ANTIOXIDANTS & REDOX SIGNALING
Volume 10, Number 9, 2008
© Mary Ann Liebert, Inc.
Comprehensive Invited Review
Endothelial Dysfunction: From Molecular Mechanisms
to Measurement, Clinical Implications, and
Michelle Le Brocq,1Stephen J. Leslie,1Philip Milliken,2and Ian L. Megson1
Reviewing Editors: Frank Faraci, Pin-Lan Li, Subramaniam Pennathur, Chandan K. Sen, and Eric Thorin
The Endothelium-Stimulation Processes
B. Neurohormonal stimuli
Endothelium-Derived Relaxing Factors
A.Nitric oxide (NO)
B. Prostanoid vasodilators
C.Endothelium-derived hyperpolarizing factor (EDHF)
D.Carbon monoxide and hydrogen sulfide
Endothelium-Derived Contracting Factors
D.Angiotensin II (Ang II)
Tissue Plasminogen Activator (t-PA)
Endothelial Activation by Inflammatory Stimuli
B. A pivotal role for NF-?B?
C.Hyperlipidemia, oxidative stress, and inflammation: a noxious triad
D.Environmental triggers of dysfunction: do infection and pollution play a part?
E. Homocysteine and endothelial dysfunction
VII. Experimental Measures of Endothelial Function
A. In vitro
1. Cell culture
2. Functional assays
B. In vivo (clinical studies)
1. In vivo study techniques
a. Measuring “endothelial dysfunction” in coronary arteries
b. Measuring “endothelial dysfunction” in peripheral arteries
c. Venous occlusion plethysmography
d. Flow-mediated dilatation with brachial artery imaging
e. Pulse-wave analysis and velocity
f. Doppler skin flowmetry
g. Serum biomarkers for endothelial dysfunction
1Health Faculty, UHI Millennium Institute, Inverness; and 2Centre for Cardiovascular Science, Queen’s Medical Research Institute, Uni-
versity of Edinburgh, Edinburgh, Scotland.
the intimal surface of the entire cardiovascular system, in-
cluding the arteries, veins, and chambers of the heart (en-
docardium); the capillary walls consist solely of endothelial
cells. The endothelium was originally considered to be sim-
ply a passive interface between the blood and tissues, but it
transpired that it performs a wide range of complex and
wide-ranging tasks. At the microvascular level, the endo-
thelium is central to control of vascular permeability, exert-
ing regulatory control over transcellular, intercellular, and
paracellular diffusion in response to environmental and mo-
lecular signals [reviewed in (438)]. Furthermore, the endo-
thelium emerged to be not simply a nonadhesive barrier be-
tween the blood and prothrombotic collagen in the
underlying basement membrane, but rather a cell layer that
actively prevents thrombosis through expression of antico-
agulants such as heparan sulfate (with properties similar to
HE ENDOTHELIUM is a monolayer of cells derived from the
embryonic mesoderm that form a continuous layer on
those of heparin) on its surface (90), together with enzymes
that destroy, for example, circulating ADP (269). Given these
properties, it is important for the endothelium to undergo
rapid repair when damaged and for apoptotic cells to be
quickly replaced by circulating endothelial progenitor cells
(EPCs), which are also central to angiogenesis throughout
our lifespan [reviewed in (434)].
However, it was a series of discoveries from the mid-1970s
onward that radically changed our perception of endothelial
cell function. Now, the endothelium is recognized to be a
highly complex “organ” that responds to physical and chem-
ical stimuli to generate a wide range of organic and inor-
ganic messenger molecules that are capable of influencing
the physiology of the surrounding tissue, particularly with
respect to blood flow. Furthermore, the endothelium is also
responsive to inflammatory activation, triggering expression
of receptors and adhesion molecules, which have a quite dif-
ferent impact on the pathophysiology of affected tissue.
Endothelial dysfunction is a widely used term to describe
any form of abnormal activity of the endothelium, encom-
LE BROCQ ET AL.1632
VIII. Endothelial Dysfunction and Aging
IX. Endothelial Dysfunction in Cardiovascular Disease
A.Atherosclerosis, coronary artery disease, and stroke
E. Heart failure
F. Ischemia–reperfusion injury
X.The Future of Endothelial Function Measurement
A.Endothelial function in predisease
B. Assessing impact of therapies in individuals by using endothelial function
C.Prognostic value of endothelial function measurement
XI.Prevention of Endothelial Dysfunction
XII. Therapies for Endothelial Dysfunction
A.Current: statins (pleiotropic effects)
B. Possible future treatments for eNOS dysfunction
C.Nitric oxide and carbon monoxide donor drugs
D.Phosphodiesterase inhibitors and activators of guanylate cyclase
F. Antioxidant therapies
I. Endothelial cell–based therapies
Endothelial dysfunction has been implicated as a key factor in the development of a wide range of cardiovas-
cular diseases, but its definition and mechanisms vary greatly between different disease processes. This review
combines evidence from cell-culture experiments, in vitro and in vivo animal models, and clinical studies to
identify the variety of mechanisms involved in endothelial dysfunction in its broadest sense. Several promi-
nent disease states, including hypertension, heart failure, and atherosclerosis, are used to illustrate the differ-
ent manifestations of endothelial dysfunction and to establish its clinical implications in the context of the range
of mechanisms involved in its development. The size of the literature relating to this subject precludes a com-
prehensive survey; this review aims to cover the key elements of endothelial dysfunction in cardiovascular dis-
ease and to highlight the importance of the process across many different conditions. Antioxid. Redox Signal. 10,
passing both dysfunctional production of messenger mole-
cules and expression of proinflammatory adhesion mole-
cules. Dysfunction is deleterious and is implicated as a key
factor in the initiation and progression of the atherogenic
process that underlies coronary artery disease, peripheral
ischemia, and some forms of stroke, although its role in
plaque rupture is less clear. Dysfunction has also been im-
plicated in the progression of other cardiovascular condi-
tions, including hypertension (337) and heart failure
(235,262). The literature on the topic is enormous, and this
review, although lengthy, provides only a glimpse of a
highly complex field.
On account of the broad range of specific definitions of
endothelial dysfunction, it is essential first to categorize the
different mechanisms and measures that are related to dys-
function, although it is important to note that many, if not
all, of the processes described separately here interact with
each other in vivo. In many cases, only one facet of endo-
thelial dysfunction is investigated in any particular study,
but it is more than likely that other forms also are present.
II. The Endothelium-Stimulation Processes
A wide range of processes and mediators are now known
to stimulate the endothelium to produce an array of factors
that mediate local vascular relaxation, contraction, platelet
function, and fibrinolysis. For the purposes of clarity, we
have distinguished endothelial stimulation (leading to in-
creased release of vasoactive agents) from activation (result-
ing in expression of adhesion molecules and progression to
a proinflammatory state). In the literature, however, these
two terms are used interchangeably.
A. Physicochemical stimuli
Shear stress–the lateral force exerted on the endothelial
cells by the passage of a semiviscous fluid over them–is a
particularly important physical stimulus for endothelium-
dependent vascular relaxation (339, 427), and it is now well
established that areas of the vasculature that experience un-
usual shear stress are particularly vulnerable to endothelial
dysfunction (402). This is particularly true in regions where
blood flow is disturbed (i.e., not laminar), including at bi-
furcations and branch points, in tortuous or curved vessels
(70), and in coronary arteries (388, 389), where heart move-
ments during the cardiac cycle contribute to unusual flow
patterns. Whereas much of the work surrounding flow cen-
ters on the stimulation of the endothelium to release relax-
ing factors, it is also worth noting that flow affects the anti-
oxidant systems of endothelial cells through induction of the
antioxidant response element (ARE) (69). Areas that experi-
ence disturbed or turbulent flow are subject to low shear
stress, leading to a failure in mechanoreceptor-mediated re-
lease of intracellular calcium, together with depression of a
range of phosphorylation pathways, including AKt and pro-
tein kinases (PK) C and G, that result in reduced generation
of endothelium-derived relaxing factors (31) (Fig. 1). Long-
term deprivation of endothelial cells of shear stress exacer-
bates the issue through downregulation of critical enzymes
involved in generating relaxing factors (e.g., nitric oxide; NO)
that are deemed to be protective, while simultaneously up-
regulating endogenous constrictors and proatherogenic
agents (e.g., endothelin-1), which promote vascular disease.
However, it is also likely that areas that experience very high
shear forces are subject to an erosive effect on the endothe-
lial cells, whereby dysfunction is precipitated through phys-
ical loss of cells (11, 116); prior apoptosis of the endothelial
cells might be a prerequisite for erosion (101).
It is important to recognize that other physical stimulators
of the endothelium exist besides shear stress. Wall stretch
also contributes to basal endothelial activity (55), whereas
some studies suggest that the level of oxygenation of the sur-
rounding tissue is important in determining the level of lo-
cal endothelium-mediated vasodilatation (211). Such a mech-
anism of oxygen sensing and local response makes
physiologic sense, but a great deal of conflicting evidence ex-
ists as to the importance of the endothelium in mediating
hypoxia-induced vasodilatation (135, 336, 472) versus, for ex-
ample, that mediated by adenosine (24, 307). If hypoxia is
involved, the consensus appears to favor prostaglandins as
the likely mediators (56, 287), rather than NO (135) or en-
dothelium-derived hyperpolarizing factor (EDHF) (136) (see
later). Some evidence, however, suggests that the endothe-
lium actually counteracts dilatation through release of vaso-
constrictors in response to hypoxia (150, 418, 473, 477). Al-
though these conflicting reports can partly be explained by
differences in experimental protocol (e.g., duration of hy-
poxic episode), this remains a controversial area.
B. Neurohormonal stimuli
A number of blood-borne messengers are potent stimula-
tors of the endothelium, primarily via G protein–coupled re-
ENDOTHELIAL DYSFUNCTION 1633
pact of insulin resistance.
Kinases and the Akt pathway: stimulators and im-
ceptors that ultimately evoke an increase in intracellular
Ca2?. Bradykinin is an important endogenous activator of
endothelial cells (181), whereas catecholamines associated
with sympathetic nerve activity also stimulate endothelial
cells, primarily via ?2and ?2adrenoceptors (151, 240, 241).
Acetylcholine (ACh) is the gold-standard stimulator of en-
dothelial cells, particularly in vitro, where it is used exten-
sively to study endothelium-dependent relaxation (133).
However, although ACh is also used regularly in clinical
studies, it is unclear to what extent ACh mediates endo-
thelium-dependent vasodilatation in vivo, given that cir-
culating anticholinesterases rapidly destroy any blood-
borne ACh, and most blood vessels, with the exception of
coronary (44) and cerebral arteries, lack parasympathetic
stimulation. Nevertheless, muscarinic [mainly M3(33), but
also other muscarinic subtypes (453,452)] ACh receptors
are expressed on endothelial cells of at least some blood
vessels, and their activation results in elevated endothelial
intracellular calcium and activation of endothelium-de-
rived relaxing factors. Other agents, such as serotonin (5-
HT), histamine, and substance P, also act through their re-
spective receptors to stimulate endothelial cells to release
relaxing factors via calcium mobilization, but insulin, lep-
tin, adiponectin, estrogen, and glucocorticoids, among oth-
ers, act via their respective receptors to stimulate NO syn-
thesis via a phosphorylation cascade, ultimately resulting
in AKt-mediated phosphorylation of endothelial NO syn-
thase (eNOS) (214) (Fig. 1).
III. Endothelium-Derived Relaxing Factors
Since the 1970s, the number of endothelium-derived re-
laxing factors (EDRFs) has proliferated, and it is now recog-
nized that an increase in endothelial Ca2?effects the release
of several relaxing factors, the relative proportion of which
varies enormously across the vascular tree. Principal among
the known factors to date are NO, prostacyclin (PGI2), and
EDHF. NO is known to predominate in large conduit ves-
sels like the aorta, whereas EDHF is dominant in resistance
arteries (377); PGI2contributes less than the other two fac-
tors, and its impact is more consistent throughout the vas-
culature. The exception to this rule, however, is the coronary
circulation, where EDHF appears to have a greater influence
in the large coronary arteries than might be predicted, par-
ticularly in porcine models (54, 57, 134, 169, 338).
A. Nitric oxide (NO)
NO is a free radical species with powerful vasodilator
properties as well as a number of other protective effects
(295). It is synthesized from the amino acid, L-arginine, by
NO synthases (NOSs) (225, 295). The endothelial isoform,
eNOS (or NOS III), is constitutive and is predominantly, al-
though not exclusively, found in endothelial cells. The en-
zyme is a homodimer, with each monomer containing a re-
ductase and a heme oxygenase domain (134, 403) (Fig. 2).
eNOS is a highly regulated protein at both transcription and
functional levels. Full function of the enzyme is dependent
on its existence as a dimer, disassociation with the membrane
protein, caveolin (129) (Fig. 3), activation through calcium-
calmodulin, and sufficient supply of substrate (L-arginine)
and cofactors, most notably, tetrahydrobiopterin (BH4) (4,
202). Activation of the enzyme results in the oxidation of
L-arginine by molecular oxygen at the heme oxygenase, to
generate NO and L-citrulline via the intermediate, N?-hy-
droxy-L-arginine (Fig. 4). NO is a small molecule that is sol-
uble in both aqueous and lipid phases, allowing it to diffuse
rapidly from its source and to cross membranes unimpeded.
The primary target of endothelium-derived NO is the en-
zyme, soluble guanylate cyclase (sGC) (300), in cells within
close proximity of the source, most notably, smooth muscle
cells, platelets, and inflammatory cells. Activation of sGC by
LE BROCQ ET AL.1634
for enzyme activity.
Disassociation of NOS from caveolin is essential
zyme, with heme oxygenase and reductase domains in both
monomers. Catalysis of L-arginine to NO occurs in the oxy-
genase domain on binding of Ca2?-calmodulin, which is
thought to provide an electron bridge for transfer of elec-
trons from NADPH via flavone nucleotides in the reductase
domain; in iNOS, Ca2?/CAM is permanently bound, hence
the very high levels of activity. BH4is an essential cofactor
for enzyme activity, probably helping to maintain the link
between the two monomers, but also perhaps maintaining
the heme iron in the high spin state necessary for activity
and also offering some antioxidant protection (229).
Nitric oxide synthase (NOS) is a homodimeric en-
NO results in catalytic conversion of GTP to cGMP, which
in turn mediates cell-specific effects via relevant cGMP-de-
pendent protein kinases (PKs). In smooth muscle cells, PKG
causes phosphorylation of myosin light-chain kinase, in-
hibits the inositol triphosphate (IP3) pathway, and activates
Ca?extrusion pumps, resulting in relaxation (300) (Fig. 5).
Furthermore, in the chronic phase, cGMP inhibits smooth
muscle mitogenesis via inhibition of the MAP kinase path-
way by preventing Ras-dependent activation of Raf-1 (481).
In platelets, the effect is to inhibit the activation processes
involved in aggregation, primarily through impedance of
Ca2?mobilization and entry. Platelets themselves have con-
stitutive eNOS (341), which is likely only to be stimulated
upon platelet activation on account of the dependence of the
enzyme on Ca2?for activity; by definition, elevation of in-
traplatelet Ca2?occurs only when the platelets are activated.
The autocrine nature of platelet-derived NO, together with
the fact that Ca2?influx is both the stimulant for NO release
and the target for its actions in this setting, can point only to
NO as a regulatory brake on the activation process that
serves to reduce the chance of inappropriate activation of
platelets by low-level stimulants.
Two other isoforms of NOS are known: a constitutive neu-
ronal isoform (nNOS, NOS I), which is a neurotransmitter in
nonadrenergic, noncholinergic nerves in the peripheral ner-
vous system and an inducible isoform (iNOS), which is usu-
ally expressed only in response to invading pathogens and
inflammatory stimuli (225, 295). Regulation of iNOS is quite
different from the constitutive isoforms; whereas eNOS and
nNOS typically generate very low concentrations of NO (in
the pico/nanomolar range) and are highly regulated by in-
tracellular calcium, iNOS generates comparatively very high
levels (?M range) of NO. The reason for the difference is that
iNOS is typically expressed in inflammatory cells, and the
NO generated is used in a noxious mix of chemicals designed
to kill invading pathogens. Such high concentrations of NO,
particularly when mixed with NAD(P)H oxidase-derived su-
peroxide to generate highly toxic peroxynitrite (ONOO?), hit
many targets besides sGC in nearby cells, including respira-
tory chain enzymes (261, 353) and DNA (53, 409), with lethal
effect. However, it is well recognized that iNOS induction is
constrained neither to the infectious state nor to inflamma-
tory cells, and its expression has been identified in a wide
range of cell types in association with inflammatory condi-
tions (361). It is also clear that iNOS does not necessarily pro-
duce “pure” NO; dysfunctional iNOS can generate ONOO?
or superoxide or both and can therefore contribute to re-
duced endothelial cell viability, increased inflammation and
peroxidation, and reduced endothelium-derived NO
The free radical nature of NO is critical to understand-
ing its physiologic impact. Radicals are naturally regarded
to be reactive species, and, although the high affinity of NO
for the Fe2?in the heme of sGC ensures that a proportion
of NO is likely to find its target, it will inevitably react with
other off-target molecules in cells. Principal among other
reactants for NO are other heme groups (e.g., in hemoglo-
bin) (148), molecular oxygen (247), and oxygen-centred free
radicals (82). The natural quenching of NO through inacti-
vating reactions is an essential component in ensuring that
its actions are localized to the vicinity of its production, but
it is becoming evident that the ultimate fate of NO is also
crucial in determining downstream activity of the moiety.
For example, the simple oxidation of NO by oxygen to
higher oxides of nitrogen generates powerful nitrosating
agents in N2O3and N2O4that can go on to nitrosate cys-
teine residues in a wide range of proteins, altering their
function (198); the reversibility of the nitrosation process
means that S-nitrosothiols constitute a dynamic NO store,
which can itself reflect endothelial function (282) and can
be altered in, for example, hypertension (125). Equally, ni-
trite, once considered to be the inert product of NO oxida-
nine to citrulline via N? ?-hydroxy- L-arginine,
resulting in NO generation.
NOS-mediated conversion of L-argi-
tion, is now regarded as being a vasoactive substance in its
own right (47, 141, 142).
Whereas the reaction of NO with oxygen at concentrations
found in the vicinity of the endothelium is likely to be rela-
tively slow, that with other radical species, and superoxide
in particular, is considerably faster, irrespective of the gen-
erally low concentrations of the reactants. Generation of re-
active oxygen species (ROS) is an inevitable consequence of
several cellular processes, not least of which is respiration
(321) (Fig. 6). In health, a battery of antioxidant defenses rep-
resents a formidable impediment to the prolonged existence
of these potentially deleterious agents. Besides protecting
cellular components from harmful peroxidation, rapid re-
moval of ROS also protects NO from inactivation in both en-
dothelial and target cells. However, in a wide range of dis-
ease states, oxidative stress can develop, whereby the
existence of ROS is prolonged either because ROS genera-
tion has increased to levels that have swamped the antioxi-
dant defenses, or because the defenses themselves have been
downregulated, are dysfunctional or depleted. Clearly, ox-
idative stress is detrimental on a number of levels, but an
immediate effect with respect to endothelial function is the
inactivation of NO, most notably by superoxide (158). The
interaction between these two radical species has led to NO
being referred to as an “antioxidant” in some quarters on ac-
count of its ability to quench superoxide. However, in our
opinion, this description is misleading with respect to NO,
at least in the sense of direct inactivation of superoxide, be-
cause the product of the reaction, peroxynitrite (ONOO?), is
itself a powerful oxidant. Although not a radical, ONOO?is
highly reactive, mediating lipid peroxidation (32) and nitra-
tion of tyrosine residues that can alter protein function (154).
Far from being protective, as the term antioxidant implies,
the reaction of NO with superoxide exacerbates endothelial
dysfunction and contributes to the atherogenic process
through the actions outlined earlier, together with cytotoxic
activity in endothelial cells. Furthermore, ONOO?inhibits
Ca2?-activated K?channels in the smooth muscle of human
coronary arterioles and contributing to impairment of
EDHF-mediated relaxation of these vessels (260) as well as
inhibiting PGI2synthase (497).
The combined effect of oxidative stress on endothelial
function and atherogenesis is compelling; not only does it
eliminate protective NO, but it also contributes to inhibition
of EDHF-mediated vasodilatation, endothelial cell death,
lipid peroxidation, as well some as yet relatively poorly eval-
uated effects on protein function through tyrosine nitration.
Although NO is commonly described as a “relaxing fac-
tor” for historic reasons, its greater importance in conduit
rather than resistance arteries might suggest that its primary
role lies in properties other than its powerful dilatory effects.
Indeed, the platelet and inflammatory cell-directed effects
LE BROCQ ET AL.1636
of vascular smooth muscle
function: an integrated sys-
tem. Ach, acetylcholine; ANG
II, angiotensin II; AC, adeny-
late cyclase; AT1, angiotensin
receptor; BK, bradykinin;
cAMP, cyclic adenosine mono-
CO, carbon monoxide; ET-1,
endothelin 1; ETNO, endothre-
lin A & B receptors; EDHF,
lariing factor; GPCR, protein
coupled receptor; HO, heme
oxygenase; NO, nitric oxide;
PCI2, prostacyclin; PGIS, pro-
staglindin 12 synthase; PGR,
prastaglandin receptor; PKs,
protein kinases; sGC, soluble
guanylate cyclase; tPA, tissue
plasminogen activator; TXA2,
thromboxane A2 VGCC, volt-
age-gated Ca2 channel.
noted earlier ensure that NO is a powerful antithrombotic
and antiinflammatory agent, whereas its ability to also in-
hibit smooth muscle cell proliferation suggests a role in de-
termining the structural composition of the vascular wall.
Taken together, these properties provide NO with a unique
antiatherogenic profile, which matches with its predomi-
nance in large conduits that are susceptible to atherosclero-
sis. The importance of the endothelium, and NO in particu-
lar, in protecting against atherosclerosis is further
emphasized by the pivotal role played by denudation or dys-
function of the endothelium, or more specifically, dysfunc-
tion in the L-arginine/NO/sGC pathway, in the initiation
and development of atherosclerotic plaques.
Dysfunction with respect to the NO/sGC pathway can oc-
cur at a number of levels (165). First, the endothelium might
be absent (e.g., after angioplasty) or the cells dead or dying in
the face of toxic stimuli, including ROS. Second, enzyme ex-
pression might be altered via transcription factors or changes
in stability of mRNA, mediated by rho-GTPases (351) (Fig. 6).
Third, enzyme activity might be depressed on account of
changes in the association with caveolin (114) (Fig. 3), reduced
availability of substrate (L-arginine) (77, 81, 356) or cofactor
(BH4) (67), all of which are dependent on complex processes
to determine intracellular levels (Fig. 7). Finally, the NO gen-
erated might be intercepted by, for example, superoxide un-
der conditions of oxidative stress, compromising its biologic
activity, while at the same time generating a prooxidant and
highly cytotoxic by-product (ONOO?) (Fig. 6). Therefore, even
if endothelial dysfunction is traced specifically to this pathway,
considerably more data are required to establish what is re-
sponsible for the dysfunction, before identifying a therapeutic
approach. It is worth noting, however, that oxidative stress has
multiple effects at different levels of this pathway, perhaps
highlighting oxidative stress as a prime target with respect to
therapeutic intervention in endothelial dysfunction.
B. Prostanoid vasodilators
The discovery of endothelium-derived prostaglandins
pre-dated that of NO by several years and provided the first
are under tight control through transcription factor–mediated alterations in protein expression. COX, cyclooxygenase; GCL,
glutamate-cysteine ligase; GPx, glutathione peroxidise; GR, glutathione reductase; GS-glutathione synthase; GSH , glu-
tathione; GSSG, glutathione (oxidised form); MPO, myeloperoxidase; NOS, nitric oxide synthase; NOX, NAD(P)H oxi-
dase,;sGC, soluble guanylate cyclise; SOD, superoxide dismutase; XO, xanthine oxidase.
Interplay between NO and reactive oxygen species (ROS). Levels of NOS and enzymes that control ROS levels
indication of the endocrine role of the endothelium (441).
Prostacyclin (PGI2) is the primary endothelium-derived
prostaglandin, although PGE2also can be generated. Like
NO, the prostaglandins are synthesised on demand in re-
sponse to an increase in intracellular Ca2?, which activates
phospholipase A2to generate arachidonic acid from phos-
pholipids (Fig. 8). Endothelial cyclooxygenase(s) (COX-1 and
possibly COX-2), together with endoperoxidases, convert
arachidonic acid to prostaglandin H2, which is finally acted
upon by the relevant synthase to generate PGI2or PGE2. The
key role for Ca2?in the process ensures that the stimuli for
generation of these prostaglandins mirror those for NO, re-
sulting in co-release of these agents. Although PGI2and NO
also share the same vascular effects in terms of vasodilata-
tion and inhibition of platelet and leukocyte function, the
mechanism of action is quite different, with PGI2acting on
cell-surface G protein–coupled receptors that activate ade-
nylate cyclase, resulting in generation of cAMP (228). Dys-
function of the system can occur through endothelial de-
nudation and alterations in expression or function of the
synthetic enzymes involved. The relative importance of PGI2
in endothelial function is eclipsed by the far greater litera-
ture relating to NO, but mounting evidence suggests that
loss of the protective effects of PGI2through endothelial dys-
function also plays a critical role in vascular disease devel-
opment. In particular, recent work relating to atherosclero-
sis suggests that PGI2is a powerful antiatherogenic agent
(226), primarily through inhibition of leukocyte and platelet
activation, whereas evidence from a knockout mouse model
suggests that PGE2is proatherogenic, or at least that dele-
tion of the gene coding for the synthase responsible for its
synthesis leads to redirection of PGH2to protective PGI2
C. Endothelium-derived hyperpolarizing factor (EDHF)
EDHF is a recently discovered endothelium-derived re-
laxing factor, which still has yet to be fully characterized [for
review, see (73, 279)]. Many candidates have been proposed
for EDHF, including epoxyeicosatrienoic acids (124), en-
dogenous cannabinoids (317), C-type natriuretic peptide
(362), or even hydrogen peroxide (230, 274, 275, 401, 480),
among others, but the consensus is beginning to settle
around a K?-mediated event that could involve gap junc-
tions (118, 123, 363), activation of which might involve any
or all of the previously mentioned stimuli (Fig. 9). In our
opinion, EDHF should not be considered to be a single “fac-
tor,” but rather a combination of mediators and processes
that are triggered on stimulation of endothelial cells, all ca-
pable of depressing intracellular K?in vascular smooth mus-
cle. The balance of the mediators and processes involved is
apparently very different, according to the specific blood
vessel and species, which accounts for the conflicting evi-
dence in the literature. Even this, however, is likely to be an
oversimplification of the phenomenon, with evidence from
human microvessels indicating that K?might not be in-
The stimuli for EDHF-mediated vasodilatation are shared
with those for NO and PGI2, as is the absolute dependence
on a healthy, functional endothelium. Given the relative
propensity for EDHF in resistance, coronary arteries (39, 121,
178) and renal afferent arterioles (454), among others, its pro-
file is increasing with respect to hypertension and coronary
artery disease. Importantly, evidence suggests that EDHF
may serve as a counterregulatory system that is upregulated
in hypertension to compensate for reduced bioavailability of
NO (386). This finding provides an interesting new insight
into the interaction between the different EDRFs, giving the
impression that their regulation is sufficiently sophisticated
to allow them to substitute for each other in diseased states.
D. Carbon monoxide and hydrogen sulfide
Recently, another somewhat surprising candidate has
emerged as a novel endothelium-derived relaxing factor.
Carbon monoxide (CO) is a highly reactive molecule
LE BROCQ ET AL. 1638
activity: impact of homocysteine. ADMA, asym-
metric dimethyl-L- arginine; BH4, tetrahydro-
biopterin; DHPR, dihydrofolate reductase; eNOS,
endothelial nitric oxide synthase; GTP, guanosine
triphosphate; GTPCH, GTP cyclohydrolase; HCys,
homocyst(e)ine; NO, nitric oxide; PTPS, 6-pyru-
voyltetrahydrobiopterin synthase; SR, sepiapterin
Substrate and cofactor control of NOS
renowned for its poisonous properties on account of irre-
versible blockade of the heme groups in hemoglobin. It is
now known that CO is generated endogenously during the
conversion of free heme to biliverdin, by the action of a fam-
ily of enzymes known as the heme oxygenases (HOs) (267).
Both the constitutive (HO-1) and inducible (HO-2) isoforms
have been found in the endothelium (305); HO-1 is upregu-
lated in response to laminar flow via an ARE-mediated pro-
cess (69). However, considerable evidence suggests that both
isoforms can also be expressed in vascular smooth muscle
cells. The roles of CO are largely analogous to those of NO,
in that it is a relaxing factor (457) with antiproliferative ef-
fects on smooth muscle (188, 248), while also inhibiting ad-
hesion of platelets (429) and inflammatory cells via activa-
tion of soluble guanylate cyclase (188) or a direct effect on
KCachannels (457) (Fig. 5), or a combination of these. How-
ever, it is recognized that CO is a less-powerful stimulator
of sGC than NO (132) and that it has a complex relationship
with NO release and NOS expression (416). Nevertheless,
CO has been shown to protect endothelial cells against apop-
tosis (19), most likely via cooperation with NF-?B (45), an im-
portant asset in protection against endothelial dysfunction.
However, the precise impact of CO on endothelial function
is disputed, with a number of studies also claiming that HO-
derived CO is instrumental in promoting dysfunction in
prostaglandin; TXA2, thromboxane A2.
COX-mediated synthesis of vasoactive prostaglandins. PLA2, phospholipase A2; COX-1, cyclo-oxygenase-1; PG,
derived hyperpolarizing factor (EDHF). AA, arachidonic
acid; CB, cannabinoid receptor; CYP, cytochrome P450; EETs,
epoxyeicosatrienoic acids; PL1%, phospholipase A2; VGCC,
voltage gated calcium channel.
Possible mechanisms of action of endothelium-
animal models of several pathologic conditions, including
metabolic syndrome and salt-induced hypertension (19, 196,
The latest gaseous messenger to emerge in the cardiovas-
cular system (and elsewhere) is hydrogen sulfide (H2S). The
balance of evidence to date suggests that the source of H2S
in blood vessels is smooth muscle rather than the endothe-
lium (495), so it should not be regarded as an EDRF per se.
Nevertheless, some evidence indicates that H2S activates re-
lease of both NO and EDHF (183, 493), although an indica-
tion also exists that H2S can downregulate NOS in the longer
term (456). NO, in turn, appears to upregulate the enzyme
responsible for H2S synthesis in vascular smooth muscle,
cystathionine ?-lyase (CSE) (495).
H2S has been shown to induce vasorelaxation at physio-
logic concentrations (493), via activation of ATP-sensitive K?
(KATP) channels (495), the opening of which hyperpolarizes
cells and closes voltage-dependent calcium channels (238).
Intravenous injection of H2S decreases the mean arterial
blood pressure of anesthetized rats by decreasing vascular
resistance (495), and daily intraperitoneal injections of D-L-
propargylglycerine (PPG, a specific blocker of CSE), for 2–3
weeks elevates systolic blood pressure, which may be a re-
sult of decreased endogenous H2S production in vascular tis-
sues (494). So it has been hypothesized that, by relaxing vas-
cular smooth muscle cells, promoting apoptosis of smooth
muscle cells (238) and inhibiting proliferation-associated vas-
cular remodelling (238), H2S modulates both the function
and structure of the circulatory system (238).
The complexity of the involvement of H2S is set to increase,
given that its precursor, cysteine, is also central to synthesis
of the intracellular antioxidant, GSH, together with its pro-
posed interaction with homocysteine, a possible mediator for
some forms of dysfunction.
IV. Endothelium-Derived Contracting Factors
Not long after the phrase “endothelium-derived relaxing
factors” was coined, an equivalent term was established to
describe humoral contracting factors (EDCFs), the identity
of whch were not yet known. In time, as with EDRFs, a num-
ber of contracting factors have been identified, ranging from
superoxide anion, which causes contraction, or more accu-
rately, attenuates relaxation, by inactivating NO (see earlier),
through to prostanoids and peptides (265).
A. Prostanoid EDCFs
As well as the prostanoids mentioned earlier that have va-
sodilatory properties, some are vasoconstrictors. The most
prominent exponents of prostanoid-mediated vasoconstric-
tion are PGH2and thromboxane A2(TXA2; Fig. 8). The for-
mer is the primary product of COX activity and an inter-
mediate in the formation of both PGE2and PGI2, whereas
TXA2requires the specific activity of thromboxane synthase
enzyme. Both are found in endothelial cells, although it is
likely that platelets in particular might represent a more
prominent source. Both act on thromboxane receptors in
smooth muscle to evoke contraction (Fig. 5).
An interesting twist to the role of prostanoid production in
the endothelium-dependent modulation of vascular tone is the
finding that activity of the COX enzyme itself can generate su-
peroxide (76). The impact of superoxide generated in this fash-
ion is twofold: first, it increases oxidative stress in the endo-
thelial cell and thereby can inactivate NO; and second, it
inhibits PGI2synthesis, without affecting that of contractile
prostanoids. Of course, COX is not the only potential source
of superoxide within endothelial cells, with the respiratory
chain, NAD(P)H oxidases, NOS itself, and endothelin (see
later) among a host of potential sources of superoxide, which,
unchecked by endogenous antioxidant systems, can mediate
vasoconstriction, primarily via inactivation of NO.
The endothelins (ET-1, -2, and -3) are a family of 21 amino
acid peptides, of which ET-1 is the most abundant and the
primary form found in the cardiovascular system (152). Sim-
ilar to the endothelium-derived relaxing factors, ET-1 is not
stored in endothelial cells; rather, it is synthesized de novo in
response to a range of stimuli, including inflammatory me-
diators (e.g., cytokines, TGF-?, hypoxia, low shear stress,
thrombin, glucose, various hormones). Endogenous inhibi-
tors include NO and PGI2, and both the stimulator- and in-
hibitor-mediated pathways ultimately act on the promoter re-
gion of the ET-1 gene in the nucleus to modulate transcription
of pre-pro ET-1 mRNA (Fig. 10). After translation into pre-
pro ET-1, the peptide is cleaved to Big ET-1 and finally by
endothelin-converting enzyme (ECE) to the mature peptide
(Fig. 10). ET-1 activates G-coupled ET receptors, which ele-
vate intracellular calcium via the phospholipase C pathway
(439). Smooth muscle cells express both subtypes of the re-
ceptor (ETAand ETB), activation of which results in potent
vasoconstriction with the downstream involvement of Rho-
kinase (376). However, endothelial cells express only ETBre-
ceptors, which cause an increase in endothelial calcium and
activation of the endothelium-derived relaxing factors de-
scribed earlier (Figs. 5 and 10). Therefore, although ET-1 re-
lease results in net vasoconstriction, the magnitude of the ef-
fect is blunted by its action on endothelial ETBreceptors, an
effect that would be diminished by endothelial cell injury or
dysfunction of the downstream relaxing factors. The effect of
endothelial dysfunction is often exacerbated with respect to
endothelin by the fact that some agents that reduce the ac-
tivity of relaxing factors are concomitant stimulators of ET-1
synthesis (e.g., superoxide). The interaction of ET-1 and ox-
idative stress is highly complex: not only does superoxide
stimulates ET-1 synthesis, but ROS also mediates some of the
downstream effects of ET-1 [e.g., JAK-2 activation (16)], as
well as being the ultimate product of ET-1–mediated proin-
flammatory effects [e.g., increase oxidative stress through in-
duction of COX-2 (406), activation of NAD(P)H oxidase (251),
and mitochondrial dysfunction (426), which apparently have
an important role in disease-mediated vascular dysfunction
in disease (111)]. Furthermore, ET-1 is seen to be a proin-
flammatory agent that contributes to vascular remodelling in
pathologic conditions (14).
ET-1 is expressed throughout the human vasculature, and
expression is increased in atheromatous tissue (486). Fur-
thermore, ET-1–activated NF-?B is a key player in the in-
flammation cascade (466). Thus, the endothelin system is im-
plicated in the pathogenesis of atherosclerosis, and several
clinical disease states, including systemic hypertension (168),
LE BROCQ ET AL.1640
pulmonary hypertension (278), and chronic heart failure
D. Angiotensin II (Ang II)
Ang II is another endothelium-derived vasoconstrictor
peptide in the sense that the enzyme responsible for its syn-
thesis from relatively inactive Ang I (angiotensin-converting
enzyme; ACE) is found predominantly in the vascular en-
dothelium (52). The primary action of Ang II is on AT1re-
ceptors, mediating a powerful vasoconstrictor effect in the
acute phase (Fig. 5), but also leading to aldosterone secre-
tion and sodium reabsorption in the kidney, the net effect of
which is fluid retention and increased blood pressure. In the
chronic phase, AT1-receptor stimulation results in hypertro-
phy and hyperplasia in vascular smooth muscle and in car-
diac myocytes, contributing to the remodelling of both blood
vessels and the ventricles of the heart in chronic conditions
such as hypertension and heart failure (42, 425). Further com-
plexity in the role of Ang II is added by the fact that this pep-
tide is well recognized to be a mediator of oxidative stress
and endothelial dysfunction (94, 237), in part through up-
regulation of NAD(P)H oxidases [NOX (155,343); see Figs. 5
and 17 for the impact of NOX].
V. Tissue Plasminogen Activator (t-PA)
t-PA is an endothelium-derived fibrinolytic agent, the pri-
mary role of which is to dissolve rapidly thrombi that form
in blood vessels. Synthesis of this serine protease is contin-
uous but is stimulated by shear stress, thrombin, and hista-
mine (163), and inhibited by plasmin (374) in a process that
might involve protein kinase C (246). t-PA is stored free in
the cytoplasm as well as in granules within endothelial cells;
cytoplasmic t-PA is released constitutively, whereas that in
storage granules requires elevated intracellular Ca2?to me-
diate exocytosis. It is postulated that the primary stimuli for
t-PA release are agonists related to the coagulation cascade,
but other recognized stimulators of endothelial function (e.g.,
bradykinin) are also known to stimulate release. Impor-
tantly, not all the recognized activators of the NO/sGC path-
way and other endothelium-derived relaxing factors are ca-
pable of stimulating t-PA release; ACh and atrial natriuretic
peptide are notable exceptions. The lack of consistency
among agents that increase intracellular Ca2?and stimulate
endothelium-dependent vasodilatation to also cause t-PA re-
lease would suggest that the release mechanism is more com-
plex than a simple Ca2?-activated response, but it has yet to
be fully elucidated.
FIG. 10. Synthesis of endothelin (ET-1).
The paradigm for t-PA function in vivo is that activation
of the coagulation cascade associated with formation of mi-
crothrombi on an eroded atherosclerotic plaque, or a full-
blown thrombus after plaque rupture, activates t-PA release
as a counterregulatory measure designed to restore the af-
fected blood vessel patency and minimize the detrimental
effect of infarction. As with endothelium-derived relaxing
factors, the effect of activated t-PA release is greatest in the
locality of the stimulus, in this case, a thrombus; systemic di-
lution and inactivation by circulating plasminogen activator
inhibitor (PAI-1) is sufficient to ensure that the impact on
global hemostasis is minimized. The full extent of the ana-
tomic distribution of tPA is not yet known, but early evi-
dence suggests that it is most abundant in large conduit ves-
sels (330, 360, 398), which is in keeping with the likely
incidence of atherothrombotic disease. Moreover, many of
these studies indicated that expression is enhanced in ath-
erosclerotic vessels, indicating the ability of this system to
anticipate the likely sites of thrombotic events. It is interest-
ing to note that t-PA is most abundant in vessels dominated
by NO, and some evidence suggests that NO might be in-
volved in t-PA release.
The reduced ability of dysfunctional endothelium to gen-
erate t-PA is evident in a wide range of cardiovascular dis-
eases, including hypertension (185) and coronary artery dis-
ease (313). Patients with hypercholesterolemia, in whom
endothelium-dependent vasodilatation is affected, do not
have impaired acute t-PA release (314). This finding is in
keeping with clinical evidence that serum cholesterol levels
do not influence the rate of patency of occluded vessels, but
is further evidence that t-PA release involves complex mech-
anisms, not all of which are shared by other endothelium-
dependent markers. The impact of smoking on t-PA is par-
ticularly interesting; basal plasma levels are often seen to be
increased, but dynamic release of t-PA in response to a stim-
ulus is dramatically impaired in smokers (313). The precise
mechanism underlying the association is not yet fully ex-
plored, but oxidative stress is sure to underpin smoking-in-
duced defects in t-PA responses.
VI. Endothelial Activation by Inflammatory Stimuli
Activation of the endothelium by inflammatory stimuli re-
sults in the expression of a wide range of proteins that alter
its function significantly. Most notable among these are vas-
cular cell-adhesion molecules (VCAM-1, ICAM), together
with selectins that are specific for endothelium, platelets, and
leukocytes (E-, P-, and L-selectin, respectively) (74, 85, 87, 96,
187). Typically, such activation would be associated with in-
jury or infection and is central to the recruitment of platelets
and lymphocytes to limit blood loss and to evoke a localized
inflammatory response. A full inflammatory response en-
sues, involving neutrophils, lymphocytes, and macrophages
under the guidance of T cells. As well as facilitating the re-
cruitment of inflammatory cells, cytokines and other in-
flammatory mediators have a profound effect on the release
of certain endothelium-derived mediators, including ET-1
(200), which has an immediate and powerful vasoconstric-
tor effect (190). In addition, cytokines induce expression of
iNOS and consequently affect generation of NO and NO-re-
lated species (e.g., ONOO?). The process ordinarily ceases
on resolution, and the adhesion molecules and selectin ex-
pression dissipate before complete resolution of inflamma-
tion. In the event that inflammation ensues for prolonged pe-
riods, secondary effects of both inflammatory cytokines and
factors such as increased ET-1 and alterations in NO gener-
ation become apparent, particularly with respect to remod-
elling of the vessel wall (Fig. 11).
Endothelial activation of the type described is recognized
as a form of dysfunction because it is central to the athero-
genic process [for review, see (88, 207)]. In this setting, ad-
hesion-molecule expression by endothelial cells is triggered
by accumulation of oxidized lipoproteins (ox-LDL) in the
subendothelial space (Fig. 11). Monocytes are captured and
translocate through the endothelial cell layer, whereupon
they differentiate into macrophages, and proliferate and in-
gest the offending ox-LDL via scavenger receptors. Unfortu-
nately, this process is not easily resolved because the lipid-
laden macrophages (now known as foam cells) accumulate,
and their demise results in lipid deposition within the ves-
sel wall–a fatty streak. The influence of inflammatory cells
in the atherogenic process is continued through their impact
on neointimal proliferation, matrix metalloproteinase ex-
pression, and fibrosis that mediate the progression of a fatty
streak to a mature atherosclerotic plaque (256, 355). Ulti-
mately, inflammation generally subsides to leave a stable
plaque; chronically inflamed plaques are commonly re-
garded to be at high risk of rupture, leading to acute throm-
botic events such as an acute coronary syndrome and stroke.
The function or otherwise of the endothelial cells that over-
lie mature atherosclerotic plaques and their role in plaque
rupture is largely unknown, but it is often surmised that it
is dysfunctional. The unusual flow patterns and wall stresses
caused by plaques might contribute to endothelial apopto-
sis (428) and erosion at critical points on the plaque surface.
Myeloperoxidase (MPO) is a leukocyte-derived heme-con-
taining enzyme (315) that has been identified as having an
important role in the atherogenic process (86, 335, 405). MPO
is secreted on activation of leukocytes (neutrophils, mono-
cytes, and some subtypes of tissue macrophages), where-
upon it converts H2O2 into potent oxidants, including
hypochlorous acid (HOCl) and nitrating species (218, 315).
Clearly MPO is an important source of oxidants in inflam-
matory conditions, and its role in the etiology of atheroscle-
rosis-related oxidative stress (Fig. 11) is well documented. It
is apparent, however, that the effects of MPO on endothelial
dysfunction are exacerbated by its transcytosization to the
subendothelial space, where it is ideally placed to intercept
NO through oxidative modification, resulting in enhanced
nitrotyrosine formation in the immediate vicinity (315).
The numerous reactive oxidants and diffusible radical
species generated from MPO (219) are capable of both initi-
ating lipid peroxidation (490, 491) and promoting an array
of posttranslational modifications to target proteins, includ-
ing halogenation, nitration, and oxidative cross-linking (170,
335). MPO also has a role in oxidative modification of HDL,
and it has been shown that apolipoprotein A-I (apoA-I), the
primary protein constituent of HDL, is a selective target for
MPO-catalyzed nitration and chlorination in vivo, resulting
in inhibition of ABCA 1–dependent cholesterol efflux from
LE BROCQ ET AL.1642
Elevated levels of leukocyte and blood MPO have also
been linked to the presence of coronary artery disease (CAD)
in humans, highlighting a potential role for MPO as an in-
flammatory marker in CAD (489).
B. A pivotal role for NF-?B?
As with inflammation in other cell types, mobilization of nu-
clear factor-?B (NF-?B) is associated with the activation pro-
cess in endothelial cells and with atherogenesis in general (75).
Despite the lack of irrefutable evidence for a causal role for
NF-?B in cardiovascular disease processes (largely on account
of the lethal effects of gene knockout associated with the NF-
?B pathway), the circumstantial evidence to support a pivotal
role for NF-?B in the process is compelling. First, activated NF-
?B is found in atherosclerotic plaques (38) but is all but absent
from the surrounding tissue. Second, oxidative stress, inflam-
mation, and dyslipidemia are central to the atherogenic pro-
cess, all of which are reputed to activate the NF-?B pathway
(36, 253). Finally, many of the risk factors associated with the
atherogenic process, including hypertension (primarily
through Ang II), diabetes (primarily as a result of hyper-
glycemia and advanced glycated end products; AGEs), hy-
perhomocysteinemia, and disturbed blood flow are linked to
NF-?B activation. Activation of NF-?B stimulates expression
of VCAM-1 and MCP-1 (75) (Fig. 12), both key players in the
recruitment of inflammatory cells to the affected region. A sen-
sory role for oxidative stress by NF-?B is disputed, particularly
in endothelial cells, but the same article suggests a requirement
for lipid peroxidation in the process instead (36).
C. Hyperlipidemia, oxidative stress, and inflammation:
a noxious triad
Oxidative stress, hyperlipidemia, and inflammation are
detrimental to cardiovascular health, even in isolation. How-
health and disease: conversion
from an anti-atherogenic, antiox-
idant protective “organ” to a pro-
inflammatory, pro-oxidant entity
that contributes to the athero-
The endothelium in
ever, the three are inextricably linked in the progression of
endothelial dysfunction (58, 394, 399) and the resultant car-
diovascular diseases. Oxidative modification of lipids plays
a central role in propagating the inflammatory response and
contributes heavily to the deposition of lipids in the vascu-
lar wall, whereas hyperlipidemia contributes to endothelial
dysfunction and activation, which in turn leads to oxidative
stress (256, 322) and inflammation. It is easy to see, there-
fore, that lipids, oxidative stress, and inflammation go hand-
in-hand in the progression of atherogenesis and that all are
legitimate therapeutic targets.
D. Environmental triggers of dysfunction: do infection and
pollution play a part?
A broad range of risk factors (age, gender, smoking, dia-
betes, hypertension, hyperlipidemia, family history) have
wholly explicable impacts on endothelial dysfunction and
cardiovascular disease, but a proportion of clinical condi-
tions related to endothelial dysfunction cannot be easily at-
tributed to conventional risk factors. The discovery that
plasma C-reactive protein is at least as good a predictor of
cardiovascular events as plasma LDL (347) suggests that in-
flammation is correlated with disease progression. A num-
ber of studies have extrapolated this finding to a connection
between bacterial infection and atherogenesis, with endo-
thelial dysfunction perhaps providing the pivotal link (434).
Some of the best data arise from work with Helicobacter py-
lori and Chlamydia pneumoniae: H. pylori has been associated
with perivascular inflammation, specifically in coronary ar-
teries, whereas C. pneumoniae has been found to be particu-
larly prevalent in carotid endarterectomy specimens, in
which it seems to predispose to thrombosis. Despite the fairly
compelling retrospective data, however, meta-analysis of 15
prospective studies did not find an association between C.
pneumoniae and cardiovascular disease, and the role for the
endothelium in any association has not yet been fully ex-
A similar link has been proposed between pollution and
atherogenesis, or plaque instability, or a combination of these.
A number of clinical studies imply that exposure to diesel ex-
haust fumes, or the nanoparticles therein, is associated with
increased risk of acute cardiovascular events, but the mecha-
nism is still poorly understood (95, 290). Nevertheless, several
preclinical studies have shown a link between pollution and
endothelial dysfunction (162, 421) or inflammation (144, 475).
In addition, recent clinical studies have indicated that endo-
thelial function, as measured by responses to the endothelium-
dependent vasodilator, bradykinin, as well as t-PA release, is
impaired after acute exposure to relevant levels of diesel soot
(291) and leads to increased risk of repeated myocardial in-
farction (451). Other clinical data point to an impact of pol-
lution on different cardiovascular diseases, including hy-
pertension and heart failure (43, 145). The implication is that
pollution causes endothelial dysfunction, but the mechanism
is unclear: is it caused indirectly via an increased inflamma-
tory or oxidative stress response on deposition in the lungs,
or do critical pollution particles enter the bloodstream as free
entities or on-board macrophages and contribute directly to
endothelial dysfunction? If the latter, is the impact of these
particles purely toxicologic, or do they influence endothelial
dysfunction through, for example, increased oxidative
stress? The answers to these questions are critical in con-
firming the link between pollution and atherosclerosis or
acute clinical events or both.
Inflammatory stimuli are emerging as independent risk
factors for vascular disease, and the profile of the endothe-
lium in mediating their effects in terms of disease progres-
sion is likely to increase as more research is conducted in
this area. Furthermore, several studies that have investigated
the effects of antibiotic therapy in patients at high risk of car-
diovascular events have shown no reduction in cardiac
events (110). Our opinion is that exposure to inflammatory
stimuli might contribute to the atherogenic process, but what
is more interesting is that these influences might well have
an impact on plaque rupture, leading to myocardial infarc-
tion and stroke. The data relating to pollution are particu-
larly interesting in this respect: epidemiologic data indicate
that the incidence of myocardial infarction is increased
within ?1 day of exposure to pollutant particles (451), im-
plying that pollution is associated with plaque rupture as
well as endothelial dysfunction and atherogenesis. If true, it
will be interesting to establish the cause of this association,
especially in view of the rapidity of the effect.
E. Homocysteine and endothelial dysfunction
Homocysteinuria is the manifestation of an autosomal re-
cessive disorder, in which patients have a defect of cys-
tathione ?-synthase, resulting in an increase in plasma ho-
mocysteine; these patients are at greater risk of premature
coronary disease (107). Furthermore, in the general popula-
tion, plasma concentrations of homocysteine appear to be as-
sociated with an increased risk of premature cardiovascular
disease. Increased homocysteine levels are associated with
endothelial dysfunction, vascular smooth muscle prolifera-
tion, increased thrombus formation, and inhibition of en-
dogenous fibrinolysis (391), although short-term increases in
LE BROCQ ET AL.1644
The impact of NF-? ?B on inflammation and anti-
homocysteine through methionine loading do not, surpris-
ingly, alter vascular stiffness (464), suggesting that the detri-
mental effects of homocysteine are slow to develop and are
associated with prolonged elevation of homocysteine.
The cause of endothelial dysfunction seen in patients with
homocysteinuria seems to include increased oxidative stress
via impaired intracellular glutathione peroxidise-1 activity
and inhibition of SOD, resulting in increased oxidation of
LDL. Other mechanisms include increased apoptosis, in-
creased ADMA (inhibiting NOS) (404), eNOS uncoupling
through reduction of intracellular BH4activity (420) (Fig. 7),
and decreased ICAM-1, VCAM-1, and E-selectin, which re-
sults in increased endothelial permeability and an increased
risk of thrombosis (392). The prothrombotic effect of homo-
cysteine might also be exacerbated by enhancement of ac-
tivity and expression of factors XII and V and reducing the
activity of protein C, thrombomodulin, and decreases the ef-
fectiveness of endogenous tPA. Platelet aggregation and ac-
tivation are both increased by homocysteine, although the
effect is not necessarily seen in brief exposure.
As noted earlier, most epidemiologic studies demonstrate
an association between plasma homocysteine and increased
cardiovascular disease (138). The magnitude of this effect
varies between 20% and 80%. With the increasing mecha-
nistic data suggesting a causal association, several random-
ized trials have been performed to test the hypothesis that
pharmacologic reduction of plasma homocysteine will re-
duce CV risk. Homocysteine concentrations can be reduced
by the simple intervention of vitamin B complex or folate.
However, the current trials of these interventions have had
mixed results; some studies have demonstrated improve-
ments in endothelial function (97, 417, 467), coronary steno-
sis (371), and cardiovascular events (371), whereas others
have shown no benefit (257, 445). One of the major issues
when performing such trials is that the size of the effect of
homocysteine is likely to be small when compared with more
traditional risk factors, such as smoking and hyperlipidemia.
Furthermore, clinically relevant reversal of endothelial dys-
function with agents such as folic acid is likely to be slow,
and therefore, adequately powered trials must be large and
have sufficiently long follow-up. Although the absolute car-
diovascular benefit of homocysteine reduction is likely to be
small, the population impact of a relatively safe intervention,
such as vitamin B complex or folate supplementation, could
yet be shown to be considerable in terms of reduced cardio-
VII. Experimental Measures of Endothelial Function
A. In vitro
1. Cell culture. Human umbilical vein endothelial cells
(HUVECs) are the most readily available endothelial cells,
although aortic, coronary, and resistance artery–derived en-
dothelial cells from both human and animal sources are also
commercially available. A vast literature exists relating to
cultured endothelial cells and, in particular, the impact of
oxidative stress and inflammation on the mechanisms un-
derlying dysfunction. The advantage of using cultured en-
dothelial cells is the potential for rapid throughput and in-
depth investigation of cell signalling, but cell culture has its
drawbacks. Endothelial cells undergo phenotypic changes,
precluding their use beyond approximately passage 7 to 8,
and even within this period, it is unclear how closely they
truly resemble human arterial endothelial cells in vivo, not
least because umbilical vein endothelial cells are (a) venous
and (b) fetal in origin. Nevertheless, the data generated from
endothelial cell culture has proved invaluable in dissecting
out cellular mechanisms involved in endothelial function
2. Functional assays. Traditional organ-bath pharmacol-
ogy and myography continue to be a mainstay of in vitro
analysis of endothelial function several decades after these
techniques proved instrumental in the discovery of EDRF
(133). The essence of these assays is a measure of endothe-
lium-dependent relaxation of arterial segments in response
to recognized agonists (e.g., ACh, bradykinin). Instruments
are available to enable force measurements in rings under
passive tension (8), whereas others measure changes in ves-
sel diameter in lengths of artery under flow conditions (per-
fusion myographs). The strength of these techniques is the
total control of the environment and the ability experimen-
tally to isolate elements of endothelial function (e.g., through
inhibitors like L-NAME and indomethacin, through removal
of the endothelium altogether, or through induction of ex-
perimental oxidative stress). The technique can also be used
as an ex vivo tool for determining the impact of disease de-
velopment on endothelial function, experimental alterations
in phenotype, and in vivo drug treatments in animal models
and human vessels removed during routine surgery. It is im-
portant, however, to recognize the limitations of the artifi-
cial nature of in vitro functional analysis of this type in ex-
trapolating results to the in vivo situation, not least with
respect to the hyperoxic conditions under which most of
these experiments are usually conducted.
An important issue relating to both cell-culture experi-
ments and in vitro functional assays is the lack of physical
stresses on the endothelial cells that would normally be ex-
perienced under flow conditions. Sophisticated experimen-
tal procedures are available to mimic at least some of these
physical parameters [e.g., shear-stress models for cell cul-
tures (458), perfusion myography for blood vessel work
(227), and isolated perfused organs (301)], but few, if any,
can satisfactorily replicate the complex combination of shear
stress and cyclical wall stretch that would be experienced in
vivo. These should be important considerations in both ex-
perimental design and interpretation of results from studies
using these models.
B. In vivo (clinical studies)
In vivo studies of endothelial function are fraught with dif-
ficulties, and currently no gold standard exists (the pros and
cons of the most popular techniques are summarized in
Table 1). Furthermore, many techniques that are purported
to measure endothelial function actually measure “vascular
reactivity,” which is used as a surrogate for endothelial dys-
function. The ability of blood vessels to dilate to either ex-
ternal stimuli (e.g., intraarterial infusion of, for example, ACh
in forearm blood-flow experiments), or to the quasi-physio-
logic stimulus of reactive hyperemia (in flow-mediated di-
latation), rely not only on the ability and function of the en-
dothelium to release relaxing factors, but also on the innate
ability of the blood vessels to dilate. The “stiffness” of blood
vessels is, therefore, an important consideration alongside
endothelial function; vessel stiffness is determined by a wide
range of factors including age, smooth muscle cell hyper-
plasia, collagen cross-linking, glycation, and fibrosis. Thus,
important structural and functional elements to vascular re-
activity are often glossed over in the published literature and
all but ignored in measures of “endothelial function.” Fur-
thermore, the potential dynamic and temporal interaction
between endothelial function and structural vascular change
are likely to differ significantly between different diseases,
patient populations, and, indeed, individual patients. Al-
though direct and comprehensive in vivo measurement of en-
dothelial function is not, therefore, currently possible, an
enormous literature relates to the impact of disease and treat-
ments on “endothelial function” assessed by vascular reac-
tivity. An appreciation of the different techniques and their
limitations is therefore necessary to put study results in con-
1. In vivo study techniques
a. Measuring “endothelial dysfunction” in coronary arteries.
In vivo coronary studies are expensive and difficult to per-
form in a large numbers of patients because of their invasive
nature. However, the response of coronary arteries to ACh
is particularly useful; it causes an endothelium-dependent
dilatation in healthy coronary arteries (462) but a converse
constriction in diseased vessels on account of a direct effect
on ACh M3receptors on vascular smooth muscle (264, 423).
Other endothelium-dependent vasodilators that have been
used in the coronary circulation to measure endothelial dys-
function include bradykinin, substance P and 5-HT (146). Re-
sponses to intracoronary drugs can be measured by quanti-
tative coronary angiography (197), intracoronary Doppler to
measure flow (485), or by using intracoronary pressure wires
(27). However, the benefits of direct measurements of coro-
nary endothelial function must be weighed against their rel-
ative difficulty, expense, and the possible health risks to pa-
tients that are associated with these techniques.
b. Measuring “endothelial dysfunction” in peripheral arteries.
Given that atherosclerotic plaque distribution is diffuse
throughout the arterial tree, it is not surprising that endo-
thelial responses in peripheral vessels correlate well with
coronary artery responses (7). Assessment of peripheral ar-
terial endothelial function has the advantage over direct
coronary measures in that it is less invasive and, therefore,
safer and less expensive. Peripheral vessel techniques are
also more amenable to complex study protocols, giving
more-detailed mechanistic data.
c. Venous occlusion plethysmography. Forearm venous
plethysmography coupled with intraarterial drug adminis-
tration can be used to investigate both endothelium-depen-
dent (e.g., ACh) and independent (e.g., sodium nitroprusside,
glyceryl trinitrate) vascular function and the direct vascular
effects of vasoconstrictor and novel substances, which can
be directly infused into one brachial artery, with the con-
tralateral limb used as a control. Forearm blood flow can be
assessed by strain-gauge plethysmography (244). However,
this is still an invasive technique involving cannulation of
the brachial artery, which is not without some risk (462). Al-
though adverse clinical events are rare, this is a specialist
technique that is not suitable for widespread clinical use.
Forearm venous plethysmography can also be coupled
with reactive hyperemia (see later) to provide an indirect
measure of endothelial function (176, 422). This may prove
to be a useful clinical test in the future, but it less suitable
for mechanistic studies.
d. Flow-mediated dilatation with brachial artery imaging. Bra-
chial artery imaging coupled with reactive hyperemia is one
of the most popular techniques for measurement of vascu-
lar function (62, 332, 387). Forearm or hand ischemia is in-
duced by a tourniquet, release of which results in hyperemia
of the distal vascular bed. This so-called reactive hyperemia
is mediated by several factors that are released in response
to ischemia, including NO (195), resulting in local vasodi-
latation and increased blood flow in both proximal and dis-
tal blood vessels. The increased blood flow in the proximal
vessel (brachial artery) results in increased shear stress and
an NO-mediated vasodilatation (flow-mediated dilatation;
FMD). The magnitude of FMD is thought to be proportional
to endothelial function, but structural alterations of blood
vessels in disease and their ability to dilate are confounding
factors. Another limitation is the degree of reactive hyper-
emia to the same stimulus, which is likely to vary in differ-
ent disease states.
The brachial artery is the most commonly used vessel to
study FMD, but other arteries, including the carotid (358),
LE BROCQ ET AL. 1646
TABLE 1.COMPARATIVE MERITS AND DISADVANTAGES OF AVAILABLE METHODS FOR ASSESSING VASCULAR FUNCTION IN VIVO
Peripheral arterial tone
Validation of technique
Ease of use
Evidence of prognostic value
Freedom from operator bias
Use for repeated studies
???, Technique performs well in this area; ??, intermediate performance; ?, technique performs poorly.
have been used for this technique. However, although it is
safe and relatively easy to perform, FMD is somewhat lim-
ited, in that it provides few mechanistic data. Furthermore,
lack of standardization and variations in positioning of the
arm cuffs and measurement of vessel diameter make com-
paring study results difficult.
e. Pulse-wave analysis and velocity. Large-vessel compli-
ance decreases with advancing age and with classic cardio-
vascular risk factors, resulting in increased arterial stiffness
and elevated systolic blood pressure. Although structural
changes in the vessel wall are a major component of arterial
stiffness (increased collagen and decreased elastin), the en-
dothelium also appears to play an important dynamic role
in arterial stiffness (216). Pulse-wave analysis and velocity
represent two related techniques for assessing the propaga-
tion of the arterial wave form and reflected wave, resulting
in a measure of arterial stiffness. High-fidelity tonometers
are used to measure the peripheral arterial wave form, the
data from which are further manipulated to establish an
“augmentation index” [see (324) for review].
f. Doppler skin flowmetry. The technique of Doppler skin
flowmetry relies on measuring the Doppler shift caused by
moving red blood cells on reflected light of a known fre-
quency. The signal (flux) is a product of the degree of
Doppler shift (speed of red cells) and strength of signal
(number of red blood cells) and thus a surrogate for blood
flow. Changes in blood flow by reactive hyperemia (30), in-
tradermal injection of substances (243), or iontophoresis of
small charged molecules (80) have all been used to assess the
skin microcirculation. The attraction of this technique is that
it is relatively noninvasive, and even when coupled with in-
tradermal injection, the technique is relatively safe, given the
extremely small doses of study drug used (243). This tech-
nique is less technically challenging than measuring brachial
artery diameter and, coupled with reactive hyperemic re-
sponses, may yet be developed into a clinically useful test of
vascular “health.” However, although vascular responses be-
tween the skin microcirculation and larger arteries are sim-
ilar, whether skin blood-flow studies are representative of
other vascular beds is unclear and requires further work.
g. Serum biomarkers for endothelial dysfunction. Use of
serum biomarkers to determine endothelial dysfunction car-
ries the advantages associated with routine sampling and
straightforward measures. Many serum biomarkers have
been shown to correlate with vascular disease and outcome,
including interleukin-6, tumor-necrosis factor-?, soluble P
selectin, and soluble intercellular adhesion molecule-1 (12,
349). The role of chronic inflammation in the atherosclerotic
process continues to fuel the interest in hsCRP as both a me-
diator and a biomarker of atherosclerosis (348). CRP can
influence a number of processes involved in endothelial dys-
function, including increasing ET-1 synthesis, downregulat-
ing eNOS (444), increasing the release of PAI-1 from endo-
thelial cells (92), and influencing endothelial progenitor cells
Cellular adhesion molecules (CAMs) are expressed on the
surface of activated endothelial cells, and elevated plasma con-
centrations of soluble CAMs are seen in patients with athero-
sclerosis (331). Furthermore, vascular extracellular superoxide
dismutase (SOD) is depressed in patients with coronary artery
disease (234). Plasma nitrite is another measure that is increas-
ingly popular in estimating endothelial function. Nitrite is a
transient oxidation product of NO, which is rapidly converted
to nitrate in the presence of red blood cells, but can be pre-
served in blood samples after rapid oxidation of red blood cell
heme or immediate centrifugation and plasma freezing. The
transient nature of the existence of nitrite ensures that its plasma
level is a dynamic measure of endothelial NO generation, as-
suming that the rate of oxidation remains relatively constant.
This measure is increasingly popular among clinicians, where
it is used in vasodilator studies invivoto confirm that any func-
tional effect seen is reflected in modulation of NO synthesis
(171, 221, 222). In addition, reduced ability of the endothelium
to secrete t-PA on activation has recently been proposed as a
reliable marker of endothelial dysfunction (325). The sensitiv-
ity of t-PA in this setting is apparently greater than that for
other markers of endothelial dysfunction, including agonist- or
flow-mediated vasodilatation, because it is measurable in some
clinical conditions (e.g., hypertension) in which agonist-in-
duced vasodilatation is unaltered. Use of these biomarkers to
measure vascular disease or prognosis is still at an early stage
in development, but great potential exists in this field.
VIII. Endothelial Dysfunction and Aging
Aging is an independent risk factor for cardiovascular dis-
ease that affects us all. In keeping with enhanced risk of car-
diovascular disease, clear evidence form both animal and hu-
man studies indicates that bioavailability of protective
endothelium-derived factors (NO, prostacyclin, and EDHF)
declines with age in both conduit and resistance vessels, with
a concomitant increase in generation of, and sensitivity to,
constrictors. Although the link between aging and dysfunc-
tion of the endothelium is undisputed, the mechanism(s) in-
volved in the phenomenon are less clear; for example, eNOS
expression has been shown to decline with age in some stud-
ies (18, 65, 84, 413, 430), but to increase in others (143, 437).
It seems likely, however, that oxidative stress is once again
the key to the association between aging and endothelial dys-
function on account of depression of antioxidant enzymes
(e.g., MnSOD) in response to prolonged exposure to ROS
(437). The endothelium itself appears to be an important
source of ROS that mediate downstream effects (143), with
increasing dysfunction of mitochondria and other well-rec-
ognized sources of ROS (e.g., xanthine oxidase, NOX) (71,
137) implicated in age-related dysfunction. ROS and a loss
of NO are also likely regulators of conversion of endothelial
cells to the senescent state, together with a reduced capacity
to regenerate and replace endothelial cells that have under-
gone apoptosis, necrosis, or removal by erosion. This effect
is, in part, due to a reduction in number of circulating en-
dothelial progenitor cells, as well as a reduced ability of such
progenitor cells to engraft and to develop full endothelial
function, particularly with respect to eNOS. In vivo, an im-
portant stimulus for the expression of eNOS is the shear
stress generated by flowing blood on the endothelial surface
(344), which increases eNOS mRNA and stability (89) and
may explain why physical training improves eNOS expres-
sion in older humans (410) and animals (413). Several other
factors, such as estrogens (223) and growth factors (34), also
can upregulate eNOS expression.
Secretion of many growth factors and hormones declines
with age. Of these, estrogens and dehydroepiandrosterone
(DHEA) have received the most attention, and DHEA has
become a widely used antiaging drug. Hard scientific evi-
dence has not been presented justifying therapy, although
treatment of middle-aged men with hypercholesterolemia
has been shown to improve endothelium-dependent flow-
mediated dilator responses (206), but it is unknown whether
long-term supplementation is able to prevent aging-induced
endothelial dysfunction. Evidence exists in rats that long-
term inhibition of the renin–angiotensin system can amelio-
rate endothelial dysfunction associated with aging through
inhibition of COX-2–derived vasoconstricting factors and su-
peroxide anion synthesis (299).
Taken together, it is evident that age-related changes in
endothelial phenotype and function are detrimental to car-
diovascular health and, indeed, mirror many of the processes
that are associated with other risk factors predisposing to
atherosclerotic disease. The inexorable march toward a dys-
functional endothelium is almost certainly a feature of even
the healthiest individuals, but the rate of decline is heavily
influenced by lifestyle; poor diet, smoking, and weight gain
top the list for aspects of lifestyle over which individuals
might exert some control in an effort to slow disease pro-
gression. Whether we will ever succeed in slowing the un-
derlying (“basal”) aging process is debatable, but a thorough
understanding of the mechanisms involved in the process
will no doubt shed further light on cardiovascular disease
processes that might simply be viewed as accelerators of, or
extensions to, the natural aging process.
IX. Endothelial Dysfunction in Cardiovascular Disease
Considerable interaction occurs between different disease
states such as diabetes, vascular disease (coronary, cerebral,
renal), systemic hypertension, pulmonary hypertension, and
chronic renal impairment, and many bold conclusions from
clinical studies about specific patient groups studied do not
take this into consideration.
A. Atherosclerosis, coronary artery disease, and stroke
Atherosclerosis is a chronic, systemic disease. Thus, the
majority of patients with clinical peripheral vascular disease
will also have coronary disease and vice versa; both groups
are also at increased risk of stroke.
The degree of coronary endothelial dysfunction appears to
be prognostically important. Impaired endothelium-depen-
dent coronary artery vasodilatation is associated with in-
creased risk of subsequent vascular events (161, 414). The same
is also true for impaired flow-mediated dilatation (366) and
cold-pressor test (367). These findings appear to be consistent
for patients with severe obstructive coronary disease, as well
as for those with angiographically normal coronary arteries
(i.e., impaired function without encroachment of atheroma on
the vessel lumen is associated with a poorer clinical outcome
in terms of future events, compared with patients with normal
coronary artery responses). Indeed, most studies in patients
with clinically significant coronary disease or stroke measure
endothelial dysfunctions in the peripheral vasculature, as these
correlate well with coronary artery responses (7).
Plaque instability is a key aspect in the development of an
acute coronary syndrome in which an acute inflammatory
process increases the likelihood of plaque rupture (256). En-
dothelial dysfunction is associated with increased oxidative
stress and inflammation (308). Plaque inflammation and rup-
ture is a complex process that is incompletely understood
but regulated in part by NO (304) and NF-?B (17). Athero-
sclerotic coronary arteries are prone to vasoconstriction, re-
sulting in Prinzmetal angina. Furthermore, coronary artery
spasm is commonly found at the site of plaque rupture and
is a major contributor to the reduced blood flow and occlu-
sion of coronary arteries during myocardial infarction. It is
likely that this is a result of local release of vasoconstrictors,
such as ET-1, but also to the impaired ability of atheroscle-
rotic arteries to vasodilate (216, 486).
Treatments that may improve endothelial function sys-
temically, such as ACE inhibitors and statins, appear to pro-
vide protection from acute clinical cardiovascular events,
with significant improvements in morbidity and mortality.
Although endothelial function is not yet easily measured
clinically, it may in the future act as a guide to the effects of
therapies on generalized endothelial function and allow tai-
lored therapy for patients with clinically significant athero-
The role of endothelial dysfunction in stroke does not ap-
pear to have attracted the same level of interest as that in
coronary artery disease, although the reason for the relative
paucity of data is not clear. Certainly one would anticipate
that atherosclerosis-related stroke at least would be associ-
ated with endothelial dysfunction, and some evidence sup-
ports this notion [see (117) for review].
Oxidative stress is central both to the progression of type
II diabetes [comprehensively reviewed by (63,113)] and to
the cardiovascular consequences of both type I and type II
diabetes. A wide range of sources of oxidative stress are
found in diabetes and in prediabetic states [insulin resistance
(184), metabolic syndrome (334)], including inflammation,
dysfunctional cellular respiration, downregulated antioxi-
dant defenses, and the impact of advanced glycated end-
The mitochondrial respiratory chain is a major site of pro-
duction of ROS within cells, with superoxide being produced
continually as a by-product of normal respiration during
synthesis of ATP (66, 342). Superoxide from mitochondria
can initiate a range of damaging reactions, from the direct
actions of the anion itself to the production of hydrogen per-
oxide (443), hydroxyl radical, and peroxynitrite, which can
damage lipids, proteins, and nucleic acids (193). It has been
suggested that production of mitochondrial ROS and subse-
quent oxidative damage during hyperglycemia may be cen-
tral to much of the pathology of diabetes (46, 319), not least
because the function of the mitochondrion itself is particu-
larly susceptible to oxidative damage, and in the pancreatic
?-cell, it plays a central role in glucose-stimulated insulin se-
cretion (359). This leads to a vicious cycle in the progression
of the disease, in which hyperglycemia leads to oxidative
damage, disrupting the ?-cell response to increases in blood
glucose, and leading to further hyperglycemia (153).
As would be expected, mitochondria have an extensive
range of antioxidant defenses, from Mn-SOD, which con-
verts superoxide to hydrogen peroxide, to its own isoforms
LE BROCQ ET AL.1648
of glutathione peroxidase and thioredoxin-dependent en-
zyme peroxiredoxin III, both of which detoxify hydrogen
peroxide (79). The mitochondrial glutathione pool is distinct
from that in the cytoplasm and is maintained in a reduced
state by an isoform of glutathione reductase, which requires
NADPH (79). Within the phospholipid bilayer, lipid-soluble
antioxidants vitamin E and coenzyme Q both help to pre-
vent lipid peroxidation (266), and an isoform of phopholipid
hydroperoxide glutathione peroxidase degrades lipid per-
oxides within the mitochondrial inner membrane (79). A
range of mechanisms repair or degrade oxidatively damaged
lipids, proteins, and DNA (21). Nevertheless, oxidative dam-
age is inevitable because some ROS produced by the mito-
chondria evade detoxification, leading to a steady-state level
of damage, which is dependent on the relative rates of dam-
age accumulation, repair, and degradation (193, 380).
The proposed consequences of hyperglycemia of partic-
ular pathologic relevance to mitochondrial dysfunction in
diabetes are formation, autooxidation, and interaction with
cell receptors of AGEs; activation of various isoforms of
protein kinase C (PKC); induction of the polyol pathway;
and increased hexosamine flux [reviewed in (153)]. Many
of these pathways have been associated with oxidative
stress, and one hypothesis is that all of these processes are
a consequence of overproduction of superoxide by the mi-
tochondria during hyperglycemia (46, 98, 319); however,
the validity of this link has not yet been demonstrated. The
evidence in support of this argument comes from experi-
ments in cultured endothelial cells, in which increased glu-
cose concentration increases cytosolic ROS production, ac-
tivation of NF-?B, formation of AGEs, and activation of
PKC, all of which were blocked with Mn-SOD, respiratory
inhibitors, or uncoupling protein-1 (319). However, this
link may turn out to be some other interaction with the mi-
tochondrion, not mediated directly by the redox state of
electron carriers (153).
Although it is tempting to suggest that the increase in mi-
tochondrial ROS in response to hyperglycemia is the proxi-
mal defect that leads to most other pathologic consequences
of the condition, this is probably too simplistic and will have
to be extended to accommodate other sites of ROS produc-
tion (461). However, it does suggest that prevention of over-
production of superoxide by mitochondria, or an increase in
the rate of decomposition of such toxic molecules by an-
tioxidants, may alleviate many of the pathologic conse-
quences of hyperglycemia (153). Only a very small propor-
tion of natural and artificial antioxidants will reach the
mitochondria, and so antioxidants that are targeted to accu-
mulate within the mitochondria may offer more protection
(153). Derivatives of the natural antioxidants, vitamin E and
coenzyme Q, specifically designed for this purpose, have
shown some promise in in vitro studies, rapidly and selec-
tively accumulating in isolated mitochondria and in intact,
isolated cells (104, 210, 384). Another possibility for treat-
ment is uncoupling proteins, such as 2,4-dinitrophenol
(DNP), which has been used extensively in the past to treat
obesity in humans (164), but unregulated administration,
abuse, and a very narrow window between efficacy and tox-
icity led to abandonment (164). Also, one of the problems as-
sociated with uncoupling proteins is the decrease in the
membrane potential of the mitochondrion, which in ?-cells
would make insulin secretion less responsive to plasma glu-
cose levels, which would be counterproductive (153). Con-
firmation of their effect has been shown in animal studies,
where treatment with DNP causes hyperglycemia (381).
AGE formation has been linked with several of the long-
term complications of diabetes, including micro- and
macrovascular disease (180, 277, 448). plasma levels of N?-
(carboxymethyl)lysine (CML) and pentosidine double in pa-
tients with advanced diabetes (412). The mechanisms by
which AGEs affect vascular function include formation of
AGE-modified LDL (130, 450). In addition, a specific trans-
membrane AGE receptor (RAGE) initiates a cascade of
events, including activation of NAD(P)H oxidase and a range
of proinflammatory mediators (cytokines and vascular cell
adhesion molecule 1; VCAM-1) (459). The consensus is that
AGE–RAGE interaction is central to the cellular and vascu-
lar dysfunction associated with diabetes complications (231,
476), but some dispute exists in the area, because AGE treat-
ment of HMEC-4 cells did not induce an inflammatory
mRNA profile (435). The precise mechanism of signal trans-
duction from RAGE to NF-?B–induced cytokine secretion
remains largely unknown, although several reports have im-
plicated p21 Ras, extracellular signal-regulated kinases
(ERK) (479) 1 and 2 (231), and protein tyrosine kinase (PTK)
in the effects (476). p38 Mitogen-activated protein kinase
[MAPK; responds to cytokines and cellular stress inducers
(479)] has also been shown to be a key downstream effector
of RAGE in THP-1 monocytes (479) and is required for NF-
?B transcriptional activation and subsequent increased se-
cretion of proinflammatory cytokines, such as tumor necro-
sis factor-? (TNF-?), interleukin 1? (IL-1?), and macrophage
chemoattractant protein-1 (MCP-1) (479). The ligation of
RAGE has been shown to induce acute tyrosine phosphory-
lation, followed by either dephosphorylation or degradation,
but no consensus kinase motif has been identified in the
RAGE intracellular domain, and so it has been suggested that
it probably couples with tyrosine kinases directly or indi-
rectly to mediate the observed tyrosine phosphorylation
A number of other receptors for AGEs have been identi-
fied, such as lactoferrin (370), oligosaccharide transferase
complex protein-48 (also know as AGE-R1) (478), 80K-H pro-
tein (AGE-R2) (478), galectin-3 (AGE-R3) (449), lysozyme
(254), macrophage-scavenger receptors (10), and CD36 (323).
Increasing evidence also shows that Amadori-modified pro-
teins have biologic effects very similar to those of AGEs, and
they may also have their own receptors, which are different
from all AGE-binding proteins, such as calnexin (shown on
mesangial cells) (468), and nucleolin (nucleophosmin and
cellular myosin heavy chain) (41), as specific binding pro-
teins for fructoselysine on various monocyte-like cells. Bind-
ing of fructoselysine to these cells induces phosphorylation
and activation of p38 and p44/42 MAPK, together with NF-
?B activation (40).
An important aspect of tissue damage and cell death as-
sociated with chronic hyperglycemia and diabetes is medi-
ated by ROS (459). Oxidative stress in this setting leads to
oxidation of sugars, nonsaturated fatty acids, and glycated
proteins, which causes an increase in glucose autoxidation
and a depression of endogenous antioxidants (25, 459). Pen-
tosidine and CML are AGEs of particular interest in the study
of oxidative stress in diabetes, as both are produced by gly-
cation and oxidation (298). It has also been shown that su-
peroxide anions and hydrogen peroxide are directly formed
through the Maillard reaction (328), although the
AGE/RAGE interaction facilitates ROS production, poten-
tially leading to apoptosis of cells and compromised cardio-
vascular function (46).
AGEs have also been shown to increase the susceptibility
of low-density lipoprotein (LDL) to oxidation (48), and this
oxidized LDL is responsible for decreased NO production,
by downregulation of NO synthase (459), contributing to de-
fective vasodilation in animal models of diabetes (49).
From the inflammatory perspective, it has been shown that
treatment of human inflammatory cells with high glucose
(159, 372) or specific AGEs (383, 450), leads to oxidative stress
and generation of proinflammatory cytokines. AGEs have
been shown to augment the inflammatory response and to
upregulate cyclooxygenase-2 (COX-2) via RAGE, which
leads to monocyte activation and vascular cell dysfunction
(372). AGEs have also been shown to lead to NF-?B activa-
tion in a process that may or may not be entirely RAGE de-
pendent (302). Cells exposed to AGEs have been previously
shown to have altered proinflammatory phenotypes; genes
affected include those that encode for IL-1? (447), TNF-?
(292), IL-6 (368), platelet-derived growth factor (PDGF) (218),
insulin-like growth factor (IGF)-1 (218), thrombomodulin
(369), vascular cell adhesion molecule (VCAM)-1 (369), and
tissue factor (TF) (25). The interaction of hyperglycemia,
AGEs, oxidative stress, and inflammation are summarized
in Fig. 13.
Oxidative stress and endothelial dysfunction go hand-in-
hand in mediating the onset and progression of diabetes-in-
duced atherosclerosis, which is ultimately the greatest sin-
gle factor responsible for premature death in patients with
diabetes. Clinically, endothelial dysfunction is associated
with insulin resistance (179, 214, 281, 378, 395) (Fig. 1), the
manifestations of which are in part mediated by increased
endogenous ET-1, leading to increased basal vasoconstric-
tion (60, 273), as well as reduced activity of eNOS (126) and
iNOS (270, 272). Increased plasma AGEs are apparently as-
sociated with endothelial dysfunction in humans (412), an
effect that might be mediated via quenching of endothelium-
derived NO (49). However, it is as yet unclear whether the
association found is causal.
The underlying pathophysiology that underpins the reci-
procal relationship between endothelial dysfunction and in-
sulin resistance is comprehensively reviewed elsewhere
(214). Clearly, the interaction between the processes is highly
complex, involving inflammation, oxidative stress, and glu-
cose toxicity. It is worth noting, however, that the AKt sig-
naling cascade is central to the expression and activation of
eNOS (Fig. 1), as well as the translocation of glucose trans-
porters (GLUT-4) that help to maintain healthy function with
respect to the endothelium and to insulin sensitivity. Evi-
dence is amassing to suggest that defects in this pathway,
driven by IKK? in response to inflammatory stimuli, could
mediate both pathophysiologic processes and might repre-
sent the key to the reciprocal nature of endothelial dysfunc-
tion and insulin resistance. Modulators of this pathway
could provide an effective means of reversing the relentless
progression toward diabetes and cardiovascular disease
once insulin resistance takes hold. Synthetic peroxisome pro-
liferator–activated receptor-? (PPAR-?) ligands (known as
glitazones) are effective insulin sensitizers that appear to im-
prove endothelial function (209). The impact of glitazones on
the endothelium is multifactorial, with evidence to support
enhancement of the PI3-kinase pathway, increased expres-
sion of adiponectin and antiinflammatory effects, as well as
depression of MAP-kinase–mediated ET-1 secretion [see
(214) for review]. It is somewhat baffling, therefore, that one
such PPAR-? ligand, rosiglitazone, has recently been at the
center of a concern surrounding increased cardiovascular
risk (320), although the findings of this meta-analysis have
received heavy criticism in the literature.
This area of research is still at a fairly early stage in de-
velopment, with many of the pathways still to be fully elu-
cidated, but clear indications suggest that specific AGEs rep-
resent a key feature of diabetes and play an important role
in initiating and propagating oxidative stress and inflam-
mation that fuels both endothelial dysfunction and the even-
tual loss of pancreatic ?-cells that is associated with late-stage
type-2 diabetes. Our opinion is that AGEs represent the vi-
tal link between oxidative stress and inflammation and there-
fore represent an as-yet-untapped therapeutic opportunity,
particularly if the AGE/RAGE interaction proves to be as
important as some suggest.
C. Systemic hypertension
Systemic hypertension is a major risk factor for coronary
heart disease (131), stroke (318), and death (9). The precise
cause of hypertension is unknown but is likely to be multi-
factorial and to involve genetic predisposition, related pri-
marily to the kidney (364) and responses to environmental
stimuli (e.g., obesity, high salt intake). Hypertension is asso-
ciated with several neuroendocrine abnormalities, including
activation of the renin–angiotensin system, sympathetic ner-
LE BROCQ ET AL.1650
into endothelial dysfunction and macrovascular disease.
Role of AGEs in translation of hyperglycemia
vous system, and increased expression of ET-1 (284, 337, 469).
Release of Ang II is reported to stimulate transcription of
pre-proendothelin-1, resulting in elevated ET-1 expression–a
form of endothelial dysfunction. However, the link between
Ang II and endothelial dysfunction is further exacerbated by
its role in instigating oxidative stress, in part through up-
regulation of NOX enzymes, either directly (233) or via ET-
1 (327). The precise role of oxidative stress in hypertension
is not yet fully understood; ROS apparently induce MAP ki-
nase activation (108), but their role in resistance vessel vaso-
constriction (that might be anticipated on account of endo-
thelial dysfunction) is not always found to be the case (108,
424). In clinical studies, endothelial dysfunction, as mea-
sured by vasodilator responses, appears to be a feature in
hypertensive patients and a factor in the progression to overt
cardiovascular disease. Several studies have shown impair-
ment of endothelium-dependent vasodilatation in hyperten-
sive patients (176, 252), and endothelial dysfunction has been
demonstrated in patients at risk of developing hypertension
even before hypertension occurs (252). It is likely that de-
creased bioavailablity of NO plays an important role in this
phenomenon, given that NOS activity is reduced in patients
with hypertension (284). Structural changes in the vessel wall
also add to an overall decrease in vascular function and in-
creased arterial stiffness (324).
Endothelial dysfunction is clearly associated with hyper-
tension but, rather than being causal in the manifestation of
the condition, it appears that it contributes to the progres-
sion of hypertension and the onset of atherosclerosis. Hy-
pertension is a good example of a condition in which many
different facets of “endothelial dysfunction” come together
to contribute to the pathology, including ET-1, Ang II, NO,
D. Pulmonary hypertension
Pulmonary hypertension is a condition of elevated pul-
monary arterial pressure that can lead to right ventricular
hypertrophy and right heart failure if untreated. Endothelial
cell proliferation and abnormal neovascularization are char-
acteristic pathologic features in idiopathic pulmonary hy-
pertension, but the triggers for these events are unknown. It
is clear, however, that the processes involved are complex
(50), and that an increase occurs in activated circulating en-
dothelial cells of unknown origin (51), reduced expression
of PGI2, and an increase in expression of smooth muscle ETB
receptors in the lungs of patients with primary pulmonary
hypertension. In addition, several other factors, such as
thromboxane, vascular endothelial growth factor, NO,
polyamines, and xanthine dehydrogenase (X-DH) have been
implicated in the development and progression of primary
Pulmonary hypertension secondary to other disease states
is widespread. Diseases that can cause pulmonary hyper-
tension include hypoxic lung disease, chronic obstructive
pulmonary disease, left heart failure, congenital heart dis-
ease, and HIV. Furthermore, a genetic component to the con-
dition may be present, with mutations in the gene encoding
bone morphogenetic protein receptor 2 (BMPR2) occurring
in the majority of patients with familial pulmonary hyper-
tension (297). Pulmonary hypertension is a complex, multi-
factorial condition involving vascular hypertrophy and ab-
normalities in the contraction and relaxation of pulmonary
arteries, facilitated by endothelial dysfunction. Various me-
diators have been implicated, most notably IL-1, IL-6 (186),
TNF-? (385), and vascular endothelial growth factor (VEGF)
(432). However, evidence is accumulating in support of ox-
idative stress as a key factor underlying the cellular changes
and endothelial dysfunction. Patients with primary and sec-
ondary pulmonary hypertension have increased plasma mal-
ondialdehyde (a marker of oxidative stress) (191) and re-
duced lung SOD expression (35). Ischemia has been shown
to stimulate ROS production in pulmonary capillaries,
mainly from endothelial cells (2); administration of antioxi-
dants protects against the increase in pulmonary artery pres-
sure (109), whereas SOD administration limits vasoconstric-
tor hypersensitivity (259). Increasing pulmonary blood
pressure upregulates p67phoxand gp91phox(259) in NOX (2),
downregulates SOD (83), and increases endothelial levels of
xanthine oxidase (166), causing impaired endothelium-de-
pendent relaxation (397), probably through inactivation of
NO (78). The footprint of resultant ONOO?(nitrotyrosine)
has been found in the endothelium of both conduit and re-
sistance pulmonary vessels (91); ONOO?also inactivates
Mn-SOD, further promoting oxidative stress (474). Further
to compound the issue, eNOS expression is reduced in the
lungs of patients with pulmonary hypertension (139); eNOS-
deficient mice exposed to hypoxia have exaggerated pul-
monary hypertension (115), but those overexpressing the en-
zyme are resistant to the condition (329). ROS also stimulate
hypoxia-inducible factor 1 (HIF-1) transcription factor, a key
component in many of the long-term changes of chronic hy-
poxia. HIF-1 regulates the expression of VEGF under hy-
poxia, and high levels of HIF-1? have been found in prolif-
erating endothelial cells of lung plexiform lesions in patients
with pulmonary hypertension (375). HIF-1?–deficient mice
exposed to prolonged hypoxia have reduced right ventricu-
lar hypertrophy and vascular remodeling. DPI and catalase
inhibit HIF-1?, signifying that superoxide and H2O2 are
linked to HIF-1? activation (149).
PGI2also helps to control pulmonary pressure. Patients
with idiopathic pulmonary hypertension or HIV-associ-
ated pulmonary hypertension (431) have reduced PGI2
synthase expression, which is likely to contribute to in-
creased platelet aggregation. PGI2generation is reduced
by hypoxia in pulmonary endothelial cells (15), and PGI2
receptor-deficient mice are hypersensitive to chronic hy-
A range of contractile mediators are also altered in pul-
monary hypertension, further contributing to increased pul-
monary pressure and remodeling. ET-1 is a potent vasocon-
strictor and co-mitogen that is elevated in the plasma of
pulmonary hypertensive patients (72) and animal models of
the disease (249). mRNA of both ETA- and ETB-receptor sub-
types is increased in animal models (249), although ET-1 va-
sodilatation through endothelial ETBreceptors is impaired
(106). The proliferative effect of ET-1 appears to be mediated
mainly through ETAreceptors (484), possibly through a path-
way involving superoxide, as ET-1 stimulates superoxide
generation in cultured pulmonary artery smooth muscle cells
(460). ET-1 also increases pulmonary endothelial Ang II se-
cretion (205), which in turn upregulates ETAexpression on
smooth muscle cells (167) and stimulates superoxide pro-
duction through NOX (155).
5-HT is another key vasoconstrictor and pulmonary
smooth muscle cell mitogen that increases superoxide pro-
duction via NOX, exacerbating vasoconstriction (258) and
stimulating c-fos (382). 5-HT is elevated in the plasma of pa-
tients with primary pulmonary hypertension (175), possibly
as a result of upregulation of angiopoietin-1, which stimu-
lates pulmonary endothelial cells to produce and secrete 5-
HT (407). Experimentally, 5-HT treatment during chronic hy-
poxia induces pulmonary hypertension, right ventricular
remodeling, and mitogenesis in pulmonary vessels in rats
(105), whereas right ventricular hypertrophy and vascular
remodeling is less evident in 5-HT1B–knockout mice (208). 5-
HT2B–receptor expression is increased in patients with pul-
monary hypertension, and 5-HT2B–receptor knockout hy-
poxic mice do not display increased pulmonary arterial
pressure or lung remodeling.
E. Heart failure
Heart failure is defined as the condition that ensues as a
result of insufficient cardiac output to meet demand, result-
ing in breathlessness and fatigue, which can be extremely
debilitating in advanced cases. The most common cause of
heart failure is ischemic heart disease as a result of coronary
artery obstruction, resulting in chronic myocardial ischemia
or myocardial infarction. However, other etiologies exist, in-
cluding viral myocarditis, drug- or toxin-induced (anthra-
cyclins, alcohol) disorders, or end-stage severe valvular heart
disease. Regardless of the initial causal etiology, chronic
heart failure is a complex neurohormonal syndrome associ-
ated with activation of the renin–angiotensin system (128)
and peripheral vasoconstriction (487). At first glance, endo-
thelial dysfunction might not be expected to play a direct
role in heart failure, and indeed much of the emphasis with
respect to oxidative stress in heart failure is focused on the
heart itself (140).
However, considerable evidence indicates that increased
peripheral vascular resistance associated with heart failure
is partly due to endothelial dysfunction (373), although hy-
peractivity of the renin–angiotensin system and the sympa-
thetic nervous system also is heavily involved in the patho-
genesis of the condition. Heart failure is a good example of
a disease in which endothelial dysfunction per se has many
facets. Both animal and human studies have shown that va-
sodilatation in response to endothelium-dependent va-
sodilators is blunted in heart failure, but the effect is likely
due both to reduced eNOS expression (262) and to increased
inactivation of NO by ROS (288), which might in turn result
in cytotoxic effects in endothelial cells through the actions of
ONOO?. Inflammation is most likely central to both eNOS
regulation and ROS production in this setting, with en-
hanced neutrophil activation and cytokine release featuring
in the disease. However, it is also important to note that clear
evidence exists for enhanced release of ET-1 in heart failure,
and that this and the renin–angiotensin system are also likely
to play important roles in the cycle of events that fuels the
progression of the disease. As mentioned earlier, Ang II it-
self promotes oxidative stress through NOX expression and
Some data suggest that endothelial dysfunction in both the
coronary (309) and peripheral (172, 204) circulation is asso-
ciated with a poorer prognosis in patients with CHF, al-
though age and renal function may have confounded these
results to some degree (172). Whether the endothelial dys-
function is a primary or secondary feature of heart failure is
unknown, although it appears to be a feature of both isch-
emic and nonischemic CHF, suggesting that it may be an im-
portant secondary feature. Regardless of its contributory role
to the development of congestive heart failure, impaired pe-
ripheral endothelial function in these patients predicts a
worse outcome and may yet become a useful clinical tool to
identify patients at higher risk and allow targeted interven-
F. Ischemia–reperfusion injury
Ischemia–reperfusion (I/R) injury is a well-recognized
phenomenon that is primarily caused by oxidative stress af-
ter reoxygenation of ischemic tissues caused, for example,
during organ transplant, bypass surgery, or recanalization
after myocardial infarction. I/R is characterized by activa-
tion of inflammatory cells [particularly neutrophils (263, 446,
465)] and reduced viability of endothelial cells, which is both
initiated and exacerbated by generation of ROS (199) in the
oxidative burst after reoxygenation (Fig. 14). It is widely ac-
knowledged that the clinical manifestations of I/R are pri-
marily underpinned by the effects of inflammatory cell acti-
vation to release inflammatory cytokines (488), ROS, and
ONOO?(255), combined with the loss of endothelial cells
that normally release protective endothelium-derived relax-
ing factors [for review, see (127)]. The resulting vasocon-
striction of the microcirculation, together with an increased
tendency for platelet aggregation, monocyte adhesion, and
leukocyte activation, is a critical limitation to organ survival
caused, at least in part, by reduced endothelial NO synthe-
sis and increased inactivation of NO by oxygen-derived free
radicals (357). Furthermore, release of inflammatory cyto-
kines by neutrophils and other inflammatory cells triggers
chronic inflammation that is often associated with I/R in-
jury. In the coronary circulation, some evidence suggests that
the microvasculature is more susceptible to endothelial dys-
function than are the epicardial coronary arteries (340).
Several strategies are used to try to mitigate against reper-
fusion injury. Organs for transplant are cooled as quickly as
possible after blood-flow cessation by perfusing with an or-
gan-preservation solution, which often contains allopurinol
to inhibit xanthine oxidase activity, together with glu-
tathione by way of antioxidant and a glucocorticoid to help
prevent inflammation (e.g., University of Wisconsin solu-
tion). Cooling the organ slows respiration and helps to pre-
vent the oxidative burst on reperfusion, but recent advances
suggest that donor blood might be oxygenated and perfused
throughout the organ-storage phase of the procedure, thus
preventing the ischemic episode and avoiding the issue of
ischemia–reperfusion altogether. However, this technique is
likely to be of merit only in transplants from a living donor,
as autologous blood is likely to be best fit for the purpose.
Endothelial function might be better protected by other ad-
ditives to cold-storage solutions: glutathione is not the ideal
antioxidant to include on the basis that it does not penetrate
membranes easily and is, therefore, likely to remain in the
extracellular environment and remote from the intracellular
source of most of the ROS. Indeed, our recent research indi-
cated that glutathione (3 mM) in the extracellular environ-
LE BROCQ ET AL.1652
ment causes a paradoxic augmentation of endothelial dys-
function and depressed endothelial survival (411).
An alternative strategy that was first used in the heart and
has since been applied to other organs, in advance of isch-
emia during surgery, is known as preconditioning. This in-
volves exposing the heart to a brief ischemic episode (a few
minutes) before the main ischemic period. Much of the ben-
efit of this approach affects the cardiac myocytes, which ap-
parently benefit from phosphorylation events in the mito-
chondria, but recently, attention has also focused on
potential benefits in the endothelial cells, most notably via
inhibition of ET-1–mediated activation of XO and NOX (99)
and via AKt-mediated survival pathways (492).
X. The Future of Endothelial Function Measurement
A. Endothelial function in predisease
Perhaps the most valuable application for endothelial-
function measurement is not the demonstration of endothe-
lial dysfunction in patients with established disease, but the
identification of asymptomatic patients with risk factors who
demonstrate endothelial dysfunction (i.e., in predisease
states). This might include patients with hypercholes-
terolemia, hypertension (293), or a family history of vascu-
lar disease, but who do not (yet) fall into a high-cardiovas-
cular-risk category. It might be envisaged in the near future
that the measurement of endothelial dysfunction could iden-
tify patients at an early stage for medical or lifestyle inter-
B. Assessing impact of therapies in individuals by using
The rationale for “evidence-based medicine” is that pop-
ulation-based outcomes can be extrapolated to the individ-
ual patient. In some cardiovascular therapies, we may mea-
sure effect (e.g., blood pressure, renal function), but the role
of the vasculature or neuroendocrine system is not moni-
tored, and thus, a proportion of patients may receive thera-
pies that are either insufficient or ineffective in that individ-
ual. Noninvasive measurement of vascular function may
identify responders to drug therapy, or allow titration of
therapy based on effect, thus facilitating tailored cardiovas-
role of reactive oxygen and reactive nitro-
gen species in mediating the inflammatory
response and endothelial dysfunction. X-
DH, xanthine dehydrogenase; XC, xanthine
oxidase; NOX, NAD(P)H oxidase; endothe-
lial eNOS, nitric oxide synthase.
Improvements in vascular function have been docu-
mented with angiotensin blockade in patients with hyper-
tension (177, 245, 433), coronary artery disease (6, 268) and
heart failure (194). Similar studies investigating the effect of
HMG-CoA inhibitors (statins) are conflicting: some studies
have demonstrated improvements in endothelial function
with statins (3, 64, 100), whereas others have shown no ef-
fect (22, 440); differences in study techniques may explain
some of these conflicting results.
C. Prognostic value of endothelial function measurement
Several studies have shown an association between mea-
sured endothelial dysfunction and outcome in patients with
heart failure (172), coronary disease in both the coronary
(161, 366, 408) and peripheral (173, 312) vasculature, hyper-
tension (333), and even in “healthy” subjects. The clinical im-
portance of this is uncertain, and the use of invasive tech-
niques to refine prognosis alone may not be clinically useful.
In summary, several techniques are used for measuring
endothelial function, but none is ideally suited for clinical
practice because they are either too invasive or too expen-
sive (coronary studies and forearm plethysmography) or are
difficult to standardize (brachial artery flow-mediated di-
latation). Simpler techniques will have to be developed and
assessed before measuring endothelial function can be used
as a reproducible diagnostic tool.
XI. Prevention of Endothelial Dysfunction
Prevention of endothelial dysfunction is clearly of great
importance in avoiding or ameliorating the development of
some aspects of vascular disease, although, given that it is a
natural process associated with aging, it might not be truly
“preventable.” An appropriate healthful diet, avoiding cer-
tain “toxic” substances (e.g., cigarette smoke), taking regular
exercise, and maintaining a health body weight clearly all
have important health benefits that include the maintenance
of a healthy endothelium. The greatest challenge that faces
public health physicians is how to encourage populations to
adopt these measures. In general, population-based inter-
ventions have been disappointingly ineffective because con-
vincing the population as a whole to undertake fundamen-
tal changes in behavior is difficult and extremely costly.
Thus, targeted intervention in “motivated” high-risk patient
groups has been adopted with some success (e.g., cardiac re-
habilitation programs after myocardial infarction). Never-
theless, population-based and fiscal interventions such as in-
creasing the tax on tobacco or banning cigarette smoking in
public places may have wider-reaching benefits to the whole
population. Early reports regarding the ban on smoking in
public places in Scotland indicated a 17% reduction in MI-
related hospital admissions since the ban came into effect in
2006, compared with a 3% reduction per annum in years pre-
ceding the ban.
XII. Therapies for Endothelial Dysfunction
As discussed at length earlier, endothelial dysfunction
can be manifest as a triggering event for disease processes
(i.e., a contributory factor to cause of disease), but can
equally be a later manifestation of disease progression that
worsens the impact (i.e., an effect of disease that is never-
theless an important facet in severity). The multifactorial
nature of cardiovascular diseases necessarily means that
treating a single aspect is unlikely to have a significant im-
pact on outcome. This concept is highlighted by the fact
that diseases like heart failure and hypertension are treated
with combinations of drugs (e.g., angiotensin converting
enzyme inhibitors/?-blockers, calcium channel blockers),
some of which themselves have multiple effects (e.g., ACE
inhibitors reduce blood volume, reduce peripheral vascu-
lar resistance, have long-term benefits in terms of cardiac
and vascular remodelling, and might be likely to have an
impact on oxidative stress by reducing NOX activity). En-
dothelial dysfunction is not an innocent bystander in any
of the disease processes detailed and therefore represents
a legitimate target for drugs that would act in concert with
existing therapies to reduce both the cause and effect of
A. Current: statins (pleiotropic effects)
Given the wealth of evidence supporting a role for endo-
thelial dysfunction in one form or another in a range of car-
diovascular conditions, including atherosclerotic disease, it
is perhaps surprising that few of the ongoing major drug tri-
als in atherosclerosis specifically target the endothelium, al-
though arguably, most might have an indirect impact on the
endothelium (326). The same is true for the existing therapy
for atherosclerosis and the related clinical conditions: statins.
This group of compounds inhibits 3-hydrox-3-methylglu-
taryl coenzyme A (HMG-CoA), which is involved in the en-
dogenous synthesis of cholesterol. Whereas lipid-lowering is
the primary target of statins, it has since been established
that they also mediate a number of so-called “pleiotropic ef-
fects.” Improved endothelial function (232) is one of these
effects, alongside antioxidant, antiplatelet, and antiinflam-
matory actions. Amelioration of endothelial dysfunction is
mediated by a number of processes, including interference
with pathways associated with oxidative stress [for review,
see (271)]. As yet, it is unclear just how much impact the
pleiotropic effects have on clinical outcome over and above
the primary benefit of lipid-lowering, but the beneficial ef-
fects of statins on endothelial function at least raise the pos-
sibility of this feature of cardiovascular disease being a le-
gitimate therapeutic target.
To date, endothelial dysfunction has not been recognized
as a primary therapeutic target in other cardiovascular dis-
eases (e.g., hypertension and heart failure), in which con-
ventional therapies concentrate on reducing volume over-
load through diuresis (diuretics) and renin–angiotensin
system–mediated effects either directly (ACE inhibitors, an-
giotensin-receptor antagonists) or indirectly via ?-adreno-
ceptor antagonism (?-blockers), although direct vasodilators
(calcium channel blockers, GTN) also have some merit in this
arena. It is not yet clear whether any of these conventional
treatments actually has an impact on improving endothelial
function; for example, the evidence relating to the effects of
antihypertensive drugs on endothelial function is conflicting
(112, 303). Given the increased interest in the profile of en-
dothelial dysfunction in these two conditions over the last
few years, it is possible that drugs specifically targeted at the
endothelium might evolve, although the market is already
LE BROCQ ET AL.1654
B. Possible future treatments for eNOS dysfunction
A number of different strategies might be adopted to over-
come the various forms of eNOS dysfunction. Ever since L-
arginine was discovered as the precursor of NO, its poten-
tial as a therapeutic agent to boost NO production has been
predicted. However, the obvious flaw in the hypothesis is
that, for supplementation of L-arginine to be effective, en-
dothelial L-arginine levels would have to be depleted to such
an extent for the enzyme to malfunction: this is particularly
unlikely in the case of eNOS, in which enzyme activity is
typically low and therefore only requires low levels of L-argi-
nine. However, unlikely as it is that L-arginine is deficient in
endothelial cells, many studies have shown a benefit of
L-arginine supplementation in terms of endothelial function
in a range of cardiovascular disease states (242). A number
of theories have been developed to explain this “arginine
paradox,” including proposals that high arginine is simply
acting in an antioxidant capacity. However, the most plau-
sible explanation has evolved since the recent discovery of
asymmetric dimethyl-L-arginine (ADMA), an endogenous
L-arginine analogue that can inhibit NOS [for review, see
(28)] and contribute to oxidative stress [reviewed in (29,
239)], possibly through invoking an imbalance in NO/O2?
generation from NOS itself (61). By the law of mass action,
increased L-arginine levels might act effectively to compete
with ADMA at both the Y?transporter responsible for its
uptake into cells and at NOS itself (Fig. 7). Conflicting re-
sults regarding the therapeutic benefit of L-arginine (or oth-
erwise) mean that the evidence is unclear as to whether, or
to what extent, L-arginine supplements might help in car-
diovascular disease. In the opinion of the authors of this re-
view, any benefits that might accrue from L-arginine sup-
plementation are not likely due to simply replenishing a
shortfall in eNOS substrate. Benefit might be achieved, how-
ever, in cases in which ADMA is a factor or, perhaps, where
dysfunctional iNOS is chronically expressed and is depleted
of substrate on account of its rapid turnover. What is clear
is that the interaction of L-arginine with NOS is dependent
as much on the activity of the enzyme responsible for its con-
version to ornithine, arginase (102), and on the synthesis and
metabolic pathways (e.g., dimethylarginine dimethylamino-
hydrolase; DDAH) relating to ADMA (23) as it is on the
plasma concentration of L-arginine (Fig. 7).
Although the precise role of BH4in NOS is still not com-
pletely clear, its importance in preventing NOS dysfunction
is without question (4, 67, 213). It follows that agents that
might act to increase BH4bioavailability in endothelial cells
would act to improve eNOS function in those conditions in
which BH4deficiency is central to the dysfunction experi-
enced. Once again, in vitro studies using BH4supplementa-
tion have shown promise in several cardiovascular disease
models. However, the therapeutic potential of this agent is
limited by its poor oral absorption and bioavailability (120),
and a number of strategies are in development to deliver
bioactive BH4analogues (350, 442) for this purpose. Alterna-
tively, as we learn more about the synthesis and metabolism
of BH4[Fig. 6; see (294) for review], new therapeutic avenues
might emerge that could exploit these pathways to maintain
or replenish BH4in individuals with depleted levels. One such
avenue is already emerging: homocysteine has long been rec-
ognized to have an impact on endothelial function, but the
reason for its detrimental effects was not obvious at the out-
set. It has emerged, however, that homocysteine reduces the
bioavailability of BH4, either via direct interaction or through
inhibition of one of the enzymes [sepiapterin reductase (SR)]
responsible for its synthesis (Fig. 7) (294). Reduction of ho-
mocysteine levels through administration of folate might,
somewhat surprisingly, increase BH4levels.
C. Nitric oxide and carbon monoxide donor drugs
Given that depressed bioavailability of NO is a prominent
cause of endothelial dysfunction, it follows that replacement
of NO from exogenous sources could be of great benefit in
ameliorating disease progression. Organic nitrates have long
been used in symptomatic treatment of angina. Strangely,
however, most studies indicate that organic nitrates do not
actually reduce mortality in patients. Since the discovery of
NO as a crucial mediator in the cardiovascular system dur-
ing the 1980s, it was widely anticipated that a wide range of
NO-donor drugs would evolve for use in cardiovascular dis-
ease. No new drugs in this class have been licensed (283,
289), prompting speculation that this is a flawed strategy.
Part of the issue surrounds the inability to target NO suit-
ably to areas of endothelial dysfunction without incurring
overwhelming vasodilatation and hypotension. Some strate-
gies are beginning to evolve for targeted NO delivery; in-
haled NO is now routinely used to help alleviate breathing
difficulties in neonates and has been proposed as a means of
limiting the effects of pulmonary hypertension (189). In ad-
dition, a number of new donor entities are in development
that effect targeted NO delivery, primarily through incor-
poration in materials used in stents and other devices that
might come into contact with the vascular wall and contrib-
ute to endothelial dysfunction (289). Despite the setbacks in
NO-based drug therapies, hopes remain high that novel
agents might yet supersede organic nitrates in protection
from, or reversal of, endothelial dysfunction.
Research into a therapeutic role for CO is still in its in-
fancy, but several CO-donor drugs have been developed
(379) and have been shown to have an impact in isch-
emia–reperfusion injury and transplant [reviewed in (306)].
Inhaled CO has also been shown to have some merit in re-
ducing arterial thrombus formation (429).
D. Phosphodiesterase inhibitors and activators of
Downstream modulators of the NO/guanylate cyclase
pathway are receiving considerable attention. Sildenafil (Vi-
agra) is a phosphodiesterase V (PDE V) inhibitor that acts to
depress cGMP breakdown by this enzyme. It was originally
developed in the wake of the discovery of the NO/sGC path-
way with a view to its use in cardiovascular conditions.
Thanks to the prevalence of PDE V in the corpus cavernosal
tissue of the penis, its development was rapidly redirected
to the lucrative market of sexual dysfunction. However, at-
tention is now reverting to the possible benefits of agents
like sildenafil that act to protect cGMP or non-NO activators
of sGC [e.g., Bay41-2272 (393)] as therapeutic agents for car-
diovascular disease. Sildenafil has potentially important ef-
fects on vascular reactivity through improved endothelial
function in the peripheral vasculature of otherwise healthy
cigarette smokers (215), as well as in patients with chronic
heart failure (286) and in the coronary circulation of patients
with coronary heart disease (160), although other studies
show no effect on endothelial function (354). Nevertheless,
treatment with sildenafil has shown promising benefits in
patients with chronic heart failure (203) and pulmonary hy-
pertension (286), alone or in combination with inhaled NO
(396), or the PGI2analogue, iloprost (463).
Prostanoid therapies have failed to make the anticipated
impact on cardiovascular disease. Pulmonary hypertension
is the most evident cardiovascular condition in which
prostanoid vasodilators, such as PGI2 and iloprost (463),
have come to the fore as credible therapeutic agents.
F. Antioxidant therapies
Given the key role of oxidative stress in many cardiovas-
cular conditions, use of broad-spectrum antioxidants might
be expected to be effective therapeutics. The in vitro data are
very encouraging for a number of natural antioxidants, in-
cluding vitamins A, C, and E, thiols [e.g., N-acetylcysteine
(NAC)], and plant-derived polyphenols (e.g., flavonoids).
The promise of such antioxidants is further supported in clin-
ical studies using standard measures of endothelial function
(157), but, unfortunately, the large clinical trials so far con-
ducted with vitamin A (174, 224, 229, 352, 419) and vitamin
C (26) have shown no clear beneficial effects. Vitamin E at
least showed some benefit in one trial (400), but others have
failed to show a clinical benefit, even in high-risk patient
populations (157, 310, 400, 482, 483). The reason(s) behind
the failure of dietary antioxidant vitamins to have the ex-
pected beneficial effect, given the overwhelming data to sup-
port antioxidants from preclinical and small-scale clinical
studies, is(are) not clear. Low doses of vitamin could explain
some of the negative effects, but even in trials of higher
doses, no positive effect was seen; the lack of effect of vita-
mins A, C, and E on cardiovascular outcomes appears to be
a consistent finding in these well-conducted large-scale clin-
ical trials. However, the effects of antioxidant vitamins may
be subtle, and investigating crude outcome measures such
as death and cardiovascular events may miss subtle benefi-
cial effects on endothelial function. Equally, the benefits
might be seen only with prolonged (life-long) treatment. Fur-
thermore, dietary vitamins, although attractive because of
their relatively low cost and high tolerability, might not be
the most suitable antioxidants for this target. Some evidence
suggests that antioxidant supplements do not necessarily
mimic the effects of whole-fruit/vegetable dietary interven-
tions, perhaps suggesting that other fruit- and vegetable-de-
rived agents are important or that either additive or syner-
gistic effects of fruit and vegetable-derived nutrients may
accrue [for reviews, see (311,419)]. Perhaps, therefore, we are
seeking a “silver bullet” that does not exist and, rather than
looking for an antioxidant supplement “quick fix” for our
inherently unhealthy lifestyle, we should default to achiev-
ing overall dietary improvements that might reduce oxida-
tive stress in the first place. What is clear to date is that in-
sufficient evidence exists to support the hypothesis that oral
vitamin supplementation is protective against cardiovascu-
lar disease in well-nourished populations (i.e., not deficient
in these vitamins). However, it is worth remembering that,
although dietary vitamins might not offer the whole answer,
an enormous amount of work is ongoing to suggest that
polyphenolic dietary antioxidants (e.g., resveratrol in red
wine and berries and catechin and epicatechin in chocolate)
might yet prove beneficial (122, 201), whereas other means
of enhancing endogenous antioxidant defenses (e.g., gene
transfer for increased expression of antioxidant enzymes)
might also prove an effective therapeutic approach (see
G. Endothelin antagonists
Intensive research has been performed into the clinical de-
velopment of several endothelin antagonists (345). However,
to date, these efforts have been disappointing in most clini-
cal conditions, with the exception of primary pulmonary hy-
pertension. Both selective ETAand combined ETA/Bhave
been investigated, but the comparative effects of these in clin-
ical trials remains unknown. The major issues with en-
dothelin antagonists to date have been the lack of significant
mortality benefits, elevation of hepatic transaminases in clin-
ical studies, and the potential for teratogenicity.
Several endothelin antagonists have delivered important
blood pressure–lowering effects in patients with systemic hy-
pertension, although given the large number of drugs already
available for this condition, they have not gained a clinical li-
cense. However, the combined ETA/Bendothelin antagonist,
bosentan, has proven clinical benefits in patients with primary
pulmonary hypertension (20) and, because of the lack of al-
ternatives, has gained a clinical license for use in these pa-
tients. To date the results in most clinical studies in acute and
chronic heart failure have been disappointing (14). Further-
more, concerns about teratogenicity with endothelin antago-
nists are likely to limit their future clinical use. Studies of en-
dothelin-converting enzyme (ECE) and combined neutral
endopeptidase (NEP) inhibitors are continuing, but their fu-
ture clinical usefulness is currently unknown.
H. Gene therapies
Endothelial dysfunction is a prime target for gene based
therapies, not least because of the accessibility of endothe-
lial cells to blood-borne agents. Gene targeting can be en-
hanced by using endothelial cell–specific promoter se-
quences to drive expression of transgenes delivered in
adenoviral vectors; flt-1 and ICAM-2 are among the most
specific promoters used to date (316). Alternative methods
of improving specificity of adenoviral vector–derived gene
transfer for endothelial cells include replacement of retro-
virus long terminal repeat with regulatory sequences from
human promoters of endothelium-specific proteins (e.g., pre-
proendothelin-1, von Willebrand factor) or genetically alter-
ing the vector itself to target endothelial cells (192, 276, 346).
Adenoviral vectors are generally regarded to be more effi-
cient means of transfer than delivery of naked plasmids or
those in cationic liposomes (285). A number of cellular tar-
gets exist for gene therapy–mediated upregulation of pro-
tein expression, most notable of which are eNOS (1, 68), en-
zymes that contribute to antioxidant defences [e.g., SOD (93,
119, 236, 250)], enzymes involved in BH4modulation (4, 5,
59), or fibrinolytic proteins (t-PA). An alternative strategy
that has been used successfully in animal models of myo-
LE BROCQ ET AL.1656
cardial infarction is to prevent the pro-inflammatory, pro-
oxidant activation of endothelial cells through activation of
NF-?B (37, 365). This can be achieved by delivery of a “de-
coy” oligonucleotide bearing the consensus binding se-
quence of NF-?B (296). The current limitations to these tech-
niques surround the effective delivery of sufficient gene
copies by a practical means for human use, but the concept
holds considerable promise if these issues can be overcome.
I. Endothelial cell–based therapies
Another approach involves use of autologous endothelial
progenitor cells (EPCs). These bone marrow–derived cells
express many endothelial cell markers and are freely circu-
lating in humans. Their isolation and culture from blood
samples is fairly straightforward, and they are highly
amenable to genetic modification through adenoviral vec-
tor–mediated gene transfer (13, 156). The concept is to iso-
late, culture, and modify endothelial cells from patients with
diseases associated with endothelial injury before reinjecting
them, in the hope that they will effect a repair to areas where
the endothelium is damaged. This approach could be par-
ticularly useful in aiding re-endothelialization after inter-
ventional procedures like angioplasty or stenting and has
also shown promise in formation of new vessels in ischemic
tissue. The number of circulating EPCs is typically very low
and is further depressed in a range of cardiovascular dis-
eases (103), but they appear to be mobilized in response to
vascular endothelial growth factor (VEGF) and granulocyte
colony-stimulating factor (G-CSF). Therapeutic elevation of
circulating EPC numbers might also be an alternative strat-
egy to helping endothelial repair. As with gene therapy, the
use of EPCs for treatment of specific conditions associated
with endothelial injury shows promise, even in small-scale
clinical studies using unmodified autologous EPCs [for re-
view, see (103)].
In view of the very wide range of functions carried out by
the endothelium, it follows that “endothelial dysfunction” is
a term that applies to an equally wide range of endothelium-
related aspects that might be considered abnormal. In the lit-
erature, it is usual for only a single parameter to be assessed
as a measure of endothelial dysfunction. Vascular response
to an endothelium-dependent vasodilator (e.g., ACh or
bradykinin) is by far the most common approach to assess-
ment of endothelial dysfunction in vitro, in vivo, and in clin-
ical studies, but this will identify only one form of the phe-
nomenon. Equally, such an approach will not provide any
information as to the reason for reduced endothelial activity
(e.g., reduced NOS expression, dysfunctional NOS, lack of
BH4, increased ADMA, increased oxidative stress, increased
ET-1, reduced prostaglandin synthesis, or EDHF activity). In
animal models, an initial observation of endothelial dys-
function can be followed up by mechanistic experiments in
vitro and in knockout models to dissect the likely cause(s) of
the effect; deeper exploration in clinical studies is consider-
ably more difficult.
Endothelial dysfunction has emerged as a contributory
factor in a wide range of cardiovascular diseases. However,
the means by which dysfunction is defined and measured
vary greatly between researchers and specific diseases. Thus,
endothelial dysfunction in atherosclerosis is driven by dif-
ferent processes, with different measurable outcomes than
that in, for example, hypertension. Whereas the term is uni-
fying in the sense that it identifies the endothelium as a cen-
tral player in many disease processes, it does not identify a
single unifying process that underlies different cardiovascu-
lar disease and, therefore, does not point to a single thera-
peutic strategy that might encompass different diseases. It is
essential, therefore, for researchers first to identify the type
of endothelial dysfunction that applies to the disease of in-
terest (e.g., is the endothelium physically removed or in-
jured? Is NOS dysfunctional? Is endothelin upregulated? Is
oxidative stress a factor? Does inflammation play a role?),
before deciding what therapeutic approach might be bene-
ficial. That said, now an enormous array of therapeutic op-
tions is available to target each of the specific factors that
might combine to constitute endothelial dysfunction. Both
pharmaceutic and neutraceutic agents are under intense
scrutiny in a drive to combat oxidative stress or to supple-
ment or replace NO, or both, whereas gene therapy and EPC
supplementation are perhaps therapeutic strategies to watch
in the future. However, the diseases in which endothelial
dysfunction contributes are all multifactorial and, although
therapies that target this facet of disease progression might
work well as an adjunct to conventional therapies, it seems
unlikely that they represent a “cure” in their own right. In
our opinion, endothelial dysfunction is a classic example of
the “prevention is better than cure” adage, in that the clas-
sic lifestyle changes that are advocated for health (e.g., exer-
cise, good diet, smoking cessation, and weight management)
are bound to have a significant and profound impact on en-
dothelial function. Moreover, the earlier lifestyle changes are
implemented, the more likely the benefit in terms of limit-
ing the endothelial dysfunction that is recognized as a key
early event in atherogenesis. Given that dysfunction is a nat-
ural aging process, it seems unlikely that we can altogether
prevent it. However, it is within each individual’s power to
limit the damaging effects though lifestyle improvements.
AA, Arachidonic acid; Ach, acetylcholine; ADMA, asym-
metric dimethyl-L-arginine; Ang II, angiotensin II; AC,
adenylate cyclase; AT1, angiotensin receptor; BH4, tetrahy-
drobiopterin; BK, bradykinin; cAMP, cyclic adenosine
monophosphate; CB, cannabinoid receptor; cGMP, cyclic
guanosine monophosphate; CO, carbon monoxide; COX, cy-
clooxygenase; CYP, cytochrome P450; DHFR, dihydrofolate
reductase; EETs, epoxyeicosatrienoic acids; ET-1, endothelin
1; ETA/B, endothelin A and B receptors; EDHF, endothelium-
derived hyperpolarizng factor; GCL, glutamate-cysteine lig-
ase; GPx, glutathione peroxidase; GPCR, G protein–coupled
receptor; GR, glutathione reductase; GS, glutathione syn-
thase; GSH, glutathione; GSSG, glutathione (oxidized form);
GTP, guanosine triphosphate; GTPCH, GTP cyclohydrolase;
HCys, homocyst(e)ine; HO, heme oxygenase; NO, nitric ox-
ide; NOS, nitric oxide synthase; NOX, NAD(P)H oxidase;
PGI2, prostacyclin; PGIS, prostaglandin I2synthase; PGR,
prostaglandin receptor; PKs, protein kinases; PLA2,
phopholipase A2; PPAR-?,_peroxisome proliferator–acti-
vated receptor-?; PTPS, 6-pyruvoyltetrahydrobiopterin syn-
thase; ROS, reactive oxygen species; sGC, soluble guanylate
cyclase; SOD, superoxide dismutase; SR, sepiapterin reduc-
tase; tPA, tissue plasminogen activator; VGCC, voltage-
gated Ca2?channel; X-DH, xanthine dehydrogenase; XO,
1. Alexander MY, Brosnan MJ, Hamilton CA, Fennell
JP, Beattie EC, Jardine E, Heistad DD, and Dominiczak
AF. Gene transfer of endothelial nitric oxide synthase but
not Cu/Zn superoxide dismutase restores nitric oxide
availability in the SHRSP. Cardiovasc Res 47: 609–617,
2. Al-Mehdi AB, Zhao G, Dodia C, Tozawa K, Costa KVM,
Ross C, Blecha F, Dinauer M, and Fisher AB. Endothelial
NADPH oxidase as the source of oxidants in lungs exposed
to ischemia or high K?. Circ Res 83: 730–737, 1998.
3. Alonso R, Mata P, De Andres R, Villacastin BP, Martinez-
Gonzalez J, and Badimon L. Sustained long-term im-
provement of arterial endothelial function in heterozygous
familial hypercholesterolemia patients treated with sim-
vastatin. Atherosclerosis 157: 423–429, 2001.
4. Alp NJ and Channon KM. Regulation of endothelial nitric
oxide synthase by tetrahydrobiopterin in vascular disease.
Arterioscler Thromb Vasc Biol 24: 413–420, 2004.
5. Alp NJ, Mussa S, Khoo J, Cai S, Guzik T, Jefferson A, Goh
N, Rockett KA, and Channon KM. Tetrahydrobiopterin-de-
pendent preservation of nitric oxide-mediated endothelial
function in diabetes by targeted transgenic GTP-cyclohy-
drolase I overexpression. J Clin Invest 112: 725–735, 2003.
6. Anderson TJ, Elstein E, Haber H, and Charbonneau F. Com-
parative study of ACE-inhibition, angiotensin II antago-
nism, and calcium channel blockade on flow-mediated va-
sodilation in patients with coronary disease (BANFF
study). J Am Coll Cardiol 35: 60–66, 2000.
7. Anderson TJ, Uehata A, Gerhard MD, Meredith IT, Knab
S, Delagrange D, Lieberman EH, Ganz P, Creager MA, and
Yeung AC. Close relation of endothelial function in the
human coronary and peripheral circulations. J Am Coll Car-
diol 26: 1235–1241, 1995.
8. Angus JA and Wright CE. Techniques to study the phar-
macodynamics of isolated large and small blood vessels. J
Pharmacol Toxicol Methods 44: 395–407, 2000.
9. Antikainen R, Jousilahti P, and Tuomilehto J. Systolic blood
pressure, isolated systolic hypertension and risk of coro-
nary heart disease, strokes, cardiovascular disease and all-
cause mortality in the middle-aged population. J Hypertens
16: 577–583, 1998.
10. Araki N, Higashi T, Mori T, Shibayama R, Kawabe Y, Ko-
dama T, Takahashi K, Shichiri M, and Horiuchi S. Macro-
phage scavenger receptor mediates the endocytic uptake
and degradation of advanced glycation end products of the
Maillard reaction. Eur J Biochem 230: 408–415, 1995.
11. Arbustini E, Dal Bello B, Morbini P, Burke AP, Bocciarelli
M, Specchia G, and Virmani R. Plaque erosion is a major
substrate for coronary thrombosis in acute myocardial in-
farction. Heart 82: 269–272, 1999.
12. Armstrong EJ, Morrow DA, and Sabatine MS. Inflamma-
tory biomarkers in acute coronary syndromes, part II:
acute-phase reactants and biomarkers of endothelial cell ac-
tivation. Circulation 113: e152–e155, 2006.
13. Asahara T, Murohara T, Sullivan A, Silver M, van der Zee
R, Li T, Witzenbichler B, Schatteman G, and Isner JM. Iso-
lation of putative progenitor endothelial cells for angio-
genesis. Science 275: 964–967, 1997.
14. Attina T, Camidge R, Newby DE, and Webb DJ. Endothe-
lin antagonism in pulmonary hypertension, heart failure,
and beyond. Heart 91: 825–831, 2005.
15. Badesch DB, Orton EC, Zapp LM, Westcott JY, Hester J,
Voelkel NF, and Stenmark KR. Decreased arterial wall
prostaglandin production in neonatal calves with severe
chronic pulmonary hypertension. Am J Resp Cell Mol Biol
1: 489–498, 1989.
16. Banes-Berceli AK, Ogobi S, Tawfik A, Patel B, Shirley A,
Pollock DM, Fulton D, and Marrero MB. Endothelin-1 ac-
tivation of JAK2 in vascular smooth muscle cells involves
NAD(P)H oxidase-derived reactive oxygen species. Vasc
Pharmacol 43: 310–319, 2005.
17. Barnes PJ and Karin M. Nuclear factor-kappaB: a pivotal
transcription factor in chronic inflammatory diseases. N
Engl J Med 336: 1066–1071, 1997.
18. Barton M, Cosentino F, Brandes RP, Moreau P, Shaw S, and
Luscher TF. Anatomic heterogeneity of vascular aging: role of
nitric oxide and endothelin. Hypertension 30: 817–824, 1997.
19. Basuroy S, Bhattacharya S, Tcheranova D, Qu Y, Regan RF,
Leffler CW, and Parfenova H. HO-2 provides endogenous
protection against oxidative stress and apoptosis caused by
TNF-alpha in cerebral vascular endothelial cells. Am J Phys-
iol Cell Physiol 291: C897–C908, 2006.
20. Bauer M, Wilkens H, Langer F, Schneider SO, Lausberg H,
and Schafers HJ. Selective upregulation of endothelin B re-
ceptor gene expression in severe pulmonary hypertension.
Circulation 105: 1034–1036, 2002.
21. Beckman KB and Ames BN. The free radical theory of ag-
ing matures. Physiol Rev 78: 547–581, 1998.
22. Beishuizen ED, Tamsma JT, Jukema JW, van de Ree MA,
van der Vijver JC, Meinders AE, and Huisman MV. The ef-
fect of statin therapy on endothelial function in type 2 di-
abetes without manifest cardiovascular disease. Diabetes
Care 28: 1668–1674, 2005.
23. Beltowski J and Kedra A. Asymmetric dimethylarginine
(ADMA) as a target for pharmacotherapy. Pharmacol Rep
58: 159–178, 2006.
24. Berne RM. The role of adenosine in the regulation of coro-
nary blood flow. Circ Res 47: 807–813, 1980.
25. Bierhaus A, Chevion S, Chevion M, Hofmann M, Quehen-
berger P, Illmer T, Luther T, Berentshtein E, Tritschler H,
Muller M, Wahl P, Ziegler R, and Nawroth PP. Advanced
glycation end product-induced activation of NF-kappaB is
suppressed by alpha-lipoic acid in cultured endothelial
cells. Diabetes 46: 1481–1490, 1997.
26. Blot WJ, Li JY, Taylor PR, Guo W, Dawsey S, Wang GQ,
Yang CS, Zheng SF, Gail M, Li GY, Yu Y, Liu B, Tangrea J,
Sun YH, Liu F, Fraumeni JF, Zhang YH Jr, and Li B. Nu-
trition intervention trials in Linxian, China: supplementa-
tion with specific vitamin/mineral combinations, cancer
incidence and disease-specific mortality in the general pop-
ulation. J Natl Cancer Inst 85: 1483–1492, 1993.
27. Blows LJ and Redwood SR. The pressure wire in practice.
Heart 93: 419–422, 2007.
28. Boger RH. Asymmetric dimethylarginine, an endogenous
inhibitor of nitric oxide synthase, explains the “L-arginine
paradox” and acts as a novel cardiovascular risk factor. J
Nutr 134: 2842S–2847S; discussion 2853S, 2004.
29. Boger RH, Vallance P, and Cooke JP. Asymmetric di-
methylarginine (ADMA): a key regulator of nitric oxide
synthase. Atherosclerosis Suppl 4: 1–3, 2003.
30. Bollinger A, Hoffmann U, and Franzeck UK. Microvascu-
lar changes in arterial occlusive disease: target for phar-
macotherapy. Vasc Med 1: 50–54, 1996.
LE BROCQ ET AL.1658
31. Boo YC and Jo H. Flow-dependent regulation of endothe-
lial nitric oxide synthase: role of protein kinases. Am J Phys-
iol Cell Physiol 285: C499–C508, 2003.
32. Botti H, Trostchansky A, Batthyany C, and Rubbo H. Re-
activity of peroxynitrite and nitric oxide with LDL. IUBMB
Life 57: 407–412, 2005.
33. Boulanger CM, Morrison KJ, and Vanhoutte PM. Media-
tion by M3-muscarinic receptors of both endothelium-de-
pendent contraction and relaxation to acetylcholine in the
aorta of the spontaneously hypertensive rat. Br J Pharmacol
112: 519–524, 1994.
34. Bouloumie A, Schini-Kerth VB, and Busse R. Vascular en-
dothelial growth factor up-regulates nitric oxide synthase
in endothelial cells. Cardiovasc Res 41: 773–780, 1999.
35. Bowers R, Cool C, Murphy RC, Tuder RM, Hopken MW,
Flores SC, and Voelkel NF. Oxidative stress in severe pul-
monary hypertension. Am J Respir Crit Care Med 169:
36. Bowie AG, Moynagh PN, and O’Neill LA. Lipid peroxida-
tion is involved in the activation of NF-kappaB by tumor
necrosis factor but not interleukin-1 in the human endo-
thelial cell line ECV304: lack of involvement of H2O2in NF-
kappaB activation by either cytokine in both primary and
transformed endothelial cells. J Biol Chem 272: 25941–25950,
37. Boyle EM Jr, Canty TG Jr, Morgan EN, Yun W, Pohlman
TH, and Verrier ED. Treating myocardial ischemia-reper-
fusion injury by targeting endothelial cell transcription.
Ann Thorac Surg 68: 1949–1953, 1999.
38. Brand K, Page S, Rogler G, Bartsch A, Brandl R, Knuechel
R, Page M, Kaltschmidt C, Baeuerle PA, and Neumeier D.
Activated transcription factor nuclear factor-kappa B is
present in the atherosclerotic lesion. J Clin Invest 97:
39. Brandes RP, Schmitz-Winnenthal FH, Feletou M, Godecke
A, Huang PL, Vanhoutte PM, Fleming I, and Busse R. An
endothelium-derived hyperpolarizing factor distinct from
NO and prostacyclin is a major endothelium-dependent va-
sodilator in resistance vessels of wild-type and endothelial
NO synthase knockout mice. Proc Natl Acad Sci U S A 97:
40. Brandt R and Krantz S. Glycated albumin (Amadori prod-
uct) induces activation of MAP kinases in monocyte-like
MonoMac 6 cells. Biochim Biophys Acta 1760: 1749–1753,
41. Brandt R, Nawka M, Kellermann J, Salazar R, Becher D,
and Krantz S. Nucleophosmin is a component of the fruc-
toselysine-specific receptor in cell membranes of Mono Mac
6 and U937 monocyte-like cells. Biochim Biophys Acta 1670:
42. Brilla CG, Rupp H, Funck R, and Maisch B. The renin-an-
giotensin-aldosterone system and myocardial collagen ma-
trix remodelling in congestive heart failure. Eur Heart J 16:
43. Brook RD, Brook JR, and Rajagopalan S. Air pollution: the
“Heart” of the problem. Curr Hypertens Rep 5: 32–39, 2003.
44. Broten TP, Miyashiro JK, Moncada S, and Feigl EO. Role
of endothelium-derived relaxing factor in parasympathetic
coronary vasodilation. Am J Physiol 262: H1579–H1584,
45. Brouard S, Berberat PO, Tobiasch E, Seldon MP, Bach FH,
and Soares MP. Heme oxygenase-1-derived carbon monox-
ide requires the activation of transcription factor NF-kappa
B to protect endothelial cells from tumor necrosis factor-al-
pha-mediated apoptosis. J Biol Chem 277: 17950–17961, 2002.
46. Brownlee M. Biochemistry and molecular cell biology of
diabetic complications. Nature 414: 813–820, 2001.
47. Bryan NS, Fernandez BO, Bauer SM, Garcia-Saura MF, Mil-
som AB, Rassaf T, Maloney RE, Bharti A, Rodriguez J, and
Feelisch M. Nitrite is a signaling molecule and regulator of
gene expression in mammalian tissues. Nat Chem Biol 1:
48. Bucala R, Makita Z, Koschinsky T, Cerami A, and Vlassara
H. Lipid advanced glycosylation: pathway for lipid oxida-
tion in vivo. Proc Natl Acad Sci U S A 90: 14: 6434–6438,
49. Bucala R, Tracey KJ, and Cerami A. Advanced glycosyla-
tion products quench nitric oxide and mediate defective en-
dothelium-dependent vasodilation in experimental dia-
betes. J Clin Invest 87: 432–438, 1991.
50. Budhiraja R, Tuder RM, and Hassoun PM. Endothelial dys-
function in pulmonary hypertension. Circulation 109:
51. Bull TM, Golpon H, Hebbel RP, Solovey A, Cool CD, Tuder
RM, Geraci MW, and Voelkel NF. Circulating endothelial
cells in pulmonary hypertension. Thromb Haemost 90:
52. Bunning P, Budek W, Escher R, and Schonherr E. Charac-
teristics of angiotensin converting enzyme and its role in
the metabolism of angiotensin I by endothelium. J Cardio-
vasc Pharmacol 8: S52–S57, 1986.
53. Burney S, Niles JC, Dedon PC, and Tannenbaum SR. DNA
damage in deoxynucleosides and oligonucleotides treated
with peroxynitrite. Chem Res Toxicol 12: 513–520, 1999.
54. Burnham MP, Bychkov R, Feletou M, Richards GR, Van-
houtte PM, Weston AH, and Edwards G. Characteriza-
tion of an apamin-sensitive small-conductance Ca(2?)-
activated K? channel in porcine coronary artery endothe-
lium: relevance to EDHF. Br J Pharmacol 135: 1133–1143,
55. Busse R and Fleming I. Pulsatile stretch and shear stress:
physical stimuli determining the production of endothe-
lium-derived relaxing factors. J Vasc Res 35: 73–84, 1998.
56. Busse R, Forstermann U, Matsuda H, and Pohl U. The role
of prostaglandins in the endothelium-mediated vasodila-
tory response to hypoxia. Pflugers Arch 401: 77–83, 1984.
57. Bychkov R, Burnham MP, Richards GR, Edwards G, We-
ston AH, Feletou M, and Vanhoutte PM. Characterization
of a charybdotoxin-sensitive intermediate conductance
Ca2?-activated K? channel in porcine coronary endothe-
lium: relevance to EDHF. Br J Pharmacol 137: 1346–1354,
58. Cai H and Harrison DG. Endothelial dysfunction in car-
diovascular diseases: the role of oxidant stress. Circ Res 87:
59. Cai S, Alp NJ, McDonald D, Smith I, Kay J, Canevari L,
Heales S, and Channon KM. GTP cyclohydrolase I gene
transfer augments intracellular tetrahydrobiopterin in hu-
man endothelial cells: effects on nitric oxide synthase ac-
tivity, protein levels and dimerisation. Cardiovasc Res 55:
60. Cardillo C, Campia U, Bryant MB, and Panza JA. Increased
activity of endogenous endothelin in patients with type II
diabetes mellitus. Circulation 106: 1783–1787, 2002.
61. Cardounel AJ, Xia Y, and Zweier JL. endogenous methy-
larginines modulate superoxide as well as nitric oxide gen-
eration from neuronal nitric-oxide synthase: differences in
the effects of monomethyl- and dimethylarginines in the
presence and absence of tetrahydrobiopterin. J Biol Chem
280: 7540–7549, 2005.
62. Celermajer DS, Sorensen KE, Gooch VM, Spiegelhalter DJ,
Miller OI, Sullivan ID, Lloyd JK, and Deanfield JE. Non-
invasive detection of endothelial dysfunction in children
and adults at risk of atherosclerosis. Lancet 340: 1111–1115,
63. Ceriello A and Motz E. Is oxidative stress the pathogenic
mechanism underlying insulin resistance, diabetes, and
cardiovascular disease? The common soil hypothesis re-
visited. Arterioscler Thromb Vasc Biol 24: 816–823, 2004.
64. Ceriello A, Taboga C, Tonutti L, Quagliaro L, Piconi L, Bais
B, Da Ros R, and Motz E. Evidence for an independent and
cumulative effect of postprandial hypertriglyceridemia and
hyperglycemia on endothelial dysfunction and oxidative
stress generation: effects of short- and long-term simvas-
tatin treatment. Circulation 106: 1211–1218, 2002.
65. Challah M, Nadaud S, Philippe M, Battle T, Soubrier F, Cor-
man B, and Michel JB. Circulating and cellular markers of
endothelial dysfunction with aging in rats. Am J Physiol 273:
66. Chance B, Sies H, and Boveris A. Hydroperoxide metabo-
lism in mammalian organs. Physiol Rev 59: 527–605, 1979.
67. Channon KM. Tetrahydrobiopterin: regulator of endothe-
lial nitric oxide synthase in vascular disease. Trends Car-
diovasc Med 14: 323–327, 2004.
68. Chen AF, Ren J, and Miao CY. Nitric oxide synthase gene
therapy for cardiovascular disease. Jpn J Pharmacol 89:
69. Chen XL, Varner SE, Rao AS, Grey JY, Thomas S, Cook CK,
Wasserman MA, Medford RM, Jaiswal AK, and Kunsch C.
Laminar flow induction of antioxidant response element-
mediated genes in endothelial cells: a novel anti-inflam-
matory mechanism. J Biol Chem 278: 703–711, 2003.
70. Cheng C, Tempel D, van Haperen R, van der Baan A,
Grosveld F, Daemen MJ, Krams R, and de Crom R. Ather-
osclerotic lesion size and vulnerability are determined by
patterns of fluid shear stress. Circulation 113: 2744–2753,
71. Chung HY, Song SH, Kim HJ, Ikeno Y, and Yu BP. Modu-
lation of renal xanthine oxidoreductase in aging: gene ex-
pression and reactive oxygen species generation. J Nutr
Health Aging 3: 19–23, 1999.
72. Cody RJ, Haas GJ, Binkley PF, Capers Q, and Kelley R.
Plasma endothelin correlates with the extent of pulmonary
hypertension in patients with chronic congestive heart fail-
ure. Circulation 85: 504–509, 1992.
73. Cohen RA. The endothelium-derived hyperpolarizing fac-
tor puzzle: a mechanism without a mediator? Circulation
111: 724–727, 2005.
74. Collins RG, Velji R, Guevara NV, Hicks MJ, Chan L, and
Beaudet AL. P-Selectin or intercellular adhesion molecule
(ICAM)-1 deficiency substantially protects against athero-
sclerosis in apolipoprotein E-deficient mice. J Exp Med 191:
75. Collins T and Cybulsky MI. NF-kappaB: pivotal mediator
or innocent bystander in atherogenesis? J Clin Invest 107:
76. Consentino F, Sill JC, and Katusië ZS. Role of superoxide
anions in the mediation of endothelium-dependent con-
tractions. Hypertension 23: 229–235, 1994.
77. Cooke JP, Singer AH, Tsao P, Zera P, Rowan RA, and
Billingham ME. Antiatherogenic effects of L-arginine in the
hypercholesterolemic rabbit. J Clin Invest 90: 1168–1172,
78. Cooper CJ, Landzberg MJ, Anderson TJ, Charbonneau F,
Creager MA, Ganz P, and Selwyn AP. Role of nitric oxide
in the local regulation of pulmonary vascular resistance in
humans. Circulation 93: 266–2671, 1996.
79. Costa NJ, Dahm CC, Hurrell F, Taylor ER, and Murphy
MP. The interactions of mitochondrial thiols with nitric ox-
ide. Antioxid Redox Signal 5: 291–305, 2003.
80. Cracowski JL, Minson CT, Salvat-Melis M, and Halliwill
JR. Methodological issues in the assessment of skin mi-
crovascular endothelial function in humans. Trends Phar-
macol Sci 27: 503–508, 2006.
81. Creager MA, Gallagher SJ, Girerd XJ, Coleman SM, Dzau
VJ, and Cooke JP. L-Arginine improves endothelium-de-
pendent vasodilation in hypercholesterolemic humans. J
Clin Invest 90: 1248–1253, 1992.
82. Crow J and Beckman J. The role of peroxynitrite in nitric
oxide-mediated toxicity. In: Koprowski H, Maeda H, eds.
The role of nitric oxide in physiology and pathophysiology.
Berlin: Springer-Verlag, 1995, pp 57–73.
83. Csiszar A, Smith KE, Koller A, Kaley G, Edwards JG, and
Ungvari Z. Regulation of bone morphogenetic protein-2 ex-
pression in endothelial cells: role of nuclear factor-kappaB
activation by tumor necrosis factor-alpha, H2O2, and high
intravascular pressure. Circulation 111: 2364–2372, 2005.
84. Csiszar A, Ungvari Z, Edwards JG, Kaminski P, Wolin MS,
Koller A, and Kaley G. Aging-induced phenotypic changes
and oxidative stress impair coronary arteriolar function.
Circ Res 90: 1159–1166, 2002.
85. Cybulsky MI, Iiyama K, Li H, Zhu S, Chen M, Iiyama M,
V D, Gutierrez-Ramos JC, Connelly PW, and Milstone DS.
A major role for VCAM-1, but not ICAM-1, in early ath-
erosclerosis. J Clin Invest 107: 1255–1262, 2001.
86. Daugherty A, Dunn JL, Rateri DL, and Heinecke JW.
Myeloperoxidase, a catalyst for lipoprotein oxidation, is ex-
pressed in human atherosclerotic lesions. J Clin Invest 94:
87. Davies MJ, Gordon JL, Gearing AJ, Pigott R, Woolf N, Katz
D, and Kyriakopoulos A. The expression of the adhesion
molecules ICAM-1, VCAM-1, PECAM, and E-selectin in
human atherosclerosis. J Pathol 171: 223–229, 1993.
88. Davignon J and Ganz P. Role of endothelial dysfunction in
atherosclerosis. Circulation 109: 11127–11132, 2004.
89. Davis ME, Cai H, Drummond GR, and Harrison DG. Shear
stress regulates endothelial nitric oxide synthase expres-
sion through c-Src by divergent signalling pathways. Circ
Res 89: 1073–1080, 2001.
90. De Agostini AI, Watkins SC, Slayter HS, Youssoufian H,
and Rosenberg RD. Localization of anticoagulantly active
heparin sulphate proteoglycans in vascular endothelium:
antithrombin binding on cultured endothelial cells and per-
fused rat aorta. J Cell Biol 111: 1293–1304, 1990.
91. Demiryurek AT, Karamsetty MR, McPhaden AR, Wads-
worth RM, Kane KA, and MacLean MR. Accumulation of
nitrotyrosine correlates with endothelial NO synthase in
pulmonary resistance arteries during chronic hypoxia in
the rat. Pulmon Pharmacol Ther 13: 157–165, 2000.
92. Devaraj S, Xu DY, and Jialal I. C-reactive protein increases
plasminogen activator inhibitor-1 expression and activity
in human aortic endothelial cells: implications for the meta-
bolic syndrome and atherothrombosis. Circulation 107:
93. Didion SP and Faraci FM. Ceramide-induced impairment
of endothelial function is prevented by CuZn superoxide
dismutase overexpression. Arterioscler Thromb Vasc Biol 25:
94. Didion SP, Kinzenbaw DA, and Faraci FM. Critical role for
CuZn-superoxide dismutase in preventing angiotensin II-
LE BROCQ ET AL.1660
induced endothelial dysfunction. Hypertension 46: 1147–
95. Donaldson K, Tran L, Jimenez LA, Duffin R, Newby DE,
Mills N, MacNee W, and Stone V. Combustion-derived
nanoparticles: a review of their toxicology following in-
halation exposure. Part Fibre Toxicol 2: 10–24, 2005.
96. Dong ZM, Brown AA, and Wagner DD. Prominent role of
P-selectin in the development of advanced atherosclerosis
in ApoE-deficient mice. Circulation 101: 2290–2295, 2000.
97. Doshi SN, McDowell IF, Moat SJ, Lang D, Newcombe RG,
Kredan MB, Lewis MJ, and Goodfellow J. Folate improves
endothelial function in coronary artery disease: an effect
mediated by reduction of intracellular superoxide? Arte-
rioscler Thromb Vasc Biol 21: 1196–1202, 2001.
98. Du XL, Edelstein D, Rossetti L, Fantus IG, Goldberg H,
Ziyadeh F, Wu J, and Brownlee M. Hyperglycaemia-in-
duced mitochondrial superoxide overproduction activates
the hexosamine pathways and induces plasminogen acti-
vator inhibitor-1 expression by increasing Sp1 glycosyla-
tion. Proc Nat Acad Sci U S A 97: 12222–12226, 2000.
99. Duda M, Konior A, Klemenska E, and Beresewicz A. Pre-
conditioning protects endothelium by preventing ET-1-in-
duced activation of NADPH oxidase and xanthine oxidase
in post-ischemic heart. J Mol Cell Cardiol 42: 400–410, 2007.
100. Dupuis J, Tardif JC, Cernacek P, and Theroux P. Choles-
terol reduction rapidly improves endothelial function after
acute coronary syndromes: the RECIFE (Reduction of Cho-
lesterol in Ischemia and Function of the Endothelium) trial.
Circulation 99: 3227–3233, 1999.
101. Durand E, Scoazec A, Lafont A, Boddaert J, Al Hajzen A,
Addad F, Mirshahi M, Desnos M, Tedgui A, and Mallat Z.
In vivo induction of endothelial apoptosis leads to vessel
thrombosis and endothelial denudation: a clue to the un-
derstanding of the mechanisms of thrombotic plaque ero-
sion. Circulation 109: 2503–2506, 2004.
102. Durante W, Johnson FK, and Johnson RA. Arginase: a crit-
ical regulator of nitric oxide synthesis and vascular func-
tion. Clin Exp Pharmacol Physiol 34: 906–911, 2007.
103. Dzau VJ, Gnecchi M, Pachori AS, Morello F, and Melo LG.
Therapeutic potential of endothelial progenitor cells in car-
diovascular diseases. Hypertension 46: 7–18, 2005.
104. Echtay KS, Murphy MP, Smith RA, Talbot DA, and Brand
MD. Superoxide activates mitochondrial uncoupling pro-
tein 2 from the matrix side: studies using targeted antiox-
idants. J Biol Chem 277: 47129–47135, 2002.
105. Eddahibi S, Raffestin B, Pham I, Launay JM, Aegerter P,
Sitbon M, and Adnot S. Treatment with 5-HT potentiates
development of pulmonary hypertension in chronically hy-
poxic rats. Am J Physiol 272: H1173–H1181, 1997.
106. Eddahibi S, Springall D, Mannan M, Carville C, Chabrier PE,
Levame M, Raffestin B, Polak J, and Adnot S. Dilator effect
of endothelins in pulmonary circulation: changes associated
with chronic hypoxia. Am J Physiol 265: L571–L580, 1993.
107. Eikelboom JW, Lonn E, Genest J Jr, Hankey G, and Yusuf
S. Homocyst(e)ine and cardiovascular disease: a critical re-
view of the epidemiologic evidence. Ann Intern Med 131:
108. Elmarakby AA, Loomis ED, Pollock JS, and Pollock DM.
NADPH oxidase inhibition attenuates oxidative stress but
not hypertension produced by chronic ET-1. Hypertension
45: 283–287, 2005.
109. Elmedal B, de Dam MY, Mulvany MJ, and Simonsen U.
The superoxide dismutase mimetic, tempol, blunts right
ventricular hypertrophy in chronic hypoxic rats. Br J Phar-
macol 141: 105–113, 2004.
110. Enos WF, Holmes RH, and Beyer J. Coronary disease
among United States soldiers killed in action in Korea: pre-
liminary report. JAMA 152: 1090–1093, 1953.
111. Ergul A, Johansen JS, Strømhaug C, Harris AK, Hutchin-
son J, Tawfik A, Rahimi A, Rhim E, Wells B, Caldwell RW,
and Anstadt MP. Vascular dysfunction of venous bypass
conduits is mediated by reactive oxygen species in diabetes:
role of endothelin-1. J Pharmacol Exp Ther 313: 70–77, 2005.
112. Erzen B, Gradisek P, Poredos P, and Sabovic M. Treatment
of essential arterial hypertension with enalapril does not
result in normalization of endothelial dysfunction of the
conduit arteries. Angiology 57: 187–192, 2006.
113. Evans JL, Goldfine ID, Maddux BA, and Grodsky GM. Ox-
idative stress and stress-activated signaling pathways: a
unifying hypothesis of type 2 diabetes. Endocr Rev 23: 599–
114. Everson WV and Smart EJ. Influence of caveolin, choles-
terol, and lipoproteins on nitric oxide synthase: implica-
tions for vascular disease. Trends Cardiovasc Med 11: 246–
115. Fagan KA, Morrissey B, Fouty BW, Sato K, Harral JW,
Morris KG Jr, Hoedt-Miller M, Vidmar S, McMurtry IF, and
Rodman DM. Upregulation of nitric oxide synthase in mice
with severe hypoxia-induced pulmonary hypertension.
Respir Res 2: 306–313, 2001.
116. Farb A, Burke AP, Tang AL, Liang TY, Mannan P, Smialek
J, and Virmani R. Coronary plaque erosion without rup-
ture into a lipid core: a frequent cause of coronary throm-
bosis in sudden coronary death. Circulation 93: 1354–1363,
117. Feigin VL, Anderson CS, and Mhurchu CN. Systemic in-
flammation, endothelial dysfunction, dietary fatty acids
and micronutrients as risk factors for stroke: a selective re-
view. Cerebrovasc Dis 13: 219–224, 2002.
118. Feletou M and Vanhoutte PM. Endothelium-derived hy-
perpolarizing factor: where are we now? Arterioscler Thromb
Vasc Biol 26: 1215–1225, 2006.
119. Fennell JP, Brosnan MJ, Frater AJ, Hamilton CA, Alexan-
der MY, Nicklin SA, Heistad DD, Baker AH, and Do-
miniczak AF. Adenovirus-mediated overexpression of ex-
tracellular superoxide dismutase improves endothelial
dysfunction in a rat model of hypertension. Gene Ther 9:
120. Fiege B, Ballhausen D, Kierat L, Leimbacher W, Goriounov
D, Schircks B, Thony B, and Blau N. Plasma tetrahydro-
biopterin and its pharmacokinetic following oral adminis-
tration. Mol Genet Metab 81: 45–51, 2004.
121. Fisslthaler B, Hinsch N, Chataigneau T, Popp R, Kiss L,
Busse R, and Fleming I. Nifedipine increases cytochrome
P4502C expression and endothelium-derived hyperpolar-
izing factor-mediated responses in coronary arteries. Hy-
pertension 36: 270–275, 2000.
122. Flammer AJ, Hermann F, Sudano I, Spieker L, Hermann
M, Cooper KA, Serafini M, Luscher TF, Ruschitzka F, Noll
G, and Corti R. Dark chocolate improves coronary vaso-
motion and reduces platelet reactivity. Circulation 116:
123. Fleming I. Myoendothelial gap junctions: the gap is there,
but does EDHF go through it? Circ. Res 86: 249–250, 2000.
124. Fleming I and Busse R. Endothelium-derived epoxye-
icosatrienoic acids and vascular function. Hypertension 47:
125. Flitney FW, Megson IL, Thomson JL, Kennovin GD, and
Butler AR. Vasodilator responses of rat isolated tail artery
enhanced by oxygen- dependent, photochemical release of
nitric oxide from iron-sulphur-nitrosyls. Br J Pharmacol 117:
126. Flowers MA, Wang Y, Stewart RJ, Patel B, and Marsden
PA. Reciprocal regulation of endothelin-1 and endothelial
constitutive NOS in proliferating endothelial cells. Am J
Physiol 269: H1988–H1997, 1995.
127. Forman MB, Puett DW, and Virmani R. Endothelial and
myocardial injury during ischemia and reperfusion: patho-
genesis and therapeutic implications. J Am Coll Cardiol 13:
2: 450–459, 1989.
128. Francis GS, Benedict C, Johnstone DE, Kirlin PC, Nicklas J,
Liang CS, Kubo SH, Rudin-Toretsky E, and Yusuf S. Com-
parison of neuroendocrine activation in patients with left
ventricular dysfunction with and without congestive heart
failure: a substudy of the Studies of Left Ventricular Dys-
function (SOLVD). Circulation 82: 1724–1729, 1990.
129. Frank PG, Woodman SE, Park DS, and Lisanti MP. Cave-
olin, caveolae, and endothelial cell function. Arterioscler
Thromb Vasc Biol 23: 1161–1168, 2003.
130. Frankel EN. Browning and glycation reaction products in
biology. In: Barnes PJ, and assoc. eds Antioxidants in food
and biology: facts and fiction. Bridgwater, The Oily Press,
131. Franklin SS, Larson MG, Khan SA, Wong ND, Leip EP,
Kannel WB, and Levy D. Does the relation of blood pres-
sure to coronary heart disease risk change with aging? The
Framingham Heart Study. Circulation 103: 1245–1249, 2001.
132. Furchgott RF and Jothianandan D. Endothelium-depen-
dent and -independent vasodilation involving cyclic GMP:
relaxation induced by nitric oxide, carbon monoxide and
light. Blood Vessels 28: 52–61, 1991.
133. Furchgott RF and Zawadzki JV. The obligatory role of en-
dothelial cells in the relaxation of arterial smooth muscle
by acetylcholine. Nature 288: 373–376, 1980.
134. Garcin ED, Bruns CM, Lloyd SJ, Hosfield DJ, Tiso M, Gach-
hui R, Stuehr DJ, Tainer JA, and Getzoff ED. Structural ba-
sis for isozyme-specific regulation of electron transfer in ni-
tric-oxide synthase. J Biol Chem 279: 37918–37927, 2004.
135. Gasser R, Koppel H, Brussee H, Grisold M, Holzmann S,
and Klein W. EDRF does not mediate coronary vasodila-
tion secondary to simulated ischemia: a study on KATP
channels and N omega-nitro-L-arginine on coronary per-
fusion pressure in isolated Langendorff-perfused guinea-
pig hearts. Cardiovasc Drugs Ther 12: 279–284, 1998.
136. Gauthier-Rein KM, Bizub DM, Lombard JH, and Rusch NJ.
Hypoxia-induced hyperpolarization is not associated with
vasodilation of bovine coronary resistance arteries. Am J
Physiol 272: H1462–H1469, 1997.
137. Geiszt M, Kopp JB, Varnai P, and Leto TL. Identification of
renox, an NAD(P)H oxidase in kidney. Proc Natl Acad Sci
U S A 97: 8010–8014, 2000.
138. Genest JJ Jr, McNamara JR, Salem DN, Wilson PW, Schae-
fer EJ, and Malinow MR. Plasma homocyst(e)ine levels in
men with premature coronary artery disease. J Am Coll Car-
diol 16: 1114–1119, 1990.
139. Giaid A and Saleh D. Reduced expression of endothelial
nitric oxide synthase in the lungs of patients with pul-
monary hypertension. N Engl J Med 333: 214–221, 1995.
140. Giordano FJ. Oxygen, oxidative stress, hypoxia, and heart
failure. J Clin Invest 115: 500–508, 2005.
141. Gladwin MT, Raat NJ, Shiva S, Dezfulian C, Hogg N, Kim-
Shapiro DB, and Patel RP. Nitrite as a vascular endocrine
nitric oxide reservoir that contributes to hypoxic signaling,
cytoprotection, and vasodilation. Am J Physiol Heart Circ
Physiol 291: H2026–H2035, 2006.
142. Gladwin MT, Schechter AN, Kim-Shapiro DB, Patel RP,
Hogg N, Shiva S, Cannon RO 3rd, Kelm M, Wink DA, Es-
pey MG, Oldfield EH, Pluta RM, Freeman BA, Lancaster
JR Jr, Feelisch M, and Lundberg JO. The emerging biology
of the nitrite anion. Nat Chem Biol 1: 308–314, 2005.
143. Goettsch W, Lattmann T, Amann K, Szibor M, Morawietz
H, Munter K, Muller SP, Shaw S, and Barton M. Increased
expression of endothelin-1 and inducible nitric oxide syn-
thase isoform II in aging arteries in vivo: implications for
atherosclerosis. Biochem Biophys Res Commun 280: 908–
144. Gojova A, Guo B, Kota RS, Rutledge JC, Kennedy IM, and
Barakat AI. Induction of inflammation in vascular endo-
thelial cells by metal oxide nanoparticles: effect of particle
composition. Environ Health Perspect 115: 403–409, 2007.
145. Goldberg MS, Burnett RT, Valois MF, Flegel K, Bailar JC
3rd, Brook J, Vincent R, and Radon K. Associations between
ambient air pollution and daily mortality among persons
with congestive heart failure. Environ Res 91: 8–20, 2003.
146. Golino P, Piscione F, Benedict CR, Anderson HV, Cappelli-
Bigazzi M, Indolfi C, Condorelli M, Chiariello M, and
Willerson JT. Local effect of serotonin released during coro-
nary angioplasty. N Engl J Med 330: 523–528, 1994.
147. Gorlach A, Brandes RP, Nguyen K, Amidi M, Deghani F,
and Busse R. A gp91phox containing NADPH oxidase se-
lectively expressed in endothelial cells is a major source of
oxygen radical generation in the arterial wall. Circ Res 87:
148. Gow AJ and Stamler JS. Reactions between nitric oxide and
haemoglobin under physiological conditions. Nature 391:
149. Goyal P, Weissmann N, Grimminger F, Hegel C, Bader L,
Rose F, Fink L, Ghofrani HA, Schermuly RT, Schmidt HH,
Seeger W, and Hanze J. Upregulation of NAD(P)H oxidase
1 in hypoxia activates hypoxia-inducible factor 1 via in-
crease in reactive oxygen species. Free Radic Biol Med 36:
150. Graser T and Vanhoutte PM. Hypoxic contraction of canine
coronary arteries: role of endothelium and cGMP. Am J
Physiol 261: H1769–H1777, 1991.
151. Gray DW and Marshall I. Novel signal transduction path-
way mediating endothelium-dependent beta-adrenoceptor
vasorelaxation in rat thoracic aorta. Br J Pharmacol 107:
152. Gray GA and Webb DJ. The endothelin system and its po-
tential as a therapeutic target in cardiovascular disease.
Pharmacol Ther 72: 109–148, 1996.
153. Green K, Brand MD, and Murphy MP. Prevention of mi-
tochondrial oxidative damage as a therapeutic strategy in
diabetes. Diabetes 53(suppl 1): S110–S118, 2004.
154. Greenacre SA and Ischiropoulos H. Tyrosine nitration: lo-
calisation, quantification, consequences for protein func-
tion and signal transduction. Free Radic Res 34: 541–581,
155. Griendling KK, Minieri CA, Ollerenshaw JD, and Alexan-
der RW. Angiotensin II stimulates NADH and NADPH ox-
idase activity in cultured vascular smooth muscle cells. Circ
Res 74: 1141–1148, 1994.
156. Griese DP, Ehsan A, Melo LG, Kong D, Zhang L, Mann MJ,
Pratt RE, Mulligan RC, and Dzau VJ. Isolation and trans-
plantation of autologous circulating endothelial cells into
denuded vessels and prosthetic grafts: implications for cell-
based vascular therapy. Circulation 108: 2710–2715, 2003.
157. Gruppo Italiano per lo Studio della Sopravvivenza nell’In-
farcto miocardico. Dietary supplementation with n-3
LE BROCQ ET AL.1662
polyunsaturated fatty acids and vitamin E after myocardial
infarction: results of the GISSI-Preventione trials. Lancet
354: 447–455, 1999.
158. Gryglewski RJ, Palmer RM, and Moncada S. Superoxide
anion is involved in the breakdown of endothelium-de-
rived vascular relaxing factor. Nature 320: 454–456, 1986.
159. Guha M, Bai W, Nadler JL, and Natarajan R. Molecular
mechanisms of tumor necrosis factor alpha gene expression
in monocytic cells via hyperglycemia-induced oxidant
stress-dependent and -independent pathways. J Biol Chem
275: 17728–17739, 2000.
160. Halcox JP, Nour KR, Zalos G, Mincemoyer RA, Waclawiw
M, Rivera CE, Willie G, Ellahham S, and Quyyumi AA. The
effect of sildenafil on human vascular function, platelet ac-
tivation, and myocardial ischemia. J Am Coll Cardiol 40:
161. Halcox JP, Schenke WH, Zalos G, Mincemoyer R, Prasad
A, Waclawiw MA, Nour KR, and Quyyumi AA. Prognos-
tic value of coronary vascular endothelial dysfunction. Cir-
culation 106: 653–658, 2002.
162. Hansen CS, Sheykhzade M, Moller P, Folkmann JK, Am-
torp O, Jonassen T and Loft S. Diesel exhaust particles in-
duce endothelial dysfunction in apoE-/- mice. Toxicol Appl
Pharmacol 219: 24–32, 2007.
163. Hanss M and Collen D. Secretion of tissue-type plas-
minogen activator and plasminogen activator inhibitor
by cultured human endothelial cells: modulation by
thrombin, endotoxin, and histamine. J Lab Clin Med 109:
164. Harper JA, Dickinson K, and Brand MD. Mitochondrial un-
coupling as a target for drug development for the treat-
ment of obesity. Obes Rev 2: 255–265, 2001.
165. Harrison DG. Cellular and molecular mechanisms of en-
dothelial cell dysfunction. J Clin Invest 100: 2153–2157, 1997.
166. Hassoun PM, Yu FS, Shedd AL, Zulueta JJ, V T, Lanzillo
JJ, and Fanburg BL. Regulation of endothelial cell xanthine
dehydrogenase xanthine oxidase gene expression by oxy-
gen tension. Am J. Physiol 266: L163–L171, 1994.
167. Hatakeyama H, Miyamori I, Yamagishi S, Takeda Y,
Takeda R, and Yamamoto H. Angiotensin II up-regulates
the expression of type A endothelin receptor in human vas-
cular smooth muscle cells. Biochem Mol Biol Int 34: 127–134,
168. Haynes WG, Hand MF, Johnstone HA, Padfield PL, and
Webb DJ. Direct and sympathetically mediated venocon-
striction in essential hypertension: enhanced responses to
endothelin-1. J Clin Invest 94: 1359–1364, 1994.
169. He GW. Hyperkalemia exposure impairs EDHF-mediated
endothelial function in the human coronary artery. Ann
Thorac Surg 63: 84–87, 1997.
170. Heinecke JW. Oxidative stress: new approaches to diagno-
sis and prognosis in atherosclerosis. Am J Cardiol 91:
171. Heiss C, Lauer T, Dejam A, Kleinbongard P, Hamada S,
Rassaf T, Matern S, Feelisch M, and Kelm M. Plasma ni-
troso compounds are decreased in patients with endothe-
lial dysfunction. J Am Coll Cardiol 47: 573–579, 2006.
172. Heitzer T, Baldus S, von Kodolitsch Y, Rudolph V, and
Meinertz T. Systemic endothelial dysfunction as an early
predictor of adverse outcome in heart failure. Arterioscler
Thromb Vasc Biol 25: 1174–1179, 2005.
173. Heitzer T, Schlinzig T, Krohn K, Meinertz T, and Munzel
T. Endothelial dysfunction, oxidative stress, and risk of car-
diovascular events in patients with coronary artery disease.
Circulation 104: 2673–2678, 2001.
174. Hennekens CH, Buring JE, Manson JE, Stampfer M, Ros-
ner B, Cook NR, Belanger C, LaMotte F, Gaziano JM, Rid-
ker PM, Willett W, and Peto R. Lack of effect of long-term
supplementation with beta carotene on the incidence of ma-
lignant neoplasms and cardiovascular disease. N Engl J Med
334: 1145–1149, 1996.
175. Herve P, Launay JM, Scrobohaci ML, Brenot F, Simonneau
G, Petitpretz P, Poubeau P, Cerrina J, Duroux P, and Drouet
L. Increased plasma serotonin in primary pulmonary hy-
pertension. Am J Med 99: 249–254, 1995.
176. Higashi Y, Sasaki S, Nakagawa K, Matsuura H, Chayama
K, and Oshima T. Effect of obesity on endothelium-depen-
dent, nitric oxide-mediated vasodilation in normotensive
individuals and patients with essential hypertension. Am J
Hypertens 14: 1038–1045, 2001.
177. Higashi Y, Sasaki S, Nakagawa K, Matsuura H, Kajiyama
G, and Oshima T. Effect of the angiotensin-converting en-
zyme inhibitor imidapril on reactive hyperemia in patients
with essential hypertension: relationship between treat-
ment periods and resistance artery endothelial function. J
Am Coll Cardiol 37: 863–870, 2001.
178. Hilgers RH, Todd J Jr, and Webb RC. Regional hetero-
geneity in acetylcholine-induced relaxation in rat vascular
bed: role of calcium-activated K? channels. Am J Physiol
Heart Circ Physiol 291: H216–H222, 2006.
179. Hogikyan RV, Galecki AT, Pitt B, Halter JB, Greene DA,
and Supiano MA. Specific impairment of endothelium-de-
pendent vasodilation in subjects with type 2 diabetes in-
dependent of obesity. J Clin Endocrinol Metab 83: 1946–1952,
180. Horiuchi S. Advanced glycation end products (AGE)-mod-
ified proteins and their potential relevance to atheroscle-
rosis. Trends Cardiovasc Med 6: 163–168, 1996.
181. Hornig B and Drexler H. Endothelial function and
bradykinin in humans. Drugs 54: 42–47, 1997.
182. Hoshikawa Y, Voelkel NF, Gesell TL, Moore MD, Morris
KG, Alger LA, Narumiya S, and Geraci MW. Prostacyclin
receptor-dependent modulation of pulmonary vascular re-
modeling. Am J Respir Crit Care 164: 314–318, 2001.
183. Hosoki R, Matsuki N, and Kimura H. the possible role of
hydrogen sulfide as an endogenous smooth muscle relax-
ant in synergy with nitric oxide. Biochem Biophys Res Com-
mun 237: 527–531, 1997.
184. Houstis N, Rosen ED, and Lander ES. Reactive oxygen
species have a causal role in multiple forms of insulin re-
sistance. Nature 440: 944–948, 2006.
185. Hrafnkelsdottir T, Ottosson P, Gudnason T, Samuelsson O,
and Jern S. Impaired endothelial release of tissue-type plas-
minogen activator in patients with chronic kidney disease
and hypertension. Hypertension 44: 300–304, 2004.
186. Humbert M, Monti G, Brenot F, Sitbon O, Portier A,
Grangeot-Keros L, Duroux P, Galanaud P, Simonneau G,
and Emilie D. Increased interleukin-1 and interleukin-6
serum concentrations in severe primary pulmonary hy-
pertension. Am J Respir Crit Care Med 151: 1628–1631, 1995.
187. Huo Y and Ley K. Adhesion molecules and atherogenesis.
Acta Physiol Scand 173: 35–43, 2001.
188. Hussain AS, Marks GS, Brien JF, and Nakatsu K. The solu-
ble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-al-
pha]quinoxalin-1-one (ODQ) inhibits relaxation of rabbit aor-
tic rings induced by carbon monoxide, nitric oxide, and
glyceryl trinitrate. Can J Physiol Pharmacol75: 1034–1037, 1997.
189. Ichinose F, Roberts JD Jr, and Zapol WM. Inhaled nitric ox-
ide: a selective pulmonary vasodilator: current uses and
therapeutic potential. Circulation 109: 3106–3111, 2004.
190. Ihling C, Szombathy T, Bohrmann B, Brockhaus M, Schae-
fer HE and Loeffler BM. Coexpression of endothelin-con-
verting enzyme-1 and endothelin-1 in different stages of
human atherosclerosis. Circulation 104: 864–869, 2001.
191. Irodova NL, Lankin VZ, Konovalova GK, Kochetov AG, and
Chazova IE. Oxidative stress in patients with primary pul-
monary hypertension. Bull Exp Biol Med 133: 580–582, 2002.
192. Jaggar RT, Chan HY, Harris AL, and Bicknell R. Endothe-
lial cell-specific expression of tumor necrosis factor-alpha
from the KDR or E-selectin promoters following retroviral
delivery. Hum Gene Ther 8: 2239–2247, 1997.
193. James AM and Murphy MP. How mitochondrial damage
affects cell function. J Biomed Sci 9: 475–487, 2002.
194. Joannides R, Bizet-Nafeh C, Costentin A, Iacob M,
Derumeaux G, Cribier A, and Thuillez C. Chronic ACE in-
hibition enhances the endothelial control of arterial me-
chanics and flow-dependent vasodilatation in heart failure.
Hypertension 38: 1446–1450, 2001.
195. Joannides R, Haefeli WE, Linder L, Richard V, Bakkali EH,
Thuillez C, and Luscher TF. Nitric oxide is responsible for
flow-dependent dilatation of human peripheral conduit ar-
teries in vivo. Circulation 91: 1314–1319, 1995.
196. Johnson RA and Johnson FK. Heme oxygenase-derived en-
dogenous carbon monoxide impairs flow-induced dilation
in resistance vessels. Shock 2007 (in press).
197. Jost S, Nolte CW, Sturm M, Hausleiter J, and Hausmann
D. How to standardize vasomotor tone in serial studies
based on quantitation of coronary dimensions? Int J Car-
diol Imaging 14: 357–372, 1998.
198. Jourd’heuil D, Jourd’heuil FL, and Feelisch M. Oxidation
and nitrosation of thiols at low micromolar exposure to ni-
tric oxide: evidence for a free radical mechanism. J Biol
Chem 278: 15720–15726, 2003.
199. Kaminski KA, Bonda TA, Korecki J, and Musial WJ. Ox-
idative stress and neutrophil activation: the two keystones
of ischemia/reperfusion injury. Int J Cardiol 86: 41–59, 2002.
200. Kanse SM, Takahashi K, Lam HC, Rees A, Warren JB, Porta
M, Molinatti P, Ghatei M, and Bloom SR. Cytokine stimu-
lated endothelin release from endothelial cells. Life Sci 48:
201. Karatzi K, Papamichael C, Karatzis E, Papaioannou TG,
Voidonikola PT, Lekakis J, and Zampelas A. Acute smok-
ing induces endothelial dysfunction in healthy smokers: is
this reversible by red wine’s antioxidant constituents? J Am
Coll Nutr 26: 10–15, 2007.
202. Katusic ZS. Vascular endothelial dysfunction: does tetrahy-
drobiopterin play a role? Am J Physiol Heart Circ Physiol 281:
203. Katz SD, Balidemaj K, Homma S, Wu H, Wang J, and May-
baum S. Acute type 5 phosphodiesterase inhibition with
sildenafil enhances flow-mediated vasodilation in patients
with chronic heart failure. J Am Coll Cardiol 36: 845–851,
204. Katz SD, Rao R, Berman JW, Schwarz M, Demopoulos L,
Bijou R, and LeJemtel TH. Pathophysiological correlates of
increased serum tumor necrosis factor in patients with con-
gestive heart failure: relation to nitric oxide-dependent va-
sodilation in the forearm circulation. Circulation 90: 12–16,
205. Kawaguchi H, Sawa H, and Yasuda H. Endothelin stimu-
lates angiotensin I to angiotensin II conversion in cultured
pulmonary artery endothelial cells. J Mol Cell Cardiol 22:
206. Kawano H, Yasue H, Kitagawa A, Hirai N, Yoshida T, Soe-
jima H, Miyamoto S, Nakano M, and Ogawa H. Dehydro-
epiandrosterone supplementation improves endothelial
function and insulin sensitivity in men. J Clin Endocrinol
Metab 88: 3190–3195, 2003.
207. Kawashima S and Yokoyama M. Dysfunction of endothe-
lial nitric oxide synthase and atherosclerosis. Arterioscler
Thromb Vasc Biol 24: 998–1005, 2004.
208. Keegan A, Morecroft I, Smillie D, Hicks MN, and MacLean
MR. Contribution of the 5-HT(1B) receptor to hypoxia-in-
duced pulmonary hypertension: converging evidence us-
ing 5-HT(1B)-receptor knockout mice and the 5-HT(1B/
1D)-receptor antagonist GR127935. Circ Res 89: 1231–1239,
209. Kelly AS, Thelen AM, Kaiser DR, Gonzalez-Campoy JM,
and Bank AJ. Rosiglitazone improves endothelial function
and inflammation but not asymmetric dimethylarginine or
oxidative stress in patients with type 2 diabetes mellitus.
Vasc Med 12: 311–318, 2007.
210. Kelso GF, Porteous CM, Coulter CV, Hughes G, Porteous
WK, Ledgerwood EC, Smith RAJ, and Murphy MP. Selec-
tive targeting of a redox-active ubiquinone to mitochondria
within cells. J Biol Chem 276: 4588–4596, 2001.
211. Kerkhof CJ, Van Der Linden PJ, and Sipkema P. Role
of myocardium and endothelium in coronary vascular
smooth muscle responses to hypoxia. Am J Physiol Heart
Circ Physiol 282: H1296–H1303, 2002.
212. Khechai F, Ollivier V, Bridey F, Amar M, Hakim J, and de
Prost D. Effect of advanced glycation end product-modi-
fied albumin on tissue factor expression by monocytes: role
of oxidant stress and protein tyrosine kinase activation. Ar-
terioscler Thromb Vasc Biol 17: 2885–2890, 1997.
213. Khoo JP, Zhao L, Alp NJ, Bendall JK, Nicoli T, Rockett K,
Wilkins MR, and Channon KM. Pivotal role for endothe-
lial tetrahydrobiopterin in pulmonary hypertension. Circu-
lation 111: 2126–2133, 2005.
214. Kim JA, Montagnani M, Koh KK, and Quon MJ. Recipro-
cal relationships between insulin resistance and endothe-
lial dysfunction: molecular and pathophysiological mech-
anisms. Circulation 113: 1888–1904, 2006.
215. Kimura M, Higashi Y, Hara K, Noma K, Sasaki S, Naka-
gawa K, Goto C, Oshima T, Yoshizumi M, and Chayama
K. PDE5 inhibitor sildenafil citrate augments endothelium-
dependent vasodilation in smokers. Hypertension 41:
216. Kinlay S, Behrendt D, Wainstein M, Beltrame J, Fang JC,
Creager MA, Selwyn AP and Ganz P. Role of endothelin-
1 in the active constriction of human atherosclerotic coro-
nary arteries. Circulation 104: 1114–1118, 2001.
217. Kiowski W, Sutsch G, Hunziker P, Muller P, Kim J, Oech-
slin E, Schmitt R, Jones R, and Bertel O. Evidence for en-
dothelin-1-mediated vasoconstriction in severe chronic
heart failure. Lancet 346: 732–736, 1995.
218. Kirstein M, Aston C, Hintz R, and Vlassara H. Receptor-
specific induction of insulin-like growth factor I in human
monocytes by advanced glycosylation end product-modi-
fied proteins. J Clin Invest 90: 439–446, 1992.
219. Klebanoff SJ. Oxygen metabolism and the toxic properties
of phagocytes. Ann Intern Med 93: 480–489, 1980.
220. Klebanoff SJ. Reactive nitrogen intermediates and anti-
microbial activity: role of nitrite. Free Radic Biol Med 14:
221. Kleinbongard P, Dejam A, Lauer T, Jax T, Kerber S, Gharini
P, Balzer J, Zotz RB, Scharf RE, Willers R, Schechter AN,
Feelisch M, and Kelm M. Plasma nitrite concentrations re-
flect the degree of endothelial dysfunction in humans. Free
Radic Biol Med 40: 295–302, 2006.
LE BROCQ ET AL.1664
222. Kleinbongard P, Dejam A, Lauer T, Rassaf T, Schindler
A, Picker O, Scheeren T, Godecke A, Schrader J, Schulz
R, Heusch G, Schaub GA, Bryan NS, Feelisch M, and
Kelm M. Plasma nitrite reflects constitutive nitric oxide
synthase activity in mammals. Free Radic Biol Med 35:
223. Kleinert H, Wallerath T, Euchenhofer C, Ihrig-Biedert I, Li
H, and Fostermann U. Estrogens increase transcription of
the human endothelial NO synthase gene: analysis of the
transcription factors involved. Hypertension 31: 582–588,
224. Knekt P, Reunanen A, Jarvinen R, Seppanen R, Heliovaara
M, and Aromaa A. Antioxidant vitamin intake and coro-
nary mortality in a longitudinal population study. Am J Epi-
demiol 139: 1180–1189, 1994.
225. Knowles RG and Moncada S. Nitric oxide synthases in
mammals. Biochem J 298: 249–258, 1994.
226. Kobayashi T, Tahara Y, Matsumoto M, Iguchi M, Sano H,
Murayama T, Arai H, Oida H, Yurugi-Kobayashi T, Ya-
mashita JK, Katagiri H, Majima M, Yokode M, Kita T, and
Narumiya S. Roles of thromboxane A(2) and prostacyclin
in the development of atherosclerosis in apoE-deficient
mice. J Clin Invest 114: 784–794, 2004.
227. Konishi C, Naito Y, Saito Y, Ohara N, and Ono H. Age-re-
lated differences and roles of endothelial nitric oxide and
prostanoids in angiotensin II responses of isolated, per-
fused mesenteric arteries and veins of rats. Eur J Pharmacol
320: 175–181, 1997.
228. Kukovetz WR, Holzmann S, Wurm A, and Poch G. Prosta-
cyclin increases cAMP in coronary arteries. J Cyclic Nucle-
otide Res 5: 469–476, 1979.
229. Kushi LH, Folsom AR, Prineas RJ, Mink PJ, Wu Y, and Bo-
stick RM. Dietary antioxidant vitamins and death from
coronary heart disease in post-menopausal women. N Engl
J Med 334: 1156–1162, 1996.
230. Lacza Z, Puskar M, Kis B, Perciaccante JV, Miller AW, and
Busija DW. Hydrogen peroxide acts as an EDHF in the
piglet pial vasculature in response to bradykinin. Am J
Physiol Heart Circ Physiol 283: H406–H411, 2002.
231. Lander HM, Tauras JM, Ogiste JS, Hori O, Moss RA, and
Schmidt AM. Activation of the receptor for advanced gly-
cation end products triggers a p21(ras)-dependent mito-
gen-activated protein kinase pathway regulated by oxidant
stress. J Biol Chem 272: 17810–17814, 1997.
232. Landmesser U, Bahlmann F, Mueller M, Spiekermann S,
Kirchhoff N, Schulz S, Manes C, Fischer D, de Groot K,
Fliser D, Fauler G, Marz W, and Drexler H. Simvastatin ver-
sus ezetimibe: pleiotropic and lipid-lowering effects on en-
dothelial function in humans. Circulation 111: 2356–2363,
233. Landmesser U, Cai H, Dikalov S, McCann L, Hwang J, Jo
H, Holland SM, and Harrison DG. Role of p47(phox) in vas-
cular oxidative stress and hypertension caused by an-
giotensin II. Hypertension 40: 511–515, 2002.
234. Landmesser U, Merten R, Spiekermann S, Buttner K,
Drexler H, and Hornig B. Vascular extracellular superox-
ide dismutase activity in patients with coronary artery dis-
ease: relation to endothelium-dependent vasodilation. Cir-
culation 101: 2264–2270, 2000.
235. Lapu-Bula R and Ofili E. From hypertension to heart fail-
ure: role of nitric oxide-mediated endothelial dysfunction
and emerging insights from myocardial contrast echocar-
diography. Am J Cardiol 99: 7D–14D, 2007.
236. Laukkanen MO, Kivela A, Rissanen T, Rutanen J,
Karkkainen MK, Leppanen O, Brasen JH and Yla-Herttuala
S. Adenovirus-mediated extracellular superoxide dismu-
tase gene therapy reduces neointima formation in balloon-
denuded rabbit aorta. Circulation 106: 1999–2003, 2002.
237. Laursen JB, Rajagopalan S, Galis Z, Tarpey M, Freeman BA,
and Harrison DG. Role of superoxide in angiotensin II-in-
duced but not catecholamine-induced hypertension. Circu-
lation 95: 588–593, 1997.
238. Leffler CW, Parfenova H, Jaggar JH, and Wang R. Carbon
monoxide and hydrogen sulphide: gaseous messengers in
cerebrovascular circulation. J Appl Physiol 100: 1065–1076,
239. Leiper J and Vallance P. Biological significance of endoge-
nous methylarginines that inhibit nitric oxide synthases.
Cardiovasc Res 43: 542–548, 1999.
240. Lembo G, Iaccarino G, Vecchione C, Barbato E, Izzo R,
Fontana D, and Trimarco B. Insulin modulation of an en-
dothelial nitric oxide component present in the alpha2- and
beta-adrenergic responses in human forearm. J Clin Invest
100: 2007–2014, 1997.
241. Lembo G, Iaccarino G, Vecchione C, Barbato E, Morisco C,
Monti F, Parrella L, and Trimarco B. Insulin enhances en-
dothelial alpha2-adrenergic vasorelaxation by a pertussis
toxin mechanism. Hypertension 30: 1128–1134, 1997.
242. Lerman A, Suwaidi JA, and Velianou JL. L-Arginine: a
novel therapy for coronary artery disease? Expert Opin In-
vest Drugs 8: 1785–1793, 1999.
243. Leslie SJ, Affolter J, Denvir MA, and Webb DJ. Validation
of laser Doppler flowmetry coupled with intra-dermal in-
jection for investigating effects of vasoactive agents on the
skin microcirculation in man. Eur J Clin Pharmacol 59:
244. Leslie SJ, Attina T, Hultsch E, Bolscher L, Grossman M,
Denvir MA, and Webb DJ. Comparison of two plethys-
mography systems in assessment of forearm blood flow. J
Appl Physiol 96: 1794–1799, 2004.
245. Leu HB, Charng MJ, and Ding PY. A double blind ran-
domized trial to compare the effects of eprosartan and
enalapril on blood pressure, platelets, and endothelium
function in patients with essential hypertension. Jpn Heart
J 45: 623–635, 2004.
246. Levin EG and Santell L. Stimulation and desensitization of
tissue plasminogen activator release from human endo-
thelial cells. J Biol Chem 263: 9360–9365, 1988.
247. Lewis RS and Deen WM. Kinetics of the reaction of nitric
oxide with oxygen in aqueous solutions. Chem Res Toxicol
7: 568–574, 1994.
248. Li Volti G, Wang J, Traganos F, Kappas A, and Abraham
NG. Differential effect of heme oxygenase-1 in endothelial
and smooth muscle cell cycle progression. Biochem Biophys
Res Commun 296: 1077–1082, 2002.
249. Li H, Chen SJ, Chen YF, Meng QC, Durand J, Oparil S, and
Elton TS. Enhanced endothelin-1 and endothelin receptor
gene expression in chronic hypoxia. J Appl Physiol 77:
250. Li L, Crockett E, Wang DH, Galligan JJ, Fink GD, and Chen
AF. Gene transfer of endothelial NO synthase and man-
ganese superoxide dismutase on arterial vascular cell ad-
hesion molecule-1 expression and superoxide production
in deoxycorticosterone acetate-salt hypertension. Arte-
rioscler Thromb Vasc Biol 22: 249–255, 2002.
251. Li L, Fink GD, Watts SW, Northcott CA, Galligan JJ,
Pagano PJ, and Chen AF. Endothelin-1 increases vascu-
lar superoxide via endothelin(A)-NADPH oxidase path-
way in low-renin hypertension. Circulation 107: 1053–
252. Li LJ, Geng SR, and Yu CM. Endothelial dysfunction in nor-
motensive Chinese with a family history of essential hy-
pertension. Clin Exp Hypertens 27: 1–8, 2005.
253. Li N and Karin M. Is NF-kappaB the sensor of oxidative
stress? FASEB J 13: 1137–1143, 1999.
254. Li YM, Tan AX, and Vlassara H. Antibacterial activity of
lysozyme and lactoferrin is inhibited by binding of ad-
vanced glycation-modified proteins to a conserved motif.
Nat Med 1: 1057–1061, 1995.
255. Liaudet L, Szabo G, and Szabo C. Oxidative stress and re-
gional ischemia-reperfusion injury: the peroxynitrite-
poly(ADP-ribose) polymerase connection. Coron Artery Dis
14: 115–122, 2003.
256. Libby P. Inflammation in atherosclerosis. Nature 420: 868–
257. Liem A, Reynierse-Buitenwerf GH, Zwinderman AH,
Jukema JW, and van Veldhuisen DJ. Secondary prevention
with folic acid: effects on clinical outcomes. J Am Coll Car-
diol 41: 2105–2113, 2003.
258. Liu JQ and Folz RJ. Extracellular superoxide enhances 5-
HT-induced murine pulmonary artery vasoconstriction.
Am J Physiol 287: L111–L118, 2004.
259. Liu JQ, Zelko IN, Erbynn EM, Sham JS, and Folz RJ. Hy-
poxic pulmonary hypertension: role of superoxide and
NADPH oxidase (gp91phox). Am J Physiol 290: L2–L10,
260. Liu Y, Terata K, Chai Q, Li H, Kleinman LH, and Gutter-
man DD. Peroxynitrite inhibits Ca2?-activated K? chan-
nel activity in smooth muscle of human coronary arteri-
oles. Circ Res 91: 1070–1076, 2002.
261. Lizasoain I, Moro MA, Knowles RG, Darley-Usmar V, and
Moncada S. Nitric oxide and peroxynitrite exert distinct ef-
fects on mitochondrial respiration which are differentially
blocked by glutathione or glucose. Biochem J 314: 877–880,
262. Lopez Farre A and Casado S. Heart failure, redox alter-
ations, and endothelial dysfunction. Hypertension 38: 1400–
263. Lucchesi BR. Complement activation, neutrophils, and oxy-
gen radicals in reperfusion injury. Stroke 24: I41–I47,1993.
264. Ludmer PL, Selwyn AP, Shook TL, Wayne RR, Mudge GH,
Alexander RW and Ganz P. Paradoxical vasoconstriction
induced by acetylcholine in atherosclerotic coronary arter-
ies. N Engl J Med 315: 1046–1051, 1986.
265. Lüscher TF, Boulanger CM, Dohu Y, and Yang ZH. Endo-
thelium-derived contracting factors. Hypertension 19: 117–
266. Maguire JJ, Wilson DS, and Packer L. Mitochondrial elec-
tron transport-linked tocoperoxyl radical reduction. J Biol
Chem 264: 21462–21465, 1989.
267. Maines MD. Heme oxygenase: function, multiplicity, reg-
ulatory mechanisms, and clinical applications. FASEB J 2:
268. Mancini GB, Henry GC, Macaya C, O’Neill BJ, Pucillo AL,
Carere RG, Wargovich TJ, Mudra H, Luscher TF, Klibaner
MI, Haber HE, Uprichard AC, Pepine CJ, and Pitt B. An-
giotensin-converting enzyme inhibition with quinapril im-
proves endothelial vasomotor dysfunction in patients with
coronary artery disease: the TREND (Trial on Reversing
ENdothelial Dysfunction) Study. Circulation 94: 258–265,
269. Marcus AJ, Broekman MJ, Drosopoulos JHF, Islam N, Aly-
onycheva TN, Safier LB, Hajjar KA, Posnett DN, Schoen-
born MA, Schooley KA, Gayle RB, and Maliszewski CR.
The endothelial cell ecto-ADPase responsible for inhibition
of platelet function is CD39. J Clin Invest 99: 1351–1360,
270. Markewitz BA, Michael JR, and Kohan DE. Endothelin-1
inhibits the expression of inducible nitric oxide synthase.
Am J Physiol 272: L1078–L1083, 1997.
271. Mason RP, Walter MF, and Jacob RF. Effects of HMG-CoA
reductase inhibitors on endothelial function: role of mi-
crodomains and oxidative stress. Circulation 109: II34–II41,
272. Mather KJ, Lteif A, Steinberg HO, and Baron AD. Interac-
tions between endothelin and nitric oxide in the regulation
of vascular tone in obesity and diabetes. Diabetes 53:
273. Mather KJ, Mirzamohammadi B, Lteif A, Steinberg HO,
and Baron AD. Endothelin contributes to basal vascular
tone and endothelial dysfunction in human obesity and
type 2 diabetes. Diabetes 51: 3517–3523, 2002.
274. Matoba T and Shimokawa H. Hydrogen peroxide is an en-
dothelium-derived hyperpolarizing factor in animals and
humans. J Pharmacol Sci 92: 1–6, 2003.
275. Matoba T, Shimokawa H, Morikawa K, Kubota H, Kuni-
hiro I, Urakami-Harasawa L, Mukai Y, Hirakawa Y, Akaike
T, and Takeshita A. Electron spin resonance detection of
hydrogen peroxide as an endothelium-derived hyperpo-
larizing factor in porcine coronary microvessels. Arterioscler
Thromb Vasc Biol 23: 1224–1230, 2003.
276. Mavria G, Jager U, and Porter CD. Generation of a high
titre retroviral vector for endothelial cell-specific gene ex-
pression in vivo. Gene Ther 7: 368–376, 2000.
277. McCance DR, Dyer DG, Dunn JA, Bailie KE, Thorpe SR,
Baynes JW, and Lyons TJ. Maillard reaction products and
their relation to complications in insulin-dependent dia-
betes mellitus. J Clin Invest 91: 2470–2478, 1993.
278. McCulloch KM, Docherty C, and MacLean MR. Endothe-
lin receptors mediating contraction of rat and human pul-
monary resistance arteries: effect of chronic hypoxia in the
rat. Br J Pharmacol 123: 1621–1630, 1998.
279. McGuire JJ, Ding H, and Triggle CR. Endothelium-de-
rived relaxing factors: a focus on endothelium-derived
hyperpolarizing factor(s). Can J Physiol Pharmacol 79: 443–
280. McIntyre CA, Buckley CH, Jones GC, Sandeep TC, An-
drews RC, Elliott AI, Gray GA, Williams BC, McKnight JA,
Walker BR, and Hadoke PW. Endothelium-derived hyper-
polarizing factor and potassium use different mechanisms
to induce relaxation of human subcutaneous resistance ar-
teries. Br J Pharmacol 133: 902–908, 2001.
281. McVeigh GE, Brennan GM, Johnston GD, McDermott BJ,
McGrath LT, Henry WR, Andrews JW, and Hayes JR. Im-
paired endothelium-dependent and independent vasodila-
tion in patients with type 2 (non-insulin-dependent) dia-
betes mellitus. Diabetologia 35: 771–776, 1992.
282. Megson IL, Holmes SA, Magid KS, Pritchard RJ, and Flit-
ney FW. Selective modifiers of glutathione biosynthesis
and ‘repriming’ of vascular smooth muscle photorelax-
ation. Br J Pharmacol 130: 1575–1580, 2000.
283. Megson IL and Webb DJ. Nitric oxide donor drugs: current
status and future trends. Expert Opin Invest Drugs 11: 587–
284. Mehta J, Lopez L, Chen L, and Cox O. Alterations in nitric
oxide activity, superoxide anion generation and platelet ag-
gregation in systemic hypertension, and effect of celipro-
lol. Am J Cardiol 74: 901–905, 1994.
285. Melo LG, Gnecchi M, Pachori AS, Kong D, Wang K, Liu X,
Pratt RE, and Dzau VJ. Endothelium-targeted gene and cell-
LE BROCQ ET AL.1666
based therapies for cardiovascular disease. Arterioscler
Thromb Vasc Biol 24: 1761–1774, 2004.
286. Michelakis ED, Tymchak W, Noga M, Webster L, Wu XC,
Lien D, Wang SH, Modry D, and Archer SL. Long-term treat-
ment with oral sildenafil is safe and improves functional ca-
pacity and hemodynamics in patients with pulmonary arte-
rial hypertension. Circulation 108: 2066–2069, 2003.
287. Michiels C, Arnould T, Knott I, Dieu M, and Remacle J.
Stimulation of prostaglandin synthesis by human endo-
thelial cells exposed to hypoxia. Am J Physiol 264: C866–
288. Miller AA, Megson IL, and Gray GA. Inducible nitric ox-
ide synthase-derived superoxide contributes to hyperreac-
tivity in small mesenteric arteries from a rat model of
chronic heart failure. Br J Pharmacol 131: 29–36, 2000.
289. Miller MR and Megson IL. Recent developments in nitric
oxide donor drugs. Br J Pharmacol 151: 305–321, 2007.
290. Mills NL, Amin N, Robinson SD, Anand A, Davies J, Patel
D, de la Fuente JM, Cassee FR, Boon NA, Macnee W, Mil-
lar AM, Donaldson K, and Newby DE. Do inhaled carbon
nanoparticles translocate directly into the circulation in hu-
mans? Am J Respir Crit Care Med 173: 426–431, 2006.
291. Mills NL, Tornqvist H, Robinson SD, Gonzalez M, Darn-
ley K, MacNee W, Boon NA, Donaldson K, Blomberg A,
Sandstrom T, and Newby DE. Diesel exhaust inhalation
causes vascular dysfunction and impaired endogenous fib-
rinolysis. Circulation 112: 3930–3936, 2005.
292. Miyata T, Hori O, Zhang J, Yan SD, Ferran L, Iida Y, and
Schmidt AM. The receptor for advanced glycation end
products (RAGE) is a central mediator of the interaction of
phagocytes via an oxidant-sensitive pathway. Implications
for the pathogenesis of dialysis-related amyloidosis. J Clin
Invest 98: 1088–1094, 1996.
293. Modena MG, Bonetti L, Coppi F, Bursi F, and Rossi R. Prog-
nostic role of reversible endothelial dysfunction in hyper-
tensive postmenopausal women. J Am Coll Cardiol 40: 505–
294. Moens AL and Kass DA. Tetrahydrobiopterin and cardio-
vascular disease. Arterioscler Thromb Vasc Biol 26: 2439–
295. Moncada S, Palmer RM, and Higgs EA. Nitric oxide: phys-
iology, pathophysiology, and pharmacology. Pharmacol Rev
43: 109–142, 1991.
296. Morishita R, Sugimoto T, Aoki M, Kida I, Tomita N,
Moriguchi A, Maeda K, Sawa Y, Kaneda Y, Higaki J, and
Ogihara T. In vivo transfection of cis element “decoy”
against nuclear factor-kappaB binding site prevents myo-
cardial infarction. Nat Med 3: 894–899, 1997.
297. Morse JH. Bone morphogenetic protein receptor 2 muta-
tions in pulmonary hypertension. Chest 121: 50S–53S, 2002.
298. Motomiya Y, Oyama N, Iwamoto H, Uchimura T, and
Maruyama I. N epsilon-(carboxymethyl)lysine in blood
from maintenance hemodialysis patients may contribute to
dialysis-related amyloidosis. Kidney Int 54: 1357–1366, 1998.
299. Mukai Y, Shimokawa H, Higashi M, Morikawa K, Matoba
T, Hiroki J, Kunihiro I, Talukder HMA, and Takeshita A.
Inhibition of rennin-angiotensin system ameliorates endo-
thelial dysfunction associated with aging in rats. Arte-
rioscler Thromb Vasc Biol 22: 1445–1450, 2002.
300. Murad F. Shattuck Lecture: nitric oxide and cyclic GMP in
cell signaling and drug development. N Engl J Med 355:
301. Musameh MD, Fuller BJ, Mann BE, Green CJ, and Motter-
lini R. Positive inotropic effects of carbon monoxide-re-
with human mononuclear
leasing molecules (CO-ROMs) in the isolated perfused rat
heart. Br J Pharmacol 149: 1104–1112, 2006.
302. Muscat S, Pelka J, Hegele J, Weigle B, Munch G, and Pis-
chetsrieder M. Coffee and Maillard products activate NF-
kappaB in macrophages via H2O2 production. Mol Nutr
Food Res 51: 525–535, 2007.
303. Nadar S, Blann AD, and Lip GY. Antihypertensive therapy
and endothelial function. Curr Pharm Des 10: 3607–3614,
304. Naghavi M, Libby P, Falk E, Casscells SW, Litovsky S, Rum-
berger J, Badimon JJ, Stefanadis C, Moreno P, Pasterkamp
G, Fayad Z, Stone PH, Waxman S, Raggi P, Madjid M,
Zarrabi A, Burke A, Yuan C, Fitzgerald PJ, Siscovick DS,
de Korte CL, Aikawa M, Juhani Airaksinen KE, Assmann
G, Becker CR, Chesebro JH, Farb A, Galis ZS, Jackson C,
Jang IK, Koenig W, Lodder RA, March K, Demirovic J,
Navab M, Priori SG, Rekhter MD, Bahr R, Grundy SM,
Mehran R, Colombo A, Boerwinkle E, Ballantyne C, Insull
W Jr, Schwartz RS, Vogel R, Serruys PW, Hansson GK,
Faxon DP, Kaul S, Drexler H, Greenland P, Muller JE, Vir-
mani R, Ridker PM, Zipes DP, Shah PK, and Willerson JT.
From vulnerable plaque to vulnerable patient: a call for
new definitions and risk assessment strategies: part I. Cir-
culation 108: 1664–1672, 2003.
305. Naik JS, O’Donaughy TL, and Walker BR. Endogenous car-
bon monoxide is an endothelial-derived vasodilator factor
in the mesenteric circulation. Am J Physiol Heart Circ Phys-
iol 284: H838–H845, 2003.
306. Nakao A, Choi AM, and Murase N. Protective effect of car-
bon monoxide in transplantation. J Cell Mol Med 10:
307. Nakhostine N and Lamontagne D. Adenosine contributes to
hypoxia-induced vasodilation through ATP-sensitive K?
channel activation. Am J Physiol 265: H1289–H1293, 1993.
308. Napoli C, de Nigris F, and Palinski W. Multiple role of re-
active oxygen species in the arterial wall. J Cell Biochem 82:
309. Neglia D, Michelassi C, Trivieri MG, Sambuceti G, Giorgetti
A, Pratali L, Gallopin M, Salvadori P, Sorace O, Carpeggiani
C, Poddighe R, L’Abbate A, and Parodi O. Prognostic role of
myocardial blood flow impairment in idiopathic left ventric-
ular dysfunction. Circulation 105: 186–193, 2002.
310. Ness A and Smith GD. Mortality in the CHAOS trial: Cam-
bridge Heart Antioxidant Study. Lancet 353: 1017–1018,
311. Ness AR and Powles JW. Fruit and vegetables, and car-
diovascular disease: a review. Int J Epidemiol 26: 1–13, 1997.
312. Neunteufl T, Heher S, Katzenschlager R, Wolfl G, Kostner
K, Maurer G, and Weidinger F. Late prognostic value of
flow-mediated dilation in the brachial artery of patients
with chest pain. Am J Cardiol 86: 207–210, 2000.
313. Newby DE, McLeod AL, Uren NG, Flint L, Ludlam CA,
Webb DJ, Fox KA, and Boon NA. Impaired coronary tissue
plasminogen activator release is associated with coronary
atherosclerosis and cigarette smoking: direct link between
endothelial dysfunction and atherothrombosis. Circulation
103: 1936–1941, 2001.
314. Newby DE, Witherow FN, Wright RA, Bloomfield P, Lud-
lam CA, Boon NA, Fox KA, and Webb DJ. Hypercholes-
terolaemia and lipid lowering treatment do not affect the
acute endogenous fibrinolytic capacity in vivo. Heart 87:
315. Nicholls SJ and Hazen SJ. Myeloperoxidase and cardio-
vascular disease. Arterioscler Thromb Vasc Biol 25: 1102–
ENDOTHELIAL DYSFUNCTION 1667
316. Nicklin SA, Reynolds PN, Brosnan MJ, White SJ, Curiel DT,
Dominiczak AF, and Baker AH. Analysis of cell-specific
promoters for viral gene therapy targeted at the vascular
endothelium. Hypertension 38: 65–70, 2001.
317. Niederhoffer N and Szabo B. Involvement of CB1 cannabi-
noid receptors in the EDHF-dependent vasorelaxation in
rabbits. Br J Pharmacol 126: 1383–1386, 1999.
318. Nielsen WB, Vestbo J, and Jensen GB. Isolated systolic hy-
pertension as a major risk factor for stroke and myocardial
infarction and an unexploited source of cardiovascular pre-
vention: a prospective population-based study. J Hum Hy-
pertens 9: 175–180, 1995.
319. Nishikawa T, Edelstein D, Du XL, Yamagishi SI, Mat-
sumura T, Kaneda Y, Yorek MA, Beebe D, Oates PJ,
Hammes HP, Giardino A, and Brownlee M. Normalizing
mitochondrial superoxide production blocks three path-
ways of hyperglycaemic damage. Nature404: 787–790, 2000.
320. Nissen SE and Wolski K. Effect of rosiglitazone on the risk
of myocardial infarction and death from cardiovascular
causes. N Engl J Med 356: 2457–2471, 2007.
321. Nohl H, Kozlov AV, Gille L, and Staniek K. Cell respira-
tion and formation of reactive oxygen species: facts and
artefacts. Biochem Soc Trans 31: 1308–1311, 2003.
322. Ohara Y, Peterson TE, and Harrison DG. Hypercholes-
terolemia increases endothelial superoxide anion produc-
tion. J Clin Invest 91: 2546–2551, 1993.
323. Ohgami N, Nagai R, Ikemoto M, Arai H, Kuniyasu A, Ho-
riuchi S, and Nakayama H. Cd36, a member of the class b
scavenger receptor family, as a receptor for advanced gly-
cation end products. J Biol Chem 276: 3195–3202, 2001.
324. Oliver JJ and Webb DJ. Noninvasive assessment of arterial
stiffness and risk of atherosclerotic events. Arterioscler
Thromb Vasc Biol 23: 554–566, 2003.
325. Oliver JJ, Webb DJ, and Newby DE. Stimulated tissue plas-
minogen activator release as a marker of endothelial func-
tion in humans. Arterioscler Thromb Vasc Biol 25: 2470–2479,
326. Opar A. Where now for new drugs for atherosclerosis? Nat
Rev Drug Discov 6: 334–335, 2007.
327. Ortiz MC, Sanabria E, Manriquez MC, Romero JC, and Jun-
cos LA. Role of endothelin and isoprostanes in slow pres-
sor responses to angiotensin II. Hypertension 37: 505–510,
328. Ortwerth BJ, James H, Simpson G, and Linetsky M. The
generation of superoxide anions in glycation reactions with
sugars, osones and 3-deoxyosones. Biochem Biophys Res
Commun 245: 1: 161–165, 1998.
329. Ozaki M, Kawashima S, Yamashita T, Ohashi Y, Rikitake
Y, Inoue N, Hirata KI, Hayashi Y, Itoh H, and Yokoyama
M. Reduced hypoxic pulmonary vascular remodeling by
nitric oxide from the endothelium. Hypertension 37: 322–
330. Padro T, Emeis JJ, Steins M, Schmid KW, and Kienast J.
Quantification of plasminogen activators and their inhibi-
tors in the aortic vessel wall in relation to the presence and
severity of atherosclerotic disease. Arterioscler Thromb Vasc
Biol 15: 893–902, 1995.
331. Parker C 3rd, Vita JA, and Freedman JE. Soluble adhesion
molecules and unstable coronary artery disease. Athero-
sclerosis 156: 417–424, 2001.
332. Patel S and Celermajer DS. Assessment of vascular disease
using arterial flow mediated dilatation. Pharmacol Rep
58(suppl): 3–7, 2006.
333. Perticone F, Ceravolo R, Pujia A, Ventura G, Iacopino S,
Scozzafava A, Ferraro A, Chello M, Mastroroberto P,
Verdecchia P, and Schillaci G. Prognostic significance of en-
dothelial dysfunction in hypertensive patients. Circulation
104: 191–196, 2001.
334. Picchi A, Gao X, Belmadani S, Potter BJ, Focardi M, Chil-
ian WM, and Zhang C. Tumor necrosis factor-alpha in-
duces endothelial dysfunction in the prediabetic metabolic
syndrome. Circ Res 99: 69–77, 2006.
335. Podrez EA, Abu-Soud HM, and Hazen SL. Myeloperoxi-
dase-generated oxidants and atherosclerosis. Free Radic Biol
Med 28: 1717–1725, 2000.
336. Pohl U and Busse R. Hypoxia stimulates release of endo-
thelium-derived relaxant factor. Am J Physiol 256: H1595–
337. Pollock DM. Endothelin, angiotensin, and oxidative stress
in hypertension. Hypertension 45: 477–480, 2005.
338. Pomposiello S, Rhaleb NE, Alva M, and Carretero OA. Re-
active oxygen species: role in the relaxation induced by
bradykinin or arachidonic acid via EDHF in isolated porcine
coronary arteries. J Cardiovasc Pharmacol 34: 567–574, 1999.
339. Pyke KE and Tschakovsky ME. The relationship between
shear stress and flow-mediated dilatation: implications for
the assessment of endothelial function. J Physiol 568: 357–
340. Quillen JE, Sellke FW, Brooks LA, and Harrison DG. Isch-
emia-reperfusion impairs endothelium-dependent relax-
ation of coronary microvessels but does not affect large ar-
teries. Circulation 82: 586–594, 1990.
341. Radomski MW, Palmer RM, and Moncada S. An L-argi-
nine/nitric oxide pathway present in human platelets reg-
ulates aggregation. Proc Natl Acad Sci U S A 87: 5193–5197,
342. Raha H and Robinson BH. Mitochondria, oxygen free rad-
icals, disease and ageing. Trends Biochem 25: 502–508, 2000.
343. Rajagopalan S, Kurz S, Munzel T, Tarpey M, Freeman BA,
Griendling KK, and Harrison DG. Angiotensin II-mediated
hypertension in the rat increases vascular superoxide pro-
duction via membrane NADH/NADPH oxidase activa-
tion: contribution to alterations of vasomotor tone. J Clin
Invest 97: 1916–1923, 1996.
344. Ranjan Z, Xiao Z, and Diamond SL. Constitutive NOS ex-
pression in cultured endothelial cells is elevated by fluid
shear stress. Am J Physiol 269: H550–H555, 1995.
345. Rich S and McLaughlin VV. Endothelin receptor blockers
in cardiovascular disease. Circulation 108: 2184–2190, 2003.
346. Richardson TB, Kaspers J, and Porter CD. Retroviral hy-
brid LTR vector strategy: functional analysis of LTR ele-
ments and generation of endothelial cell specificity. Gene
Ther 11: 775–783, 2004.
347. Ridker PM. C-reactive protein and the prediction of car-
diovascular events among those at intermediate risk: mov-
ing an inflammatory hypothesis toward consensus. J Am
Coll Cardiol 49: 2129–2138, 2007.
348. Ridker PM. Clinical application of C-reactive protein for
cardiovascular disease detection and prevention. Circula-
tion 107: 363–369, 2003.
349. Ridker PM, Rifai N, Rose L, Buring JE, and Cook NR. Com-
parison of C-reactive protein and low-density lipoprotein
cholesterol levels in the prediction of first cardiovascular
events. N Engl J Med 347: 1557–1565, 2002.
350. Riethmuller C, Gorren AC, Pitters E, Hemmens B, Habisch
HJ, Heales SJ, Schmidt K, Werner ER, and Mayer B. Acti-
vation of neuronal nitric-oxide synthase by the 5-methyl
analog of tetrahydrobiopterin: functional evidence against
reductive oxygen activation by the pterin cofactor. J Biol
Chem 274: 16047–16051, 1999.
LE BROCQ ET AL.1668
351. Rikitake Y and Liao JK. Rho GTPases, statins, and nitric ox-
ide. Circ Res 97: 1232–1235, 2005.
352. Rimm EB, Stampfer MJ, Ascherio A, Giovannucci E, Colditz
GA, and Willett WC. Vitamin E consumption and the risk
of coronary heart disease in men. N Engl J Med 328:
353. Riobo NA, Clementi E, Melani M, Boveris A, Cadenas E,
Moncada S, and Poderoso JJ. Nitric oxide inhibits mito-
chondrial NADH:ubiquinone reductase activity through
peroxynitrite formation. Biochem J 359: 139–145, 2001.
354. Robinson SD, Ludlam CA, Boon NA, and Newby DE. Phos-
phodiesterase type 5 inhibition does not reverse endothe-
lial dysfunction in patients with coronary heart disease.
Heart 92: 170–176, 2006.
355. Ross R. Atherosclerosis: an inflammatory disease. N Engl J
Med 340: 115–126, 1999.
356. Rossitch E Jr, Alexander E 3rd, Black PM, and Cooke JP. L-
Arginine normalizes endothelial function in cerebral ves-
sels from hypercholesterolemic rabbits. J Clin Invest 87:
357. Rubanyi GM and Vanhoutte PM. Superoxide anions and
hyperoxia inactivate endothelium-derived relaxing factor.
Am J Physiol 250: H822–H827, 1986.
358. Rubenfire M, Cao N, Smith DE, and Mosca L. Carotid artery
reactivity to isometric hand grip exercise identifies persons
at risk and with coronary disease. Atherosclerosis 160: 241–
359. Sakai K, Matsumoto K, Nishikawa T, Suefuji M, Nakamaru
K, Hirashima Y, Kawashima J, Shirotani T, Ichinose K,
Brownlee M, and Araki E. Mitochondrial reactive oxygen
species reduce insulin secretion by pancreatic beta-cells.
Biochem Biophys Res Commun 300: 216–222, 2003.
360. Salame MY, Samani NJ, Masood I, and deBono DP. Ex-
pression of the plasminogen activator system in the human
vascular wall. Atherosclerosis 152: 19–28, 2000.
361. Salvemini D and Marino MH. Inducible nitric oxide synthase
and inflammation. Expert Opin Invest Drugs 7: 65–75, 1998.
362. Sandow SL and Tare M. C-type natriuretic peptide: a new
endothelium-derived hyperpolarizing factor? Trends Phar-
macol Sci 28: 61–67, 2007.
363. Sandow SL, Tare M, Coleman HA, Hill CE, and Parking-
ton HC. Involvement of myoendothelial gap junctions in
the actions of endothelium-derived hyperpolarizing factor.
Circ Res 90: 1108–1113, 2002.
364. Satoh T, Owada S, and Ishida M. Recent aspects in genetic
renal mechanisms involved in hypertension. Intern Med 38:
365. Sawa Y, Morishita R, Suzuki K, Kagisaki K, Kaneda Y,
Maeda K, Kadoba K, and Matsuda H. A novel strategy for
myocardial protection using in vivo transfection of cis ele-
ment “decoy” against NFkappaB binding site: evidence for
a role of NFkappaB in ischemia-reperfusion injury. Circu-
lation 96: II280–II284, 1997.
366. Schachinger V, Britten MB, and Zeiher AM. Prognostic im-
pact of coronary vasodilator dysfunction on adverse long-
term outcome of coronary heart disease. Circulation 101:
367. Schindler TH, Hornig B, Buser PT, Olschewski M,
Magosaki N, Pfisterer M, Nitzsche EU, Solzbach U, and Just
H. Prognostic value of abnormal vasoreactivity of epicar-
dial coronary arteries to sympathetic stimulation in pa-
tients with normal coronary angiograms. Arterioscler
Thromb Vasc Biol 23: 495–501, 2003.
368. Schmidt AM, Hasu M, Popov D, Zhang JH, Chen J, Yan
SD, Brett J, Kuwabara K, and Costache G. Receptor for ad-
vanced glycation end products (AGEs) has a central role in
vessel wall interactions and gene activation in response to
circulating AGE proteins. Proc Natl Acad Sci U S A 91:
369. Schmidt AM, Hori O, Chen JX, Crandall J, Zhang J, Cao R,
Yan SD, Brett J, and Stern D. Advanced glycation end-
products interacting with their endothelial receptor induce
expression of vascular cell adhesion molecule-1 (VCAM-1)
in cultured human endothelial cells and in mice: a poten-
tial mechanism for the accelerated vasculopathy of dia-
betes. J Clin Invest 96: 1395–1403, 1995.
370. Schmidt AM, Vianna M, Gerlach M, Brett J, Ryan J, Kao J,
Esposito C, Hegarty H, Hurley W, and Clauss M. Isolation
and characterization of two binding proteins for advanced
glycosylation end products from bovine lung which are
present on the endothelial cell surface. J Biol Chem 267:
371. Schnyder G, Roffi M, Pin R, Flammer Y, Lange H, Eberli
FR, Meier B, Turi ZG, and Hess OM. Decreased rate of coro-
nary restenosis after lowering of plasma homocysteine lev-
els. N Engl J Med 345: 1593–1600, 2001.
372. Shanmugam N, Reddy MA, Guha M, and Natarajan R.
High glucose-induced expression of proinflammatory cy-
tokine and chemokine genes in monocytic cells. Diabetes 52:
373. Sharma R and Davidoff MN. Oxidative stress and endo-
thelial dysfunction in heart failure. Congest Heart Fail 8:
374. Shi GY, Hau JS, Wang SJ, Wu IS, Chang BI, Lin MT, Chow
YH, Chang WC, Wing LY, and Jen CJ. Plasmin and the reg-
ulation of tissue-type plasminogen activator biosynthesis in
human endothelial cells. J Biol Chem 267: 19363–19368, 1992.
375. Shimoda LA and Semenza GL. Functional analysis of the
role of hypoxia-inducible factor 1 in the pathogenesis of hy-
poxic pulmonary hypertension. Methods Enzymol 381: 121–
376. Shimokawa H and Takeshita A. Rho-kinase is an impor-
tant therapeutic target in cardiovascular medicine. Arte-
rioscler Thromb Vasc Biol 25: 1767–1775, 2005.
377. Shimokawa H, Yasutake H, Fujii K, Owada MK, Nakaike
R, Fukumoto Y, Takayanagi T, Nagao T, Egashira K, Fu-
jishima M, and Takeshita A. The importance of the hyper-
polarizing mechanism increases as the vessel size decreases
in endothelium-dependent relaxations in rat mesenteric cir-
culation. J Cardiovasc Pharmacol 28: 703–711, 1996.
378. Shinozaki K, Ayajiki K, Kashiwagi A, Masada M, and Oka-
mura T. Malfunction of vascular control in lifestyle-related
diseases: mechanisms underlying endothelial dysfunction
in the insulin-resistant state. J Pharmacol Sci 96: 401–405,
379. Shiohira S, Yoshida T, Shirota S, Tsuchiya K, and Nitta K.
Protective effect of carbon monoxide donor compounds in
endotoxin-induced acute renal failure. Am J Nephrol 27:
380. Sies H. Strategies of antioxidant defense. Eur J Biochem 215:
381. Simkins S. Dinitrophenol and desiccated thyroid in the
treatment of obesity. JAMA 108: 2210–2217, 1937.
382. Simon AR, Severgnini M, Takahashi S, Rozo L, Andrahbi
B, Agyeman A, Cochran BH, Day RM, and Fanburg BL. 5-
HT induction of c-fos gene expression requires reactive
oxygen species and Rac1 and Ras GTPases. Cell Biochem Bio-
phys 42: 263–276, 2005.
383. Singh R, Barden A, Mori T, and Beilin L. Advanced glyca-
tion end-products: a review. Diabetologia 44: 129–146, 2001.
384. Smith RAJ, Porteous CM, Coulter CV, and Murphy MP.
Targeting and antioxidant to mitochondria. Eur J Biochem
263: 709–716, 1999.
385. Smith RM, McCarthy J, and Sack MN. TNF alpha is re-
quired for hypoxia-mediated right ventricular hypertro-
phy. Mol Cell Biochem 219: 139–143, 2001.
386. Sofola OA, Knill A, Hainsworth R, and Drinkhill M.
Change in endothelial function in mesenteric arteries of
Sprague-Dawley rats fed a high salt diet. J Physiol 543:
387. Sorensen KE, Celermajer DS, Spiegelhalter DJ, Geor-
gakopoulos D, Robinson J, Thomas O, and Deanfield JE.
Non-invasive measurement of human endothelium de-
pendent arterial responses: accuracy and reproducibility.
Br Heart J 74: 247–253, 1995.
388. Soulis JV, Farmakis TM, Giannoglou GD, and Louridas GE.
Wall shear stress in normal left coronary artery tree. J Bio-
mech 39: 742–749, 2006.
389. Soulis JV, Giannoglou GD, Chatzizisis YS, Farmakis TM,
Giannakoulas GA, Parcharidis GE, and Louridas GE. Spa-
tial and phasic oscillation of non-newtonian wall shear
stress in human left coronary artery bifurcation: an insight
to atherogenesis. Coron Artery Dis 17: 351–358, 2006.
390. Spence JD and Norris J. Infection, inflammation, and ath-
erosclerosis. Stroke 34: 333–334, 2003.
391. Splaver A, Lamas GA, and Hennekens CH. Homocysteine
and cardiovascular disease: biological mechanisms, obser-
vational epidemiology, and the need for randomized trials.
Am Heart J 148: 34–40, 2004.
392. Stangl V, Gunther C, Jarrin A, Bramlage P, Moobed M,
Staudt A, Baumann G, Stangl K, and Felix SB. Homocys-
teine inhibits TNF-alpha-induced endothelial adhesion
molecule expression and monocyte adhesion via nuclear
factor-kappaB dependent pathway. Biochem Biophys Res
Commun 280: 1093–1100, 2001.
393. Stasch JP, Becker EM, Alonso-Alija C, Apeler H, Dem-
bowsky K, Feurer A, Gerzer R, Minuth T, Perzborn E, Pleiss
U, Schroder H, Schroeder W, Stahl E, Steinke W, Straub A,
and Schramm M. NO-independent regulatory site on sol-
uble guanylate cyclase. Nature 410: 212–215, 2001.
394. Steinberg D. Atherogenesis in perspective: hypercholes-
terolemia and inflammation as partners in crime. Nat Med
8: 1211–1217, 2002.
395. Steinberg HO, Chaker H, Leaming R, Johnson A, Brechtel
G, and Baron AD. Obesity/insulin resistance is associated
with endothelial dysfunction: implications for the syn-
drome of insulin resistance. J Clin Invest 97: 2601–2610,
396. Steiner MK, Preston IR, Klinger JR, and Hill NS. Pulmonary
hypertension: inhaled nitric oxide, sildenafil and natriuretic
peptides. Curr Opin Pharmacol 5: 245–250, 2005.
397. Steinhorn RH, Russell JA, Lakshminrusimha S, Gugino SF,
Black SM, and Fineman JR. Altered endothelium-depen-
dent relaxations in lambs with high pulmonary blood flow
and pulmonary hypertension. Am J Physiol 280: H311–
398. Steins MB, Padro T, Li CX, Mesters RM, Ostermann H,
Hammel D, Scheld HH, Berdel WE, and Kienast J. Over-
expression of tissue-type plasminogen activator in athero-
sclerotic human coronary arteries. Atherosclerosis 145: 173–
399. Stenvinkel P. Endothelial dysfunction and inflammation-is
there a link? Nephrol Dial Transplant 16: 1968–1971, 2001.
400. Stephens NG, Parsons A, Schofield PM, Kelly F, Cheese-
man K, and Mitchinson MJ. Randomised controlled trial of
vitamin E in patients with coronary disease: Cambridge
Heart Antioxidant Study (CHAOS). Lancet 347: 781–786,
401. Stone JR and Yang S. Hydrogen peroxide: a signaling mes-
senger. Antioxid Redox Signal 8: 243–270, 2006.
402. Stone PH, Coskun AU, Yeghiazarians Y, Kinlay S, Popma
JJ, Kuntz RE, and Feldman CL. Prediction of sites of coro-
nary atherosclerosis progression: in vivo profiling of en-
dothelial shear stress, lumen, and outer vessel wall char-
acteristics to predict vascular behavior. Curr Opin Cardiol
18: 458–470, 2003.
403. Stuehr DJ, Santolini J, Wang ZQ, Wei CC, and Adak S. Up-
date on mechanism and catalytic regulation in the NO syn-
thases. J Biol Chem 279: 36167–36170, 2004.
404. Stühlinger MC, Tsao PS, Her JH, Kimoto M, Balint RF, and
Cooke JP. Homocysteine impairs the nitric oxide synthase
pathway: role of asymmetric dimethylarginine. Circulation
104: 2569–2575, 2001.
405. Sugiyama S, Okada Y, Sukhove GK, Virmani R, Heinecke
JW, and Libby P. Macrophage myeloperoxidase regulation
by granulocyte macrophage colony-stimulating factor in
human atherosclerosis and implications in acute coronary
syndromes. Am J Pathol 158: 879–891, 2001.
406. Sugiyama T, Yoshimoto T, Sato R, Fukai N, Ozawa N,
Shichiri M, and Hirata Y. Endothelin-1 induces cyclooxy-
genase-1 expression and generation of reactive oxygen
species in endothelial cells. J Cardiovasc Pharmacol 44(suppl
1): S332–S335, 2004.
407. Sullivan CC, Du L, Chu D, Cho AJ, Kido M, Wolf PL,
Jamieson SW, and Thistlethwaite PA. Induction of pul-
monary hypertension by an angiopoietin 1/TIE2/serotonin
pathway. Proc Natl Acad Sci U S A 100: 12331–12336, 2003.
408. Suwaidi JA, Hamasaki S, Higano ST, Nishimura RA,
Holmes DR Jr, and Lerman A. Long-term follow-up of pa-
tients with mild coronary artery disease and endothelial
dysfunction. Circulation 101: 948–954, 2000.
409. Szabo C and Ohshima H. DNA damage induced by per-
oxynitrite: subsequent biological effects. Nitric Oxide 1:
410. Taddei S, Galetta F, Virdis A, Ghiadoni L, Salvetti G, Fran-
zoni F, Giusti C, and Salvetti A. Physical activity prevents
age-related impairment in nitric oxide availability in el-
derly athletes. Circulation 101: 2896–2901, 2000.
411. Tambyraja AL, Mitchell R, Driscoll PJ, Deans C, Parks RW,
Rahman I, and Megson IL. Glutathione supplementation to
University of Wisconsin solution causes endothelial dys-
function. Transplant Immunol 18: 146–150, 2007.
412. Tan KC, Chow WS, Ai VH, Metz C, Bucala R, and Lam KS.
Advanced glycation end products and endothelial dys-
function in type 2 diabetes. Diabetes Care 25: 1055–1059,
413. Tanabe T, Maeda S, Miyauchi T, Iemitsu M, Takanashi M,
Irukayama-Tomobe Y, Yokota T, Ohmori H, and Matsuda
M. Exercise training improves ageing-induced decrease in
eNOS expression of the aorta. Acta Physiol Scand 178: 3–10,
414. Targonski PV, Bonetti PO, Pumper GM, Higano ST,
Holmes DR Jr, and Lerman A. Coronary endothelial
dysfunction is associated with an increased risk of
cerebrovascular events. Circulation
415. Teran FJ, Johnson RA, Stevenson BK, Peyton KJ, Jackson
KE, Appleton SD, Durante W, and Johnson FK. Heme oxy-
genase-derived carbon monoxide promotes arteriolar en-
dothelial dysfunction and contributes to salt-induced hy-
LE BROCQ ET AL.1670
pertension in Dahl salt-sensitive rats. Am J Physiol Regul In-
tegr Comp Physiol 288: R615–R622, 2005.
416. Thorup C, Jones CL, Gross SS, Moore LC, and Goligorsky
MS. Carbon monoxide induces vasodilation and nitric ox-
ide release but suppresses endothelial NOS. Am J Physiol
Renal Physiol 277: F882–F889, 1999.
417. Title LM, Cummings PM, Giddens K, Genest JJ Jr, and Nas-
sar BA. Effect of folic acid and antioxidant vitamins on en-
dothelial dysfunction in patients with coronary artery dis-
ease. J Am Coll Cardiol 36: 758–765, 2000.
418. Toda N, Matsumoto T, and Yoshida K. Comparison of hy-
poxia-induced contraction in human, monkey, and dog
coronary arteries. Am J Physiol 262: H678–H683, 1992.
419. Todd S, Woodward M, Tunstall-Pedoe H, and Bolton-
Smith C. Dietary antioxidant vitamins and fiber in the eti-
ology of cardiovascular disease and all-cause mortality: re-
sults from the Scottish Heart Health Study. Am J Epidemiol
150: 1073–1080, 1999.
420. Topal G, Brunet A, Millanvoye E, Boucher JL, Rendu F, De-
vynk MA, and David-dufilho M. Homocysteine induces ox-
idative stress by uncoupling of NO synthase activity
through reduction of tetrahydrobiopterin. Free Radic Biol
Med 36: 1532–1541, 2004.
421. Tornqvist H, Mills NL, Gonzalez M, Miller MR, Robinson
SD, Megson IL, Macnee W, Donaldson K, Soderberg S,
Newby DE, Sandstrom T, and Blomberg A. Persistent en-
dothelial dysfunction following diesel exhaust inhalation
in man. Am J Respir Crit Care Med 176: 395–400, 2007.
422. Tousoulis D, Antoniades C, Tentolouris C, Tsioufis C,
Toutouza M, Toutouzas P, and Stefanadis C. Effects of com-
bined administration of vitamins C and E on reactive hy-
peremia and inflammatory process in chronic smokers.
Atherosclerosis 170: 261–267, 2003.
423. Tousoulis D, Davies G, Lefroy DC, Haider AW, and Crake
T. Variable coronary vasomotor responses to acetylcholine in
patients with normal coronary arteriograms: evidence for lo-
calised endothelial dysfunction. Heart 75: 261–266, 1996.
424. Touyz RM. Reactive oxygen species, vascular oxidative
stress, and redox signaling in hypertension: what is the clin-
ical significance? Hypertension 44: 248–252, 2004.
425. Touyz RM, Tabet F, and Schiffrin EL. Redox-dependent sig-
nalling by angiotensin II and vascular remodelling in hy-
pertension. Clin Exp Pharmacol Physiol 30: 860–866, 2003.
426. Touyz RM, Yao G, Viel E, Amiri F, and Schiffrin EL. An-
giotensin II and endothelin-1 regulate MAP kinases
through different redox-dependent mechanisms in human
vascular smooth muscle cells. J Hypertens 22: 1141–1149,
427. Traub O and Berk BC. Laminar shear stress: mechanisms
by which endothelial cells transduce an atheroprotective
force. Arterioscler Thromb Vasc Biol 18: 677–685, 1998.
428. Tricot O, Mallat Z, Heymes C, Belmin J, Leseche G, and
Tedgui A. Relation between endothelial cell apoptosis and
blood flow direction in human atherosclerotic plaques. Cir-
culation 101: 2450–2453, 2000.
429. True AL, Olive M, Boehm M, San H, Westrick RJ,
Raghavachari N, Xu X, Lynn EG, Sack MN, Munson PJ,
Gladwin MT, and Nabel EG. Heme oxygenase-1 deficiency
accelerates formation of arterial thrombosis through ox-
idative damage to the endothelium, which is rescued by in-
haled carbon monoxide. Circ Res 101: 893–901, 2007.
430. Tschudi MR, Barton M, Bersinger NA, Moreau P, Cosentino
F, Noll G, Malinski T, and Luscher TF. Effect of age on ki-
netics of nitric oxide release in rat aorta and pulmonary
artery. J Clin Invest 98: 899–905, 1996.
431. Tuder RM, Cool CD, Geraci MW, Wang J, Abman SH,
Wright L, Badesch D, and Voelkel NF. Prostacyclin syn-
thase expression is decreased in lungs from patients with
severe pulmonary hypertension. Am J Respir Crit Care Med
159: 1925–1932, 1999.
432. Tuder RM, Flook BE, and Voelkel NF. Increased gene ex-
pression for VEGF and the VEGF receptors KDR/Flk and
Flt in lungs exposed to acute or to chronic hypoxia: mod-
ulation of gene expression by nitric oxide. J Clin Invest 95:
433. Uehata A, Takase B, Nishioka T, Kitamura K, Akima T, Ku-
rita A, and Isojima K. Effect of quinapril versus nitrendip-
ine on endothelial dysfunction in patients with systemic
hypertension. Am J Cardiol 87: 1414–1416, 2001.
434. Urbich C and Dimmeler S. Endothelial progenitor cells:
characterization and role in vascular biology. Circ Res 95:
435. Valencia JV, Mone M, Zhang J, WEetall M, Buxton FP, and
Hughes TE. Divergent pathways of gene expression are ac-
tivated by the RAGE ligands S100b and AGE-BSA. Diabetes
53: 743–751, 2004.
436. Vallance P, Collier J, and Bhagat K. Infection, inflamma-
tion, and infarction: does acute endothelial dysfunction
provide a link? Lancet 349: 1391–1392, 1997.
437. van der Loo B, Labugger R, Skepper JN, Bachschmid M,
Kilo J, Powell JM, Palacios-Callender M, Erusalimsky JD,
Quaschning T, Malinski T, Gygi D, Ullrich V, and Luscher
TF. Enhanced peroxynitrite formation is associated with
vascular aging. J Exp Med 192: 1731–1744, 2000.
438. van Hinsbergh VWM. Endothelial permeability for macro-
molecules: mechanistic aspects of pathophysiological mod-
ulation. Arterioscler Thromb Vasc Biol 17: 1018–1023, 1997.
439. Van Renterghem C, Vigne P, Barhanin J, Schmid-Alliana
A, Frelin C, and Lazdunski M. Molecular mechanism of ac-
tion of the vasoconstrictor peptide endothelin. Biochem Bio-
phys Res Commun 157: 977–985, 1988.
440. van Venrooij FV, van de Ree MA, Bots ML, Stolk RP, Huis-
man MV, Banga JD, and DALI Study Group. Aggressive
lipid lowering does not improve endothelial function in
type 2 diabetes: the Diabetes Atorvastatin Lipid Interven-
tion (DALI) Study: a randomized, double-blind, placebo-
controlled trial. Diabetes Care 25: 1211–1216, 2002.
441. Vane JR, Bunting S, and Moncada S. Prostacyclin in phys-
iology and pathophysiology. Int Rev Exp Pathol 23: 161–207,
442. Vasquez-Vivar J, Duquaine D, Whitsett J, Kalyanaraman B,
and Rajagopalan S. Altered tetrahydrobiopterin metabo-
lism in atherosclerosis: implications for use of oxidized
tetrahydrobiopterin analogues and thiol antioxidants. Ar-
terioscler Thromb Vasc Biol 22: 1655–1661, 2002.
443. Vasquez-Vivar J, Kalyanaraman B, and Kennedy MC. Mi-
tochondrial aconitase is a source of hydroxyl radical: an
electron spin resonance investigation. J Biol Chem 275:
444. Verma S, Wang CH, Li SH, Dumont AS, Fedak PW, Badi-
wala MV, Dhillon B, Weisel RD, Li RK, Mickle DA, and
Stewart DJ. A self-fulfilling prophecy: C-reactive protein at-
tenuates nitric oxide production and inhibits angiogenesis.
Circulation 106: 913–919, 2002.
445. Vermeulen EG, Stehouwer CD, Twisk JW, van den Berg M,
de Jong SC, Mackaay AJ, van Campden CM, Visser FC,
Jakobs CA, Bulterjis EJ, and Rauwerda JA. Effect of homo-
cysteine-lowering treatment with folic acid plus vitamin B6
on progression of subclinical atherosclerosis: a randomised,
placebo-controlled trial. Lancet 355: 517–522, 2000.
446. Vinten-Johansen J. Involvement of neutrophils in the
pathogenesis of lethal myocardial reperfusion injury. Car-
diovasc Res 61: 481–497, 2004.
447. Vlassara H, Brownlee M, Manogue KR, Dinarello CA, and
Pasagian A. Cachetin, TNF and IL-1 induced by glucose-
modified proteins: role in normal tissue remodelling. Sci-
ence 240: 1546–1548, 1988.
448. Vlassara H, Fuh H, Makita Z, Krungkrai S, Cerami A, and
Bucala R. Exogenous advanced glycosylation end products
induce complex vasculat dysfunction in normal animals: a
model for diabetic and aging complications. Proc Natl Acad
Sci U S A 89: 12043–12047, 1992.
449. Vlassara H, Li YM, Imani F, Wojciechowicz D, Yang Z, Liu
FT, and Cerami A. Identification of glaectin-3 as a high-
affinity binding protein for advanced glycation end prod-
ucts (AGE): a new member of the AGE-receptor complex.
Mol Med 1: 634–646, 1995.
450. Vlassara H and Palace MR. Diabetes and advanced glyca-
tion endproducts. J Intern Med 251: 87–101, 2002.
451. von Klot S, Peters A, Aalto P, Bellander T, Berglind N,
D’Ippoliti D, Elosua R, Hormann A, Kulmala M, Lanki T,
Lowel H, Pekkanen J, Picciotto S, Sunyer J, Forastiere F and
health effects of particles on susceptible subpopulations
(HEAPSS) study group: ambient air pollution is associated
with increased risk of hospital cardiac readmissions of
myocardial infarction survivors in five European cities. Cir-
culation 112: 3073–3079, 2005.
452. Waid DK, Chell M, and El-Fakahany EE. M(2) and M(4)
muscarinic subtypes couple to activation of endothelial ni-
tric oxide synthase. Pharmacology 61: 37–42, 2000.
453. Walch L, Gascard JP, Dulmet E, Brink C, and Norel X. Ev-
idence for a M1muscarinic receptor on the endothelium of
human pulmonary veins. Br J Pharmacol 130: 73–78, 2000.
454. Wang D, Borrego-Conde LJ, Falck JR, Sharma KK, Wilcox
CS, and Umans JG. Contributions of nitric oxide, EDHF,
and EETs to endothelium-dependent relaxation in renal af-
ferent arterioles. Kidney Int 63: 2187–2193, 2003.
455. Wang M, Zukas AM, Hui Y, Ricciotti E, Pure E, and
FitzGerald GA. Deletion of microsomal prostaglandin E
synthase-1 augments prostacyclin and retards atherogene-
sis. Proc Natl Acad Sci U S A 103: 14507–14512, 2006.
456. Wang R.Two’s company, three’s a crowd: can H2S be the
third endogenous gaseous transmitter? FASEB J 16: 1792–
457. Wang R, Wang Z, and Wu L. Carbon monoxide-induced
vasorelaxation and the underlying mechanisms. Br J Phar-
macol 121: 927–934, 1997.
458. Warabi E, Wada Y, Kajiwara H, Kobayashi M, Koshiba N,
Hisada T, Shibata M, Ando J, Tsuchiya M, Kodama T, and
Noguchi N. Effect on endothelial cell gene expression
of shear stress, oxygen concentration, and low-density
lipoprotein as studied by a novel flow cell culture system.
Free Radic Biol Med 37: 682–694, 2004.
459. Wautier JL and Guillausseau PJ. Advanced glycation end
products, their receptors and diabetic angiopathy. Diabetes
Metab 27: 535–542, 2001.
460. Wedgwood S, McMullan DM, Bekker JM, Fineman JR, and
Black SM. Role for endothelin-1-induced superoxide and
peroxynitrite production in rebound pulmonary hyperten-
sion associated with inhaled nitric oxide therapy. Circ Res
89: 357–364, 2001.
461. West IC. Radicals and oxidative stress in diabetes. Diabet
Med 17: 171–180, 2000.
462. Widlansky ME, Gokce N, Keaney JF Jr, and Vita JA. The
clinical implications of endothelial dysfunction. J Am Coll
Cardiol 42: 1149–1160, 2003.
463. Wilkens H, Guth A, Konig J, Forestier N, Cremers B, Hen-
nen B, Bohm M, and Sybrecht GW. Effect of inhaled ilo-
prost plus oral sildenafil in patients with primary pul-
monary hypertension. Circulation 104: 1218–1222, 2001.
464. Wilkinson IB, Megson IL, MacCallum H, Rooijmans DF,
Johnson SM, Boyd JL, Cockcroft JR, and Webb DJ. Acute
methionine loading does not alter arterial stiffness in hu-
mans. J Cardiovasc Pharmacol 37: 1–5, 2001.
465. Williams FM. Neutrophils and myocardial reperfusion in-
jury. Pharmacol Ther 72: 1–12, 1996.
466. Wilson SH, Simari RD, and Lerman A. The effect of en-
dothelin-1 on nuclear factor kappa B in macrophages.
Biochem Biophys Res Commun 286: 968–972, 2001.
467. Woo KS, Chook P, Lolin YI, Sanderson JE, Metreweli C,
and Celermajer DS. Folic acid improves arterial endothe-
lial function in adults with hyperhomocystinemia. J Am Coll
Cardiol 34: 2002–2006, 1999.
468. Wu VY, Shearman CW, and Cohen MP. Identification of
calnexin as a binding protein for Amadori-modified gly-
cated albumin. Biochem Biophys Res Commun 284: 602–606,
469. Xiao J and Pang P. Does a general alteration in the nitric
oxide synthesis system occur in spontaneously hyperten-
sive rats? Am J Physiol 266: H272–H278, 1994.
470. Xu B, Chibber R, Ruggiero D, Kohner E, Ritter J, and Ferro
A. Impairment of vascular endothelial nitric oxide synthase
activity by advanced glycation end products. FASEB J 17:
471. Xu B, Ji Y, Yao K, Cao YX, and Ferro A. Inhibition of hu-
man endothelial cell nitric oxide synthesis by advanced gly-
cation end products but not glucose: relevance to diabetes.
Clin Sci 109: 439–446, 2005.
472. Xu XP, Pollock JS, Tanner MA, and Myers PR. Hypoxia ac-
tivates nitric oxide synthase and stimulates nitric oxide pro-
duction in porcine coronary resistance arteriolar endothe-
lial cells. Cardiovasc Res 30: 841–847, 1995.
473. Xu XP, Tanner MA, and Myers PR. Prostaglandin-medi-
ated inhibition of nitric oxide production by bovine aortic
endothelium during hypoxia. Cardiovasc Res 30: 345–350,
474. Yamakura F, Taka H, Fujimura T, and Murayama K. Inac-
tivation of human manganese-superoxide dismutase by
peroxynitrite is caused by exclusive nitration of tyrosine 34
to 3-nitrotyrosine. J Biol Chem 273: 14085–14089, 1998.
475. Yamawaki H and Iwai N. Mechanisms underlying nano-
sized air-pollution-mediated progression of atherosclero-
sis: carbon black causes cytotoxic injury/inflammation and
inhibits cell growth in vascular endothelial cells. Circ J 70:
476. Yan SD, Schmidt AM, Anderson GM, Zhang J, Brett J, Zou
YS, Pinsky D, and Stern D. Enhanced cellular oxidant stress
by the interaction of advanced glycation end products with
their receptors/binding proteins. J Biol Chem 269: 9889–
477. Yang BC and Mehta JL. Critical role of endothelium in sus-
tained arterial contraction during prolonged hypoxia. Am
J Physiol 268: H1015–H1020, 1995.
478. Yang Z, Makita Z, Horii Y, Brunelle S, Cerami A, Sehajpal
P, Suthanthiran M, and Vlassara H. Two novel rat lever
membrane proteins that bind advanced glycosylation end-
products: relationship to macrophage receptor for glucose-
modified proteins. J Exp Med 174: 515–524, 1991.
479. Yeh CH, Sturgis L, Haidacher J, Zhang XN, Sherwood SJ,
Bjercke RJ, Juhasz O, Crow MT, Tilton RG, and Denner L.
Requirement for p38 and p44-p42 mitogen-activated pro-
tein kinases in RAGE-mediated nuclear factor-kappaB tran-
LE BROCQ ET AL.1672
scriptional activation and cytokine secretion. Diabetes 50:
480. You J, Golding EM, and Bryan RM Jr. Arachidonic acid
metabolites, hydrogen peroxide, and EDHF in cerebral ar-
teries. Am J Physiol Heart Circ Physiol 289: H1077–H1083,
481. Yu SM, Hung LM, and Lin CC. cGMP-elevating agents sup-
press proliferation of vascular smooth muscle cells by in-
hibiting the activation of epidermal growth factor signal-
ing pathway. Circulation 95: 1269–1277, 1997.
482. Yusuf S, Dagnenais G, Pogue J, Bosch J, and Sleight P. Vi-
tamin E supplementation and cardiovascular events in
high-risk patients: the Heart Outcomes Prevention Evalu-
ation Study Investigators. N Engl J Med 342: 154–160, 2000.
483. Yusuf S, Sleight P, Pogue J, Bosch J, Davies R, and Dage-
nais G. Effects of an angiotensin-converting-enzyme inhib-
itor, ramipril, on cardiovascular events in high-risk pa-
tients: the Heart Outcomes Prevention Evaluation Study
Investigators. N Engl J Med 342: 145–153, 2000.
484. Zamora MA, Dempsey EC, Walchak SJ, and Stelzner TJ.
BQ123, an ETA receptor antagonist, inhibits endothelin-1-
mediated proliferation of human pulmonary artery smooth
muscle cells. Am J Respir Cell Mol Biol 9: 429–433, 1993.
485. Zeiher AM, Drexler H, Wollschlager H, and Just H. Endo-
thelial dysfunction of the coronary microvasculature is as-
sociated with coronary blood flow regulation in patients
with early atherosclerosis. Circulation 84: 1984–1992, 1991.
486. Zeiher AM, Goebel H, Schachinger V, and Ihling C. Tissue
endothelin-1 immunoreactivity in the active coronary ath-
erosclerotic plaque: a clue to the mechanism of increased
vasoreactivity of the culprit lesion in unstable angina. Cir-
culation 91: 941–947, 1995.
487. Zelis R and Flaim SF. Alterations in vasomotor tone in con-
gestive heart failure. Prog Cardiovasc Dis 24: 437–459, 1982.
488. Zhang C, Xu X, Potter BJ, Wang W, Kuo L, Michael L, Bagby
GJ, and Chilian WM. TNF-alpha contributes to endothelial
dysfunction in ischemia/reperfusion injury. Arterioscler
Thromb Vasc Biol 26: 475–480, 2006.
489. Zhang R, Brennan ML, Fu X, Aviles RJ, Pearce GL, Penn
MS, Topol EJ, Sprecher DL, and Hazen SL. Association be-
tween myeloperoxidase levels and risk of coronary artery
disease. JAMA 286: 2136–2142, 2001.
490. Zhang R, Brennan ML, Shen Z, MacPherson JC, Schmitt D,
Molenda CE, and Hazen SL. Myeloperoxidase functions as
a major enzymatic catalyst for initiation of lipid peroxida-
tion at sites of inflammation. J Biol Chem 277: 46116–46122,
491. Zhang R, Shen Z, Nauseef WM, and Hazen SL. Defects in
leukocyte-mediated initiation of lipid peroxidation in
plasma as studied in myeloperoxidase-deficient subjects:
systematic identification of multiple endogenous diffusible
substrates for myeloperoxidase in plasma. Blood 99: 1802–
492. Zhang Y, Park TS, and Gidday JM. Hypoxic precondition-
ing protects human brain endothelium from ischemic
apoptosis by Akt-dependent survivin activation. Am J Phys-
iol Heart Circ Physiol 292: H2573–H2581, 2007.
493. Zhao W and Wang R. H2S-induced vasorelaxation and un-
derlying cellular and molecular mechanisms. Am J Physiol
Heart Circ Physiol 283: H474–H480, 2002.
494. Zhao W, Zhang J, Lu Y, and Wang R. Modulation of en-
dogenous production of H2S in rat tissues. Can J Physiol
Pharmacol 81: 848–853, 2003.
495. Zhao W, Zhang J, Lu Y, and Wang R. The vasorelaxant ef-
fect of H2S as a novel endogenous gaseous KATPchannel
opener. EMBO J 20: 6000–6016, 2001.
496. Zheng L, Nukuna B, Brennan ML, Sun M, Goormastic M,
Settle M, Schmitt D, Fu X, Thomson L, Fox PL, Ischiro-
poulos H, Smith JD, Kinter M, and Hazen SL. Apolipopro-
tein A-I is a selective target for myeloperoxidase-catalyzed
oxidation and functional impairment in subjects with car-
diovascular disease. J Clin Invest 114: 529–541, 2004.
497. Zou MH. Peroxynitrite and protein tyrosine nitration of
prostacyclin synthase. Prostaglandins Other Lipid Mediat 82:
Address reprint requests to:
Prof. Ian L Megson
Free Radical Research Facility, Department of Diabetes
UHI Millennium Institute
The Green House, Beechwood Business Park North
Inverness, UK IV2 3BL
Date of first submission to ARS Central, December 10, 2007;
date of final revised submission, March 18, 2008; date of ac-
ceptance, March 18, 2008.
Page 44 Download full-text