated rats and prevention of this adverse effects by nebivolol
therapy can be indirectly measured by the plasma levels of the he-
moglobin-nitric oxide complex by EPR, which was decreased in
hypertensive rats and increased over control by nebivolol therapy.
The reason for this beneficial action of nebivolol is based on the
potent inhibition of the NADPH oxidase by the -blocker, thereby
eliminating an important source for the “kindling radicals” (see
preceding paragraphs). Similarly, PETN via induction of HO-1
prevents eNOS uncoupling in hypertensive rats as demonstrated by
the increase in endothelial superoxide formation by AT-II infusion
and normalization of this process by PETN but not isosorbide-5-
mononitrate (ISMN) [81]. The method of oxidative fluorescence
microtopography using dihydroethidine as a fluorescence probe for
endothelial superoxide formation, which is applied here is highly
reliable. In combination with L-NAME this assay is highly specific
to detect an uncoupled NOS. L-NAME blocks nitric oxide forma-
tion from intact eNOS and thereby increases the free, detectable
superoxide (DHE signal), by preventing the break-down reaction of
superoxide with nitric oxide to yield peroxynitrite. Reciprocally, L-
NAME blocks superoxide formation from uncoupled eNOS and
thereby decreases the free, detectable superoxide (DHE signal). A
very similar technique is based on the modulation of aortic superox-
ide release (measured by lucigenin-derived chemiluminescence) by
L-NAME in intact vessel segm ents: The principle of action is just
similar to the one explained for oxidative fluorescence microtopog-
raphy. We applied this assay to demonstrate increased eNOS un-
coupling in aortic tissue from diabetic rats, compatible with the L-
NAME-induced decrease in lucigenin ECL signal (blockade of
eNOS-derived superoxide formation) [39]. Vice versa, telmisartan
therapy normalized the eNOS coupling state, compatible with the
L-NAME-induced increase in lucigenin ECL signal (blockade of
eNOS-derived nitric oxide formation). Another example for a nor-
malization of eNOS coupling state or function by a cardiovascular
drug is based on the detection by the eNOS dimer/monomer ratio
mentioned before (an indirect read-out), which was decreased in
aorta from diabetic rats and normalized by atorvastatin treatment
[84]. Finally, the already described eNOS S-glutathionylation rep-
resents another indirect read-out for the eNOS coupling or func-
tional state and was in creased in aorta from nitrate tolerant, nitro-
glycerin-treated rats and was normalized by telmisartan therapy
[39].
All of these assays to d etect eNOS-derived superoxide to assess
its coupling state are hampered under conditions of iNOS induction
as the iNOS-derived nitric oxide will lead to confusing L-NAME
effects. As shown in Figure 7, AT-II in fusion in AMPK deficient
mice results in severe expression of iNOS and high nitric oxide
formation rates (seen by dramatically increased plasma nitrite and
aortic nitric oxide EPR signal) [120]. iNOS mimics a functional
eNOS with respect to nitric oxide release and especially L-NAME
effects (blockade of nitric oxide formation and increase in superox-
Fig. (7). Detection of eNOS-derived superoxide formation in the pres-
ence of iNOS. More complicated is the detection of eNOS uncoupling in the
presence of iNOS-derived high fluxes of nitric oxide as observed in AMPK
deficient mice with AT-II infusion [120]. The infusion of AT-II should
result in eNOS uncoupling with subsequent superoxide formation, which is
sensitive to L-NAME challenges – blockade of uncoupled eNOS should
reduce the superoxide-induced 2-hydroxyethidium (2-HE) signal. In the
presence of iNOS, the high fluxes of nitric oxide will mask the eNOS-
derived superoxide formation via consumption to yield peroxynitrite. L-
NAME will block superoxide formation from uncoupled eNOS but at the
same time will inhibit high nitric oxide fluxes from iNOS and thereby the
consumption of superoxide to yield peroxynitrite, which will result in the
overall increase in superoxide-induced 2-HE signal (also from other ROS
sources). Therefore, aortic tissue with uncoupled eNOS and iNOS expres-
sion will show similar results as control tissues with respect to the L-NAME
sensitive superoxide assay. It is recommended to either use specific inhibi-
tors for eNOS and iNOS to characterize the contribution of each isoform or
to use indirect methods instead that are based on eNOS specific antibodies
(e.g. S-glutathionylation or Thr495/Tyr657 phosphorylation). The color
version of the figure is available in the electronic copy of the article.
ide signal). Indicative for the contribution of iNOS to this process
were the significantly increased levels of 3-nitrotyrosine-positiv e
proteins. eNOS uncoupling under these conditions is most success-
fully documented by indirect methods based on specific eNOS anti-
bodies (e.g. dimer/monomer ratio, S-glutathionylation or Thr495/
Tyr657 phosphorylation).
Novel strategies to recouple eNOS are based on targeting the
drugs to the endothelium. Previous work has demonstrated that
administration of extracellular SOD, which directly bind to the

3586 Current Pharmaceutical Design, 2014, Vol. 20, No. 22 Karbach et al.
endothelium, is highly protective in many states of cardiovascular
disease [121]. Likewise, deletion of extracellular SOD impaired
vascular function and hemodynamics [121]. Therefore, the targeting
of extracellular SOD to the endothelium is a promising strategy to
prevent endothelial (vascular) dysfunction. In a recent publication,
Shuvaev et al. have demonstrated that targeting of SOD but not
catalase to the endothelium improved AT-II induced endothelial
dysfunction [122]. The antioxidant enzymes superoxide dismutase
(SOD) and catalase were conjugated with an antibody to platelet-
endothelial cell adhesion molecule-1 (PECAM-1) to ensure endo-
thelial binding. The results of this study once more demonstrate that
superoxide is the more harmful species in the vascular system than
hydrogen peroxide. The covalent binding of antioxidants (e.g. SOD
mimics) to heparin may be another promising attempt [123]. This
strategy seems to be highly attractive, since the endothelium has
heparin-binding sites.
The scheme in Figure 8 provides a summary of the oxidant
mechanisms that contribute to vascular dysfunction [124]. Possible
antioxidant therapeutic interventions are also provided within the
scheme. Despite the fact that most controlled and large clinical
trials did not support a beneficial role of oral antioxidant therapy for
cardiovascular disease, there is ample evidence that antioxidant
therapy may be protective when directed to specific sites and ad-
ministrated acutely. In addition, some of the most promising thera-
peutic targets have not been exploited so far (e.g. mitochondrial
pores or NADPH oxidases) or pleiotropic antioxidant properties of
established drugs have not been recognized up to now. Besides the
here presented data and the discussed ideas on the development of
new synthetic th erapeutics, there is the whole field of gen e therap y
which opens many new pathways with respect to future strategies
for antioxidant therapy (e.g. silencing of oxidant producing systems
or inducing antioxidant systems) and which is just at its beginning
after the discovery of microRNAs and antagomirs.
CARDIOVASCULAR DISEASE, NADPH OXIDASES AND
THE IMMUNE SYSTEM
As described in the preceding paragraphs, endothelial dysfunc-
tion, arterial hypertension as well as other cardiovascular diseases
Fig. (8). Scheme illustrating the mechanisms underlying vascular (endothelial) dysfunction by oxidative stress. Known cardiovascular risk factors (e.g.
smoking, hypertension, hyperlipidemia, diabetes) activate the renin-angiotensin-aldosterone-system (RAAS) leading to elevated AT-II levels as well as in-
creased endothelial and smooth muscle superoxide (O2
•-) formation from NADPH oxidase activation by protein kinase C (PKC) and from the mitochondria.
Superoxide reacts with •NO, thereby decreases •NO bioavailability in favor of peroxynitrite (ONOO-) formation. Peroxynitrite causes uncoupling of endothe-
lial NOS due to oxidation of tetrahydrobiopterin (BH4) to BH2 and nitration/inactivation of prostacyclin synthase (PGI2S). Direct proteasome-dependent deg-
radation of the BH4 synthase GTP-cyclohydrolase (GTP-CH) further contributes to eNOS uncoupling. Uncoupled NOS produces superoxide instead of •NO
and nitrated PGI2S produces no prostacyclin (PGI2) but activated cyclooxygenase-2 (due to increased peroxide tone) generates vasoconstrictive prostaglandin
H2. Inhibition of smooth muscle soluble guanylylcyclase (sGC) by superoxide and peroxynitrite contributes to vascular dysfunction as well as increased inacti-
vation of cyclic GMP (cGMP) by phosphodiesterases (PDE) and oxidative stress increases the sensitivity to vasoconstrictors such as endothelin-1 (ET-1).
Mitochondrial ROS formation is modulated by oxidative activation of ATP-dependent potassium channels (KATP) leading to altered mitochondrial membrane
potential and permeability. Upon uncoupling of mitochondrial respiratory complexes, the mitochondrial permeability transition pore (mPTP) may be oxida-
tively opened allowing mtROS to escape to the cytosol activating the PKC-NADPH-Ox system. Modified from Münzel et al., Circ. Res. 2005 [115]. With
permission of Wolters Kluwer Health. Copyright © 2011. Reproduced from Chen, Chen, Daiber, Faraci, Li, Rembold and Laher, Clinical Science 2012 [124].
With permission of Portland Press. Copyright © 2011. The color version of the figure is available in the electronic copy of the article.

Vascular Dysfunction, eNOS Uncoupling and Inflammation Current Pharmaceutical Design, 2014, Vol. 20, No. 22 3587
are usually accompanied and at least partially mediated by in-
creased levels of oxidative stress in form of increased RONS for-
mation: An elevated RONS formation by NADPH oxidases or by
the uncoupled eNOS goes in hand in hand with an insufficient neu-
tralization of RONS leading to a dysequilibrium between RONS
formation and degradation.
A recent advance in the understanding of the development of
endothelial dysfunction consists of the integration of the immune
system (e.g. inflammatory cells) in this concept (Fig. 9): Besides
endothelial cells and vascular smooth muscle cells, inflammatory
cells such as neutrophil granulocytes and macrophages (myelo-
monocytic cells) but also T-cells and dendritic cells provide func-
tional NADPH oxidases and are capable to produce RONS or at
least to activate phagocytic cells leading to an “oxidative burst” of
the blood [11, 125]. Different immune cells have been reported to
contribute to the development of cardiovascular disease but since
they interact with each other, the individual impact of each cell type
on the development of cardiovascular disease remains elusive.
It has been shown that mice without B- and T-cells (RAG-1-/-)
mice show a less pronounced superoxide production and a less
severe development of hypertension in response to AT-II [11]. Ab-
lation of myelomonocytic cells also attenuated the AT-II-induced
blood pressure increase and ROS/RNS production [13]. As a possi-
ble link to these important observations, it has been described that
immune suppressive treatment of patients with rheumatoid arthritis
or psoriasis leads to reduction of systolic blood pressure [126],
conferring more clinical impact to this new hypothesis of immune
system triggered vascular dysfunction.
MYELOMONOCITIC CELLS IN HYPERTENSION
Macrophages/monocytes and especially neutrophil granulocytes
are capable to produce large amounts of ROS/RNS within a short
period of time for host defense with in the innate immediate immune
response [127] – this process is called “oxidative burst”. Both, neu-
trophils and macrophages/monocytes can be activated and stimu-
lated by AT-II over angiotensin receptor type 1 (Agtr1). AT-II leads
to the recru itment of CD45+ leuco cytes to the aortic wall [14] – and
one part of these cells consists of infiltrating myelomonocytic cells
[13]. By selective ablation of the LysM-positive myelomonocytic
cells with low-dose diphteria toxin using the CreLox approach, the
AT-II induced endothelial dysfunction and hypertension as well as
the AT-II induced oxidative burst in the blood was suppressed.
Vascular dysfunction was reestablished by adoptive cell transfer of
monocytes but not of neutrophils [13]. The changes in ROS forma-
tion were accompanied by an up-regulation of the catalytic subunit
of the phagocytic NADPH oxidase Nox2 under ATII and by a
decrease of NOX2 in the ATII treated mice depleted of the
myelomonocytic cells [13].
Neutrophils are also implicated to be important in the develop-
ment of endothelial dysfunction: Neutrophil granulocytes in spon-
taneously hypertensive rats (SHR) provided an elevated oxidative
burst (whereas the phagocytic potential was comparable), an in-
creased iNOS expression and an enhanced myeloperoxidase
(MPO)-catalysis [128]. In addition, a subendothelial MPO accumu-
lation by infiltrated neutrophils was described as a major sink for
oxidative nitric oxide depletion with subsequent development of
endothelial dysfunction [129-131]. This prompts the speculation
that neutrophils are at least partially involved in the development of
oxidative stress, vascular dysfunction and hypertension. Aside from
that, elevated concentrations of MPO in peripheral blood, which is
the most abundant enzyme stored in the granules of neutrophils
[132] and directly related to neutrophil number [133], are connected
to coronary artery disease [134, 135]. It was shown that MPO se-
rum levels in patients with ACS predict an increased risk for subse-
quent cardiovascular events and by this way extend the prognostic
information we get with the help of traditional biochemical markers
[129]. There is in addition striking evidence that the NOS inhibitor
asymmetrical dimethylarginine (ADMA), which is associated with
Fig. (9). Scheme illustrating the role of the immune system in the development of endothelial dysfunctio n. Dendritic cells in the vascular wall (possibly
presenting neo antigens as answer to hypertensive stimuli like in atherosclerosis [163]) produce IL-6, IL-1 and TGF-. Thus CD4 T cells are activated and
switch to Th17 cell. IL-17A then activates T cells themselves but also endothelial cells, smooth muscle cells and macrophages. Endothelial cells can produce
IL-6 and also G-CSF when activated. Macrophages secrete IL-6 and TNF- as answer to IL-17A contributing to neutrophil activation and recruitment to the
site of inflammation. Processes like this can take place in all kinds of locations and also in the vascular wall leading to an upregulation of inflammatory cells in
the vasculature. Retraced from Iwakura et al., Immunity 2011 [138]. The color version of the figure is available in the electronic copy of the article.

3588 Current Pharmaceutical Design, 2014, Vol. 20, No. 22 Karbach et al.
endothelial dysfunction, leads to an elevated adhesion of neutro-
phils to endothelial cells and to an increased superoxide generation
and release of MPO by neutrophils [136].
INTERLEUKIN-17A AND IL-6 IN ENDOTHELIAL DYS-
FUNCTION
IL-17A is an important cytokine which is responsible for neu-
trophil recruitment and macrophage activation [137, 138]. IL-17A
is mainly produced by the T helper 17 subset of CD4+ T cells
(Th17) and by  TCR expressing T cells ( cells). IL-6, which is
produced by the innate immunity cells (e.g. dendritic cells, mono-
cytes, macrophages, mast cells, B-cells as well as endothelial cells,
keratinocytes and fibroblasts), is an essential differentiation factor
of Th17 cells [137]. Furthermore, IL-6 is secreted together with
TNF-, IL-1 and IL-12 by macrophages which are triggered by IL-
17A, being part of an inflammatory amplification loop [138]. Thus,
IL-6 is located upstream, but also downstream of IL-17A. IL-17A
has been described to be involved in vascular inflammation of athe-
rosclerosis and hypertensive disease [139]. Madhur et al. demon-
strated that IL-17A plays a leading role in the development of AT-
II-induced vascular dysfunction: The aortas of IL17A deficient
mice showed only a mild impairment of the endothelial function
under AT-II in comparison to AT-II infused control mice. In addi-
tion, superoxide production was reduced in IL-17A-/- [14]. Madhur
et al showed that AT-II increases the IL-17A production of T-cells
and that chronic AT-II infusion over 4 weeks led to a reduced T-
cell infiltration into the aortic wall of IL-17A-/- mice in comparison
to control mice with the same treatment. Although the initial blood
pressure increase after 4 weeks of AT-II treatment was similar in
IL-17A-/- and control mice, IL-17A-/- deficiency ameliorated the
elevated blood pressure levels averaging 30 mm Hg lower as com-
pared to AT-II infused control mice. It was shown before using IL-
6 knock-out mice that IL-6 is important for the onset of AT-II-
induced endothelial dysfunction and superoxide production [140]:
AT-II-triggered endothelial dysfunction in carotid arteries from IL-
6-/- mice was less pronounced as compared to AT-II-treated control
mice and a similar observation was made for aortic superoxide
levels measured by lucigenin enhanced chemiluminescence [140].
Increased levels of IL-17 were found in patients with myocar-
dial infarction respectively and patients with unstable angina [106,
139, 141], as well as in patients with hypertension [14] as compared
to healthy controls and patients with stable angina. Overall, plasma
IL-6 levels were described to correlate positively with the progres-
sion of cardiovascular disease [142].
IL-17A is not only involved in neutrophil and macrophage acti-
vation and recruitment but also triggers the pro-inflammatory re-
sponse and ROS-production of the vascular smooth muscle cells
(VSMC) [143]: IL-17A leads to an increased ROS-production in
murine aortic VSMC which could be abolished by the NADPH-
oxidase inhibitor apocynin. Treatment with siRNA against Nox2
led to a significant reduction of the IL-17A stimulated ROS-
formation demonstrating that Nox2 plays an important role in the
IL-17A induced oxidative stress in VSMCs. In addition, IL-17A
treatmen t of VSMCs led to an increase of IL-6, Granulocyte Col-
ony-Stimulating Factor (G-CSF) and Granulocyte-Macrophage
Colony-Stimulating Factor (GM-CSF) in the supernatants of the
cell culture, and all of these cytokines play an important role in
neutrophil and macrophage activation and recruitment [143].
Even more importantly, IL-17 was shown to be able to induce
cell death in human endothelial cells suggesting its involvement in
human acute coronary syndrome (ACS) by contributing to plaque
destabilization [144]. It has been demonstrated that IL-17A induces
disruption of the blood-brain-barrier by induction of NADPH-
derived production of ROS by the human brain endothelial cell
layer. This led to a down-regulation of the tight junction molecule
occludin between the endothelial cells and by this mechanism to a
disruption of the blood-brain-barrier [145]. This could be avoided
either by application of an IL-17A blocking antibody or by inhibi-
tion of ROS formation [145]. Finally, IL-17 is an important activa-
tor of the endothelium, which leads to an induction of endothelial
adhesion molecules favoring the adhesion and transmigration of
neutrophils [146].
T CELLS IN HYPERTENSION
Patients treated for cancer with infusion of autologous, acti-
vated lymphocytes from the peripheral blood exhibited an increased
blood pressure within the first hours of infusion pointing to a possi-
ble role of T cells in the development of hypertension [147].
As summarized above IL-17A, produced by Th17 and  T
cells, plays an important role in the development of endothelial
dysfunction. In accordance with these observations, Guzik et al
demonstrated that the elevation of blood pressure caused by AT-II
was markedly reduced in mice without B and T cells (RAG-1-/-
mice) [11]. The development of vascular dysfunction, vascular
hypertrophy as well as vascular superoxide production was likewise
suppressed in AT-II-treated RAG-1-/- mice comp ared to AT-II-
treated control mice. Adoptive cell transfer of functional T-cells
(but not of B cells) restored the hypertensive response to AT-II as
well as the AT-II-induced increase in vascular superoxide produc-
tion. Transfer of T cells lacking the AT-II receptor type 1 (Agtr1) or
a functional NADPH oxidase only partially reduced the AT-II-
mediated hypertensive response [11]. There is an infiltration of T
cells to the aortic adventitia and the periadventitial fat under AT-II-
treatment which could be proved by FACS-analysis of the aortic
vessel [11]. RAG-1-/- mice had a similar reduction in the hyperten-
sive response and the vascular superoxide production compared to
control mice when blood pressure was induced with the DOCA-salt
model, which shows that the reduced hypertensive response in
RAG-1-/- mice was not sp ecific to AT-II.
Blood-derived T cells themselves produce AT-II when treated
with anti-CD3 [148]. This could be prevented by application of the
angiotensin-converting enzyme inhibitor (ACE-inhibitor) perindo-
pril. Furthermore T cells seem to express the angiotensin-conver-
ting enzyme, renin and both the AT-II receptor type 1 and type 2.
Thus, they provide all enzymes of the renin-angiotensin-aldosterone
system (RAAS) – meaning that T cells have an own, endogenous
RAAS [148, 149]. T cells produce TNF- as response to exogenous
and endogenous AT-II. Endogenously-produced AT-II induces
ROS-production in T cells most likely via activation of NADPH-
oxidases, and ROS themselves stimulate T cells to produce TNF-
[148]. Treatment with etanercept, the soluble TNF- receptor anti-
body, during AT-II-infusion led to a reduction of AT-II-mediated
vascular superoxide production and prevented hypertension [148,
149]. The latter may provide a link between thrombosis and hyper-
tension and thereby the basis for atherothrombotic events in CAD
patients.
The role of T cells in the development of pulmonary arterial
hypertension remains ambiguous: RAG-1-/- mice did not develop
pulmonary artery hypertension induced by monocrotaline in con-
trast to wild type mice where monocrotaline leads to pulmonary
vascular endothelial cell damage followed by a perivascular in-
flammatory response, also with infiltration of CD4 T cells. Transfer
of CD4+ T cells into RAG-1-/- mice via adoptive T cell transfer
restored the effect of monocrotaline causing vascular damage in the
pulmonary artery [150]. However, it has been reported that athymic
nudes rats which lack T cells more easily develop severe pulmonary
hypertension when treated with the vascular endothelial growth
factor receptor blocker than euthymic rats [151]. In this case, injec-
tions of splenic immune cells provided protection [151]. So we are
here probably faced with a double-edged problem.
Regulatory T cells (Tregs) are CD4+ T cells that express con-
stantly the IL-2 receptor CD25 and the transcription factor X-linked
forkhead/winged helix (Foxp3). They are generally described to
suppress innate as well as adaptive immune responses [152, 153]

Vascular Dysfunction, eNOS Uncoupling and Inflammation Current Pharmaceutical Design, 2014, Vol. 20, No. 22 3589
and they also seem to confer improvement in hypertension: The
AT-II-induced hypertension was significantly normalized in mice
with adoptive transfer of CD4+ CD25+ T cells (and not by the adop-
tive transfer of CD4+ CD25- T cells) [154]. Also the AT-II-evoked
oxidative stress of the aortic vessel was reduced by adoptive Treg
transfers shown by DHE staining and normalization of the NADPH
oxidase activity [154]. This fits to the discovery that IL-10 secreted
by Tregs attenuates NADPH oxidase activity [155]. The number of
Tregs detected in the aorta was very low in control mice and not
altered by AT-II-treatment nor by adoptive transfer of Tregs or
effector T cells. But there were Foxp3+ cells detectable in the renal
cortex of control mice shown by immunofluorescence – and the
number was decreased under AT-II treatment and significantly
elevated in AT-II-treated mice with adoptive Treg transfer [154]. In
line with this the adoptive transfer of Treg s also prevented the al-
dosterone-induced vascular injury [156].
DENDRITIC CELLS (DCS) IN HYPERTENSION
As the T cell response is controlled by antigen-presenting cells
(APCs) like dendritic cells (DCs), it may be assumed that DCs are
also involved in the development of endothelial dysfunction. DCs
are responsible to prime naïve T cells [157, 158] and to initiate the
adaptive immune response after being activated themselves by in-
nate stimuli. Among various mechanisms, naïve T cells are acti-
vated by the binding of the T cell CD28 to the B7 ligands CD80
and CD86 on DCs [159]. It was shown that the percentage of
CD11c+ cells expressing the activation marker CD86 was upregu-
lated in the spleen and lymphnodes of mice treated with AT-II
which could point to a possible maturation of DCs under AT-II
[160]. It had been shown previously that DCs have an own RAAS
and that AT-II can modulate functionality and activation of DCs
[161]. Blockade of CD28 interaction with the B7 ligands by cyto-
toxic T lymphocyte antigen 4 immunoglobulin (CTLA4-Ig) led to a
significant reduction of the AT-II-induced hypertension [160]: Be-
sides, there was no increased superoxide production in aortas from
mice treated with CTLA4-Ig under AT-II whereas in aortas from
AT-II-treated mice that had not been treated with CTLA4-Ig the
superoxide production was 3-fold increased. Also the aortic infiltra-
tion with T cells under AT-II was reduced by CTLA4-Ig treatment
showing the importance of the interaction between T cells and DCs
in the development of AT-II-induced endothelial dysfunction [160].
In parallel it was shown that aldosterone increased the capacity of
DCs to activate CD8+ T cells and aldosterone-treated DCs pro-
moted a polarization of CD4+ T cells to Th17 cells [162] which are
important in the development of hypertension as previously de-
scribed. It is being discussed that hypertensive stimuli could lead to
the formation of neoantigens like in atherosclerosis [163] which
APCs like DCs present to T cells for induction of T cell activation
[160]. Activated T cells then are involved in the development of
hypertension as already described above.
B CELLS IN HYPERTENSION
Up to now, only very little is known on the involvement of B
cells in the development of arterial hypertension and endothelial
dysfunction. Aside from the classical arterial hypertension, B cells
have been described to contribute to the development of hyperten-
sion in response to placental ischemia during pregnancy [164] and
that peripheral activated B cells are involved the pathogenesis of
idiopathic pulmonary arterial hypertension [165].
As only the adoptive cell transfer of T-cells but not of B cells
could restore the hypertensive response and superoxide production
to AT-II in mice RAG-1-/- mice which lack per definition B and T
cells [11], B cells do not seem to be as important in the develop-
ment of hypertension as T cells. But it has nevertheless been shown
that B cell deficiency reduced and even abolished the cold-induced
blood pressure elevation [166] and that there is an up-regulated
immunoglobulin formation and secretion in B cells from hyperten-
sive patients [167]. Therefore, it could be interesting to further elu-
cidate the role of B cells in the development of hypertension.
CHRONIC AUTOIMMUNE DISEASES ASSOCIATED WITH
CARDIOVASCULAR DISEASE
Several chronic autoimmune diseases have been reported to be
linked with an increased risk for cardiovascular ev ents: Rheumatoid
arthritis, systemic lupus erythematodes and also severe psoriasis
have been described to be connected to cardiovascular disease [168-
171]. Psoriasis has even been described to be a new risk factor in-
dependent of the classical cardiovascular risk factors [172]. Con-
cerning the elevated cardiovascular risk of patients with arthritis the
European League against Rheumatism has published recommenda-
tions for cardiovascular risk management in inflammatory arthritis
(including rheumatoid arthritis, psoriatic arthritis and ankylosing
arthritis) [173].
Preceding the cardiovascular ev ents, a reduced vascular func-
tion was associated with these diseases: So, the carotid intima-
media thickness in patients with early rheumatoid arthritis did not
differ from the control patients at the beginning of the disease – but
18 months after the first evaluation there was already a significant
increase in the intima-media thickness compared to control group
[174]. It was also shown that psoriasis patients have an impaired
endothelial function measured by flow-mediated dilatation and an
increased intima-media thickness of the carotid artery compared to
healthy controls [175].
The existing connection between the autoimmune diseases
mentioned above and cardiovascular disease is certainly depending
on many different factors (also differing in the existing primary
diseases. All these diseases present the situation of a chronic in-
flammation with at least locally elevated cytokine levels and acti-
vated inflammatory cells: In the pathogenesis of psoriasis, the IL-
17/IL-23 axis plays a pivotal role [176-178]. Also in patients with
systemic lupus erythematodes, IL-17A production is, besides other
cytokines, increased [179]. In rheumatoid arthritis IL-6, TNF- and
also IL-17A are of big importance [180, 181]. All these cytokines
are, as mentioned above, of relevance in the development of endo-
thelial dysfunction. Thus, this could be the link between the devel-
opment of cardiovascular disease and the described chronic auto-
immune diseases [168, 170]. However, further clinical studies and
mechanistic analyses are required.
CONCLUSION AND CLINICAL IMPLICATIONS
Cardiovascular disease, the most important reason for death in
industrial nations, is a multifactorial complication involving a lot of
different mechanisms as uncoupling of eNOS, reactive oxygen
species formation, adverse calcium homeostasis and signaling asso-
ciated with a deleterious phosphorylation pattern as well as futile
counter-regulatory mechanisms at the humoral and cellular/tissue
structure level (to mention some of the important contributors).
According to more recent data, also immune cells and inflammatory
mechanisms play an essential role in cardiovascular disease. The
present review wants to highlight the link between the immune
system and cardiovascular disease which may translate to more
integrative therapies using multidisciplinary expertise and simulta-
neously curing the underlying multifactorial complications.
CONFLICT OF INTEREST
The authors confirm th at this article content has no conflicts of
interest.
ACKNOWLEDGEMENTS
We thank Margot Neuser and Thilo Weckmüller for graphical
assistance. The present work was supported by generous financial
support by the Federal Ministry of Education and Research (BMBF
01EO1003 to P.W., T.M. and A.D.). Susanne Karbach holds a grant

3590 Current Pharmaceutical Design, 2014, Vol. 20, No. 22 Karbach et al.
of the “Margarethe-Waitz-Stiftung” and a MAIFOR-grant. All
authors are supported by the “Stiftung Mainzer Herz”.
ABBREVIATIONS
ADMA = Asymmetric dimethylarginine
ACE = Angiotensin-converting-enzyme
Agtr1 = Angiotensin receptor type 1
AT-II = Angiotensin-II
BH4 = Tetrahydrobiopterin
CAD = Coronary artery disease
CTLA4-Ig = Cytotoxic T lymphocyte antigen 4 immunoglo-
bulin
DDAH = Dimethylarginine dimethylamino hydrolase
DC = Dendritic cell
DHE = Dihydroethidine
DOCA = Deoxycorticosterone acetate
ECL = Enhanced chemiluminescence
EDRF = Endothelium-derived relaxing factor
EPR = Electron spin resonance
eNOS = Endothelial nitric oxide synthase
FAD = Flavin adenine dinucleotide
FMN = Flavin mononucleotide
Foxp3 = Transcription factor X-linked forkhead/winged
helix
 cells = Gamma delta T cells
G-CSF = Granulocyte colony-stimulating factor
GM-CSF = Granulocyte-macrophage colony-stimulating
factor
HO = Heme oxygenase (HO-1, inducible isoform;
HO-2, constitutive isoform)
IL-6 = Interleukin-6
IL-17A = Interleukin-17A
iNOS = Inducible nitric oxide synthase
L-NAME = NG-nitro-L-arginine methyl ester
MI = Myocardial infarction
mPTP = Mitochondrial permeability transition pore
nNOS = Neuronal nitric oxide synthase
Nox = NADPH oxidase
PETN = Pentaerithrityl tetranitrate
PKC = Protein kinase C
PYK = Protein tyrosine kinase
RAG-1-/- mice = Recombination activating gene-1 knockout
mice
RONS = Reactive oxygen and nitrogen species
RNS = Reactive nitrogen species
ROS = Reactive oxygen species
SOD = Superoxide dismutase
SHR = Spontaneously hypertensive rats
Th17 = T helper 17 subset of CD4+ T cells
TNF- = Tumor necrosis factor 
VSMC = Vascular smooth muscle cells
XDH = Xanthine dehydrogenase
XO = Xanthine oxidase
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Received: July 19, 2013 Accepted: October 21, 2013