Current Pharmaceutical Design, 2008, 14, 1761-1770 1761

Endothelial Function Assessment in Complicated Hypertension

A. Virdis*, L. Ghiadoni, D. Versari, C. Giannarelli, A. Salvetti and S. Taddei

Department of Internal Medicine, University of Pisa, Pisa, Italy

Abstract: A large body of evidence indicates that patients with essential hypertension, and even more those with complicated hyperten-
sion, are characterized by endothelial dysfunction characterized by impaired NO availability secondary to oxidative stress production. A
dysfunctioning endothelium is an early marker of the development of atherosclerotic changes and can also contribute to cardiovascular
events. Vascular reactivity tests represent the most widely used methods in the clinical assessment of endothelial function. In the last two
decades, many studies have evaluated the endothelium in hypertensive patients, using different techniques. Several methodologies were
developed to study microcirculation (resistance arteries and arterioles) and macrocirculation (conduit arteries), both in coronary and pe-
ripheral vascular districts.
This review will centre on the most relevant available techniques in the research on endothelial dysfunction in essential hypertension,
their advantages and limitations, focusing on available data on endothelial dysfunction which characterizes patients with complicated hy-
pertension.
No available test to assess endothelial function has sufficient sensitivity and specificity to be used in clinical practice. Therefore, the op-
timal methodology for investigating the multifaceted aspects of endothelial dysfunction is still under debate. Only the growing concor-
dant results from different reproducible and reliable methods exploring endothelial function with different stimuli will support and
strengthen experimental findings, thus providing conclusive answers in this area of research.
Key Words: Endothelium, hypertension, nitric oxide, large arteries, microcirculation, cardiovascular risk factors.

INTRODUCTION opportunity to detect early disease, quantify risk, judge response to

Since the pioneering report by the 1998 Nobel Prize-winner
Robert Furchgott on the obligatory role of endothelium in vascular
relaxation in response to acetylcholine [1], an impressive array of
evidence has made it possible to state today that vascular endothe-
lium plays a primary role in the control of vascular function and
structure by the production of nitric oxide (NO) [2]. NO derives
from the transformation of L-arginine into citrulline by the activity
of the constitutive endothelial enzyme NO synthase (eNOS). NO is
produced and released either basally or under the influence of ago-
nists, such as acetylcholine, bradykinin, substance P, serotonin and
others acting on specific endothelial receptors, and by mechanical
forces, such as shear stress [2]. Other endothelium-derived relaxing
factors include prostacyclin and the production of different endo-
thelium-derived hyperpolarizing factors (EDHFs), which represent
a compensatory vasodilating pathway acting in those clinical condi-
tions characterized by a reduced NO availability [3, 4] In several
pathological conditions, activation of endothelial cells can lead to
the production and release of contracting factors including cy-
clooxygenase-derived endothelium-dependent contracting factors,
represented by prostanoids (thromboxane A2 and prostaglandin H2)
[2], which counteract the relaxing activity of NO, and reactive oxy-
gen species (ROS) which impair endothelial function by causing
NO breakdown [2]. In particular ROS, mainly superoxide anion,
traps NO to form substances including peroxynitrites, which are
thought to alter vascular function and structure [5]. Of importance,
NO and EDCFs not only exert an opposite effect on vascular tone,
but also respectively inhibit and activate those mechanisms, such as
platelet aggregation, vascular smooth muscle cell proliferation and
migration, monocyte adhesion, and adhesion molecule expression,
which exert an important role in the pathogenesis of thrombosis and
atherosclerotic plaque, as summarized in Fig. (1). Appreciation of
the central role played by endothelium in the atherosclerotic disease
process has led to the development of a range of methods to assess
different aspects of endothelial function. These have provided not
only novel insights into patho-physiology, but also a clinical
*Address correspondence to this author at the Department of Internal Medi-
cine, University of Pisa, Via Roma, 67, 56100 Pisa, Italy; Tel: +39-50-
992558; Fax: +39-50-551110; E-mail: a.virdis@med.unipi.it
interventions designed to prevent progression of early disease, and
reduce later adverse events in patients [6, 7]. It should be noticed
that endothelial function, unlike measures of vessel wall morphol-
ogy, has intrinsic biological variability, and thus a single measure-
ment, may give only a snapshot and limited information. Neverthe-
less, several longitudinal studies have shown that endothelial dys-
function is an early indicator of atherosclerotic damage and has a
prognostic value in patients with established coronary disease and
at high cardiovascular risk [8].
A large body of evidence indicates that patients with essential
hypertension, and further more those with complicated hyperten-
sion, are characterized by endothelial dysfunction, an alteration
detected at the level of macro- and microcirculation, including
coronary and peripheral vascular districts [9]. This review will cen-
tre on different available techniques to study endothelial function in
essential hypertension, their advantages and limitations, focusing on
endothelial dysfunction in patients with complicated hypertension.
ASSESSMENT OF ENDOTHELIAL FUNCTION IN HY-
PERTENSION
Vascular Reactivity Tests
The autocrine/paracrine activity of endothelial cells makes very
difficult to investigate endothelial function in clinical research.
Usually, it is tested by vascular reactivity studies [6]. Indeed, it is
possible to activate or inhibit endothelial cells in several vascular
regions and measure the vessel changes induced by experimental
perturbation. Endothelial cells can be activated by agonists operat-
ing through specific receptors or by increasing shear stress. In addi-
tion, it is possible to block pathways involved in endothelial re-
sponses such as NOS activity by NG-mono-methyl-L-arginine (L-
NMMA), hyperpolarization of vascular smooth muscle cells (by
ouabain), cyclooxygenase activity (by indomethacin) or oxidative
stress (by antioxidants such as vitamin C). In describing the ap-
proach to the evaluation of endothelium-dependent mechanisms in
humans, it is crucial to consider the type of vascular bed to be in-
vestigated. It must to be distinguished between macrocirculation
(large arteries) and microcirculation. These two vessel types are
differently regulated and therefore results obtained in large arteries
cannot be extrapolated to microvascular regions. Valuable large

1381-6128/08 $55.00+.00 © 2008 Bentham Science Publishers Ltd.

1762 Current Pharmaceutical Design, 2008, Vol. 14, No. 18
Fig. (1). Vascular effects of nitric oxide (NO) and endothelium-dependent contracting factors (EDCFs).
arteries include brachial (most frequently), radial, femoral, and
epicardial arteries. Microcirculation can be evaluated in the periph-
eral muscle (usually forearm), in the subcutaneous tissue, skin and
in the coronary circulation.
Evaluation of Macrocirculation
The most widely used technique for assessing endothelial func-
tion is the so called “flow-mediated dilation” (FMD). In this non
invasive method, introduced in 1992 [10], brachial artery diameter
is measured before and after an increase in shear stress induced by
reactive hyperemia. When a sphygmomanometer cuff placed on the
forearm distal to the brachial artery is inflated to 200 mmHg and
subsequently released 5 minutes later, FMD occurs as a result of
local endothelial release of NO [11, 12]. Endothelium-independent
dilator response can be tested by low dose sublingual nitroglycerin
[13]. FMD has been studied widely in clinical research as it enables
serial evaluation of young subjects, including children. It also per-
mits testing of lifestyle and pharmacological interventions on endo-
thelial biology at an early preclinical stage, when the disease proc-
ess is most likely to be reversible [7]. However, some practical
challenges need to be overcome before this technique could be
suitable for use in routine clinical practice [6, 7, 14]. These chal-
lenges include the need for highly trained operators, the expense of
the equipment, and also the care required to minimize the effect of
environmental or physiological influences [15]. It is important to
note that variations in technique, such as the position of the occlud-
ing cuff and duration of inflation, may produce results that are less
representative of local NO activity, since FMD is also determined,
in part, by the magnitude of post-ischemic forearm vasodilatation,
which is a measure of microcirculatory function [7]. Furthermore,
due to the variability and degree of response, large study population
are required for clinical studies [6, 7, 14].
The use of camp to held and to adjust probe position, as well as
of computerized system to measure automatically brachial artery
diameter are currently require to obtain the best reproducibility of
this non invasive technique [6, 16, 17].
Other alternative non invasive approaches have been also re-
cently suggested. The
2 agonist salbutamol reduce arterial stiffness
Virdis et al.
and wave reflection measured by pulse wave analysis (radial artery
tonometry), in an NO-dependent manner without significant reduc-
tion in blood pressure when given by inhaler at standard clinical
doses [18]. Reactive hyperemia elicit changes in arterial pulse wave
velocity and digital pulse volume which can be measured by oscil-
lometry to identify limb arterial pulse pressure, wave form, timing,
and also digital pulse amplitude tonometry [19]. However, when
using these techniques, the relative contribution of macro- and mi-
crocirculation, as well as structural alterations in the vessel wall
versus endothelial response remains uncertain. Further validation is
therefore required, which should include a wider study of their re-
producibility in different age groups and stages of disease, as well
as clarification of their relationships with other established meas-
ures of endothelial function.
Endothelium-dependent response can be evaluated in the coro-
nary circulation at level of pericardial vessels (usually in the left
descending artery) by quantitative angiography allows assessment
of coronary blood flow during the intracoronary infusion of sub-
stances which can influence either endothelium-dependent (acetyl-
choline) or endothelium-independent (nitrates, papaverine) vasodi-
lation of the epicardial vessels. This experimental model provided
important insight into the vascular effects of risk factors. In particu-
lar, it was clearly evidenced the presence of endothelial dysfunc-
tion, show as vasoconstriction instead of vasodilation to acetylcho-
line in epicardial arteries of essential hypertensive patients in ab-
sence of angiographycally detectable lesions [20-22]. A FMD can
be also obtained in the epicardial arteries by increasing distal mi-
crocirculatory flow with papaverine administration [23]. Endothe-
lial dysfunction in epicardial arteries have been shown to be of
prognostic value in patients with established coronary disease [23,
24].
Evaluation of Microcirculation
In the microcirculation endothelial function can be evaluated in
functionally isolated vascular districts, such as the forearm and
coronary circulations [6, 7].
The isolated and perfused forearm technique is the most often
used approach in the assessment of endothelial function in periph-
Endothelial Function Assessment in Complicated Hypertension Current Pharmaceutical Design, 2008, Vol. 14, No. 18 1763

eral microcirculation. This technique is a minimally invasive test,
and it requires brachial artery cannulation by a small polyethylene
cannula which is connected to a pressure transducer, for systemic
blood pressure and heart rate monitoring and for intra-arterial infu-
sion of endothelial agonists. The forearm blood flow (FBF) is
measured by strain-gauge venous plethysmography. This device
allows to infuse drugs at systemically ineffective rates through
separate ports via three-way stopcocks.
When using this technique, the changes in blood flow are repre-
sentative of modification in local vascular resistance (increase in
flow = vasodilation). Endothelium-dependent vasodilation is com-
monly estimated by the dose-response curve to intra-arterial acetyl-
choline, or other endothelial agonists, such as bradykinin, meth-
acholine etc. Endothelium-independent vasodilation is assessed by
the dose-response curve to intra-arterial sodium nitroprusside, a
direct smooth muscle relaxant. This technique easily allows to
evaluate NO availability by infusing acetylcholine in the presence
of L-NMMA, or oxidative stress production by infusion of vitamin
C. For these reasons, the forearm technique allows the possibility to
explore the mechanisms of disease. Thus, by this methodology, a
very large body of evidence documented the presence of impaired
endothelium-dependent relaxation in the forearm microcirculation
of patients with essential hypertension [25-30], an alteration charac-
terized by impaired NO availability attributable to oxidative stress
production, which causes NO breakdown [29].
In hypertensive patients, cyclooxygenase was shown to be a
main source of oxidative stress [31]. As already above mentioned,
endothelial cells are able to produce and release different EDHFs
[3, 4]. Experimental studies observed that the cytochrome P-450
2C9 (CYP 2C9) might be one of the main sources of EDHF [32].
By utilizing the forearm technique, it was recently demonstrated in
essential hypertensive patients that vascular responses to acetylcho-
line and mainly bradykinin are inhibited by sulfaphenazole, a CYP
2C9 selective inhibitor [33]. Moreover, in these patients the acute
inhibition of oxidative stress by vitamin C restores the blunting
effect of L-NMMA, thus abrogating the inhibitory effect of sul-
faphenazole [33]. Taken together, these findings demonstrate that
the CYP 2C9-derived EDHF acts as a partial compensatory mecha-
nism to sustain endothelium-dependent vasodilation in those condi-
tions, such as that occurring in essential hypertension, characterized
by absent NO availability secondary to oxidative excess.
As shown in Table 1, the main advantage of this technique in-
clude the possibility to assess many mechanisms accounting for
endothelial dysfunction in hypertensive patients and the strict rela-
tionship between an altered endothelial function in such vascular
district and cardiovascular outcome [34]. On the other hand, the
limitation of the forearm technique is mainly represented by its
invasiveness, which limits the number of patients enrolled and the
possibility to repeat testing frequently (see Table 1).
Subcutaneous microcirculation can be studied using the Mul-
vany myograph device, an in vitro ex vivo technique [35]. This
technique allows to investigate morphological and functional char-
acteristics of isolated resistance arterioles (lumen diameter 150 to
300
m), taken from subcutaneous tissue obtained by skin biopsies.
Once cleaned of adherent connective tissue, vessels can be investi-
gated with the “wire myograph” or the “pressurized myograph”.
Briefly, the first technique implies that two wires are threaded
through the vessel, while in the pressurized system the artery is
slipped into two glass microcannulae and exposed to a constant
pressure [36]. Independently of the approached used, in this vascu-
lar district a reduced endothelial function in essential hypertensive
patients was clearly documented [37-41]. Because of an in vitro

Table 1. Advantages and Disadvantages of Main Techniques Utilized to Assess Endothelial Function

Vascular District Advantages Disadvantages

Coronary circulation
Epicardial artery (Angiography) - Target atherosclerotic vascular district
- Highly invasive (limited follow-up)
Microcirculation (Doppler) - Association with CV prognosis
Peripheral large arteries
- Not invasive - Reproducibility
Brachial artery
- Large repeatability - Low degree of response (large sample)
(FMD by ultrasound)
- Association with CV prognosis - Mechanisms not valuable
- Macro or microcirculation?
Radial artery - Contribution of structural alterations?
- Not invasive
(PWA change by 2-agonist on tonometry) - Variability of 2 agonists response
- No studies on CV prognosis
- Contribution of structural alterations ?
Reactive hyperemia - Other mediators than NO ?
- Not invasive
(digital PAT signal) - Endothelium-independent response not valuable
- No studies on CV prognosis
Peripheral small vessels
- Highly reproducible
- Easy to investigate mechanisms
Muscle microcirculation (forearm plethysmography) - Invasive (limited follow-up)
- Limited sample size needed
- Association with TOD and CV prognosis
Subcutaneous small vessels - Direct vessel evaluation - Invasive (limited follow-up)
(myograph technique) - Easy to investigate mechanisms - Clinical relevance?
- Not yet satisfactory reproducibility
Skin microcirculation (laser digital Doppler) - Not invasive
- No studies on association with TOD or CV prognosis

CV, cardiovascular; FMD, flow-mediated dilation; PWA, pulse wave analysis; TOD, target organ damage.
1764 Current Pharmaceutical Design, 2008, Vol. 14, No. 18
technique, such methodology allows to explore several pathways
and to test many compounds non valuable in vivo, although the
prognostic value of endothelial dysfunction in isolated small vessels
from hypertensive patients is still under debate (Table 1) [42].
The use of the laser digital Doppler technique provides a non-
invasive approach to the evaluation of the skin microcirculation.
The reactive response to local application of endothelium-
dependent vasodilators (i.e. acetylcholine), usually by iontophore-
sis, provides useful information about endothelial function in this
vascular bed. However, inter-assay and intra-assay reproducibility
of this technique is not yet satisfactory; for this reason its value is
limited and should be taken with care (Table 1) [6]
Finally, coronary microcirculation can be evaluated by measur-
ing coronary blood flow with Doppler flow wire and quantitative
angiography during the intracoronary infusion of various substances
which can influence either endothelium-dependent or endothelium-
independent vasodilation. Responses to a wide range of endothelial
agonists including acetylcholine and substance P have been meas-
ured, while sodium nitroprusside and nitroglycerin are usually used
to assess endothelium-independent vasodilation. In addition, intra-
coronary infusion of L-NMMA has defined the contribution of NO
to these vasomotor responses [43]. This experimental model has
provided important insight into the vascular effects of risk factors.
In particular, it was clearly evidenced the presence of endothelial
dysfunction of coronary microcirculation in essential hypertensive
patients with angiographycally normal coronary arteries [20-22].
This alteration is due to a reduction in agonist-induced NO produc-
tion and, in some hypertensive patients with more marked endothe-
lial dysfunction, also to production of cyclooxygenase-dependent
EDCFs [44]. Although this test directly assesses the coronary circu-
lation, its invasive nature limits the use to patients with advanced
disease, and precludes repeated testing during serial follow-up (Ta-
ble 1). Nevertheless, it was shown that endothelial function in coro-
nary microcirculation can be of prognostic value in patients with
established coronary disease [23].
Circulating Markers of Endothelial Function
The possibility of utilizing circulating markers of endothelial
function was recently introduced [7, 45]. It includes direct products
of endothelial cells that change when the endothelium is activated,
such as measures of NO biology, inflammatory cytokines, adhesion
molecules, as well as markers of endothelial damage and repair.
Many of these circulating markers are difficult and expensive to
measure, and currently are only used in research settings. As a re-
sult of biological and assay availability and variability, these factors
currently have only a very limited role in the assessment of individ-
ual patients.
Circulating levels of nitrites and nitrosylated proteins in part
reflect endothelial generation of NO, but are difficult to measure
and may not always represent endothelial NO production [46].
Asymmetric dimethylarginine (ADMA) is an endogenously derived
competitive NOS antagonist, which levels are elevated in subjects
with CVD risk factors and are associated with a reduction in NO
bioavailability in both animal and clinical studies [47]. Because
ADMA levels have been linked to preclinical atherosclerotic dis-
ease and an adverse outcome, they may well prove to be a useful
measure of endothelial status and a potential marker of risk in clini-
cal practice [48]. At present, however, the assay remains challeng-
ing and expensive.
Circulating endothelial cells (CEC) that detach in the context of
endothelial activation and loss of integrity can be measured in the
circulation by both flow cytometry and a combination of magnetic
bead selection and fluorescent microscopy [49]. Circulating endo-
thelial progenitor cells (CEPC) can be characterized by the expres-
sion of characteristic surface markers, which are detectable by flow
cytometry, but because a wide range of hematopoietic progenitor
cells, which include abundantly present myeloid precursors, has the
Virdis et al.
potential to adopt an endothelial phenotype, the specificity of these
measurements is controversial [50]. Further methods to characterize
CEPC biology include quantification of the potential to differentiate
into an endothelial cell phenotype, as well as determination of func-
tional characteristics, which include migration toward a chemical
stimulus adhesion, formation of vascular tubules, and the ability to
attenuate ischemia in animal models [51]. Thus, measurement of
CEC and CEPC levels provides a novel and exciting means to fol-
low the determinants of endothelial injury and repair. Although the
balance of these two cell populations has already been linked to
other in vivo measures of endothelial function, and has been shown
to be associated with future cardiovascular events [52], these novel
measures still remain far from clinical use.
ENDOTHELIAL FUNCTION IN PATIENTS WITH COM-
PLICATED HYPERTENSION
The importance of subclinical and clinical target organ damage
which characterize the complicated hypertensive patients, is widely
recognized, and recently underlined by the 2007 European Hyper-
tension Guidelines [53], as an intermediate stage in the continuum
of vascular disease and as a determinant of total cardiovascular risk.
The main subclinical organ damage include vascular atherosclero-
sis, detected by ultrasound scanning, left ventricular hypertrophy
assessed by electrocardiography or by the more sensitive echocar-
diography, and hypertension-induced renal damage, as based on the
finding of a reduced renal function and/or the detection of elevated
urinary albumin excretion. Such complicated patients have been
investigated in terms of endothelial function by several reports, here
summarized.
Carotid Intima-Media Thickness
Increased intima-media thickness (IMT) of the common carotid
artery is a non-invasive marker of atherosclerosis which signifi-
cantly correlates with coronary or cerebrovascular disease [54, 55].
The first observation of a relationship between increased carotid
IMT and endothelial dysfunction was detected in the forearm mi-
crocirculation of untreated hypertensive patients [56]. Indeed, ca-
rotid IMT was inversely related to the response to acetylcholine, but
not to sodium-nitroprusside. The majority of following studies have
shown a correlation between carotid IMT and brachial artery FMD.
Hashimoto et al. [57] investigated 34 atherosclerotic men and 33
age-matched subjects without clinical atherosclerosis but with simi-
lar cardiovascular risk factors. The authors showed that patients
with atherosclerosis had increased IMT and reduced FMD, respec-
tively, as compared to controls. A significant negative correlation
between IMT and FMD in the whole group was also reported, dem-
onstrating a significant relationship between IMT and FMD and
thereby supporting the concept that endothelial dysfunction is sig-
nificantly related to atherosclerosis [57]. Jounala M et al. [58] re-
ported similar results in a larger population of 2109 young healthy
adults from the Cardiovascular Risk in Young Finns Study. FMD
was inversely associated with IMT in a multivariate model adjusted
for age, sex, brachial, vessels size and several risk variables. In
addition, the number of risk factors was correlated with increased
IMT in subjects with impaired FMD response but not in subjects
with preserved endothelial function [58]. Different results emerged
from the study of Jan et al. [59], who showed no relationship be-
tween brachial artery FMD and carotid IMT in 1,578 middle-aged
men without known cardiovascular disease, although both FMD
and IMT were correlated with the presence of cardiovascular risk
factors. Interestingly, Campunzano et al. [60] observed that in 106
patients without cardiovascular disease, besides the relationship
between the presence of cardiovascular risk factors, increased IMT
and reduced FMD, these vascular alterations were associated with
decreased coronary flow reserve assessed non-invasively by echo-
graphy [60].
Finally, Rundek et al. [61] reported that endothelial dysfunction
of the conduit artery, measured as brachial FMD, was independent-
Endothelial Function Assessment in Complicated Hypertension
ly associated to carotid plaque in a multi-ethnic population of elder-
ly men and women [61]. In summary, these data demonstrate an
association of endothelial dysfunction, detected either in peripheral
large arteries and in the forearm microcirculation, with subclinical
markers of carotid damage.
Left Ventricular Hypertrophy
The increase of left ventricular mass is a feature of hypertensive
heart disease and predicts independently an increased risk for car-
diovascular disease [62]. Accordingly, regression of left ventricular
hypertrophy has a positive prognostic impact [63]. Several findings
suggest that left ventricular hypertrophy (LVH) is associated with
the presence of endothelial dysfunction. In particular, Treasure
et al. [21] demonstrated an impaired endothelium-dependent vaso-
dilation in the coronary vessels of 10 patients with LVH secondary
to essential hypertension, an alteration which may contribute to the
ischemic manifestations of hypertensive heart disease. Perticone
et al. [64] investigated endothelial function in the forearm microcir-
culation of 65 never-treated hypertensive patients to assess the rela-
tionship between endothelial dysfunction and LVH. In hypertensive
patients with LVH endothelium-dependent vasodilation was more
pronounced than in patients without LVH. In particular, left ven-
tricular mass was inversely related to FBF response to intra-brachial
acetylcholine. Moreover, a higher decrease in peak response to
acetylcholine was evident in patients with concentric pattern of
LVH [64]. Similar results were found by Ercan et al. [65], who
investigated endothelial function non-invasively by FMD of the
brachial artery in 32 hypertensive patients with LVH, in 28 hyper-
tensive patients without LVH and in 29 normotensive controls.
Results showed that FMD was significantly lower in hypertensive
patients as compared to controls, and that endothelial dysfunction
was significantly higher in hypertensive patients with LVH as com-
pared to patients without LVH [65].
Different results were found by Muiesan et al. [66] in 78 hyper-
tensive patients with essential hypertension investigating endothe-
lial function. Patients were divided in four groups according to the
presence of LVH and left ventricular geometry and no significant
difference in FMD was observed between patients with or without
LVH or among patients with different geometric changes of the left
ventricle [66]. Recently, Sciacqua et al. [67] reported that both
LVH and endothelial dysfunction, measured as response to acetyl-
choline in the forearm microcirculation, are independent predictors
of cardiovascular events in a group of 324 never-treated hyperten-
sive patients. A major novelty of this study concerns the demonstra-
tion that the co-existence of both LVH and endothelial dysfunction
significantly increase the risk for future cardiovascular events in
hypertensive patients [67].
Microalbuminuria
The loss of albumin in urines is a marker of impaired glomeru-
lar permeability for plasma proteins and is thought to represent an
integrated marker of subclinical organ damage in primary hyperten-
sion. Several epidemiological data demonstrated that the presence
of microalbuminuria is an independent predictor of renal events as
well as cardiovascular mortality and morbidity after adjustment for
other conventional cardiovascular risk factors[68]. Moreover, in the
LIFE trial, the levels of albumin excretion at randomization were
independent predictor of outcome also in non-diabetic hypertensive
patients with left ventricular hypertrophy, also for range of albu-
minuria below the threshold to define microalbuminuria [69]. A
reduced endothelial function has been demonstrated in the presence
of microalbuminuria in type 1 and 2 diabetic patients compared
with normoalbuminuric diabetic patients or healthy subjects [69].
Moreover, the levels of albumin excretion are inversely related to
endothelium-dependent response in several diabetic and non-
diabetic populations [69]. Both microalbuminuria and endothelial
dysfunction are expressions of an endothelial pathology, however
Current Pharmaceutical Design, 2008, Vol. 14, No. 18 1765
whether they are interrelated or are two phenomena caused in paral-
lel by the cardiovascular risk burden is still uncertain. Indeed, some
published studies failed to demonstrate a relation between endothe-
lial function and urine albumin excretion. Diercks et al. [70] evalu-
ated the relation between microalbuminuria and endothelial dys-
function, assessed as FMD of the brachial artery in 654 apparently
healthy subjects. The authors did not find any reduction in FMD or
nitroglycerin-mediated vasodilation in microalbuminuric patients,
nor were the levels of albuminuria related to FMD [70]. Consis-
tently with these data, our group found similar results also in the
forearm microcirculation of essential hypertensive patients with and
without microalbuminuria [71]. Indeed, no correlation between
urinary albumin excretion and vasodilatation in response to acetyl-
choline or to sodium nitroprusside in the forearm microcirculation
was found. Taken together, these data seem to suggest either that no
direct connection between systemic endothelial function and albu-
min excretion exists or that impaired endothelial function precedes
the development of microalbuminuria.
Kidney Disease
Renal disease, through the activation of several mechanisms
reducing NO availability [72], leads to impairment of endothelial
function which, accordingly, is severely reduced in the presence of
advanced renal failure [73-75]. It is well known that renal disease is
an invariable complication of hypertension which, as already de-
scribed, represents an important determinant of oxidative stress-
driven endothelial dysfunction. However, the reduced renal func-
tion can per se impair endothelial function, as demonstrated by the
presence of endothelial dysfunction in uremic children who are not
dyslipidemic, diabetic or hypertensive [74]. The pathophysiology of
endothelial dysfunction in the presence of moderate-severe renal
insufficiency is complex and not fully characterized. Firstly, with
the increasing loss of the clearing capacity of the kidney, a progres-
sive accumulation of substances which are potentially noxius for
the endothelium occurs. Among these, a prominent role is ascribed
to ROS. Indeed, in the presence of renal disease systemic levels of
oxidative stress are extremely high and proportional to the degree
of renal dysfunction [73, 76]. Moreover, it is important to note that
in these patients the levels of oxidative stress are also inversely
correlated to the endothelial function [73, 77] and the administra-
tion of antioxidant vitamin C is able to ameliorate the endothelium-
dependent vasodilation [73]. The increased oxidative stress in the
presence of renal failure is the consequence of the activation of
several pathways, including NAD(P)H-oxidase, xanthine-oxidase,
mitochondrial oxidases, and myeloperoxidase [72]. NAD(P)H-
oxidase is probably the most important source of ROS and it is
stimulated by Angiotensin II [78]. Indeed, activation of the renin-
angiotensin system occurs in many forms of renal disease. Angio-
tensin II stimulates NAD(P)H-oxidase, which leads to generation of
superoxide anion and contributes to endothelial dysfunction, vascu-
lar remodeling and growth [79]. Of importance, when Angiotensin
II acts through the AT-1 receptors, the consequent generation of
ROS leads to upregulation of inflammatory mediators, which in-
clude cytokines, chemokines, adhesion molecules and plasminogen
activator inhibitor 1. These events, together with the mechanisms
already mentioned, promote endothelial dysfunction, vascular re-
modeling and the progression of atherosclerosis [80]. Of note, be-
sides the activation of enzymes, the reduced clearing capacity of the
kidney causes an accumulation of toxic substances which reduce
NO availability, via both increasing oxidative stress and other
mechanisms [72]. Indeed, the failing kidneys become progressively
unable to clear homocysteine and the resultant hyperhomocys-
teinemia causes endothelial dysfunction through ROS generation
[30]. Another substance that accumulates in the presence of renal
failure is ADMA, able to contribute in reducing NO availability
independently from oxidative stress [81, 82]. Taken in conjunction,
these findings allow to hypothesize that endothelial dysfunction
1766 Current Pharmaceutical Design, 2008, Vol. 14, No. 18
might be one of the principal pathophysiological mechanisms in-
volved in the accelerated atherosclerotic process and cardiovascular
morbidity and mortality which characterize patients with renal dis-
ease [83]. Although, as noted, in advanced renal disease endothelial
dysfunction is constantly present and its degree is correlated to the
degree of glomerular filtration rate decrease [73], the relation be-
tween endothelial and renal function is still uncertain in the pres-
ence of mild renal insufficiency. So far, available data are not con-
sistent. In a small population of healthy individual no relation was
found between glomerular filtration rate and endothelium-
dependent vasodilation in the brachial artery [73]. On the contrary,
it was shown that in a population of non-complicated essential hy-
pertensive patients with serum creatinine within normal range, the
endothelium-dependent vasodilation to acetylcholine in the forearm
district was directly correlated with the estimation of glomerular
filtration rate by the MDRD formula [84]. The authors speculated
that before the onset of overt nephropathy the presence of endothe-
lial dysfunction can per se influence the kidney hemodynamics.
More recently, no correlation between the endothelium-dependent
vasodilation to methacholine in internal mammary arteries and
glomerular filtration rate estimate was found in a group of patients
with severe coronary atherosclerosis and normal or mildly reduced
renal function [85]. In line with these results, preliminary data from
our laboratory failed to find an independent role for reduced endo-
thelial function, either in the peripheral microcirculation or macro-
circulation, in predicting the glomerular filtration rate estimate de-
crease in a unselected population of normal subjects and patients
with cardiovascular risk factors and serum creatinine within normal
limits. On the contrary, we found that the presence of a reduced
endothelium-independent vasodilation to sodium nitroprusside in
the forearm, an index of structural alteration in the peripheral mi-
crocirculation, was associated with initial impairment of renal func-
tion.
In summary, advanced kidney disease invariably causes or
worsen endothelial dysfunction through several mechanism which
impair NO bioavailability. On the contrary, the overall data seem to
suggest that mild renal insufficiency is not able to influence periph-
eral endothelial function, nor vice versa but both renal and periph-
eral circulations are targets of the action of the traditional risk fac-
tors.
CLINICAL SIGNIFICANCE OF ENDOTHELIAL DYS-
FUNCTION
Endothelial dysfunction is a common alteration of the major
cardiovascular risk factors, including hypertension. Moreover, the
association of different cardiovascular risk factors is associated
with a progressive increase in endothelial dysfunction [86]. Such
alteration is a common pathogenetic mechanism determining athe-
rosclerotic disease and cardiovascular events in patients with car-
diovascular risk factors. This possibility is in line with the evidence
that NO and EDCFs not only exert an opposite effect on vascular
tone but also respectively inhibit and activate those mechanisms,
summarized in Fig. (1), such as platelet aggregation, vascular
smooth muscle cell proliferation and migration, monocyte adhesion
and adhesion molecule expression, which exert an important role in
the genesis of thrombosis and atherosclerotic plaque [87]. Endothe-
lial dysfunction is thus a mechanism deeply affecting vascular func-
tion and structure, including vasomotricity alteration or promotion
of atherosclerosis and thrombosis, thereby contributing to cardio-
vascular events. This interpretation is supported by mounting evi-
dence, which demonstrates the association of endothelial dysfunc-
tion with markers of vascular damage and with cardiovascular
events, both in patients with essential hypertension and, more in
general, in patients with atherosclerotic disease. In a recent meta-
analysis by Lerman and Zeiher, the available longitudinal studies
evaluating the prognostic impact of endothelial dysfunction were
evaluated [8]. This analysis included around 2500 patients with
atherosclerotic coronary disease or characterized by high cardiovas-
Virdis et al.
cular risk. The outcomes, evaluated in a wide follow-up range
(from 1 to 92 months) included major cardiovascular events. The
authors observed that endothelial dysfunction, evaluated either in
the coronary district or in the peripheral circulation by the FMD or
the forearm techniques, significantly predicted cardiovascular
events, independently of traditional cardiovascular risk factors [8].
These studies describe a prognostic relevance of endothelial dys-
function in high risk patients characterized by coronary artery dis-
ease.
As concern patients with low cardiovascular risk profile, a re-
cent report from the Framingham study revealed a progressive in-
verse relation between endothelium-dependent relaxation, evaluated
by FMD, and the increasing cardiovascular risk score, evaluated
according to tables from “Framingham risk score” [88]. A recent
meta-analysis, performed in over 200 available studies in which
peripheral conduit artery FMD was evaluated according to cardio-
vascular risk factors analyzed with “Framingham risk score” tables,
observed that the relationship between FMD and risk factors was
more evident in patient with a lower cardiovascular risk, whereas in
patients with a moderate-high risk such relationship disappeared
[54]. The authors concluded that endothelial dysfunction, evaluated
by FMD technique, may have a prognostic significance only in low-
risk populations. However, when analyzing these data, the small
numbers of patients recruited in some of these studies clearly
emerged. Moreover, in many studies the criteria of patients selec-
tion cannot allow to extrapolate the results obtained to a common
general population. Finally, the methodological approach to FMD
assessment was much different among studies. Taken in conjunc-
tion, although not allowing to obtain definitive conclusions in low-
risk populations, such findings seem to confirm a great impact ex-
erted by endothelial dysfunction on cardiovascular events. Further
large-scale studies, conducted with reproducible and reliable meth-
ods to assess endothelial function will clarify a direct relationship
between endothelial dysfunction and cardiovascular events in low-
risk populations.
HOW TO INTERPRET ENDOTHELIAL RESPONSES
In evaluating the role of endothelium on vascular tone modula-
tion, the degree of agonist-induced vasodilation is considered a
measure of endothelial function, so that greater is the vasodilation,
higher is endothelial function. By this approach, it is mandatory to
compare the responses evoked by endothelial agonists with those
obtained by endothelium-independent relaxant compounds, in order
to exclude the possibility that an altered smooth muscle cells con-
tractility may influence the endothelium-dependent response. In-
deed, it is crucial to obtain comparable degrees of vasodilation by
the different agonists infused.
In contrast to what observed in the coronary or peripheral mi-
crocirculation, where a dysfunctioning endothelium reflects a re-
duced agonist-induced vasodilation, in epicardial large arteries
acetylcholine injection induces vasoconstriction in subjects with
endothelial dysfunction, as a result of a direct muscarinic smooth
muscle vasoconstrictor effect [89].
Another important aspect in evaluating results from vascular
reactivity in humans involves methodological rules. Thus, a re-
sponse to a pharmacological tool such as acetylcholine or FMD
may be influenced by several factors, such as the selection criteria.
Since most of cardiovascular risk factors, ageing above all [28], can
induce endothelial dysfunction, it is crucial that the populations
selected for the reactivity studies are well-matched. In addition, a
crucial point that should be kept in mind in interpreting the results
is that the endothelial response to an endothelial agonist is an inte-
grated mechanism and different pathways and mediators besides
NO account for endothelium-dependent vasodilation. In the forearm
of healthy subjects NO release contributes greatly to the vasodilator
effect of acetylcholine or bradykinin, since it is substantially
blunted by L-NMMA [4, 90, 91]. In contrast, in hypertensive pa-
Endothelial Function Assessment in Complicated Hypertension
tients, the response to both agonists is not only blunted but proven
to be resistant to L-NMMA blockade, thus suggesting that a differ-
ent pathway, likely hyperpolarization, accounts for the endothelial
response in the presence of impaired NO availability in this popula-
tion [4, 26, 29, 31]. The NO-dependent component of endothelium-
dependent vasodilation can be quantified only by compounds such
as L-NMMA. Accordingly, when a treatment increases a depressed
endothelial response, caution should be exercised in attributing such
a result to increased NO availability.
DOES A BETTER TECHNIQUE TO EXPLORE ENDOTHE-
LIAL FUNCTION EXIST?
The above mentioned available evidence strongly suggests that
the determination of impaired endothelium-dependent vasodilation
might represent a measurable intermediate endpoint in patient with
essential hypertension. Unfortunately, despite the considerable
evidence, at the present time several issues make this suggestion
very implausible.
First, attention must focus on the choice of an appropriate
method for measuring endothelial function in clinical practice. The
endothelium is an autocrine/paracrine organ and its func-
tion/dysfunction can vary depending on which vascular district is
explored or which stimulus is employed. For instance, in hyperten-
sive patients the presence of oxidative stress has been documented
by the utilization of vitamin C in the peripheral microcirculation
[29] and in epicardial arteries [92], but it has not been confirmed in
the coronary microcirculation [92]. Moreover, whereas ACE inhibi-
tors seem to be effective in reversing endothelial dysfunction in
peripheral conduit arteries, they are devoid of effect in the micro-
circulation, and vice versa occurred when calcium antagonists are
assessed [93]. These different responses indicate that it is difficult
to make generalizations regarding the concept of endothelial func-
tion.
Moreover, as already commented, each test employed to assess
endothelial function has specific limitations that hinder its applica-
bility in large-scale screening (Table 1). On the other hand, studies
assessing the degree of correlation among measures of endothelial
function, as evaluated in different vascular beds, have demonstrated
a poor relationship, and, while statistically significant, the findings
Fig. (2). Scatterplot showing the correlation between brachial artery flow-mediated dilation and vasodilation to acetylcholine in the forearm microcirculation
of healthy individuals and patients with essential hypertension. The lack of correlation between the two parameters, when analysed in a single population, is
clearly manifested. Only if the results are analysed in the global population does the correlation increase and reach statistical significance, albeit of negligible
clinical significance (S. Taddei et al., unpublished observations). Total population r=0.38, P<0.01; normotensive population r =0.17; P= not significant.
Current Pharmaceutical Design, 2008, Vol. 14, No. 18 1767
are of no clinical relevance [94], Fig. (2). For these reasons, no one
test can be considered a surrogate for another (e.g., results obtained
in the peripheral circulation cannot be translated to the coronary
vascular bed).
In conclusions, at the present time no definitive conclusions can
be taken by a single technique assessing endothelial function. Only
the growing concordant results from different reproducible and
reliable methods exploring endothelial function with different stim-
uli will support and strength experimental findings.
CONCLUSIONS
Endothelium plays a central role in the maintenance of vascular
homeostasis. Tests have been developed to study endothelial func-
tion in hypertensive population, able to assess its changes in rela-
tion with subclinical and clinical target organ damage as well as
after therapy. Endothelial dysfunction in essential hypertension may
be of particular clinical relevance since it can be a promoter of athe-
rosclerotic and thrombotic damage, typical complications of hyper-
tension. However, endothelial function cannot yet be included
among the surrogate endpoints that need to be measured for cardio-
vascular risk stratification in hypertensive patients. This limitation
springs from the fact that no available tests to assess endothelial
function has sufficient sensitivity and specificity to be used in clini-
cal practice. In addition, it is important to underline the concept that
the study of vascular reactivity in humans needs rigorous experi-
mental conditions, such as the criteria for population to be enrolled.
Moreover, despite considerable evidence that impaired endothe-
lium-dependent vasodilation can be improved by appropriate anti-
hypertensive treatment [93], further large-scale clinical trials are
required to prove conclusively whether the reversal of endothelial
dysfunction offers a clinical advantage in essential hypertensive
patients.
Only after a specific and affordable test has been validated, and
definite evidence becomes available to demonstrate that endothelial
dysfunction must be a target of pharmacological treatment, will
evaluation of endothelium-dependent vasodilation be included in
the list of clinical examinations that define cardiovascular risk in
patients with essential hypertension.
1768 Current Pharmaceutical Design, 2008, Vol. 14, No. 18
REFERENCES
[1] Furchgott RF, Zawadzki JV. The obligatory role of endothelial
cells in the relaxation of arterial smooth muscle by acetylcholine.
Nature 1980; 288: 373-6.
[2] Luscher TF, Barton M. Biology of the endothelium. Clin Cardiol
1997; 20: II-3-10.
[3] Cohen RA, Vanhoutte PM. Endothelium-dependent hyperpolariza-
tion. Beyond nitric oxide and cyclic GMP. Circulation 1995; 92:
3337-49.
[4] Taddei S, Ghiadoni L, Virdis A, Buralli S, Salvetti A. Vasodilation
to bradykinin is mediated by an ouabain-sensitive pathway as a
compensatory mechanism for impaired nitric oxide availability in
essential hypertensive patients. Circulation 1999; 100: 1400-5.
[5] Cai H, Harrison DG. Endothelial dysfunction in cardiovascular
diseases: the role of oxidant stress. Circ Res 2000; 87: 840-4.
[6] Deanfield J, Donald A, Ferri C, Giannattasio C, Halcox J, Halligan
S, et al. Endothelial function and dysfunction. Part I: Methodologi-
cal issues for assessment in the different vascular beds: a statement
by the Working Group on Endothelin and Endothelial Factors of
the European Society of Hypertension. J Hypertens 2005; 23: 7-17.
[7] Deanfield JE, Halcox JP, Rabelink TJ. Endothelial function and
dysfunction: testing and clinical relevance. Circulation 2007; 115:
1285-95.
[8] Lerman A, Zeiher AM. Endothelial function: cardiac events. Circu-
lation 2005; 111: 363-8.
[9] Taddei S, Salvetti A. Endothelial dysfunction in essential hyperten-
sion: clinical implications. J Hypertens 2002; 20: 1-4.
[10] Celermajer DS, Sorensen KE, Gooch VM, Spiegelhalter DJ, Miller
OI, Sullivan ID, et al. Non-invasive detection of endothelial dys-
function in children and adults at risk of atherosclerosis. Lancet
1992; 340: 1111-5.
[11] Joannides R, Haefeli WE, Linder L, Richard V, Bakkali EH,
Thuillez C, et al. Nitric oxide is responsible for flow-dependent
dilatation of human peripheral conduit arteries in vivo. Circulation
1995; 91: 1314-9.
[12] Ghiadoni L, Versari D, Magagna A, Kardasz I, Plantinga Y, Gian-
narelli C, et al. Ramipril dose-dependently increases nitric oxide
availability in the radial artery of essential hypertension patients. J
Hypertens 2007; 25: 361-6.
[13] Ghiadoni L, Huang Y, Magagna A, Buralli S, Taddei S, Salvetti A.
Effect of acute blood pressure reduction on endothelial function in
the brachial artery of patients with essential hypertension. J Hyper-
tens 2001; 19: 547-51.
[14] Corretti MC, Anderson TJ, Benjamin EJ, Celermajer D, Charbon-
neau F, Creager MA, et al. Guidelines for the ultrasound assess-
ment of endothelial-dependent flow-mediated vasodilation of the
brachial artery: a report of the International Brachial Artery Reac-
tivity Task Force. J Am Coll Cardiol 2002; 39: 257-65.
[15] Ghiadoni L, Donald AE, Cropley M, Mullen MJ, Oakley G, Taylor
M, et al. Mental stress induces transient endothelial dysfunction in
humans. Circulation 2000; 102: 2473-8.
[16] Gemignani V, Bianchini E, Faita F, Giannarelli C, Plantinga Y,
Ghiadoni L, et al. Ultrasound measurement of the brachial artery
flow-mediated dilation without ECG gating. Ultrasound Med Biol
2008; 34(3): 385-91.
[17] Gemignani V, Faita F, Ghiadoni L, Poggianti E, Demi M. A system
for real-time measurement of the brachial artery diameter in B-
mode ultrasound images. IEEE Trans Med Imaging 2007; 26: 393-
404.
[18] Wilkinson IB, Hall IR, MacCallum H, Mackenzie IS, McEniery
CM, van der Arend BJ, et al. Pulse-wave analysis: clinical evalua-
tion of a noninvasive, widely applicable method for assessing endo-
thelial function. Arterioscler Thromb Vasc Biol 2002; 22: 147-52.
[19] Chowienczyk PJ, Kelly RP, MacCallum H, Millasseau SC, Anders-
son TL, Gosling RG, et al. Photoplethysmographic assessment of
pulse wave reflection: blunted response to endothelium-dependent
beta2-adrenergic vasodilation in type II diabetes mellitus. J Am
Coll Cardiol 1999; 34: 2007-14.
[20] Quyyumi AA, Cannon RO 3rd, Panza JA, Diodati JG, Epstein SE.
Endothelial dysfunction in patients with chest pain and normal
coronary arteries. Circulation 1992; 86: 1864-71.
[21] Treasure CB, Klein JL, Vita JA, Manoukian SV, Renwick GH,
Selwyn AP, et al. Hypertension and left ventricular hypertrophy are
Virdis et al.
associated with impaired endothelium-mediated relaxation in hu-
man coronary resistance vessels. Circulation 1993; 87: 86-93.
[22] Egashira K, Suzuki S, Hirooka Y, Kai H, Sugimachi M, Imaizumi
T, et al. Impaired endothelium-dependent vasodilation of large
epicardial and resistance coronary arteries in patients with essential
hypertension. Different responses to acetylcholine and substance P.
Hypertension 1995; 25: 201-6.
[23] Schachinger V, Britten MB, Zeiher AM. Prognostic impact of
coronary vasodilator dysfunction on adverse long-term outcome of
coronary heart disease. Circulation 2000; 101: 1899-906.
[24] Suwaidi JA, Hamasaki S, Higano ST, Nishimura RA, Holmes DR
Jr, Lerman A. Long-term follow-up of patients with mild coronary
artery disease and endothelial dysfunction. Circulation 2000; 101:
948-54.
[25] Panza JA, Quyyumi AA, Brush JE, Epstein SE. Abnormal endothe-
lium-dependent vascular relaxation in patients with essential hyper-
tension. N Engl J Med 1990; 323: 22-7.
[26] Panza JA, Casino PR, Kilcoyne CM, Quyyumi AA. Role of endo-
thelium-derived nitric oxide in the abnormal endothelium-
dependent vascular relaxation of patients with essential hyperten-
sion. Circulation 1993; 87: 1468-74.
[27] Taddei S, Virdis A, Mattei P, Salvetti A. Vasodilation to acetylcho-
line in primary and secondary forms of human hypertension. Hy-
pertension 1993; 21: 929-33.
[28] Taddei S, Virdis A, Mattei P, Ghiadoni L, Gennari A, Fasolo CB,
et al. Aging and endothelial function in normotensive subjects and
patients with essential hypertension. Circulation 1995; 91: 1981-7.
[29] Taddei S, Virdis A, Ghiadoni L, Magagna A, Salvetti A. Vitamin C
improves endothelium-dependent vasodilation by restoring nitric
oxide activity in essential hypertension. Circulation 1998; 97:
2222-9.
[30] Virdis A, Ghiadoni L, Cardinal H, Favilla S, Duranti P, Birindelli
R, et al. Mechanisms responsible for endothelial dysfunction in-
duced by fasting hyperhomocystinemia in normotensive subjects
and patients with essential hypertension. J Am Coll Cardiol 2001;
38: 1106-15.
[31] Taddei S, Virdis A, Ghiadoni L, Magagna A, Salvetti A. Cy-
clooxygenase inhibition restrores nitric oxide activity in essential
hypertension. Hypertension 1997; 29: 274-9.
[32] Fleming I. Cytochrome P450 epoxygenases as EDHF synthase(s).
Pharmacol Res 2004; 49: 525-33.
[33] Taddei S, Versari D, Cipriano A, Ghiadoni L, Galetta F, Franzoni
F, et al. Identification of a cytochrome P450 2C9-derived endothe-
lium-derived hyperpolarizing factor in essential hypertensive pa-
tients. J Am Coll Cardiol 2006; 48: 508-15.
[34] Heitzer T, Schlinzig T, Krohn K, Meinertz T, Munzel T. Endothe-
lial dysfunction, oxidative stress, and risk of cardiovascular events
in patients with coronary artery disease. Circulation 2001; 104:
2673-8.
[35] Mulvany MJ, Baumbach GL, Aalkjaer C, Heagerty AM, Korsgaard
N, Schiffrin EL, et al. Vascular remodeling. Hypertension 1996;
28: 505-6.
[36] Schiffrin EL. Vascular structure in NG-nitro-L-arginine methyl
ester-induced hypertension: methodological considerations for
studies of small arteries in hypertension. J Hypertens 1995; 13:
817-21.
[37] Schiffrin EL, Deng LY. Structure and function of resistance arter-
ies of hypertensive patients treated with a beta-blocker or a calcium
channel antagonist. J Hypertens 1996; 14: 1247-55.
[38] Rizzoni D, Muiesan ML, Porteri E, Castellano M, Zulli R, Bettoni
G, et al. Effects of long-term antihypertensive treatment with lisi-
nopril on resistance arteries in hypertensive patients with left ven-
tricular hypertrophy. J Hypertens 1997; 15: 197-204.
[39] Rizzoni D, Porteri E, Castellano M, Bettoni G, Muiesan ML, Ti-
berio G, et al. Endothelial dysfunction in hypertension is independ-
ent from the etiology and from vascular structure. Hypertension
1998; 31: 335-41.
[40] Schiffrin EL, Park JB, Intengan HD, Touyz RM. Correction of
arterial structure and endothelial dysfunction in human essential
hypertension by the angiotensin receptor antagonist losartan. Circu-
lation 2000; 101: 1653-9.
[41] Endemann DH, Pu Q, De Ciuceis C, Savoia C, Virdis A, Neves
MF, et al. Persistent remodeling of resistance arteries in type 2 dia-
Endothelial Function Assessment in Complicated Hypertension
betic patients on antihypertensive treatment. Hypertension 2004;
43: 399-404.
[42] Rizzoni D, Porteri E, De Ciuceis C, Boari GE, Zani F, Miclini M,
et al. Lack of prognostic role of endothelial dysfunction in subcu-
taneous small resistance arteries of hypertensive patients. J Hyper-
tens 2006; 24: 867-73.
[43] Goodhart DM, Anderson TJ. Role of nitric oxide in coronary arte-
rial vasomotion and the influence of coronary atherosclerosis and
its risks. Am J Cardiol 1998; 82: 1034-9.
[44] Marzilli M, Taddei S, Virdis A, Ghiadoni L, Salvetti A. Endothelial
function and coronary microcirculaiton in essential hypertension. J
Hypertens 1998; 16(Suppl 8): S59-63.
[45] Versari D, Lerman LO, Lerman A. The importance of reendothe-
lialization after arterial injury. Curr Pharm Des 2007; 13: 1811-24.
[46] Rassaf T, Feelisch M, Kelm M. Circulating NO pool: assessment of
nitrite and nitroso species in blood and tissues. Free Radic Biol
Med 2004; 36: 413-22.
[47] Vallance P, Leiper J. Cardiovascular biology of the asymmetric
dimethylarginine: dimethylarginine dimethylaminohydrolase path-
way. Arterioscler Thromb Vasc Biol 2004; 24: 1023-30.
[48] Boger RH, Maas R, Schulze F, Schwedhelm E. Elevated levels of
asymmetric dimethylarginine (ADMA) as a marker of cardiovascu-
lar disease and mortality. Clin Chem Lab Med 2005; 43: 1124-9.
[49] Goon PK, Boos CJ, Lip GY. Circulating endothelial cells: markers
of vascular dysfunction. Clin Lab 2005; 51: 531-8.
[50] Fujiyama S, Amano K, Uehira K, Yoshida M, Nishiwaki Y, No-
zawa Y, et al. Bone marrow monocyte lineage cells adhere on in-
jured endothelium in a monocyte chemoattractant protein-1-
dependent manner and accelerate reendothelialization as endothe-
lial progenitor cells. Circ Res 2003; 93: 980-9.
[51] Murohara T, Asahara T, Silver M, Bauters C, Masuda H, Kalka C,
et al. Nitric oxide synthase modulates angiogenesis in response to
tissue ischemia. J Clin Invest 1998; 101: 2567-78.
[52] Werner N, Kosiol S, Schiegl T, Ahlers P, Walenta K, Link A, et al.
Circulating endothelial progenitor cells and cardiovascular out-
comes. N Engl J Med 2005; 353: 999-1007.
[53] Mancia G, De Backer G, Dominiczak A, Cifkova R, Fagard R,
Germano G, et al. Guidelines for the management of arterial hyper-
tension: The task force for the management of arterial hypertension
of the European Society of Hypertension (ESH) and of the Euro-
pean Society of Cardiology (ESC). J Hypertens 2007; 25: 1105-87.
[54] Witte DR, Westerink J, de Koning EJ, van der Graaf Y, Grobbee
DE, Bots ML. Is the association between flow-mediated dilation
and cardiovascular risk limited to low-risk populations? J Am Coll
Cardiol 2005; 45: 1987-93.
[55] Chambless LE, Heiss G, Folsom AR, Rosamond W, Szklo M,
Sharrett AR, et al. Association of coronary heart disease incidence
with carotid arterial wall thickness and major risk factors: the Athe-
rosclerosis Risk in Communities (ARIC) Study, 1987-1993. Am J
Epidemiol 1997; 146: 483-94.
[56] Ghiadoni L, Taddei S, Virdis A, Sudano I, Di Legge V, Meola M,
et al. Endothelial function and common carotid artery wall thicken-
ing in patients with essential hypertension. Hypertension 1998; 32:
25-32.
[57] Hashimoto M, Eto M, Akishita M, Kozaki K, Ako J, Iijima K, et al.
Correlation between flow-mediated vasodilatation of the brachial
artery and intima-media thickness in the carotid artery in men. Ar-
terioscler Thromb Vasc Biol 1999; 19: 2795-800.
[58] Juonala M, Viikari JS, Laitinen T, Marniemi J, Helenius H, Ron-
nemaa T, et al. Interrelations between brachial endothelial function
and carotid intima-media thickness in young adults: the cardiovas-
cular risk in young Finns study. Circulation 2004; 110: 2918-23.
[59] Yan RT, Anderson TJ, Charbonneau F, Title L, Verma S, Lonn E.
Relationship between carotid artery intima-media thickness and
brachial artery flow-mediated dilation in middle-aged healthy men.
J Am Coll Cardiol 2005; 45: 1980-6.
[60] Campuzano R, Moya JL, Garcia-Lledo A, Tomas JP, Ruiz S, Me-
gias A, et al. Endothelial dysfunction, intima-media thickness and
coronary reserve in relation to risk factors and Framingham score
in patients without clinical atherosclerosis. J Hypertens 2006; 24:
1581-8.
[61] Rundek T, Hundle R, Ratchford E, Ramas R, Sciacca R, Di Tullio
MR, et al. Endothelial dysfunction is associated with carotid
Current Pharmaceutical Design, 2008, Vol. 14, No. 18 1769
plaque: a cross-sectional study from the population based Northern
Manhattan study. BMC Cardiovasc Disord 2006; 6: 35.
[62] Levy D, Salomon M, D'Agostino RB, Belanger AJ, Kannel WB.
Prognostic implications of baseline electrocardiographic features
and their serial changes in subjects with left ventricular hypertro-
phy. Circulation 1994; 90: 1786-93.
[63] Verdecchia P, Angeli F, Borgioni C, Gattobigio R, de Simone G,
Devereux RB, et al. Changes in cardiovascular risk by reduction of
left ventricular mass in hypertension: a meta-analysis. Am J Hyper-
tens 2003; 16: 895-9.
[64] Perticone F, Maio R, Ceravolo R, Cosco C, Cloro C, Mattioli PL.
Relationship between left ventricular mass and endothelium-
dependent vasodilation in never-treated hypertensive patients. Cir-
culation 1999; 99: 1991-6.
[65] Ercan E, Tengiz I, Ercan HE, Nalbantgil I. Left ventricular hyper-
trophy and endothelial functions in patients with essential hyper-
tension. Coron Artery Dis 2003; 14: 541-4.
[66] Muiesan ML, Salvetti M, Monteduro C, Corbellini C, Guelfi D,
Rizzoni D, et al. Flow-mediated dilatation of the brachial artery
and left ventricular geometry in hypertensive patients. J Hypertens
2001; 19: 641-7.
[67] Sciacqua A, Scozzafava A, Pujia A, Maio R, Borrello F, Andreozzi
F, et al. Interaction between vascular dysfunction and cardiac mass
increases the risk of cardiovascular outcomes in essential hyperten-
sion. Eur Heart J 2005; 26: 921-7.
[68] Ochodnicky P, Henning RH, van Dokkum RP, de Zeeuw D. Mi-
croalbuminuria and endothelial dysfunction: emerging targets for
primary prevention of end-organ damage. J Cardiovasc Pharmacol
2006; 47(Suppl 2): S151-62; discussion S172-56.
[69] Wachtell K, Ibsen H, Olsen MH, Borch-Johnsen K, Lindholm LH,
Mogensen CE, et al. Albuminuria and cardiovascular risk in hyper-
tensive patients with left ventricular hypertrophy: the LIFE study.
Ann Intern Med 2003; 139: 901-6.
[70] Diercks GF, Stroes ES, van Boven AJ, van Roon AM, Hillege HL,
de Jong PE, et al. Urinary albumin excretion is related to cardio-
vascular risk indicators, not to flow-mediated vasodilation, in ap-
parently healthy subjects. Atherosclerosis 2002; 163: 121-6.
[71] Taddei S, Virdis A, Mattei P, Ghiadoni L, Sudano I, Arrighi P,
et al. Lack of correlation between microalbuminuria and endothe-
lial function in essential hypertensive patients. J Hypertens 1995;
13: 1003-8.
[72] Schiffrin EL, Lipman ML, Mann JF. Chronic kidney disease: ef-
fects on the cardiovascular system. Circulation 2007; 116: 85-97.
[73] Ghiadoni L, Cupisti A, Huang Y, Mattei P, Cardinal H, Favilla S,
et al. Endothelial dysfunction and oxidative stress in chronic renal
failure. J Nephrol 2004; 17: 512-9.
[74] Barnes PJ, Karin M. Nuclear factor-kappaB: a pivotal transcription
factor in chronic inflammatory diseases. N Engl J Med 1997; 336:
1066-71.
[75] Morris ST, McMurray JJ, Rodger RS, Jardine AG. Impaired endo-
thelium-dependent vasodilatation in uraemia. Nephrol Dial Trans-
plant 2000; 15: 1194-200.
[76] Richard MJ, Arnaud J, Jurkovitz C, Hachache T, Meftahi H,
Laporte F, et al. Trace elements and lipid peroxidation abnormali-
ties in patients with chronic renal failure. Nephron 1991; 57: 10-5.
[77] Annuk M, Zilmer M, Lind L, Linde T, Fellstrom B. Oxidative
stress and endothelial function in chronic renal failure. J Am Soc
Nephrol 2001; 12: 2747-52.
[78] Touyz RM, Yao G, Quinn MT, Pagano PJ, Schiffrin EL. p47phox
associates with the cytoskeleton through cortactin in human vascu-
lar smooth muscle cells: role in NAD(P)H oxidase regulation by
angiotensin II. Arterioscler Thromb Vasc Biol 2005; 25: 512-8.
[79] Touyz RM, Schiffrin EL. Reactive oxygen species in vascular
biology: implications in hypertension. Histochem Cell Biol 2004;
122: 339-52.
[80] Schiffrin EL, Touyz RM. From bedside to bench to bedside: role of
renin-angiotensin-aldosterone system in remodeling of resistance
arteries in hypertension. Am J Physiol Heart Circ Physiol 2004;
287: H435-46.
[81] Vallance P, Leone A, Calver A, Collier J, Moncada S. Accumula-
tion of an endogenous inhibitor of nitric oxide synthesis in chronic
renal failure. Lancet 1992; 339: 572-5.
[82] Sydow K, Schwedhelm E, Arakawa N, Bode-Boger SM, Tsikas D,
Hornig B, et al. ADMA and oxidative stress are responsible for en-
1770 Current Pharmaceutical Design, 2008, Vol. 14, No. 18
dothelial dysfunction in hyperhomocyst(e)inemia: effects of L-
arginine and B vitamins. Cardiovasc Res 2003; 57: 244-52.
[83] Sarnak MJ, Levey AS, Schoolwerth AC, Coresh J, Culleton B,
Hamm LL, et al. Kidney disease as a risk factor for development of
cardiovascular disease: a statement from the American heart asso-
ciation councils on kidney in cardiovascular disease, high blood
pressure research, clinical cardiology, and epidemiology and pre-
vention. Circulation 2003; 108: 2154-69.
[84] Perticone F, Maio R, Tripepi G, Zoccali C. Endothelial dysfunction
and mild renal insufficiency in essential hypertension. Circulation
2004; 110: 821-5.
[85] van der Harst P, Smilde TD, Buikema H, Voors AA, Navis G, van
Veldhuisen DJ, et al. Vascular function and mild renal impairment
in stable coronary artery disease. Arterioscler Thromb Vasc Biol
2006; 26: 379-84.
[86] Vita JA, Treasure CB, Nabel EG, McLenachan JM, Fish RD, Ye-
ung AC, et al. Coronary vasomotor response to acetylcholine re-
lates to risk factors for coronary artery disease. Circulation 1990;
81: 491-7.
[87] Ross R. Atherosclerosis--an inflammatory disease. N Engl J Med
1999; 340: 115-26.
[88] Benjamin EJ, Larson MG, Keyes MJ, Mitchell GF, Vasan RS,
Keaney JF Jr, et al. Clinical correlates and heritability of flow-
Virdis et al.
mediated dilation in the community: the Framingham heart study.
Circulation 2004; 109: 613-9.
[89] Ludmer PL, Selwyn AP, Shook TL, Wayne RR, Mudge GH, Alex-
ander RW, et al. Paradoxical vasoconstriction induced by acetyl-
choline in atherosclerotic coronary arteries. N Engl J Med 1986;
315: 1046-51.
[90] Taddei S, Virdis A, Mattei P, Ghiadoni L, Fasolo CB, Sudano I,
et al. Hypertension causes premature aging of endothelial function
in humans. Hypertension 1997; 29: 736-43.
[91] John S, Schmieder RE. Impaired endothelial function in arterial
hypertension and hypercholesterolemia: potential mechanisms and
differences. J Hypertens 2000; 18: 363-74.
[92] Solzbach U, Hornig B, Jeserich M, Just H. Vitamin C improves
endothelial dysfunction of epicardial coronary arteries in hyperten-
sive patients. Circulation 1997; 96: 1513-9.
[93] Taddei S, Virdis A, Ghiadoni L, Sudano I, Salvetti A. Effects of
antihypertensive drugs on endothelial dysfunction: clinical implica-
tions. Drugs 2002; 62: 265-84.
[94] Anderson TJ, Uehata A, Gerhard MD, Meredith IT, Knab S, Dela-
grange D, et al. Close relation of endothelial function in the human
coronary and peripheral circulations. J Am Coll Cardiol 1995; 26:
1235-41.