Cardiovascular Research 69 (2006) 916 – 924
www.elsevier.com/locate/cardiores
17h-estradiol increases volume, apical surface and elasticity of human
endothelium mediated by Na+/H+ exchange
U. Hillebrand a,*, M. Hausberg a, C. Stock b, V. Shahin b, D. Nikova b, C. Riethmüller b,
K. Kliche b, T. Ludwig c, H. Schillers b, S.W. Schneider d, H. Oberleithner b
a
Department of Internal Medicine D, University of Muenster, Germany
b
Institute of Physiology II, University of Muenster, Germany
c
Department of Cellular and Molecular Physiology, Yale University, New Haven, United States
d
Department of Dermatology, University of Muenster, Germany
Received 12 July 2005; received in revised form 9 November 2005; accepted 20 November 2005
Available online 17 January 2006
Time for primary review 12 days
Abstract
Objective: 17h-estradiol is known to delay the onset of atherosclerosis in women but cellular mechanisms are still unclear. Estrogens bind to
specific receptors and initiate a signaling cascade that involves the activation of plasma membrane Na+/H+ exchange. We hypothesized that
estrogens interfere with ion transport across the plasma membrane and thus control endothelial structure and function. Therefore, we investigated
the effects of the sex steroids 17h-estradiol, progesterone, and testosterone on volume, apical surface and elasticity in human endothelium.
Methods: The atomic force microscope was used as an imaging tool and as an elasticity sensor. We applied the antiestrogen tamoxifen, the
Na+/H+ exchange blocker cariporide and the epithelial Na+channel blocker amiloride to elucidate the role of transmembrane ion transport in
hormone-treated human umbilical vein endothelial cells (HUVEC).
Results: Incubation with 17h-estradiol for 72 h led to a dose-dependent increase of endothelial cell volume (41%), apical cell surface (22%),
and cell elasticity (53%) as compared to non-17h-estradiol treated controls. Block of the 17h-estradiol receptor by tamoxifen and of plasma
membrane Na+/H+ exchange by cariporide prevented the hormone-induced changes. Progesterone and testosterone were ineffective.
Conclusions: 17h-estradiol increases HUVEC water content and HUVEC elasticity mediated by activated estrogen receptors. The estrogen
response depends on the activation of plasma membrane Na+/H+ exchange. The increase in endothelial cell elasticity could be one of the
vasoprotective mechanisms postulated for 17h-estradiol.
D 2005 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.
Keywords: HUVEC; 17h-estradiol; Atomic force microscopy; Cariporide; Tamoxifen
1. Introduction
There is a substantial body of evidence that endogenous
estrogens have protective effects on the cardiovascular
system in women. After menopause women rapidly develop
an increased risk of cardiovascular diseases. Both clinical
* Corresponding author. Klinik und Poliklinik für Innere Medizin D,
Albert-Schweitzer-Str. 33, 48149 Münster, Germany. Tel.: +49 251
8347535; fax: +49 251 8349643.
E-mail address: [email protected] (U. Hillebrand).
and experimental data suggest that this loss of protection is
caused by a deficiency of endogenous estrogens [1– 5].
Potential mechanisms include positive effects of estrogens on lipid profile and inhibition of cholesterol deposition
in the arterial wall [2,6]. However, epidemiological data
suggest that the favourable effects of estrogens on lipid
metabolism account for only 30 –50% of the observed
reduction in adverse cardiovascular events [2]. Therefore,
other mechanisms must contribute to the protective effects
of estrogens on the vasculature.
In vivo the endothelium serves as a physical barrier
protecting the underlying components of the blood vessel,
0008-6363/$ - see front matter D 2005 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.cardiores.2005.11.025
U. Hillebrand et al. / Cardiovascular Research 69 (2006) 916 – 924
as well as a docking station for monocytes, leukocytes, and
neutrophils. It also has important functions in hemostasis
and blood flow through the vessel. Elastic deformation of
the endothelial cell layer is crucial for the preservation of a
non-turbulent blood flow [7]. The relationship between cell
surface and volume determines endothelial cell shape and
thus, among other factors, cell elasticity.
Although there is evidence for estrogen action on
endothelium only little is known about underlying cellular
mechanisms. In epithelia changes in ion flow across plasma
membrane, mediated by estrogens, have been reported [8].
Plasma membrane Na+/H+ exchange was disclosed as a
major molecular target. Since any change in activity of this
membrane transporter is likely to change cell volume and
thus cell structure we applied a novel experimental
approach, atomic force microscopy (AFM) in order to
measure volume, surface and elasticity in endothelial cells
exposed to estrogens and, for comparison, to progesterone
and testosterone. AFM is a tactile instrument that images
three-dimensional surfaces and measures cell elasticity with
a mechanical sensor [9]. Here, we report for the first time on
these single cell parameters in response to sex steroids.
2. Methods
2.1. Endothelial cell culture
According to the Declaration of Helsinki, human
umbilical vein endothelial cells (HUVEC) were isolated
and grown as described previously [10,11]. The culture
medium (M199, Gibco, Karlsruhe, Germany) contained
10% heat-inactivated fetal calf serum (Roche, Mannheim,
Germany), antibiotics (penicillin 100 U/ml, streptomycin
100 Ag/ml), 5 U/ml heparin (Biochrom KG, Berlin,
Germany) and 1 ml/100 ml growth supplement derived
from bovine retina [12]. Cells (passage 0) were cultivated in
T25 culture flasks coated with 0.5% gelatine (Sigma-Aldrich
Chemie GmbH, Steinheim, Germany). After reaching
confluence cells were split using trypsin and then cultured
(passage 1) on thin glass coverslips (diameter = 15 mm)
coated with 0.5% gelatine, cross-linked with 2% glutaraldehyde. Glass coverslips with cells were placed in Petri
dishes filled with culture medium supplemented with
various chemicals and incubated at 37 -C and 5% CO2 for
24 h. The sex steroids 17h-estradiol, progesterone and
testosterone as well as the anti-estrogen tamoxifen (from
Sigma-Aldrich Chemie GmbH, Steinheim, Germany) were
dissolved in ethanol (stock solutions = 1 nmol/l, stored at
4 -C for two weeks). Final concentrations in the
experiments ranged from 1 to 15 nmol/l for 17hestradiol, 1 to 100 nmol/l for progesterone and testosterone, and 10 to 150 nmol/l for tamoxifen. For coincubation of 17h-estradiol and tamoxifen, the latter was used
in 10-fold higher concentration than 17h-estradiol. Coincubation of 17h-estradiol and progesterone was done
917
with 17h-estradiol in concentration of 15 nmol/l with
progesterone added in a range of 1 to 100 nmol/l.
Furthermore, we used the plasma membrane Na+/H+ exchange inhibitor cariporide (HOE 642; gift of Aventis Pharma
Deutschland GmbH, Frankfurt, Germany), dissolved in
water (stock solution = 1 mmol/l), at a final concentration of
10 Amol/l and the epithelial Na+channel blocker amiloride
(Sigma-Aldrich), dissolved in water at increased temperature (about 40 -C), using a final concentration of 1 Amol/l.
Both inhibitors were added to 15 nmol/l 17h-estradiol
treated cells 1 h prior to fixation. In corresponding control
experiments we added only the solvent (ethanol 0.1%) to
the media. For cell volume and apical surface measurements
HUVEC were fixed with glutaraldehyde (final concentration = 0.5%) gently added to the medium after appropriate
time periods. Fixed cells remained undisturbed for the next
45 min in the incubator. Then the medium was exchanged
by HEPES buffered solution (mmol/l: 140 NaCl; 5 KCl;
1 MgCl2; 1 CaCl2; 10 HEPES (N-2-hydroxyethylpiperazineNV-2-ethanesulfonic acid); pH = 7.4). Cells were stored in
HEPES buffer under sterile conditions at 4 -C. Fixed
HUVEC could be kept in fluid at least for a week without
measurable changes in morphology or cell volume. With
fixed cells two subsequent series of AFM experiments were
carried out: The first series was performed in fluid (HEPES
buffered solution) to quantitatively obtain total cell volume
and apical surface. The second series was performed in air
using the same samples after a Fwash and dry_ procedure to
obtain the Fwater-free_ volume of the cells. In another series
of experiments, cell elasticity measurements were performed
in living HUVEC bathed in HEPES buffered saline.
2.2. Atomic force microscopy
AFM is a scanning probe technique that can collect
quantitative three-dimensional information of adherent cells
at nanometer resolution [13]. The method of measuring cell
volume in fixed adherent HUVEC and the measurement of
cellular elasticity by AFM were previously described [14 –
18]. Thin glass coverslips carrying the cells were glued to
thick microscope glass slides and mounted in the Ffluid cell_
of the AFM. For fluid experiments care was taken to keep
the HUVEC in fluid at all times. AFM was performed in
contact mode using a Nanoscope III Bioscope-AFM (Digital
Instruments, Santa Barbara, California, USA) with a type
8505GN 2 scanner (maximal scan area: 100 100 Am). Vshaped silicon nitride cantilevers with spring constants of
0.06 N/m (Digital Instruments) were used for scanning.
Surface profiles (512 512 pixels) were obtained with scan
sizes of 6,400 Am2 (80 80 Am) at a scan rate of 3.05 Hz.
Further settings were: height mode, gains between 2 and 11,
interaction force between AFM tip and sample surface less
than 5 nN. 15 to 20 images from individual samples were
analysed using the Nanoscope III software (Digital Instruments). For measurement of endothelial cell volume in fluid
and air (i.e., wet and dry state, respectively), the obtained
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U. Hillebrand et al. / Cardiovascular Research 69 (2006) 916 – 924
image was plane-fitted (order 1) and the volume of the total
image (about 7 to 12 cells per image) was then analysed
using the ‘‘bearing’’ software feature (Digital Instruments).
A mean single cell volume was obtained in dividing the
volume of the total image by the number of cells. To obtain
the percentage of the volume increase under different
concentrations the relative cell volume was calculated by
dividing each cell volume by the mean value of the
respective control.
Furthermore, the three-dimensional information enables
the measurement of the apical surface area of adherent cells
using the ‘‘roughness’’ software feature (Digital Instruments). After analysing the apical surface of the total image,
the mean single apical surface was obtained by mathematically dividing the total apical surface by the number of cells
per image. The Frelative_ apical surface per cell was
obtained by dividing each single apical cell surface by the
mean value of the respective control.
Measurements of cell elasticity were performed with
AFM using the same equipment as described above except
that softer cantilevers were used (MLCT-contact microlevers, spring constant: 0.01N/m; Digital Instruments). For
elasticity measurements the endothelial monolayers were
prepared as described above. However, these experiments
were performed in living HUVEC applying HEPES
buffered saline.
Technical details on elasticity measurements have been
published previously [14,19 –21]. In principle, the AFM is
used as a mechanical sensor. The AFM tip is pressed against
the cell so that the membrane is indented. At the same time,
the AFM cantilever that serves as a soft spring is distorted.
Force – distance curves quantify the force (in Newton)
necessary to indent the membrane for a given distance (in
meter). Force –distance curves were made on individual
cells identified by AFM in the HUVEC monolayer. The
elastic (Young’s) modulus was estimated using the Hertz
model that describes the indentation of elastic material [22],
defined as follows: F = y2 (2 / k) [E / (1 r2 )] tan(a),
where F is the applied force (calculated from the spring
constant (0.01N/m) multiplied by the measured cantilever
deflection), E is the elastic modulus (kPa), m is the Poisson’s
ratio assumed to be 0.5 because the cell was considered
incompressible, a is the opening angle of the AFM tip (35-),
and y is the indentation depth (300 nm). Data on elasticity
were expressed in relative terms, i.e., experimental data were
related to the respective control (control value = 100%).
2.3. Statistical analysis
For each experimental series endothelial cells obtained
from one individual umbilical cord were used. Thus, each
experiment has its respective control. This was necessary
due to considerable biological scatter among different
umbilical cords. Data obtained from one individual experiment were related to its own control. Therefore, mean
control values were set as 100% for each experiment. Data
of experiments are given as mean values (T S.E.M.). Data
were tested for normal distribution with the Chi-square-test.
Significance was tested employing analysis of variance with
post hoc tests (Fisher’s PLSD) if allowed or the Kruskal –
Wallis-test if normal distribution failed. Results were
considered to be statistically significant if p < 0.05 (indicated
by asterisks).
3. Results
3.1. 17b-estradiol
Single cell volume significantly increased when HUVEC
were treated with 17h-estradiol for three days. Fig. 1 shows
AFM images of endothelial cells maintained under control
conditions and in presence of 15 nmol/l 17h-estradiol. This
increase in cell volume was accompanied by a significant
augmentation in apical cell surface. Single cell volume
increased by about 41% and apical surface by about 22% as
compared to controls. The response to the steroid was
clearly dependent on the respective concentration applied.
Data are summarized in Tables 1 and 2.
Elasticity measurements in living HUVEC using the
AFM as a mechanical instrument gave striking results. Cell
elasticity increased in response to 17h-estradiol by some
53%. In more detail, the force needed to indent the apical
cell membrane by 300 nm was only about half of the force
needed to indent a control cell (control: 3.2 T 0.2 kPa, 17hestradiol: 1.7 T 0.1 kPa; both n = 15; p < 0.05; Fig. 2).
3.2. 17b-estradiol and tamoxifen
Application of the competitive estrogen receptor antagonist tamoxifen did not by itself change endothelial cell
volume and surface (Tables 1 and 2). However, the drug was
effective in preventing the 17h-estradiol response. Neither
single cell volume nor surface increased when tamoxifen
was added in a 10-fold higher concentration as compared to
17h-estradiol. Furthermore, tamoxifen also abolished the
effects of 17h-estradiol on endothelial cell elasticity (17hestradiol + tamoxifen: 3.3 T 0.4 kPa; n = 10; Fig. 2).
Fig. 3a exhibits the relative cell volume after a 72 h
treatment with 17h-estradiol, tamoxifen and both substances.
Fig. 3b shows the relative apical cell surface. Three different
17h-estradiol concentrations were tested, in combination
with 10-fold higher tamoxifen concentrations. Clearly,
HUVEC respond to 17h-estradiol with increases of both
volume and surface when 10 nmol/l concentrations are
applied.
3.3. Amiloride and cariporide
Volume and surface of cells are determined by the
abundance of ion channels and transporters in plasma
membrane. Since we observed marked volume-surface
U. Hillebrand et al. / Cardiovascular Research 69 (2006) 916 – 924
919
Fig. 1. Atomic force microscopy (top view AFM images) of human umbilical vein endothelial cells (HUVEC). (a) AFM images of HUVEC under control
conditions (no hormone) and after 72 h treatment with 15 nmol/l 17h-estradiol. (b) Height profiles according to the profile lines in the respective images.
changes in response to 17h-estradiol, we applied the
epithelial sodium channel blocker amiloride and the Na+/
H+ exchange inhibitor cariporide to 17h-estradiol-treated
endothelial cells one hour prior to fixation. We tested the
hypothesis whether the apical entry of sodium via one of
these transport pathways could explain the observed
changes. Indeed, the 17h-estradiol-induced increase in
volume, apical surface and endothelial elasticity was
Table 1
Volume of human endothelial cells in response to sex steroids
17h-estradiol n = 20
Tamoxifen n = 20
17h-estradiol + Tamoxifen n = 20
17h-estradiol + Progesterone n = 20
17h-estradiol + Inhibitor n = 15
Progesterone n = 15
Testosterone n = 15
Final concentration
Single apical cell volume [fl/cell]
Control
1 nmol/l
5 nmol/l
10 nmol/l
15 nmol/l
Control
10 nmol/l
50 nmol/l
100 nmol/l
150 nmol/l
Control
5 nmol/l + 50 nmol/l
10 nmol/l + 100 nmol/l
15 nmol/l + 150 nmol/l
Control
15 nmol
15 nmol/l + 1 nM Progesterone
15 nmol/l + 10 nM Progesterone
15 nmol/l + 100 nM Progesterone
Control
15 nmol/l 17h-estradiol
15 nmol/l + 1 Amol/l Amiloride
15 nmol/l + 10 Amol/l Cariporide
Control
1 nmol/l
10 nmol/l
100 nmol/l
Control
1 nmol/l
10 nmol/l
100 nmol/l
2408 T 94.1
2394 T 81.2
2585 T 90.6
3315 T 111.7
3397 T 139.5
2975 T 112.3
3055 T 81.6
3019 T 194.2
2991 T115.0
3003 T 109.7
2713 T 84.4
2673 T 81.5
2782 T 103.6
2765 T 88.2
3098 T 67.7
4276 T 84.5
4358 T 120.5
4247 T 113.2
4329 T 110.8
1879 T 68.1
2624 T 92.2
2585 T 56.8
1889 T 37.4
2110 T 40.1
2155 T 77.1
2080 T 50.9
2121 T 221.7
2051 T 58.8
2079 T 39.7
2043 T 64.4
2073 T 72.3
P
n. s.
n. s.
p < 0.05
p < 0.05
n.
n.
n.
n.
s.
s.
s.
s.
n. s.
n. s.
n. s.
p < 0.05
p < 0.05
p < 0.05
p < 0.05
p < 0.05
p < 0.05
n. s.
n. s.
n. s.
n. s.
n. s.
n. s.
n. s.
920
U. Hillebrand et al. / Cardiovascular Research 69 (2006) 916 – 924
Table 2
Apical surface of human endothelial cells in response to sex steroids
17h-estradiol n = 20
Tamoxifen n = 20
17h-estradiol + Tamoxifen n = 20
17h-estradiol + Progesterone n = 20
17h-estradiol + Inhibitor n = 15
Progesterone n = 15
Testosterone n = 15
Final concentration
Single apical cell surface [Am2/cell]
Control
1 nmol/l
5 nmol/l
10 nmol/l
15 nmol/l
Control
10 nmol/l
50 nmol/l
100 nmol/l
150 nmol/l
Control
5 nmol/l + 50 nmol/l
10 nmol/l + 100 nmol/l
15 nmol/l + 150 nmol/l
Control
15 nmol
15 nmol/l + 1 nM Progesterone
15 nmol/l + 10 nM Progesterone
15 nmol/l + 100 nM Progesterone
Control
15 nmol/l 17h-estradiol
15 nmol/l + 1 Amol/l Amiloride
15 nmol/l + 10 Amol/l Cariporide
Control
1 nmol/l
10 nmol/l
100 nmol/l
Control
1 nmol/l
10 nmol/l
100 nmol/l
1362 T 37.7
1363 T 37.6
1474 T 37.4
1671 T 70.6
1659 T 64.1
1822 T 78.8
1840 T 62.6
1793 T 69.9
1868 T 48.5
1847 T 81.5
1514 T 42.7
1482 T 40.5
1458 T 60.3
1504 T 41.0
1713 T 83.9
2240 T 70.6
2265 T 63.8
2290 T 117.2
2314 T 59.4
1156 T 48.3
1602 T 51.8
1596 T 45.1
1138 T 20.9
1586 T 56.9
1554 T 57.7
1654 T 60.7
1671 T164.4
1632 T 43.7
1631 T 44.4
1626 T 45.7
1632 T 36.9
completely abolished after the application of cariporide
while amiloride was ineffective (17h-estradiol + cariporide:
3.1 T 0.3 kPa; 17h-estradiol + amiloride: 2.0 T 0.2 kPa; n = 9;
Fig. 2). The data for cell volume and surface are
summarized in Tables 1 and 2, and in Fig. 4. They indicate
P
n. s.
n. s.
p < 0.05
p < 0.05
n.
n.
n.
n.
s.
s.
s.
s.
n. s.
n. s.
n. s.
p < 0.05
p < 0.05
p < 0.05
p < 0.05
p < 0.05
p < 0.05
n. s.
n. s.
n. s.
n. s.
n. s.
n. s.
n. s.
that for the action of 17h-estradiol on endothelial cells the
plasma membrane Na+/H+ exchange is of crucial importance.
3.4. Progesterone and testosterone
In order to test for similar responses in volume, apical
surface and elasticity, we applied other sex steroids to the
endothelial cells. The results were clearly negative. Neither
progesterone nor testosterone treatment of HUVEC in
concentrations from 1 to 100 nmol/l over 72 h resulted in
any significant changes of volume and apical surface (Fig.
5). To test wether the combination of estradiol and
progesterone potentiates the volume and apical surfaceincreasing effects co-incubation with 15 nmol/l 17hestradiol and progesterone in concentration from 1 to 100
nmol/l were carried out. No further effects on endothelial
cell volume and apical surface could be measured (Tables
1 and 2).
3.5. Intracellular water and 17b-estradiol
Fig. 2. Relative elasticity of HUVEC after a 72-h treatment with
15 nmol/l 17h-estradiol (E2), E2 + 150 nmol/l tamoxifen (E2 + T), E2 + 1
Amol/l amiloride (E2 + A) and E2 + 10 Amol/l cariporide (E2 + C) as
compared to controls. The asterisk indicates a significant difference
(P < 0.05) compared to control and to the other mean values of this figure.
In order to estimate intracellular water content in HUVEC
before and after treatment with 17h-estradiol we performed
another series of AFM experiments. In a first step, we
scanned HUVEC in HEPES buffered saline in order to obtain
U. Hillebrand et al. / Cardiovascular Research 69 (2006) 916 – 924
921
p < 0.05 compared to control; n = 20). The data indicate that
the major increase in cell volume mediated by 17h-estradiol is
due to the increase in cell water.
4. Discussion
The aim of the study was to investigate the effects of the
sex steroids, 17h-estradiol, progesterone and testosterone
applied in physiological concentrations, on human umbilical
vein endothelial cells. Our findings are: (i) Treatment of
HUVEC with 17h-estradiol causes an increase of cell
volume by 41%, of apical cell surface by 22% and of cell
elasticity by 53% as compared to non-estradiol treated
controls. (ii) Inhibition of the 17h-estradiol receptor by
tamoxifen prevents the changes. (iii) Block of plasma
membrane Na+/H+ exchange but not of epithelial Na+channels prevents the 17h-estradiol response. (iv) Progesterone
and testosterone are ineffective. (v) Co-incubation of 17hestradiol and progesterone has no further effect. (vi) 17h-
Fig. 3. Relative volume (a) and apical surface (b) per cell after 72-h exposure
to 17h-estradiol (E2), tamoxifen (T) and 17h-estradiol + tamoxifen (E2 + T)
at different concentrations. The asterisks indicate significant differences
(P < 0.05) compared to control and to the other two mean values of an
individual triplet in this figure.
Ftotal_ cell volume. In a second step, we scanned the same
samples, but this time in air (after a wash and dry procedure of
the samples) in order to measure cell volume lacking cell
water. This Fpartial_ cell volume represents exclusively
Fwater-free_ organic cell matter. Since cells were fixed by
glutaraldehyde prior to the drying procedure only cell water
was removed while cell matter remained in place. Fig. 6
shows the results. As expected, single cell volume significantly increased when HUVEC were treated with 17hestradiol for three days. Similar, as already reported, the
volume increase was concentration-dependent (Single cell
volume in fluid: control = 3097 T 67.7 fl; 1 nmol/l 17hestradiol = 3294 T 79.9 fl; 5 nmol/l 17h-estradiol = 3186 T 87.6
87.6 fl, 10 nmol/l 17h-estradiol =3756 T 105.2 fl, p < 0.05
compared to control; 15 nmol/l 17h-estradiol = 4275 T 84.5 fl,
p < 0.05 compared to control; n = 20). After drying 77 – 79%
of volume turned out to be water and the remaining
portion to be organic cell matter (control = 719 T 18.1 fl;
1 nmol/l 17h-estradiol = 732 T 17.9 fl; 5 nmol/l 17h-estradiol =
748 T 24.6 fl; 10 nmol/l 17h-estradiol = 803 T 16.7 fl, p < 0.05
compared to control; 15 nmol/l 17h-estradiol = 904 T 31.5 fl,
Fig. 4. Relative volume per cell (a) and relative apical surface per cell (b)
after 72 h of exposure to 15 nmol/l 17h-estradiol (E2), 15 nmol/l 17hestradiol + 1 Amol/l amiloride (E2 + A) and 15 nmol/l 17h-estradiol + 10
Amol/l cariporide (E2 + C). The asterisks indicate significant differences
(P < 0.01) compared to control and to the other mean values of this figure.
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U. Hillebrand et al. / Cardiovascular Research 69 (2006) 916 – 924
deformability, etc, are biophysical cell parameters more or
less closely related to each other. They include properties of
the plasma membrane, the cytoskeletal elements and the cell
organells. In the present study, we could not quantify the
contributions of the different components. We only state and
quantify that 17h-estradiol increases the elasticity of
endothelial cells.
4.2. Sex hormone concentrations
In the experiments, we used physiological concentrations
of the sex hormones. The most effective natural compound
of estrogens is 17h-estradiol. In females physiological
concentrations fluctuate from 0.1 to 10 nmol/l during the
menstrual cycle and up to micromolar concentrations during
pregnancy whereas in males estrogen levels remain below
0.1 nmol/l. After the menopause, however, the 17h-estradiol
blood levels of females fall to those of males. Progesterone
blood concentrations reach 100 nmol/l in the luteal phase in
females. Except for this phase concentrations are comparably low (< 5 nmol/l) in both genders. Testosterone levels
vary between 5 and 100 nmol/l in males, with 10-fold lower
blood levels in females [23].
4.3. Estrogen receptors
Fig. 5. Relative volume per cell (a) and relative apical surface per cell (b)
after 72 h of exposure to progesterone and testosterone. Different hormone
concentrations were applied. No significant differences between individual
mean data could be observed.
In the classical pathway of estrogen action, 17h-estradiol
binds to the estrogen receptor, a ligand-activated transcription factor in the nucleus that regulates gene transcription by
binding to DNA regulatory sequences. In endothelial cells
the effects of 17h-estradiol are also mediated via rapid nongenomic actions that depend on membrane-bound estrogen
receptors [24]. These receptors may act coordinately with
estradiol-mediated cell volume expansion is mainly due to
the increase in cell water.
4.1. Cell elasticity and cell shape
The increase in the 17h-estradiol-mediated elasticity of
the endothelial cells can be explained by the sustained
change in cell shape: according to the law of Laplace the
mechanical tension (i.e., the inverse of elasticity) of a
spherical structure correlates positively with the radius. In
other words, when an endothelial cell increases its volume
to surface ratio (i.e., more gain in volume than in surface as
it occurred after 17h-estradiol treatment) cell shape changes
from flat to spherical. This leads to a decrease in radius if an
endothelial cell is modelled as a sphere’s segment. Then a
decrease in cell tension (i.e., an increase in elasticity) is
expected. Such changes were indeed observed indicating
that an altered cell shape due to volume uptake could be
responsible for the increase in elasticity. Cell membrane
tension, cell membrane elasticity, intracellular pressure, cell
Fig. 6. The volume per cell 72 h after exposure to increasing concentrations
of 17h-estradiol (E2). Volume was measured at two different conditions:
cells scanned either in HEPES-buffered solution (Fin fluid_) or scanned in
air, after a wash and dry procedure (Fafter drying_). The asterisks indicate
significant differences (P < 0.05) compared to the corresponding control
values.
U. Hillebrand et al. / Cardiovascular Research 69 (2006) 916 – 924
other membrane proteins and trigger a rapid intracellular
response. Both genomic and non-genomic mechanisms lead
to alterations in gene transcription and are sensitive to
tamoxifen [25]. Our experiments do not allow to distinguish
whether the observed effects of 17h-estradiol are attributable
to the classical genomic or to the non-genomic pathways.
4.4. Estradiol and the mechanisms of volume regulation
One of the most prominent volume regulatory proteins
located in the plasma membrane is the Na+/H+ exchanger
(NHE) [26 –28]. The plasmalemmal NHEs catalyze the
antiport of intracellular H+ and extracellular Na+. To date at
least seven NHE isoforms have been identified and cloned
[29]. NHE1 is expressed in the plasma membrane of most, if
not all, mammalian cells.
Most likely one of the molecular targets of estrogens is
indeed NHE1. Cariporide (also known as HOE642), a
specific NHE1 inhibitor [30], completely abolished the 17hestradiol effects on the endothelium. In contrast, the
epithelial sodium channel blocker amiloride had no effect.
The differential effects of the two drugs clearly differs from
the response of endothelial cells after treatment with the
mineralocorticoid hormone aldosterone. The latter steroid
induces an amiloride sensitivity in endothelial cells [17]
while no activity change was observed after application of
cariporide [15]. However, both hormones alter cell volume.
A likely explanation is that similar as in renal collecting
duct, aldosterone inserts epithelial sodium channels into the
apical plasma membrane and thus renders the cells sensitive
to amiloride (Oberleithner NIPS 2004). In contrast, estrogens stimulate plasma membrane Na+/H+ exchange and thus
render cells sensitive to cariporide. The different pathways
of apical sodium influx could have important consequences
on the elasticity of endothelial cells. Aldosterone stiffens the
endothelium [31] while estrogens makes it more elastic. The
reason for the opposing effects of the two steroids on
elasticity could be found in the difference of the surface to
volume ratios of the cells when exposed to either one of the
steroids. While aldosterone only mildly increases cell
volume but strongly increases apical surface (i.e., cells
flatten), estrogens strongly increase volume but only mildly
increase apical cell surface (i.e., cells round up). A large and
flat cell (= large radius) is, according to the Laplace law
(tension å pressure radius) expected to be stiffer than a
more round-shaped cell (= small radius). Taken together, it is
likely that cell shape controlled by steroids contributes to
endothelial elasticity.
923
a cytoplasmic modifier site of the Na+/H+ exchanger was
postulated some years before [36] and recently visualized in
the crystal structure of the antiporter [37]. Action of estrogens
on Na+/H+ exchange possibly involves protein kinase C as an
intracellular mediator [38] or, even more directly, could
involve calcium ions released by intracellular stores or
entering the cells through cation channels across plasma
membrane. Sex steroid hormones are known to act via
ionized calcium in vascular smooth muscle [38,39]. In
endothelia it is rather unlikely, since estrogens do not promote
increases in intracellular Ca2+in this tissue [40].
Our data indicate that a change in set-point led to the
increased cell volume. This argument is based on the
persistance of the cell volume increase over days and on the
rapid reversibility upon Na+/H+ exhange inhibition by
cariporide. A sustained change in set-point will counteract
a regulatory volume decrease; then the cell remains volume
expanded. The present observation that the absolute
intracellular water content rises in parallel with estrogeninduced cell swelling strongly supports this view. It is
interesting to note that the percentage of cell water in total
cell volume remained constant, between 77% and 79%. This
indicates that the organic material has also increased in
parallel, albeit at much lower level. The increase in organic
material could be explained by de novo protein synthesis
and plasma membrane augmentation, as observed in
response to 17h-estradiol.
4.6. Possible physiological relevance
The underlying mechanisms of the estradiol-induced
increase in volume and apical surface of endothelial cells
can be summarized as follows: administration of 17hestradiol to endothelial cells leads to an activation of the
NHE activity with an increased import of sodium followed
by an osmotic influx of water. Cells Fround up_ and,
consequently, increase elasticity. Using such a mechanism,
estrogens improve vascular compliance by the appropriate
adjustment of endothelial mechanical properties.
Acknowledgements
We thank Mrs. Marianne Wilhelmi for her excellent
technical assistance. The study was supported by the
Deutsche Forschungsgemeinschaft, SFB 629 A6 and DFG
Re 1284/2-1. Support was also provided by EU grant
FTips4cells_. The technical support of Digital Instruments
(VEECO, Mannheim) is gratefully acknowledged.
4.5. Possible set-point change of the Na+/H+ exchanger
Steroid hormones can change the set-point of the Na+/H+
exchanger, i.e., the antiport remains active even at more
alkaline intracellular pH. As a consequence, cells alkalinize
and expand their volume. This has been shown first in kidney
tubules [32,33], frog skin [34] and lymphocytes [35]. Indeed,
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