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INTRODUCTION |
Rac1 belongs to the small (21 kDa) Rho GTPase family that binds to
and hydrolyzes guanosine triphosphate (GTP). Rho proteins have been
shown to be central regulators of the actin cytoskeleton. Rho proteins
function as transducers between mechanical forces, cell morphology, and
gene regulation. In its active GTP-bound state, Rac1 plays an important
role in the regulation of cell shape, adhesion, movement, endocytosis,
secretion, and growth (1, 2).
In the cardiovascular system, activation of Rac1 is necessary for the
release of reactive oxygen species
(ROS)1 in the vessel wall
(3-5). Oxygen radicals impair endothelial function and accelerate the
progression of atherosclerotic lesions by promoting lipid oxidation,
the expression of proinflammatory genes, and by oxidative inactivation
of endothelial nitric oxide (6-8). The NAD(P)H oxidase complex in
vascular smooth muscle cells is regarded the most important source of
the primordial oxygen radical, superoxide, in the vessel wall (9). Rac1
GTPase plays a pivotal role in the assembly and activation of the
NAD(P)H enzymatic system, which is composed of several subunits
including p22phox, the flavoprotein p91phox (or its homologues, such as nox1 in VSMC), and the cytoplasmic subunits p47phox and p67phox (3,
10). Consequently, inhibition of Rac1 activity has been shown to
inhibit oxygen radical release in vascular smooth muscle and
endothelial cells as well as in phagocytes (3, 5). In addition to
vascular superoxide production, activation of Rac1 signaling leads to
cellular hypertrophy cardiac myocytes (11).
Despite the importance of Rac1 GTPase for vascular ROS release, the
regulation of Rac1 in the cardiovascular system is only partially
understood. It is thought that the reduced prevalence of cardiovascular
disease in women is based on atheroprotective effects of estrogens. The
latter are potentially mediated directly through binding to vascular
estrogen receptors (12-17). Although the antioxidative properties of
estrogens are among the most prominent vasoprotective functions of sex
steroids, the underlying molecular mechanisms are only partially known.
Furthermore, it is not known whether small GTPases are regulated by
steroid hormones. We hypothesized that 17
-estradiol may regulate
Rac1 GTPase expression and activity and, thereby, inhibit the release
of ROS from VSMC.
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MATERIALS AND METHODS |
Materials--
Angiotensin II, L-mevalonate and
chemicals were purchased from Sigma. [32P]dCTP and Hybond
N-nylon membranes were obtained from Amersham Biosciences.
[35S]GTP
S was supplied by PerkinElmer Life Sciences.
H2DCF-DA was purchased from Molecular Probes (Eugene, OR).
Antibiotics, calf serum, and cell culture medium were obtained from
Invitrogen. RNA-clean was purchased from AGS (Heidelberg, Germany).
Clostridium sordellii lethal toxin was kindly provided by K. Aktories (Freiburg, Germany) (1). RhoN19 and RacN17 were a kind gift
from A. Hall (London, UK) (2).
Cell Culture--
VSMC were isolated from female rat thoracic
aorta (strain, male Sprague-Dawley, 6-10 weeks old, Charles River Wega
GmbH, Sulzfeld, Germany) by enzymatic dispersion and cultured over
several passages. Cells were grown in a 5% CO2 atmosphere
at 37 °C in Dulbecco's modified Eagles medium without phenol
supplemented with 100 units/ml of penicillin, 100 µg/ml streptomycin,
1% nonessential amino acids (100×), and 10% fetal calf serum (free
of steroid hormones, S-15-M, c.c.pro GmbH). Experiments were
performed with cells from passage 5-10. Cells were kept in quiescent
medium without fetal calf serum 24 h prior to treatment.
Cellular viability under all treatment conditions was determined by
cell count, morphology, and trypan blue exclusion.
Animal Treatment--
Female, spontaneously hypertensive rats
put on a standard chow and were ovariectomized or sham-operated
(control group) 16 weeks after birth. For treatment, 17
-estradiol
pellets (containing 1.7 mg of estradiol each, 60-day release,
Innovative Research) were implanted subcutaneously. E2 levels were
determined by radioimmunoassay (DPC Biermann, Bad Nauheim,
Germany). The thoracic aorta was harvested 5 weeks after surgery. All
animal experiments were conducted in accordance to the German animal
protecting law.
Transfection--
Female VSMC were harvested and resuspended in
electroporation medium (Optimem 1, Invitrogen) at a concentration of
5 × 107 cells/ml. The following constructs were
transfected: insertless vector (pcDNA3) as control,
pRK5-myc-Rac1-L61 (constitutively active Rac1 mutant), and
pRK5-myc-Rac1-N17 (dominant-negative Rac1 mutant) (18, 19). 20 µg of
plasmid DNA and 200 µl of cell suspension were placed in a 0.4-cm
cuvette, mixed, and incubated for 30 min on ice. After incubation at
37 °C for 30 s, the cuvette was pulsed with 300 V and 500 µF
(Electro Cell Manipulator, BioRad). The pulse length was determined by
the electroporator based on capacitance, field strength, and resistance
of the medium. Upon electroporation the cuvette was incubated at room
temperature for an additional 30 min. The cells were plated at tissue
culture plates and cultured for 48 h before treatment with
angiotensin II, E2, and vehicle as indicated.
Western Blotting--
Immunoblotting was performed using Rac1
monoclonal antibody (Santa Cruz Biotechnology Inc., 1:250 dilution).
ER
and ER
monoclonal antibodies were from Dianova (1 µg/ml).
-Actin was used to control for equal protein loading (Santa Cruz
Actin H-196 polyclonal antibody, 1:250 dilution).
Northern Blotting--
Northern blotting in the presence and
absence of 5,6-dichlorobenzimidazole riboside (DRB) (Sigma) using
[32P]dCTP-labeled, full-length Rac1 cDNA and p22phox
cDNA was performed as described previously (19).
Measurement of Reactive Oxygen Species--
Intracellular
reactive oxygen species production was measured by
2',7'-dichlorofluorescein (DCF) fluorescence using confocal laser
scanning microscopy techniques. Dishes of subconfluent cells were
washed and incubated in the dark for 30 min in the presence of 10 mmol/liter 2',7'-dichloro-dihydro-fluorescein-diacetate (H2DCF-DA). Culture dishes were transferred to a Zeiss
Axiovert 135 inverted microscope (Carl Zeiss, Jena, Germany), equipped with a 25×, numerical aperture 0.8, oil-immersion objective
(Plan-Neofluar, Carl Zeiss) and Zeiss LSM 410 confocal attachment, and
reactive oxygen species generation was detected as a result of the
oxidation of H2DCF (excitation, 488 nm; emission longpass
LP515-nm filter set). 512 × 512 pixel images were collected by
single rapid scans, and identical parameters, such as contrast and
brightness, were used for all samples. Five groups of 25 cells for each
sample were randomly selected from the image, and fluorescent intensity was taken. The relative fluorescence intensity are average values of
all experiments.
For measurement of superoxide release in intact vessel segments, aortas
were excised carefully and placed in chilled, aerated Krebs-HEPES
buffer as described (20). Chemiluminescence of aortic rings was
assessed over 10 min in the presence of 5 µmol/liter lucigenin in a
scintillation counter (Lumat LB 9501, Berthold, Bad Wildbad, Germany)
in 1-min intervals. Superoxide release is expressed as relative
chemiluminescence per mg of aortic tissue.
NAD(P)H Oxidase Activity Assay--
NADH or NADPH oxidase
activity was measured by a lucigenin-enhanced chemiluminescence assay
in a 50-mmol liter
1 phosphate buffer (buffer A), pH 7.0, containing 1 mmol liter
1 EGTA, protease inhibitors
(Complete®, Roche Molecular Biochemicals), 150 mmol
liter
1 sucrose, 5 µmol liter
1 lucigenin,
and either 100 µmol liter
1 NADH or 100 µmol
liter
1 NADPH as substrate (21). Cell cultures were
treated as indicated, washed twice with ice-cold phosphate-buffered
saline, pH 7.4, and scraped from the dishes. After a step of low spin
centrifugation, the pellet was resuspended in ice-cold buffer A,
lacking lucigenin and substrate. Then, the cells were mechanically
lysed by using a glass/Teflon potter on ice. The total protein
concentration was determined using the Bradford assay (BioRad) and
adjusted to 1 mg ml
1. 100-µl aliquots of the protein
sample were measured over 10 min in quadruplicates using NADH or NADPH
as substrate in a scintillation counter (Berthold Lumat LB 9501) in
1- min intervals.
Real-time RT-PCR--
Real-time quantitative reverse
transcription-polymerase chain reaction (RT-PCR) was performed with the
TaqMan system (Prism 7700 Sequence Detection System, PE Biosystems).
For rat Rac1 the primers were 5'-GTA AAA CCT GCC TGC TCA TC and 5'-GCT
TCG TCA AAC ACT GTC TTG. The nox1 primers were 5'-CCC GCA ACT GTT CAT ACT C and 5'-CAT TGT CCC ACA TTG GTC TC. For 18 S
the primers were 5'-TTG ATT AAG TCC CTG CCC TTT GT and 5'-CGA TCC GAG
GGC CTA ACTA. For quantification, Rac1 mRNA expression was
normalized to the expressed housekeeping gene 18 S.
Rac1 GST-PAK Pull Down Assay--
A
glutathione-S-transferase (GST)-PAK-CD (PAK-CRIB domain)
fusion protein, containing the Rac1 binding region from human PAK1B (22) was used to determine Rac1 activity as described (23). Escherichia coli transformed with the GST-PAK-CD construct
were grown at 37 °C to an absorbance of 0.3. The construct was a
kind gift of R. C. Roovers and J. G. Collard, The Netherlands
Cancer Institute, Amsterdam, The Netherlands. Expression of recombinant protein was induced by addition of 0.1 mmol/liter isopropyl
thiogalactoside for 2 h. Cells were harvested, resuspended in
lysis buffer (50 mmol/liter Tris-HCl, pH 8, 2 mmol/liter
MgCl2, 0.2 mmol/liter Na2S2O, 10% glycerol, 20% sucrose,
2 mmol/liter dithiothreitol, 1 µg/ml leupeptin, 1 µg/ml pepstatin,
and 1 µg/ml aprotinin), and then sonicated. Cell lysates were
centrifuged at 4 °C for 20 min at 45,000 × g, and
the supernatant was incubated with glutathione-coupled Sepharose 4B
beads (Amersham Biosciences) for 30 min at 4 °C. Protein bound to
the beads was washed three times in lysis buffer, and the amount of
bound fusion protein was estimated using Coomassie-stained SDS gels.
Vascular smooth muscle cells were treated as indicated and washed with
ice-cold phosphate-buffered saline, incubated 5 min on ice in lysis
buffer (50 mmol/liter Tris-HCl, pH 7.4, 2 mmol/liter MgCl2,
1% Nonidet P-40, 10% glycerol, 100 mmol/liter NaCl, 1 mmol/liter benzamidine, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 µg/ml
aprotinin), and then centrifuged for 5 min at 21,000 × g at 4 °C. Aliquots were taken from the supernatant to
compare protein amounts. Equal amounts of supernatant protein were
incubated with the bacterially produced GST-PAK-CD fusion protein bound
to glutathione-coupled Sepharose beads at 4 °C for 30 min. The beads
and proteins bound to the fusion protein were washed three times in an
excess of lysis buffer, eluted in Laemmli sample buffer (60 mmol/liter
Tris, pH 6.8, 2% SDS, 10% glycerin, 0.1% bromphenol blue), and then analyzed for bound Rac1 molecules by Western blotting.
Nuclear Run-on Assays--
Vehicle- and E2-treated VSMC were
collected and washed. After lysis for 10 min on ice, nuclei were
isolated by centrifugation through 0.6 mol/liter sucrose (4, 24). The
nuclei (~3-5 × 108/reaction) were used to carry
out the in vitro transcription in a reaction mixture
containing 40% glycerol, 50 mmol/liter Tris/HCl, 5 mmol/liter
MgCl2, 0.1 mmol/liter EDTA, 0.5 mmol/liter levels of CTP,
GTP, ATP, and UTP at 30 °C for 30 min. Reactions were terminated by
addition of RNA-clean. Immediately before transcription a sample of
each condition was removed. Total RNA before and after transcription
was isolated and Rac1 and 18 S mRNA were quantitated using
real-time RT-PCR (see above). The extent of Rac1 mRNA transcription was determined by subtracting the amount of Rac1 mRNA standardized to 18 S mRNA prior to transcription from the amounts post
transcription. For some experiments, transcription was performed in the
presence of 0.2 µmol/liter [32P]UTP (>3000
µCi/mmol). The transcribed radioactive RNA was hybridized with nylon
membranes dotted with linearized pKS+ BlueScript, Rac1, and
glyceraldehyde-3-phosphate dehydrogenase cDNA, 5 µg of each, as
described in detail previously (4, 24). Quantification using dot-blots
did not differ from quantifications by real-time RT-PCR.
Human Mononuclear Cells--
Blood samples of patients from the
gynecology outpatient clinic scheduled for planned in vitro
fertilization were investigated. Controlled ovarian hyperstimulation
following the long-protocol was initiated in all patients with
the gonadotropin-releasing hormone analogue triptorelin, 0.1 mg
subcutaneously daily, starting in the midluteal phase of the
previous cycle until pituitary desensitization was achieved. Then
gonadotropin therapy (recombinant follicle-stimulating hormone 150-200
IE subcutaneously daily, Gonal-F; Serono) was given to induce
follicular growing. Gonadotropin-releasing hormone analogue injection
was continued up to and including the day of ovulation induction (day
10-12). 30 ml of EDTA plasma were taken before and after 6-10 days of
follicle-stimulating hormone treatment. Estradiol levels were
evaluated, and mononuclear cells were separated immediately by standard
Ficoll gradient centrifugation.
Data Analysis--
Band intensities were analyzed by
densitometry. All values are expressed as mean ± S.E. compared
with controls. Paired and unpaired Student's t tests and
analysis of variance for multiple comparisons were employed. Post-hoc
comparisons were performed with the Newman-Keuls test.
Differences were considered significant at p < 0.05.
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RESULTS |
Inhibition of Rac1-dependent ROS Release in Vascular
Smooth Muscle Cells by E2--
To test the effect of Rac1 and E2 on
ROS release, VSMC were treated with angiotensin II, 1 µM
for 3 h. DCF fluorescence laser microscopy showed a 2-fold
up-regulation of ROS production (208 ± 22%, p < 0.005), which was prevented by pretreatment with E2, 100 nM. Pretreatment with E2 for 6, 12, 24, 36, and 48 h
time dependently inhibited angiotensin II-induced ROS release (167 ± 22, 135 ± 17, 99 ± 8, 95 ± 23, and 79 ± 23%
of control, respectively) (representative microscopic scan is shown in
Fig. 1A, data analysis in
B). E2 alone had no significant effect on basal ROS
production. In addition, VSMC intracellular superoxide anion formation
in the presence of NADPH and NADH was detected by lucigenin assays as
described by Griendling et al. (21). Angiotensin II mediated an up-regulation of both NADH (243 ± 109%) and NADPH oxidase
activity (307 ± 126%), which was inhibited after pretreatment
with E2 (100 nM, 16 h) (n = 3, *
p < 0.05) (Fig. 1C).

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Fig. 1.
A, representative DCF
fluorescence microscopic scan showing the effects of angiotensin II
(Ang, 1 µM, 3 h) alone or after
preincubation with 17 -estradiol (E, 100 nM,
16 h) on ROS production in VSMC. B, quantification of
the time-dependent effects of 17 -estradiol (100 nM) (n = 3-8; *, p < 0.05). C, effects of angiotensin II (1 µM, 3 h)
alone and 17 -estradiol (100 nM, 16 h) on NADH and
NADPH oxidase activity (n = 3; *, p < 0.05).
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Clostridium sordellii lethal toxin inhibits Rac1 activity by
specific glucosylation (1). Treatment with lethal toxin (200 ng/ml, 16 h) completely abolished angiotensin II-stimulated oxygen radical release (Fig. 2A).
Similarly, overexpression of the dominant-negative RacN17 reduced
angiotensin-mediated ROS production (Fig. 2B). Transfection
with the constitutively active mutant RacL61 increased ROS release by
2-fold (Fig. 2B). E2 completely reversed angiotensin II-mediated ROS release in cells transfected with empty vector but had
no significant effect after transfection with RacL61. These data show
that E2 inhibits angiotensin II-stimulated free radical release from
VSMC and that Rac1 activity is both necessary and sufficient for ROS
production. The experiments suggest that Rac1 is involved in E2-induced
decrease of oxidative stress.

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Fig. 2.
A, free oxygen radical production by
angiotensin II (Ang, 1 µM, 3 h) alone and
after inhibition of Rac1 activity by C. sordellii lethal
toxin (LT, 200 ng/ml, 16 h) (n = 3; *,
p < 0.05). B, ROS production in VSMC
transfected with dominant-negative RacN17 (N17),
constitutively active RacL61 (L61), and empty pcDNA3
vector (C) in the presence of angiotensin II (1 µM, 3 h) alone or after preincubation with
17 -estradiol (E, 100 nM, 16 h)
(n = 4; *, p < 0.05).
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Down-regulation of Rac1 Expression by E2--
Western analysis
demonstrated time-dependent down-regulation of Rac1
expression by E2, 100 nM, by 28 ± 12%, 39 ± 6.7%, 46 ± 6%, 61 ± 7%, and 78 ± 10% after 6, 12, 24, 36, and 48 h, respectively (p < 0.05 after
12 h) (Fig. 3, A and
B). Similarly, treatment with E2 (0.01-10 µM)
for 24 h concentration dependently reduced Rac1 protein levels to
91 ± 8.4%, 54 ± 6%, 33 ± 4%, and 49 ± 10% of control, respectively (p < 0.05 for E2
100 nM) (Fig. 3C). As shown previously, angiotensin
II (1 µM, 3 h) increased Rac1 protein expression by
2-fold (4). In the presence of E2, 100 nM, the angiotensin
II effect was completely abolished (Fig. 3, D and
E). Next, the effects of E2 on Rac1 mRNA levels were
studied. Northern blots showed significant
concentration-dependent as well as
time-dependent down-regulation of Rac1 mRNA levels
similar to the effects of E2 on Rac1 protein (Fig.
4, A-C).

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Fig. 3.
Effects of
17 -estradiol (E) on Rac1 protein
expression. (E, 100 nM). A,
representative Western blot of time-dependent effects of E
(100 nM) on Rac1 with corresponding -actin expression
and, B, quantification. C,
concentration-dependent effects of E after 24 h.
D and E, effects of angiotensin II (1 µM, 3 h) alone or in the presence of E (100 nM, 16 h). n = 5-6 each; *,
p < 0.05.
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Fig. 4.
A, representative Northern blot of Rac1
mRNA expression with corresponding 18 S below and B,
quantification of the time-dependent effects of
17 -estradiol (E, 100 nM). C,
Concentration-dependent effects of E on Rac1
mRNA after 24 h. D, effect of 17 -estradiol (1 µM, 24 h) on nox1 mRNA and E, on
p22phox mRNA expression. n = 4-5 each; *,
p < 0.05.
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In contrast to the down-regulation of Rac1 mRNA, the expression the
NAD(P)H oxidase subunits p22phox and nox1 was not significantly altered
by E2 (1 µM, 16 h, n = 5) (Fig. 4,
D and E).
Down-regulation of Rac1 Activity by E2--
Rac1 activity was
assessed using GST-PAK Crib domain pull down assays (22, 23).
Angiotensin II (1 µM, 3 h) up-regulated Rac1
activity to 175 ± 21% (Fig. 5),
which was time dependently inhibited by pretreatment with 100 nM E2 for 6, 12, and 24 h. E2 alone (24 h)
down-regulated basal Rac1 GTP-binding activity by 52 ± 18%
(p < 0.05).

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Fig. 5.
Rac1 PAK-binding activity in the presence of
angiotensin II (Ang, 1 µM, 3 h) alone or after
preincubation with 17 -estradiol
(E, 100 nM) for the indicated time-points
(n = 3-5; *, p < 0.05).
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Inhibition of Rac1 Gene Transcription by E2--
To elucidate the
mechanism of down-regulation of Rac1 mRNA expression by E2, the
rate of Rac1 gene transcription and mRNA stability were studied in
the presence and absence of E2 (1 µM, 16 h). Nuclear
run on assays showed a reduction of Rac1 transcription to one-third
compared with untreated cells (34.5 ± 10%, p < 0.05) (Fig. 6A). In contrast,
DRB studies showed no significant alteration of Rac1 mRNA half-life
in the presence of estrogen (Fig. 6B). Down-regulation of
Rac1 expression by E2 is mediated by inhibition of gene
transcription.

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Fig. 6.
A, nuclear run on assays showing Rac1
mRNA transcription in vehicle (C) compared with
17 -estradiol (E, 1 µM, 24 h)-treated
VSMC (n = 3, *p < 0.005).
B, effects of 17 -estradiol (E,
dotted line, 1 µM) on Rac1 mRNA stability
compared with vehicle-treated cells (n = 4).
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Down-regulation of Rac1 Mediated by Estrogen Receptor--
To
study whether the effects of E2 on Rac1 were receptor-mediated, VSMC
were treated with E2 (0.01-1 µM, 16 h) in the
presence of ICI 182.780, 1 µM. Co-treatment with ICI
showed complete inhibition of E2-induced down-regulation of Rac1
expression, suggesting receptor-mediated signaling (Fig.
7A). To verify the expression
of estrogen receptor
(ER
) and
(ER
) in vascular smooth
muscle cells, Western analysis was performed. Both receptor subtypes
were expressed abundantly. Treatment with E2 (0.01-10
µM, 16 h) lead to
concentration-dependent up-regulation of ER
and ER
expression (Fig. 7, B and C).

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Fig. 7.
A, effects of 17 -estradiol
(E, 1 µM, 16 h) in the presence of ICI
182.780, 1 µM, 16 h (n = 4; *,
p < 0.05) on Rac1 protein expression. C and
D, concentration-dependent effects of
17 -estradiol after 24 h on ER- and ER- protein expression
(n = 3; *, p < 0.05).
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Inhibition of Rac1 Expression by E2 in Vivo--
To investigate a
possible regulation of Rac1 by E2 in vivo, spontaneously
hypertensive rats (SHR) were ovariectomized and were treated with
17
-estradiol pellets (containing 1.7 mg of estradiol each, 60-day
release) for 5 weeks. E2 plasma levels dropped to 1.6 ± 0.5 pg/ml
in ovariectomized SHR compared with 35.7 ± 12 pg/ml in
sham-operated rats and to 61 ± 21 pg/ml after estrogen
replacement. Aortic superoxide production (20) was up-regulated
(160 ± 27%, p < 0.05) in the ovariectomized
animals, which was completely reversed by E2 replacement (Fig.
8A). To test the effects of E2
on Rac1 expression in vivo, real-time PCRs were performed in
the aortas of these animals (Fig. 8B). In E2-deficient rats,
there was a trend toward up-regulation of Rac1 mRNA (126 ± 33% of control, n = 5, p = non-significant). Treatment of ovariectomized rats with E2
down-regulated vascular Rac1 expression (58 ± 19%, n = 4, p < 0.05). Similarly, aortic
protein expression was reduced (42 ± 12%) after treatment with
E2 (n = 3, p < 0.05) (Fig.
8C). These data suggest that estrogen regulates vascular
Rac1 expression in vivo.

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Fig. 8.
Effects of 17 -estradiol treatment in
ovariectomized and sham-operated spontaneously hypertensive rats on
A, aortic superoxide production and B, Rac1
mRNA expression (*, p < 0.05). C,
representative Western blot for Rac1 and corresponding -actin
expression (n = 3; *, p < 0.05).
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Inhibition of Rac1 Expression in Mononuclear Cells of Women with
Elevated E2 Levels--
To assess whether the cell culture and animal
studies may have significance in humans, mononuclear cells were
collected from women before and during controlled ovarian
hyperstimulation prior to in vitro fertilization, leading to
significant increase of 17
-estradiol blood levels (Fig.
9A). Real-time PCR showed
down-regulation of Rac1 mRNA levels to 51 ± 36% in the
presence of elevated estrogen levels (n = 6, p < 0.05) (Fig. 9B).

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Fig. 9.
A,
17 -estradiol serum levels and
B, corresponding Rac1 mRNA expression of
mononuclear cells in women undergoing ovarian hyperstimulation
(n = 6; *, p < 0.05).
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 |
DISCUSSION |
This study shows that 17
-estradiol inhibits the expression and
activity of Rac1 GTPase leading to inhibition of free radical production in vascular smooth muscle cells. Similar effects were observed in the vessel wall in vivo. Down-regulation of Rac1
by E2 was not limited to VSMC but was observed in mononuclear cells of
women with elevated E2 levels after controlled ovarian hyperstimulation.
An important step in the pathogenesis of endothelial dysfunction and
the progression of atherosclerosis is the activation of NAD(P)H oxidase
enzyme complex in VSMC by angiotensin II, the primary source of
superoxide production in the vessel wall (25). Rac1 GTPase plays a
pivotal role during the assembly of the NAD(P)H system (3, 10, 26).
Here we show, using overexpression of dominant-negative and active Rac1
mutants, that Rac1 activity is both necessary for ROS production in
vascular smooth muscle cells and sufficient for ROS release. In
agreement with previous studies (27), E2 effectively and completely
inhibited angiotensin II-mediated ROS release. More specifically, E2
prevents angiotensin II-mediated NADH and NADPH oxidase activity. But
E2 did not significantly reduce ROS after transfection with the active
RacL61, pointing toward a role of Rac1 for the anti-oxidative effects
of E2. Indeed, Western and Northern analyses demonstrated that E2
concentration and time dependently down-regulated Rac1 protein and
mRNA expression, both alone and in the presence of angiotensin II.
Similarly, E2 inhibited basal and stimulated Rac1 activity. The
molecular mechanism is the inhibition of Rac1 gene transcription,
whereas E2 had no significant effect on Rac1 mRNA half-life. The
estrogen receptors
and
were abundantly expressed in VSMC and
up-regulated by treatment with E2. Down-regulation of Rac1 expression
by E2 was completely blocked in the presence of the nonselective
estrogen receptor antagonist ICI 182.780, demonstrating a
receptor-mediated event.
To test the relevance of these findings in vivo, a well
characterized animal model of estrogen deficiency by ovariectomy and E2
replacement therapy was studied (20). In the aortas of ovariectomized SHR significant down-regulation of Rac1 mRNA and protein expression by E2 was observed. Depression of Rac1 by estrogen replacement strongly
correlates with reduced vascular oxidative stress. The presented cell
culture data assign an essential role to Rac1 in NAD(P)H
oxidase-mediated radical release. Thus, it may be suggested that
estrogen-induced inhibition of Rac1 reduces production of ROS in
vitro as well as in vivo.
To further extend these findings to the human situation, mononuclear
cells of young women with elevated estrogen levels undergoing controlled ovarian hyperstimulation prior to in vitro
fertilization were studied. Elevation of serum 17
-estradiol
correlated with a decrease of Rac1 mRNA expression. These data
suggest that estrogen may regulate Rac1 GTPase in humans, but
additional studies are needed before conclusions regarding a potential
effect of estrogen replacement therapy, especially in combination with
progesterone, should be drawn (27).
In the vascular wall, estrogens exert anti-oxidant effects in addition
to the inhibition of Rac1 GTPase in VSMC, which are primarily located
in the media of the arterial wall. ROS release from the endothelium as
well as the adventitia may play an important role in vivo.
3-nitrotyrosine immunoreactivity as well as expression of the NAD(P)H
oxidase subunit gp91(phox) have been shown to increase in the
endothelium and adventitia of mice treated with angiotensin II (28,
29). Importantly, recent work by Wagner et al. shows that E2
decreases the function of the NAD(P)H oxidase in endothelial cells,
which is mediated by down-regulation gp91phox (30). The expression of
its homologue in vascular smooth muscle cells, nox1, was not
significantly altered by E2, suggesting a potential dichotomy between
endothelial cells and VSMC, which may help to address the cell-specific
function of NAD(P)H oxidases in different cell types in further
studies. Down-regulation of gp91phox in the endothelium and Rac1 in the
media are likely complementary effects in vivo. In addition,
estrogen reduces oxidative stress by down-regulation of the AT1
receptor (7, 12, 27). The estrogen-induced reduction of ROS is closely
connected to another beneficial action of estrogen on vascular cells,
namely the up-regulation of endothelial nitric-oxide synthase activity
(12, 30, 31). Therefore, the well established increase of NO
bioavailability is caused by increased NO production and decreased
superoxide release.
It is thought that the putative vasoprotective effects of estrogens are
at least in part mediated via reduction of oxidative stress. Decreased
Rac1 expression and activity may resemble a novel and important
mechanism by which estrogens interfere with free radical production. In
addition, recent evidence suggests an important role for Rac1 GTPase
for the control of oxygen radical release outside the vascular wall in
several cell types, including leukocytes, fibroblasts, and cardiac
myocytes (10, 32). Inhibition of Rac1 by expression of
dominant-negative N17rac1 has been shown to protect from
hypoxia/reoxygenation-induced cell death in a variety of cell types
including vascular smooth muscle cells, fibroblasts, endothelial cells,
and ventricular myocytes (33). In cardiomyocytes, Rac1 has been
identified as a mediator of hypertrophy (11, 34, 35). Inhibition of
Rac1 activity in the heart, e.g. by inhibition of Rac1
isoprenylation using HMG-CoA reductase inhibitors, has been
shown to prevent the hypertrophic phenotype as well as cardiac ROS
production (36, 37). Interestingly, estrogen has been reported to
prevent cardiac hypertrophy by a mechanism yet unknown (16, 38). We
speculate that the antihypertrophic effects of estrogen could at least
in part be mediated by regulation of Rac1 GTPase.
In summary, Rac1 GTPase gene transcription and activity are regulated
by E2, which may be an important molecular mechanism contributing to
the cardiovascular effects of estrogens.