Improvement of Endothelial Dysfunction by Selective
Estrogen Receptor-␣ Stimulation in Ovariectomized SHR
Julian Widder,* Theo Pelzer,* Christine von Poser-Klein, Kai Hu, Virginija Jazbutyte,
Karl-Heinrich Fritzemeier, Christa Hegele-Hartung, Ludwig Neyses, Johann Bauersachs
Abstract—Both known estrogen receptors, ER␣ and ER␤, are expressed in blood vessels. To gain further insight into the
role of ER␣ in a functional setting, we investigated the effect of the novel highly selective ER␣ agonist Cpd1471 on
vascular reactivity in ovariectomized spontaneously hypertensive rats (SHR). After ovariectomy or sham operation,
12-week-old female SHR received either 17␤-estradiol (E2, 2 ␮g/kg body wt per day), the selective ER␣ agonist
Cpd1471 (30 ␮g/kg body wt per day), or placebo. Acetylcholine-induced endothelium-dependent vasorelaxation was
significantly blunted in aortas from ovariectomized rats (Rmax, 53%⫾3% versus sham, 79%⫾2%; P⬍0.001). Treatment
with E2 or Cpd1471 significantly augmented acetylcholine-induced relaxation in ovariectomized rats (Rmax, 70%⫾2%;
resp, 73%⫾2%). Endothelium-independent relaxation induced by sodium nitroprusside was not different among the four
groups. The contractile response induced by the nitric oxide (NO) synthase inhibitor N␻-nitro-L-arginine, an index of
basal NO formation, was significantly lower in ovariectomized rats compared with sham-operated animals (53⫾2%
versus 77%⫾5%; P⬍0.01) and was normalized by both E2 (70%⫾2%) and Cpd1471 (70%⫾3%). Aortic endothelial
NO synthase (eNOS) expression and phosphorylation of the vasodilator-stimulated phosphoprotein, an index of
NO/cGMP-signaling, was reduced in ovariectomized SHR and normalized by E2 and Cpd1471. In SHR after
ovariectomy, endothelium-dependent NO-mediated vasorelaxation and eNOS expression are attenuated. The novel
selective ER␣ agonist Cpd1471 prevented these pathophysiological changes to a similar extent as E2. Thus, the
pharmacological principle of selective ER␣ activation mediates positive vascular effects. (Hypertension. 2003;42:991996.)
Key Words: estrogen 䡲 endothelium 䡲 nitric oxide 䡲 nitric oxide synthase 䡲 rats, spontaneously hypertensive
G
ender differences in the risk for cardiovascular diseases are well recognized, with premenopausal
women exhibiting a lower risk than age-matched men. The
advantage of women over men in cardiovascular morbidity
disappears after menopause, suggesting that estrogen plays
an important role in cardiovascular health.1 Estrogens are
known to exert beneficial effects on the vascular wall.
Long-term estrogen treatment improves endothelial dysfunction, a major contributor to the pathophysiology of
cardiovascular disease, through upregulation of endothelial cell genes, such as endothelial nitric oxide synthase
(eNOS).2– 4 Furthermore, estrogen has rapid nongenomic
effects on the vascular endothelium, including activation
of nitric oxide (NO) synthesis.5,6 However, despite the
positive effects on vascular function in animal models7–9
and humans,10 –14 estrogen replacement therapy with 17␤estradiol or mixtures of equine estrogens as in the Heart
and Estrogen/progestin Replacement Study (HERS) has
failed to protect from cardiovascular diseases in large
controlled clinical trials.15–18 Therefore, in recent years,
research has focused on selective estrogen receptor modulation as a possible new pharmacological principle.19
Estrogen effects occurring at physiological hormone concentrations are mediated by estrogen receptors, which are
transcription factors belonging to the family of steroid hormone receptors. Both estrogen receptor subtypes known,
ER␣ and ER␤, are encoded by different genes and are
expressed in vascular endothelial and smooth muscle
cells.20,21 Positive cardiovascular effects and especially the
increase in NO production by estrogen have been reported to
involve ER␣22–24 as well as ER␤.25,26
To bring the differential effects of ER␣ and ER␤ closer to
the clinical situation, we investigated for the first time the
influence of a novel highly selective ER␣ agonist on vasorelaxation and eNOS expression in isolated aortas from spontaneously hypertensive rats (SHR) after ovariectomy. In
addition, we measured the phosphorylation status of the
vasodilator-stimulated phosphoprotein (VASP) to determine
Received June 17, 2003; first decision July 8, 2003; revision accepted September 18, 2003.
From Medizinische Klinik der Julius-Maximilians-Universität (J.W., T.P., C.V.P.-K., K.H., V.J., J.B.), Würzburg, Germany; Schering AG (K.-H.F.,
C.H.-H.), Berlin, Germany; and the University of Manchester, Manchester Royal Infirmary (L.N.), Manchester, UK.
*Both authors contributed equally to this study.
Correspondence to Priv Doz Dr Johann Bauersachs, Medizinische Universitätsklinik, Josef-Schneider-Str 2, D-97080 Würzburg, Germany. E-mail
bauersachs_j@klinik.uni-wuerzburg.de
© 2003 American Heart Association, Inc.
Hypertension is available at http://www.hypertensionaha.org
DOI: 10.1161/01.HYP.0000098661.37637.89
991
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Hypertension
November 2003
the functionality of the NO/cGMP-mediated vasodilatory
pathway in vivo.27
Methods
Study Protocol, Hemodynamic Measurements
Twelve-week-old female SHR (Charles River) were ovariectomized
or sham-operated under isoflurane anesthesia. Starting 1 day after
ovariectomy, animals were randomly selected for daily subcutaneous
injection of 17␤-estradiol (E2, 2 ␮g/kg body wt per day), the
selective ER␣ agonist Cpd1471 (30 ␮g/kg body wt per day), or
placebo. All animals had free access to standard rat chow and water.
E2 and Cpd1471 were dissolved in ethanol and injected with peanut
oil used as a carrier substance; placebo animals received ethanol/
peanut oil alone. After 4 weeks of treatment, blood pressure was
recorded as described previously28 and animals were euthanized.
Estradiol serum levels were measured by radioimmunoassay
(DPC-Biermann).
Pharmacological Properties of Cpd1471
The binding affinity of Cpd1471 to ER␣ and ER␤ was determined by
competition experiments, using cytosol preparations from rat uterus
(containing ER␣) or rat prostate (containing ER␤). [3H]-17␤estradiol served as the radioactive ligand; unlabeled 17␤-estradiol
was used as the reference. The relative binding affinity of Cpd1471
to ER␣ proved to be 125-fold higher compared with the relative
binding affinity to ER␤. Transactivation assays with Cpd1471 were
performed with U2-OS human osteosarcoma cells transfected with
either ER␣ or ER␤ and an estrogen-sensitive reporter gene (EREERE-luciferase).29 Cpd1471 exhibited 200-fold higher relative potency (compared with the reference 17␤-estradiol) in cells transfected with ER␣ compared with ER␤.
Vascular Reactivity Studies
The descending thoracic aorta was dissected and carefully cleaned of
connective tissue. A 10-mm segment was immediately frozen in
liquid nitrogen for Western blot analysis. Another segment was cut
into rings (3 mm in length), which were mounted in an organ bath
(Föhr Medical Instruments) for isometric force measurements. The
rings were equilibrated for 30 minutes under a resting tension of 2 g
in oxygenated (95% O2; 5% CO2) Krebs-Henseleit solution (NaCl
118 mmol/L, KCl 4.7 mmol/L, MgSO4 1.2 mmol/L, CaCl2
1.6 mmol/L, KH2PO4 1.2 mmol/L, NaHCO3 25 mmol/L, glucose
12 mmol/L; pH 7.4, 37°C) containing diclofenac (1 ␮mol/L) to
exclude influences of cyclooxygenase products. Rings were repeatedly contracted by KCl (100 mmol/L) until reproducible responses
were obtained. Thereafter, the relaxant response to cumulative doses
of acetylcholine after preconstriction with 50 mmol/L KCl was
assessed. Contractile response of aortic rings induced by KCl was
not different among the four groups of SHR.
To evaluate the formation of basal NO, the contraction induced by
45 minutes of incubation with the NO-synthase inhibitor N␻-nitroL-arginine (L-NA, 100 ␮mol/L) was measured in ring segments
preconstricted with phenylephrine to ⬇10% of maximum contraction. Endothelium-independent relaxation was assessed with the use
of sodium nitroprusside.
Materials
All biochemicals were obtained in the highest purity available from
Sigma. The selective ER␣ agonist Cpd1471 was kindly provided by
Schering, AG (Berlin, Germany).
Statistics
Relaxant responses were given as percentage relaxation relative to
the preconstriction level. Values are expressed as mean⫾SEM of n
experiments with segments from different animals. Statistical analysis was performed by 1-way ANOVA for repeated measurements,
followed by a post hoc Bonferroni test. Probability values ⬍0.05
were considered statistically significant.
Results
Global Parameters
Mean arterial blood pressure did not differ between ovariectomized and sham-operated SHR (193⫾5 resp, 188⫾4 mm Hg),
but it tended to be slightly lower in ovariectomized rats supplemented with either E2 or Cpd1471 (173⫾5 resp,
171⫾7 mm Hg; NS). Uterus weight, a long-term parameter of
ER␣ activation, was lower in ovariectomized placebo-treated
compared with sham-operated rats (91.8⫾12.1 resp,
288.1⫾16.5 mg; P⬍0.001) and increased substantially by treatment with E2 (173.0⫾17.8 mg) and Cpd1471 (160.5⫾17.9 mg).
Body weight was increased in ovariectomized placebo-treated
compared with sham-operated rats (244⫾2 resp, 199⫾3 g;
P⬍0.001) and lowered by treatment with E2 (212⫾4 g) and
Cpd1471 (225⫾3 g). Estradiol serum levels were significantly
reduced in ovariectomized SHR treated with placebo or
Cpd1471 (24.7⫾0.9 resp, 28.4⫾1.8 pg/mL; P⬍0.01) compared
with sham-operated animals (74.8⫾31.7 pg/mL) and normalized
by E2 treatment (103.6⫾10.4 pg/mL).
Vascular Reactivity in Aortic Rings
In preconstricted aortic rings, acetylcholine induced a
concentration-dependent relaxation that was blunted in aortas
from ovariectomized rats (Figure 1). The concentrationrelaxation curves to the endothelium-independent relaxant
sodium nitroprusside were not different between shamoperated and ovariectomized rats (Figure 2). Treatment with
either the selective ER␣ agonist Cpd1471 or E2 significantly
augmented the acetylcholine-induced relaxation of aortic
rings from ovariectomized rats (Figure 1) without affecting
the response to sodium nitroprusside (Figure 2).
The NO synthase inhibitor L-NA induced a significantly
higher contraction in sham-operated than in ovariectomized
animals (Figure 3), indicating a reduced basal NO formation
in rings from ovariectomized SHR. L-NA–induced contraction in aortic rings from ovariectomized SHR was significantly augmented either by Cpd1471 or E2, suggesting a
higher NO formation in these segments.
Western Blot Analysis
Aortic tissue was homogenized in ice-cold RIPA buffer. These crude
protein extracts were subjected to SDS–PAGE electrophoresis and
transferred to nitrocellulose membranes. Proteins were detected by
using their specific antibodies and visualized by enhanced chemiluminescence (eNOS) or with the Odyssey Infrared Imaging System
(LI-COR) (VASP).
A more detailed description of the Western blot analysis can be found
in an online supplement available at http://www.hypertensionaha.org.
Expression of Endothelial NO Synthase in
Rat Aorta
eNOS protein expression was decreased in ovariectomized
rats as compared with sham-operated rats (Figure 4). Treatment with either the selective ER␣ agonist Cpd1471 or E2
normalized eNOS protein expression in aortas from ovariectomized rats.
Widder et al
Figure 1. Acetylcholine-induced relaxation in aortic rings from
placebo-treated ovariectomized (ovex) SHR (䢇) was diminished
compared with sham-operated animals (sham, 䡩). Treatment
with selective ER␣ agonist Cpd1471 (䡵) or with 17␤-estradiol
(E2, ‘) enhanced acetylcholine-induced relaxation in aortic rings
from ovariectomized rats. Results are expressed as mean⫾SEM
from 6 to 10 separate experiments. *P⬍0.001 vs sham;
⫹P⬍0.001 vs ovex placebo.
Phosphorylation of VASP Serine239 in Rat Aorta
VASP phosphorylation at serine239, a biochemical marker of
the functionality of cyclic nucleotide–mediated vasodilatory
pathways in the vessel wall,27 was reduced in aortas from
ovariectomized SHR compared with sham-operated animals.
Treatment with the selective ER␣ agonist Cpd1471 as well as
with E2 normalized VASP phosphorylation at serine239 in the
aortas from ovariectomized rats (Figure 5). Total VASP
expression was similar among the four groups.
Figure 2. Sodium nitroprusside–induced relaxation in aortic
rings from sham-operated (sham, 䡩) and ovariectomized SHR
(ovex) treated with placebo (䢇), selective ER␣ agonist Cpd1471
(䡵), or 17␤-estradiol (E2, ‘). Results are expressed as
mean⫾SEM from 6 to 10 separate experiments.
ER␣ Stimulation and Endothelial Function
993
Figure 3. Contraction induced by NO synthase inhibitor L-NA as
an index of basal NO formation in aortic rings from ovariectomized (ovex) or sham-operated (sham) SHR. Ovariectomized
animals were treated with placebo, 17␤-estradiol (E2), or selective ER␣ agonist Cpd1471. Aortic rings were slightly preconstricted with phenylephrine to 10% of maximum contraction.
Contraction induced by L-NA is shown as percentage of maximum phenylephrine-induced contraction. Results are expressed
as mean⫾SEM from 5 to 9 separate experiments. *P⬍0.01 vs
sham; #P⬍0.05 vs ovex E2 and ⫹P⬍0.05 versus ovex
Cpd1471.
Discussion
In the present study, we demonstrated for the first time that a
subtype-selective ER␣ agonist prevents endothelial dysfunction and downregulation of eNOS protein expression in
ovariectomized SHR to the same extent as non–subtypeselective ER␣ and ER␤ stimulation by 17␤-estradiol.
Figure 4. Representative Western blot showing eNOS protein
expression in aorta from sham-operated (sham) or ovariectomized SHR (ovex). Ovariectomized animals were treated with
placebo, 17␤-estradiol (E2), or selective ER␣ agonist Cpd1471.
Densitometric analysis is expressed as mean⫾SEM (n⫽6 to 8
per group, *P⬍0.05).
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November 2003
Figure 5. Representative Western blot showing phospho-VASP
and total-VASP in aorta from ovariectomized (ovex) or shamoperated (sham) SHR. ␣-Actinin was used as control protein.
Ovariectomized animals were treated with placebo, 17␤estradiol (E2), or selective ER␣ agonist Cpd1471. Intensities of
phospho-VASP and total-VASP bands are expressed as
phospho-VASP/total-VASP ratio. Mean phospho-VASP/totalVASP ratio of aortas from sham SHR was set at 100%. Data are
expressed as mean⫾SEM (n⫽4 per group, *P⬍0.05).
Estrogen has an important role in the maintenance of
cardiovascular health in women. After menopause, the natural state of estrogen deficiency, women are at higher risk for
cardiovascular diseases.30 Estrogen is a potent vasoprotective
molecule exerting indirect and direct effects on cardiovascular tissues. The indirect effects such as lowering of plasma
lipoprotein levels and modulating the fibrinolytic system
account for approximately one third of the beneficial cardiovascular effects. Direct actions of estrogen on the vascular
wall, including changes in endothelial cell gene expression
and function, substantially contribute to its cardioprotective
effects. Estrogens exert their biological effects on vascular
cells by rapid nongenomic and classic genomic mechanisms31: rapid activation of eNOS induces acute vasodilation;
upregulation of eNOS expression confers long-term benefit.
As confirmed in the present study in SHR, estrogen deficiency induces endothelial dysfunction in animal models as
well as in humans,13,32 which can be restored by estrogen
supplementation.12,33,34 Observational studies have shown
that women without documented cardiovascular disease taking hormone replacement therapy have a reduced risk of
major cardiovascular events compared with untreated women.35–37 However, large controlled clinical trials, such as
HERS, failed to show a beneficial effect on secondary
prevention in women with heart disease and even suggested
an increased risk of cardiovascular events.15–18 In the HERS
trial, hormone replacement therapy was associated with a
significantly increased risk of cardiovascular events within
the first year and thromboembolic events (deep venous
thrombosis and pulmonary embolism). Several mechanisms
may account for the disappointing results in these end point
trials, for example, concomitant use of medroxyprogesterone
may mask positive effects of conjugated estrogens, and the
study population in the HERS trial was significantly older
than the average postmenopausal women prescribed hormone
replacement therapy. However, there is no clear explanation
for the negative results in these clinical end point studies.
With the development of selective estrogen receptor modulators acting as estrogen receptor antagonist in the breast and
uterus and as estrogen receptor agonist in the vasculature,
such as raloxifene, the hope was raised to induce only the
positive effects and exclude the negative side effects of the
nonselective estrogen receptor stimulator E2 such as deep
venous thrombosis and pulmonary embolism. Indeed, studies
in animals as well as in humans revealed that selective
estrogen receptor modulation can mediate positive cardiovascular effects, such as improving endothelium-dependent
vasodilation.38 – 42
Besides selective receptor modulation, selective activation
of ER␣ and ER␤, both of which differ in tissue-specific
expression and biological function, appears as a novel pharmacological principle to improve the safety and efficiency of
estrogens in cardiovascular disease. To gain further insight
into the role of ER␣ in a functional setting and to identify
novel compounds for clinical application, we investigated the
influence of a novel and highly selective ER␣ agonist on
endothelial function in SHR. The essential new finding of the
present study is the preservation of endothelium-dependent
vasorelaxation after ovariectomy in SHR by a chronic treatment with the novel subtype ER␣ selective agonist Cpd1471.
The SHR is a well-known and widely used animal model of
hypertension with documented aggravation of endothelial
dysfunction after ovariectomy compared with sham-operated
animals.43 Therefore, ovariectomized SHR constitute a suitable model to study estrogen deficiency and the effect of
replacement therapy. Indeed, after ovariectomy, endotheliumdependent vasorelaxation was significantly reduced in
placebo-treated SHR. Furthermore, we found a diminished
contraction induced by the NO synthase inhibitor L-NA in
aortic rings from ovariectomized placebo-treated rats, indicating a reduced basal NO formation in these rats. To further
elucidate the functionality of NO/cGMP signaling in the
aorta, we measured VASP phosphorylation at serine239 by
using a phosphorylation-specific antibody. NO induces vasorelaxation by stimulating the soluble guanylyl cyclase,
increasing the level of cGMP and activating cGMPdependent protein kinases type I (cGK-I). VASP, a protein
localized at actin filaments, focal adhesions, and dynamic
membrane regions, is a validated substrate of cGK-I. Hence,
phosphorylation of VASP at serine239 is a suitable biochemical marker for cGK-I activity and has been shown to reflect
NO bioavailability in the vascular wall under physiological as
Widder et al
well as pathophysiological conditions.27,44 Estrogen deficiency after ovariectomy resulted in attenuation of VASP
phosphorylation in the aortas of SHR that could be restored
by either E2 or the selective ER␣ agonist Cpd1471. Taken
together, our data suggest that the reduced protein expression
of eNOS in estrogen-deficient SHR diminishes NO/cGMP
signaling in the vessel wall, leading to blunted endotheliumdependent relaxation. Although rapid nongenomic effects
were not the focus of the present study, the demonstration that
long-term E2 therapy increased eNOS protein expression in
aorta from SHR is in line with previous observations obtained
in cultured endothelial cells.2– 4
The relative importance of ER␣ and ER␤ in cardiovascular
disease is still a matter of debate, although the genomic
effects of estrogen have been attributed mainly to stimulation
of ER␣ by estrogen.23,24 To date, it was unclear whether a
subtype ER␣-selective agonist would be as effective in
inducing beneficial vascular effects as E2. Biochemical
studies demonstrated a 125-fold-higher binding affinity of
Cpd1471 for ER␣ than for ER␤. In transactivation assays,
Cpd1471 exhibited 200-fold-higher relative potency if acting
through ER␣ compared with ER␤. Prevention of endothelial
dysfunction by Cpd1471 in SHR after ovariectomy in the
present study shows that selective ER␣ stimulation is sufficient to mediate beneficial vascular effects. Indeed, Cpd1471
increased eNOS protein expression and VASP phosphorylation to the same extent as E2.
Perspectives
It is established that estrogen has beneficial cardiovascular
effects. However, there is a striking discrepancy between
animal and human studies showing positive effects on vascular wall morphology and function and large controlled
clinical end point trials demonstrating increased cardiovascular events by hormone replacement therapy. The role of the
two estrogen receptors, ER␣ and ER␤, which may mediate
positive cardiovascular effects, to date was only investigated
by cell culture studies or by knockout models. Using an in
vivo model of estrogen deficiency, we for the first time
demonstrated that chronic treatment with a novel highly
selective ER␣ agonist mediates positive vascular effects.
Although we do not rule out that ER␤ effects also contribute
to E2-mediated vasoprotection, our data suggest that pharmacological ER␣ stimulation alone is sufficient enough to
prevent endothelial dysfunction elicited by estrogen deficiency. Further studies will be necessary to investigate
whether ER␣ stimulation is superior to E2 treatment regarding negative side effects of hormone replacement therapy
such as thrombotic events. However, the pharmacological
principle of subtype-specific estrogen receptor stimulation
gives the hope for a new treatment option for cardiovascular
protection.
Acknowledgments
This work was supported by the Deutsche Forschungsgemeinschaft
(SFB 355, B10). Ludwig Neyses was supported by an International
Appointee Grant from the Medical Research Council, UK.
ER␣ Stimulation and Endothelial Function
995
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