Cardiovascular Research 53 (2002) 597604

www.elsevier.com / locate / cardiores

Review

Endothelial function, vascular reactivity and gender differences in the

cardiovascular system
Mark A. Sader a,b , David S. Celermajer a,b , *
a

Department of Cardiology, Royal Prince Alfred Hospital, Missenden Road, Camperdown, 2050 NSW, Australia
b
Department of Medicine, University of Sydney, Camperdown, 2006 NSW, Australia

Keywords: Atherosclerosis; Endothelial function; Gender

1. Introduction

2. Normal endothelial function

A marked gender difference exists in the prevalence and

severity of cardiovascular disease, even after adjustment
for traditional vascular risk factors, which are more
prevalent in males [1]. This has led to an interest in the
role of sex steroids themselves in the promotion or
inhibition of atherogenic events. As endothelial dysfunction is an early and important event in atherogenesis [2]
and appears to predict adverse coronary outcomes [3],
gender differences in endothelial function and the effects
of hormonal therapy on vascular function have been the
focus of considerable research interest.
The normal vascular endothelium regulates arterial tone,
platelet and leukocyte interactions, coagulation, fibrinolysis
and vascular growth [4]. Measurement of endothelial
function in the systemic arteries has become established as
an important method for the detection of early (pre-symptomatic) arterial abnormalities in humans [5]. Ultrasounddetected endothelial dysfunction in peripheral arteries
correlates significantly with coronary endothelial dysfunction [6], as well as with the extent and severity of coronary
atherosclerosis [7]. The availability of a non-invasive
method for the reproducible measurement of endotheliumdependent dilatation has facilitated the study of the interactions between sex hormones and arterial function, in both
males and females.

The normal vascular endothelium is only one cell layer

thick, separating the blood and vascular smooth muscle.
The endothelium responds to physical and chemical stimuli
via the synthesis and / or release of regulatory substances
affecting vascular tone and growth, thrombosis and thrombolysis and platelet and leukocyte interactions with the
endothelium. Substances released by the endothelium
include nitric oxide (NO), prostacyclin, endothelins, interleukins, endothelial growth factors, adhesion molecules,
plasminogen inhibitors and von Willebrand factor [4,8
10].
The pivotal role of the endothelium in regulating
vascular smooth muscle tone has only recently been
appreciated [11]. The existence of an endothelium-derived
relaxing factor (EDRF) was first postulated by Furchgott
and Zawadzki [12], when it was observed that rabbit aortic
rings relaxed to acetylcholine only in the presence of an
intact endothelium. NO, the endothelium-derived relaxing
factor, was subsequently shown to maintain a low resting
arterial tone in both the peripheral [13] and pulmonary [14]
circulations.
NO is synthesized from L-arginine by the enzyme, nitric
oxide synthase (NOS) [15], a reaction which can be
specifically blocked by arginine analogues, such as N G monomethyl-L-arginine (L-NMMA). NO release is stimulated by increased blood flow (resulting in increased shear
stress on the endothelium) [16] and by a variety of agents
in the circulation, including acetylcholine, serotonin,
bradykinin and thrombin, via the activation of specific
endothelial cell membrane receptors. NO release results in
vascular smooth muscle relaxation, through a reduction in
intracellular calcium levels mediated via cyclic GMP [17].

*Corresponding author. Tel.: 161-2-9515-6519; fax: 161-2-95506262.

E-mail address: davidc@card.rpa.cs.nsw.gov.au (D.S. Celermajer).

Time for primary review 40 days.

0008-6363 / 02 / $ see front matter 2002 Elsevier Science B.V. All rights reserved.
PII: S0008-6363( 01 )00473-4

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Received 23 May 2001; accepted 21 August 2001

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M. A. Sader, D.S. Celermajer / Cardiovascular Research 53 (2002) 597 604

3. Mechanisms of hormonal regulation of endothelial

function

4. Assessment of the effects of gender and hormones

on endothelial function in vivo
Gender differences and hormonal effects on endothelial
function have been assessed predominantly using functional methods examining NO-dependent vasomotion, both
in the coronary and peripheral circulations. The acute

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Hormonal regulation of endothelial function may be the

consequence of receptor-dependent or receptor-independent mechanisms. Oestrogen receptors (ER), progesterone
receptors (PR) and androgen receptors (AR) have all been
identified in human vascular endothelium [1822]. ER and
AR have also been identified in smooth muscle cells,
macrophages and platelets [2025].
Gender differences in hormone receptor expression have
been demonstrated in some cell lines [26], as has the
hormonal regulation of hormone receptor expression
[25,27]. Indeed, a man with a disruptive mutation of the
oestrogen receptor gene was demonstrated to have endothelial dysfunction [28].
Oestrogen has been demonstrated to upregulate endothelial nitric oxide synthase (eNOS) activity via a receptormediated system [29]. Oestrogen also upregulates prostacyclin synthase and has beneficial effects in regards to
the response to vascular injury and on atherosclerosis, by
upregulating the expression of vascular endothelial growth
factor (VEGF) and inhibiting endothelial cell apoptosis, as
well as smooth muscle cell migration and proliferation
[30]. Androgens have also been demonstrated to upregulate eNOS and VEGF [30,31].
Receptor-independent pathways include non-genomic,
anti-oxidant effects and effects mediated by hypothalamic
pituitary feedback inhibition. Non-genomic effects occur
early (within minutes) with a rapid onset and offset,
occurring too quickly to be the result of altered gene
expression and subsequent protein synthesis. These nongenomic effects are possibly mediated through membrane
receptors or protein interactions with steroid hormone
receptors [23] and involve common second messengers,
such as intracellular calcium and cyclic AMP. Physiologic
concentrations of oestrogen have been demonstrated to
activate calcium-dependent potassium channels [32,33]
and result in a rapid increase in human endothelial cell
basal NO release [34]. Oestrogens are known to have
anti-oxidant potential [35] and this may improve redox
balance in the wall, improving local NO bioavailability
and consequently enhance endothelium-dependent dilatation [36]. Natural hormone production is regulated by the
hypothalamicpituitary axis, hence exogenous hormone
therapy, such as with oestrogen, will result in suppression
of oestrogen and androgen production.

effects of hormones have been assessed usually using

supraphysiological doses and longer-term studies have
targeted specific subpopulations with natural or induced
conditions of hormonal deficiency or excess, such as postmenopausal women or transsexuals.
Functional methods of assessing endothelial function
examine the endotheliums ability to release NO and cause
vasodilation in response to pharmacological and physiological stimuli. The majority of studies assessing the effects of
hormones on endothelial function have utilised either
invasive coronary artery testing or non-invasive peripheral
artery ultrasound. Non-invasive techniques are more widely applicable to asymptomatic subjects, especially when
assessing the effects of long-term hormonal therapy.
Invasive coronary artery testing in humans assesses
coronary artery responses to intravenous endothelium-dependent (e.g., acetylcholine) and endothelium-independent
(e.g., nitroprusside), using quantitative angiography and
Doppler wires or catheters [37,38].
Non-invasive assessment of brachial artery endothelial
function uses high resolution external vascular ultrasound
[2]. Arterial diameter is measured at rest, during reactive
hyperaemia (leading to flow-mediated dilatation, FMD, an
endothelium-dependent response) and after sublingual nitroglycerin (GTN, an endothelium-independent dilator).
Reactive hyperaemia responses are assessed following
temporary
brachial
artery
occlusion
with
a
sphygmomanometer placed below the target artery. Arterial FMD measurements reflect endothelial vasodilator function, predominantly due to nitric oxide release [39],
correlate significantly with coronary endothelial function
[6] and coronary atherosclerosis [7].
Other functional methods assessing endothelial function
include: plethysmography, a method requiring the intraarterial infusion of endothelium-dependent and independent vasodilator substances; and positron emission tomography which allows the non-invasive assessment of coronary endothelial function, but is expensive and involves
exposure to radiation [40].
As the endothelium influences many parameters other
than vasomotor control, other methods of assessing endothelial function have been utilized, including assays for
measuring NO and circulating markers of normal endothelial activity. Assays for measuring NO in plasma and
urine, however, are generally not suitable for routine
clinical use due to multiple non-vascular sources of nitrates
[41]. Circulating markers of endothelial function (and
dysfunction) include asymmetric dimethylarginine,
endothelin-1, Von Willebrand factor, tissue plasminogen
activator, plasminogen activator inhibitor-1 and cell adhesion molecules. These assays, however, also have limitations with poor sensitivity and specificity for endothelial
dysfunction, considerable overlap of normal and abnormal
levels and uncertainty in regards to whether changes in
circulatory markers parallel changes in other tests of
endothelial function [40].

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599

5. Gender, hormones and endothelial function

6.2. Age

The effects of sex hormones on endothelial function

may depend on subject gender, as well as the dose,
duration and route of hormone delivery. Secondary effects
of hormones, such as alteration in the lipid profile, may in
turn alter endothelial function [42,43]. Assessment of the
independent effects of hormones on endothelial function
involves adjustment for known vascular risk factors,
including lipids, as oestrogens in physiological doses
favorably augment the lipid profile [44] and androgens in
supraphysiological doses have been associated with reduced HDL levels [45].
Additionally, when considering the effects of androgens,
one must remember that testosterone is converted intracellularly to dihydrotestosterone (a more potent and nonaromatizable androgen) by 5a-reductase, but also to oestrogen by aromatase, therefore may result in both androgenic and oestrogenic effects.

Aging is associated with progressive endothelial

dysfunction in both genders [47,48]. The age-related
impairment in endothelial function, however, appears to
occur earlier in men than women [49]. A steep decline in
FMD in women is observed, however, around the time of
the menopause, consistent with a protective effect of
physiological oestrogen levels on endothelial function.

The effect of the menstrual cycle on endothelial function

was assessed by Hashimoto et al., using brachial artery
ultrasound during the three phases of the menstrual cycle.
Endothelium-dependent vasodilatation was maximal during
the follicular and luteal phases of the menstrual cycle,
corresponding to elevated levels in serum oestradiol (Fig.
1) [50]. In the menstrual phase, endothelial function in the
women studied was similar to that in healthy men.

7. Pharmacological doses of sex hormones effects

on endothelial function

6.1. Gender
7.1. Oestrogens in post-menopausal women
Normal ranges have been established for FMD (an
endothelium-dependent) and GTN-mediated (endotheliumindependent) responses of systemic arteries [46] in healthy
young adults, without identifiable atherogenic risk factors.
In age-matched subjects, females show significantly greater FMD and GTN values than males; however the differences in FMD and GTN responses are completely accounted for by the smaller vessel size of females, rather
than due to hormonal differences.

Physiologic oestrogen levels potentiate endothelium-dependent vasodilatation in both the coronary and systemic
circulations (Fig. 2) [51,52] and the acute parenteral
administration of high-dose oestrogen in women with
coronary artery disease attenuates the acetylcholine-induced coronary artery vasoconstriction [53]. Also with
sublingual administration, oestradiol has been demonstrated to have direct vasodilatory properties, with anti-is-

Fig. 1. The effect of the menstrual cycle on endothelial function. Endothelial function was maximal during the follicular and luteal phases of the menstrual
cycle, corresponding to elevated levels in serum oestradiol, consistent with a beneficial effect of physiological levels of oestrogens on vascular reactivity
(adapted from Hashimoto et al. [50]). P,0.01.

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6. Effects of physiological levels of sex hormones on

endothelial function

6.3. Menstrual cycle

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M. A. Sader, D.S. Celermajer / Cardiovascular Research 53 (2002) 597 604

Fig. 2. Oestrogen replacement in post-menopausal women is associated with improved endothelial function (adapted from Lieberman et al. [52]). P,0.05.

7.2. Oestrogens in men

The short-term beneficial effects of oestrogen on endo-

thelial function in women have not been paralleled in men.

Collins et al. found that, although 17-b oestradiol attenuated the acetylcholine-induced coronary vasoconstriction in females, it did not do so in males [53]. Similarly,
the transdermal application of oestradiol for 36 h improved
arterial endothelium-dependent vasodilatation in women,
but not in men [65], and the acute sublingual administration of oestradiol in young men did not improve FMD,
despite resulting in supraphysiological serum oestradiol
levels [66].
Longer-term oestrogen therapy in men has, however,
been associated with improvements in endothelial function.
Two cross-sectional studies assessing the effects of longterm high dose oral oestrogens in genetic males (male to
female transsexuals) demonstrated enhanced arterial reactivity compared with age-matched male controls (Fig. 3)
[67,68]. These unusual populations possessed several
potential confounding factors, including variations in the
type of castrative therapy, dose and route of oestrogen
administration, use of progestins, and a high percentage of
smokers in the groups studied. Nevertheless, a subsequent
prospective study in healthy young men on depot subcutaneous testosterone therapy, with or without oestrogen,
demonstrated an improvement in endothelial function
correlating with serum oestradiol levels [69].
Further support of the potential longer-term benefit of
oestrogens on endothelial function in men, was the improvement in endothelial function seen following the
commencement of Tamoxifen (a SERM) [70], and the
cessation of testalactone treatment (an aromatase inhibitor,
which inhibits the conversion of testosterone to oestradiol)
in male subjects [71].

7.3. Androgens in men

Acutely administered testosterone results in arterial
vasodilatation in both human and animal studies, through
an endothelium-independent mechanism, probably involving ATP sensitive potassium channels on smooth muscle
cells [7274].
Regarding endothelial function, supraphysiological

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chaemic effects in older women with coronary disease

[54].
Differential effects of oral and transdermal oestrogen
replacement therapy in post-menopausal women have been
observed, with oral oestrogen, but not transdermal preparations, improving endothelial function [55].
Combined hormone replacement therapy (oestrogen plus
progesterone) has also been associated with beneficial
effects on endothelial function [56,57], although there have
been conflicting results [58], with androgenic progestagens, such as norethisterone. This raises the concern that
the type of progesterone used, as well as the mode of
delivery, might influence any beneficial effects of unopposed oestrogen. Recently, however, Koh et al. demonstrated no difference in the vascular effects of natural or
synthetic progestagens, when combined with oestrogen, in
healthy post-menopausal women [59].
Selective oestrogen receptor modulators (SERMs, including Tamoxifen and Raloxifene) are agents with high
affinity for the oestrogen receptor and display a tissueselective profile, with oestrogen-agonist activities in some
tissues (such as bone) and oestrogen-antagonist in others
(such as uterus and breast) [60]. Raloxifene induced
coronary arterial relaxation in male and female coronary
arteries by an endothelium-dependent and oestrogen receptor-dependent mechanism involving nitric oxide [61].
The influence of various SERMs on endothelial function in
humans is presently being assessed, with preliminary
results indicating an improvement in endothelial function
in post-menopausal women on Raloxifene [62].
Phytooestrogens are plant oestrogens found mainly in
soybean products, principally as isoflavanoids, and are
being increasingly used to relieve menopausal symptoms.
Although animal studies have indicated a protective effect
of genistein [63], phytooestrogen intake by healthy postmenopausal women have not been demonstrated to have
any beneficial effect on endothelial function [64].

M. A. Sader, D.S. Celermajer / Cardiovascular Research 53 (2002) 597 604

601

Fig. 3. High-dose oestrogen use in genetic males is associated with improved arterial endothelial function (adapted from McCrohon et al. [67]). P50.001.

peak serum testosterone levels [78]. The impairment in

endothelial function occurred despite significant elevations
in serum oestradiol (from testosterone aromatisation),
consistent with a deleterious androgenic effect. This may
vary with age, however, as dihydrotestosterone administration in older men does not appear to alter endotheliumdependent dilatation [79].

7.4. Androgens in women

The use of high dose parenteral testosterone by young
genetic females (female to male transsexuals) has been
associated with impaired arterial reactivity and an increase
in arterial size [80], but no significant difference in FMD
was demonstrated. The study power was limited, however,
by a small number of subjects and a high percentage of
smokers. Recently, however, low dose parenteral testosterone supplementation in post-menopausal women taking
hormone replacement therapy has been associated with an
improvement in endothelium-dependent and endotheliumindependent vasodilatation [81]. Once again, suggesting
that differences in subject age and dose / combination of

Fig. 4. Androgen deprivation therapy (for prostate cancer) is associated with improved endothelial function in elderly men (adapted from Herman et al.
[76]). P,0.001. FMD flow-mediated dilatation of the brachial artery.

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doses of the anabolic steroid nandrolone in an animal

model resulted in impairment of both endothelium-dependent and endothelium-independent dilatation (of rabbit
aortic rings) [75].
In human studies, however, androgens have had varied
effects on endothelial function. Androgen deprivation
therapy in elderly men with prostate cancer was associated
with enhanced endothelial function, suggesting a deleterious effect of physiological levels of androgens (Fig. 4)
[76]. A recent study assessing the effects of long-term
androgenic anabolic steroid use in competition bodybuilders, however, did not reveal any significant difference in
arterial reactivity compared to bodybuilders who had never
used anabolic steroids [77]. Interestingly, both bodybuilding groups had significantly impaired arterial reactivity
compared to sedentary controls, suggesting the intense
exercise undertaken may have had a deleterious effect.
The above studies were both cross-sectional, with the
potential for unknown confounding factors. We recently
studied prospectively a group of otherwise healthy hypogonadal males on long-term testosterone replacement
therapy, with subjects serving as their own controls,
finding a significant impairment in endothelial function at

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M. A. Sader, D.S. Celermajer / Cardiovascular Research 53 (2002) 597 604

sex hormones may influence the effects on endothelial

function.
Polycystic ovarian syndrome is associated with endothelial dysfunction [82] and more extensive and severe
coronary artery disease [83], and features mild hyperandrogenism. In addition to the effects of androgens, however, this syndrome is also characterized by other potentially pro-atherogenic metabolic abnormalities, including
insulin resistance, which may confound any potential
linking of endothelial dysfunction with androgens in this
select subpopulation.

8. Conclusion

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