Dehydroepiandrosterone protects against oxidative stress-induced endothelial dysfunction in ovariectomized rats

Article (PDF Available)inThe Journal of Physiology 589(Pt 10):2585-96 · May 2011with29 Reads
DOI: 10.1113/jphysiol.2011.206078 · Source: PubMed
Abstract
Cardiovascular disease is less frequent in premenopausal women than in age-matched men or postmenopausal women. Moreover, the marked age-related decline in serum dehydroepiandrosterone (DHEA) level has been associated to cardiovascular disease. The aim of this study was to evaluate the effects of DHEA treatment on vascular function in ovariectomized rats. At 8 weeks of age, female Wistar rats were ovariectomized (OVX) or sham (SHAM) operated and 8 weeks after surgery both groups were treated with vehicle or DHEA (10mg kg⁻¹ week⁻¹) for 3 weeks. Aortic rings were used to evaluate the vasoconstrictor response to phenylephrine (PHE) and the relaxation responses to acetylcholine (ACh) and sodium nitroprusside (SNP). Tissue reactive oxygen species (ROS) production and SOD, NADPH oxidase and eNOS protein expression were analysed. PHE-induced contraction was increased in aortic rings from OVX compared to SHAM, associated with a reduction in NO bioavailability. Furthermore, the relaxation induced by ACh was reduced in arteries from OVX, while SNP relaxation did not change. The incubation of aortic rings with SOD or apocynin restored the enhanced PHE-contraction and the impaired ACh-relaxation only in OVX. DHEA treatment corrected the increased PHE contraction and the impaired ACh-induced relaxation observed in OVX by an increment in NO bioavailability and decrease in ROS production. Besides, DHEA treatment restores the reduced Cu/Zn-SOD protein expression and eNOS phosphorylation and the increased NADPH oxidase protein expression in the aorta of OVX rats. The present results suggest an important action of DHEA, improving endothelial function in OVX rats by acting as an antioxidant and enhancing the NO bioavailability.
J Physiol 589.10 (2011) pp 2585–2596 2585
The Journal of Physiology
Dehydroepiandrosterone protects against oxidative
stress-induced endothelial dysfunction in ovariectomized
rats
Jo
˜
ao Paulo Gabriel Camporez
1
, Eliana Hiromi Akamine
2
,AnaPaulaDavel
1,3
, Celso Rodrigues Franci
4
,
Luciana Venturini Rossoni
1
and Carla Roberta de Oliveira Carvalho
1
1
Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of S
˜
ao Paulo, SP, Brazil
2
Department of Pharmacology, Institute of Biomedical Sciences, University of S
˜
ao Paulo, SP, Brazil
3
Department of Anatomy, Cellular Biology and Physiology & Biophysics, State University of Campinas, SP, Brazil
4
Depart ment of Physiology, Medical School of Riber
˜
ao Preto, University of S
˜
ao Paulo, SP, Brazil
Non-technical summary It is well known that cardiovascular disease is more frequent
in postmenopausal than in premenopausal women. Moreover, it has been shown that
dehydroepiandrosterone (DHEA), a steroid hormone secreted by adrenal glands, reduces during
ageing. Its reduced plasma level has been related to increased prevalence of obesity, insulin
resistance and cardiovascular disease. We show that DHEA treatment in ovariectomized rats,
an experimental model of menopause, reduces blood pressure and improves vascular function.
Furthermore, DHEA reduced reactive oxygen species (ROS), correcting the reduced protein
expression of Cu/Zn-SOD, an antioxidant protein, and increased protein expression of NADPH
oxidase, a pro-oxidant protein. This work shows the potential effect of DHEA upon correction of
endothelial dysfunction observed on oestrogen deprivation.
Abstract Cardiovascular disease is less frequent in premenopausal women than in age-matched
men or postmenopausal women. Moreover, the marked age-related decline in serum
dehydroepiandrosterone (DHEA) level has been associated to cardiovascular disease. The aim of
this study was to evaluate the effects of DHEA treatment on vascular function in ovariectomized
rats. At 8 weeks of age, female Wistar rats were ovariectomized (OVX) or sham (SHAM) operated
and 8 weeks after surgery both groups were treated with vehicle or DHEA (10 mg kg
1
week
1
)
for 3 weeks. Aortic rings were used to evaluate the vasoconstrictor response to phenylephrine
(PHE) and the relaxation responses to acetylcholine (ACh) and sodium nitroprusside (SNP).
Tissue reactive oxygen species (ROS) production and SOD, NADPH oxidase and eNOS protein
expression were analysed. PHE-induced contraction was increased in aor tic rings from OVX
compared to SHAM, associated with a reduction in NO bioavailability. Furthermore, the
relaxation induced by ACh was reduced in arteries from OVX, while SNP relaxation did
not change. The incubation of aortic rings with SOD or apocynin restored the enhanced
PHE-contraction and the impaired ACh-relaxation only in OVX. DHEA treatment corrected
the increased PHE contraction and the impaired ACh-induced relaxation observed in OVX by
an increment in NO bioavailability and decrease in ROS production. Besides, DHEA treatment
restores the reduced Cu/Zn-SOD protein expression and eNOS phosphorylation and the increased
NADPH oxidase protein expression in the aorta of OVX rats. The present results suggest an
important action of DHEA, improving endothelial function in OVX rats by acting as an anti-
oxidant and enhancing the NO bioavailability.
(Received 24 November 2010; accepted after revision 11 March 2011; first published online 14 March 2011)
Corresponding author C. R. O. Carvalho: Department of Physiology and Biophysics, Institute of Biomedical Sciences,
University of S
˜
ao Paulo, 05508-900 S
˜
ao Paulo, S.P., Brazil. Email: croc@icb.usp.br
Abbreviations ACh, acetylcholine; DHEA, dehydroepiandrosterone; NO, nitric oxide; ORT, oestrogen replacement
therapy; OVX, ovariectomized rats; PHE, phenylephrine; ROS, reactive oxygen species; SNP, sodium nitroprusside.
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2586 J. P. G. Camporez and others J Physiol 589.10
Introduction
It is known that oestrogen has profound effects on
the c ardiovascular system (White, 2002). Indeed, cardio-
vascular disease is much less frequent in premenopausal
women than in age-matched men or postmenopausal
women (Burt e t al. 1995).
Endothelial dysfunction is a common factor in
the development of many cardiovascular diseases. In
line w ith this, endothelial function was shown to be
impaired in ovariectomized women (Virdis et al. 2000)
and hypertensive female rats (Wassmann et al. 2001;
Widder et al. 2003). Indeed, it has been observed that
ovariectomy reduces vascular response to acetylcholine in
Sprague–Dawley rats (Squadrito et al. 2000; Lam et al.
2006), female hypertensive rats (Wassmann et al. 2001;
Widder e t al. 2003), and Wistar rats (Paredes-Cabejal
et al. 1995). Ovariectomy also increases vascular response
to phenylephrine in Sprague–Dawley rats (Lam et al.
2006), and Wistar r ats (Paredes-Cabejal et al. 1995). This
impairment in endothelial function was associated with an
increment in superoxide anion production (Wassmann
et al. 2001; Lam e t al. 2006) as well as a reduction in
eNOS protein expression (Widder et al. 2003). Moreover,
ovariectomy promoted an increment in blood pressure of
normotensive women (Mercuro et al. 2004), as well as in
female Wistar rats (Mendonc¸a et al. 2007), and in hyper-
tensive rats (Ito et al. 2006).
Oestrogen replacement therapy (ORT) in
ovariectomized women and female rats prevents
the deleterious effect of ovariectomy by reducing
blood pressure and increasing antioxidant defence and
eNOS protein expression. However, large controlled
clinical trials have not shown benefits of ORT, or
have even shown deleterious effects, such as an
increment of venous thromboembolic disease, stroke
and breast cancer incidence (Prentice et al. 2009).
Therefore, new therapeutic strategies to prevent
oestrogen deprivation-induced deleterious effects on the
cardiovascular system will be valuable.
Dehydroepiandrosterone (DHEA) has been considered
a potential alternative hor mone for ORT, due to its
apparent safe, antioxidant and metabolic effects (Labrie
et al. 2005). DHEA and its sulfated form (DHEAS) are the
most abundant cholesterol-derived hormones in humans
and their plasma concentrations progressively decline
with age. An inverse relation between plasma DHEA
concentration and obesity, diabetes and incidence of
cardiovascular disease has been observed (Bar rett-Connor
et al. 1986; Nestler et al. 1988; Schriock e t al. 1988).
Moreover, DHEA treatment protects heart of diabetic rats
against oxidative stress (Aragno et al. 2008); it furthermore
protects endothelial cells against apoptosis (Liu et al.
2007), and stimulates ser1177 eNOS phosphorylation
(Formoso et al. 2006).
It is well known that ovariectomized female rats present
endothelial dysfunction and high blood pressure levels
(Wassmann et al. 2001; Widder et al. 2003). Therefore,
DHEA evokes beneficial mechanisms that could reduce
endothelial dysfunction and reduce the cardiovascular
risk present after surgical menopause in rats. Thus,
the present work hypothesizes that DHEA could be an
alternative hormonal therapy, and provide benefits to the
vascular system of postmenopausal women. To evaluate
this hypothesis, the aim of the present study was to assess
the effects of DHEA treatment on endothelium-dependent
relaxation, focusing on the mechanisms involved, mainly
related to NO and oxidative stress, in an experimental
menopause models in rats.
Methods
Animal care and protocol
All experimental procedures were approved by and
conducted in accordance with the guidelines of the
Animal Care and Use Committee of the Biomedical
Institute of Sao Paulo University, according to the
guidelines of the Brazilian Societies of Experimental
Biology. Seven-week-old female Wistar rats were
obtained from colonies maintained at the Institute of
Biomedical Sciences of the University of S
˜
ao Paulo.
At 8 weeks of age, the rats were anaesthetized with a
ketamine–xylazine–acepromazine mixture (64.9, 3.2 and
0.78 mg kg
1
, respectively, I.P.) and were ovariectomized
(OVX) or sham-operated (SHAM). After 8 weeks of
surgery, OVX and SHAM rats were treated with vehicle
(soybean oil) or DHEA (10 mg kg
1
week
1
, S.C.) for
3 weeks. The DHEA administration protocol was based
on studies published by us and others (Campbell et al.
2004; Medina et al. 2006; Jacob et al. 2009). During
all the procedures rats were housed in a constant room
temperature environment, with 12:12 h light–dark cycle,
with free access to standard rat chow and tap water. Rats
were randomly div ided into four groups: (1) SHAM, (2)
SHAM+DHEA, (3) OVX, and (4) OVX+DHEA.
Biometrics characteristics
At the end of the treatment period, the body weight
gain, retroperitoneal fat pad and uterus weight were
determined.
Arterial blood pressure measurement
Rats were anaesthetized with a ketamine–xylazine–
acepromazine mixture (64.9, 3.2 and 0.78 mg kg
1
,
respectively ,
I.P.) and allowed to breathe room air
spontaneously. The right carotid artery was cannulated
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2011 The Physiological Society
J Physiol 589.10 Dehydroepiandrosterone reduces reactive oxygen species in aorta 2587
with a polyethylene catheter (PE-50 with heparinized
saline) that was exteriorized in the mid-scapular region.
After 24 h, arterial pressure and heart rate were measured
in conscious animals by a pressure transducer (model TRA
021, PanLab, Barcelona, Spain) and recorded by using
an interface and software for computer data acquisition
(Power Lab 4/25; ADInstruments, Sydney, Australia).
Heart rate was determined from the interbeat intervals.
Vascular reactivity study
Vascular function was evaluated in aortic rings from all
groups studied. We used the aortic rings as a model to
study v ascular function because in this artery the main
endothelium-dependent vasodilator factor is NO. Thus,
since the aim of the present study is to assess the role of
NO and reactive oxygen species on the DHEA treatment
in OVX rats, the aortic rings are the best model to answer
our questions without misinterpretation. Segments of the
thoracic aorta (4 mm in length), free of connective tissue,
were mounted in an isolated tissue chamber containing
Krebs–Henseleit solution (containing (in m
M): 118 NaCl,
4.7KCl,25NaHCO
3
,2.5CaCl
2
.2H
2
O, 1.2 KH
2
PO
4
,1.2
MgSO
4
.7H
2
O, 11 glucose, and 0.01 EDTA), gassed with
95% O
2
–5% CO
2
, and maintained resting tension of 1 g
at 37
C at pH 7 .4 as previously published by our group
(Davel et al. 2008). In order to analyse the influence of
the endothelium on vascular responses, the endothelial
layer was mechanically removed in certain experiments by
rubbing the lumen with a needle. Isometric tension was
recorded by using an isometric force transducer (Letica
TRI 210, Barcelona, Spain) connected to an acquisition
system (MP100, Biopac Systems, Goleta, CA, USA).
After 30 min equilibration, concentration–response
curves to the α
1
-adrenoceptor agonist phenylephrine
(PHE, 10
10
–10
4
mol l
1
), to the endothelium-
dependent vasodilator acetylcholine (ACh,
10
11
–10
5
mol l
1
) and to the nitric oxide donor
sodium nitroprusside (SNP, 10
11
–10
5
mol l
1
)were
obtained. At the end of the experiment, the presence
or absence of functional endothelium was verified in all
aortic rings by observing whether relaxation occurred on
exposure to ACh (10
5
mol l
1
) or not. The endothelium
was considered intact if the aortic ring relaxed more than
80% to ACh, while endothelial denudation was confirmed
by less than 10% relaxation. At the end of experiments,
aortic rings were allowed to dry for at least 24 h and
then were weighed. Vasoconstrictor responses to PHE are
expressed as gram (g) of tension per milligram (mg) of
tissue. Vasodilator concentration–response curves were
obtained in the aortic r ings pre-contracted with PHE
(10
6
mol l
1
), which is approximately 80% of maximal
contraction. Relaxation induced by ACh and SNP was
expressed as the percentage of the tonus obtained with
PHE.
The role of NO and superoxide anion in the
response to PHE and ACh was evaluated by incubating
some aortic rings with the non-selective NOS
inhibitor
L-N
G
-nitro-L-arginine methyl ester (L-NAME;
100 μmol l
1
), the superoxide anion scavenger superoxide
dismutase (SOD; 150 U ml
1
) or the inhibitor of NADPH
oxidase, apocynin (100 μmol l
1
), added 30 min before
the construction of the concentration–response curves to
PHE or ACh.
For each concentration–response curve to PHE and
ACh, the maximal response (R
max
) and the agonist
concentration log resulting in 50% of the R
max
(log
EC
50
) were calculated using non-linear regression analysis
(GraphPad Prism software, USA).
Reactive oxygen species measurement in aorta
Thoracic aortas were carefully dissected out and cleaned
of connective tissue. Aortas were then divided into
cylindrical segments 4 mm in length and were first
immersed in an embedding medium (tissue freezing
medium) and then frozen and kept at 80
C until super-
oxide anion was measured. The oxidative fluorescent
dye hydroethidine was used to evaluate the in situ
production of reactive oxygen species (ROS) as pre-
viously described (Hernanz et al. 2004). Transverse aortic
sections (10 μm) were obtained in a cryostat from
previously frozen aorta and collected on glass slides
and left to reach equilibrium for 30 min at 37
Cin
phosphate-buffered saline (PBS). Fresh buffer containing
hydroethidine (5 μmol l
1
) was topically applied to each
tissue section and coverslipped. Slides were incubated
in a light-protected, humidified chamber at 37
C for
30 min. Control sections received the same volume of
PBS. Images were obtained with a Nikon E1000 micro-
scope equipped for epifluorescence (excitation at 488 nm;
emission at 610 nm). Fluorescence was detected with a
585 nm long-pass filter. The fluorescence intensity was
quantified using ImageJ software (NIH) and was expressed
as arbitrary units.
Western blot analysis
For analysis of eNOS, ser1177 phosphorylated eNOS,
SOD and NADPH oxidase protein expression, aortas were
dissected out, cleaned of connective tissue, frozen in liquid
nitrogen and kept at 70
C until the day of analysis.
They were homogenized in RIPA lysis buffer containing
protease inhibitor cocktail (1:5000 dilution), sodium
fluoride (100 m
M), sodium pyrophosphate (10 mM),
sodium orthovanadate (100 m
M), and PMSF (10 mM).
Proteins from homogenized aorta (75 μgofprotein
extracts) were elect rophoretically separated by 7.5% or
12% SDS-PAGE and then transferred to p olyvinylidene
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2588 J. P. G. Camporez and others J Physiol 589.10
Table 1. Body weight gain, retroperitoneal fat pad and uterus weight from ovariectomized
(OVX) or sham-operated (SHAM) rats treated with vehicle or DHEA for 3 weeks
SHAM SHAM + DHEA OVX OVX + DHEA
Body weight gain (g) 47.8 ± 3.049.2 ± 2.683.3 ± 4.3
79.3 ± 4.5
Retroperitoneal fat pad
(mg(100gBW)
1
)
0.5 ± 0.04 0.6 ± 0.06 1.1 ± 0.09
1.1 ± 0.10
Uterus weight (mg) 613 ± 103 594 ± 50 176 ± 14
178 ± 24
The values are expressed as means ± SEM. Two-way ANOVA:
P < 0.01 in comparison to
SHAM and SHAM+DHEA.
Table 2. Changes in systolic (SBP) and diastolic blood pressure (DBP) and in heart
rate from ovariectomized (OVX) or sham-operated (SHAM) rats treated with vehicle
or DHEA for 3 weeks
SHAM SHAM + DHEA OVX OVX + DHEA
SBP (mmHg) 129.2 ± 2.5 130.5 ± 2.6 139.6 ± 1.8
128.4 ± 2.7
DBP (mmHg) 91.5 ± 1.090.2 ± 2.6 100.2 ± 2.3
94.0 ± 1.6
Heart rate (bpm) 356.8 ± 9.1 365.2 ± 14.6 384.0 ± 11.9 354.5 ± 10.0
The values are expressed as mean ± SEM. Two-way ANOVA:
P < 0.05 in comparison
to SHAM and P < 0.05 in comparison to OVX.
difluoride membranes overnight at 4
C using a Mini
Trans-Blot Cell system (Bio-Rad) containing 25 m
M Tr is ,
190 m
M glycine, 20% methanol and 0.05% SDS.
After blockade of non-specific sites with 5% non-fat
dry milk, membranes were incubated overnight at
4
C with the following primary antibodies: anti-eNOS
(1:1000, BD Transduction Laboratories, Lexington, KY,
USA), anti-phospho eNOS
ser1177
(1:1000, Cell Sig naling
Technology, Inc., Danvers, MA, USA), anti-Cu/Zn
SOD (1:2000, Sigma-Aldrich, Germany), anti-Mn SOD
(1:2000, Sigma-Aldrich), anti-gp91phox (1:1000, Upstate
Biotechnology, Inc., Lake Placid, NY, USA) and
anti-p22phox (1:500, Santa Cruz Biotechnology, Inc.,
Santa Cruz, CA, USA). After washing (10 m
M Tr is,
100 m
M NaCl, and 0.1% Tween 20), membranes
were incubated with horseradish peroxidase-conjugated
anti-rabbit IgG antibody (1:1500, Bio-Rad Laboratories,
Hercules, CA, USA) for gp91phox, p22phox, Mn-SOD
and phopho eNOS, and with anti-mouse IgG (1:1500,
Bio-Rad) for Cu/Zn-SOD and eNOS. The same
membranes were used to determine α-actin protein
expression using a mouse monoclonal antibody (1:1500,
Sigma-Aldrich). Membranes were thoroughly washed,
and immune complexes were detected using an
enhanced luminol chemiluminescence system (ECLPlus,
Amersham GE Healthcare, UK) and subjected to
MF-Chemibis-Bio-Imageing System (BioAmerica Inc.,
Miami, FL, USA). Signals on the immunoblot were
quantified with Scion Image software.
Total expression of the proteins is given as the ratio
between the optical density for the specific protein and
the signal for α-actin protein expression.
Drugs
Dehydroepiandrosterone, phenylephrine hydrochloride,
acetylcholine hydrochloride, sodium nitroprusside, SOD
(bovine erythrocyte),
L-NAME and apocynin were
purchased from Sigma-Aldr ich (USA); stock solutions
(10 mmol l
1
) were prepared in distilled water, except for
DHEA, which wassuspended in soybean oil (10 mg ml
1
).
Statistical analysis
Results were expressed as means ± SEM, and n represents
the numbers of animals used in each experiment. All
results were analysed by two-way ANOVA followed by
the Bonferroni post hoc test. A value of P < 0.05 was
considered significant.
Results
Biometrics and haemodynamic parameters
OVX rats displayed elevated body weight gain (SHAM:
47.8 ± 3.0 vs. OVX: 83.3 ± 4.3 g; n = 10, P < 0.01)
and retroperitoneal fat pad (SHAM: 0.5 ± 0.04 vs.
OVX: 1.1 ± 0.09 mg (100 g BW)
1
; n = 10, P < 0.01)
(Table 1). However, they presented reduced uterus
weight (SHAM: 613 ± 103 vs. OVX: 176 ± 14 mg;
n = 10, P < 0.01) as compared to SHAM rats (Table 1).
DHEA treatment did not modify these parameters;
body weight gain (SHAM+DHEA: 49.2 ± 2.6 and
OVX+DHEA: 79.3 ± 4.5 g; n = 10), retroperitoneal fat
pad (SHAM+DHEA: 0.6 ± 0.06 and OVX+DHEA:
1.1 ± 0.10 mg (100 g BW)
1
; n = 10) and uterus
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J Physiol 589.10 Dehydroepiandrosterone reduces reactive oxygen species in aorta 2589
weight (SHAM+DHEA: 594 ± 50 and OVX+DHEA:
178 ± 24 mg; n = 10), compared to respective groups
treated with vehicle (Table 1).
Table 2 shows that OVX rats presented higher systolic
and diastolic blood pressure as compared to SHAM r ats,
while heart rate was not different in different groups.
DHEA treatment corrected the systolic and diastolic blood
pressure in OVX rats (Table 2). However, it did not change
the blood pressure levels in SHAM rats as well as heart rate
in both groups (Table 2).
Vascular reactivity
The contraction induced by PHE was increased in aorta
from OVX rats compared with SHAM (Fig. 1A)(R
max
:
SHAM 0.84 ± 0.03 vs. OVX 1.27 ± 0.07 g of tension per
mg of tissue; n = 11, P < 0.01). On the other hand, the
endothelium-dependent relaxation induced by ACh was
reduced in aorta from OVX rats (Fig. 1B)(R
max
:SHAM
91.6 ± 2.7 vs. OVX 74.1 ± 2.7% of relaxation; n = 9,
P < 0.01), while there was no change in the relaxation
response to the NO donor SNP (Fig. 1C). DHEAtreatment
in OVX rats corrected both the increased PHE-induced
contraction (Fig. 1A)(R
max
:OVX+DHEA 0.90 ± 0.04 g
of tension per mg of tissue; n = 11) and the impaired
ACh-induced relaxation (Fig. 1B)(R
max
:OVX+DHEA
90.3 ± 2.4% of relaxation; n = 9); while DHEA treatment
in SHAM rats did not change either PHE-induced
contraction (Fig. 1A)(R
max
:SHAM+DHEA 0.86 ± 0.06 g
of tension per mg of tissue; n = 11) or ACh-induced
relaxation (Fig. 1B)(R
max
:SHAM+DHEA 95.7 ± 3.7% of
relaxation; n = 9). There were no changes in EC
50
values
among groups for each drug studied (data not shown).
Effects of DHEA treatment on the endothelial and NO
modulation of phenylephrine-induced contraction
in OVX rats
Endothelium and NO modulation of PHE-induced
contraction were evaluated by endothelium denudation
or
L-NAME incubation, respectively. An increase in R
max
to PHE was observed in all groups of animals after
endothelium removal (Table 3) or
L-NAME incubation
(Table 3). It is important to note that in the absence of
endothelial cells or in the presence of nitric oxide inhibitor
(
L-NAME) there were no differences among groups. EC
50
values were similar in all conditions (data not shown).
Effects of DHEA treatment on the role of superoxide
anion in the phenylephrine-induced contraction
in OVX rats
To evaluate the possible role of superoxide anion
modulation, derived from NADPH oxidase, on
PHE-induced contraction, SOD and apocynin were
incubated in some aortic rings 30 min before the
concentration–response cur ves to PHE. Both SOD and
apocynin significantly reduced the R
max
to PHE in aorta
from OVX rats, whereas they did not have any effects in
Figure 1. Vascular reactivity to phenylephrine, acetylcholine
and sodium nitroprusside
Concentration–response curve to phenylephrine (A), acetylcholine
(B) and sodium nitroprusside (C) in endothelium-intact aortic rings
from SHAM, OVX and DHEA-treated rats. Results are expressed as
means ± SEM. Two-way ANOVA:
P < 0.01 in comparison to SHAM
(n = 9–13).
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2590 J. P. G. Camporez and others J Physiol 589.10
Table 3. Effect of endothelium denudation (E
), N
G
-nitro-L-arginine methyl
ester (
L-NAME), SOD and apocynin (Apo) on the maximal response (R
max
)to
phenylephrine
SHAM SHAM + DHEA OVX OVX + DHEA
E
+
0.84 ± 0.03 0.86 ± 0.06 1.27 ± 0.07
0.90 ± 0.04
E
2.69 ± 0.17 2.71 ± 0.25 2.54 ± 0.10 2.66 ± 0.16
L-NAME 2.53 ± 0.13 2.37 ± 0.07 2.33 ± 0.11 2.42 ± 0.10
SOD 0.84 ± 0.05 0.80 ± 0.02 0.90 ± 0.02 0.84 ± 0.04
Apo 0.85 ± 0.09 0.74 ± 0.09 0.88 ± 0.11 0.82 ± 0.07
The values are expressed as mean ± SEM (g of tension per mg
of tissue). Two-way ANOVA:
P < 0.01 in comparison to SHAM,
P < 0.01 in comparison to OVX, P < 0.01 in comparison to E
+
rings
(endothelium intact).
Table 4. Effect of SOD and apocynin (Apo) on the maximal
response (R
max
) to acetylcholine
SHAM SHAM + DHEA OVX OVX + DHEA
E
+
91.6 ± 2.795.7 ± 3.774.1 ± 2.7
90.3 ± 2.4
SOD 93.4 ± 2.595.7 ± 1.696.5 ± 2.2 94.9 ± 1.6
Apo 92.5 ± 3.994.4 ± 1.693.4 ± 1.7 90.5 ± 2.3
The values are expressed as mean ± SEM (% of relaxation).
Two-way ANOVA:
P < 0.01 in comparison to SHAM, P < 0.01
in comparison to OVX, P < 0.01 in comparison to E
+
rings (end-
othelium intact).
aorta from SHAM and DHEA-treated rats (Table 3). There
were no differences among groups in EC
50
in any situation
(data not shown).
Effects of DHEA treatment on the role of superoxide
anion in the acetylcholine-induced relaxation
in OVX rats
The involvement of superoxide anion in ACh-induced
relaxation was also evaluated. Both SOD and apocynin
significantly enhanced the R
max
to ACh in aortic rings
from OVX rats, whereas they did not have any effect in
aorta from SHAM and DHEA-treated rats (Table 4). In
addition, there were no differences among groups in EC
50
to ACh in the presence of SOD or apocynin (data not
shown).
Reactive oxygen species p roduction
In order to evaluate tissue reactive oxygen species
(ROS) production, dihydroethidium (DHE) staining
wasperformedinaorta.AsobservedinFig.2
dihydroethidium-emitted fluorescence intensity was
higher in aorta from OVX rats than in SHAM, indicating
increased ROS production (Fig. 2). Treatment with DHEA
for 3 weeks did not change ROS production in aorta
from SHAM rats, although it corrected the enhanced ROS
production in aorta from OVX rats (Fig. 2).
Western blot analysis of endothelial nitric oxide
synthase, superoxide dismutase and subunits
of NADPH oxidase
Ovariectomy or DHEA treatment did not change end-
othelial nitric oxide synthase (eNOS) protein expression in
aorta (Fig. 3A). However, in aorta f rom OVX rats there was
observed a reduction on eNOS phosphorylation compared
to SHAM (Fig. 3B ). Furthermore, DHEA treatment in
OVX rats corrected this reduction (Fig. 3B).
In addition, Cu/Zn-SOD protein expression was
reduced in aorta from OVX rats and corrected by
DHEA treatment (Fig. 4A), while there was no change
in Mn-SOD protein expression among groups (Fig. 4B).
Besides reduction of Cu/Zn-SOD protein expression, the
aorta from OVX rats displayed an increased protein
expression of gp91phox and p22phox (Fig. 5) NADPH
oxidase subunits. Moreover, DHEA treatment of OVX rats
corrected the increased of both NADPH oxidase subunits
in aorta (Fig. 5).
Discussion
It has b een shown that oestrogens have protective
effects on the vascular system (White, 2002), based
on epidemiological, clinical and molecular findings. In
line with this affirmation, the loss of oestrogens may
have profound effects on the cardiovascular system, with
increase in blood pressure and endothelial dysfunction,
both in women (Virdis e t al. 2000; Mercuro et al. 2004) and
in experimental models of menopause (Wassmann et al.
2001; Ito et al. 2006). In the present study we demonstrated
that DHEA treatment reduces blood pressure and protects
against oxidative stress-induced endothelial dysfunction
in ovariectomized rats.
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The favourable biological effects of oestrogen in the
cardiovascular system have been recognized, in spite
of the fact that several r a ndomized clinical trials have
failed to observe a benefit of hormone replacement
therapy on the incidence of cardiovascular events in post-
menopausal women (Hulley et al. 1998; Rossouw et al.
2002; Prentice et al. 2009). On the other hand, oestrogen
replacement therapy corrected, in ovariectomized women,
endothelial dysfunction (Virdis et al. 2000) and increased
blood pressure (Mercuro et al. 2004). It is well known
that ovar iectomized rats display endothelial dysfunction,
showing reduction in vascular response to ACh and
increase in vascular response to PHE (Wassmann et al.
2001; Wong et al. 2006). Furthermore, Mendonc¸a et al.
(2007) found increased blood pressure after 11 weeks of
ovariectomy in female Wistar rats. Interestingly, oestrogen
seems to be important not only for females, since
aromatize knockout male mice displayed reduced vascular
response to ACh (Kimura et al. 2003). In line with
those observations, our study shows an enhancement in
blood pressure, increased vascular contraction to PHE
and reduced endothelium-dependent relaxation to ACh
following ovariectomy in rats.
Supporting the hypothesis of the present study, 3 weeks
of DHEA treatment reduced the systolic and diastolic
blood pressure, and corrected vascular responses to PHE
and ACh in ovariectomized rats. The plasma DHEA level
reduces during the ageing process and this has been
related to the prevalence of body weight gain, obesity
and insulin resistance, and mor tality by cardiovascular
disease (Barrett-Connor et al. 1986; Nestler et al. 1988;
Schriock et al. 1988). Both aged men and aged women
submitted to DHEA treatment for 6 months displayed
visceral and subcutaneous fat reduction, besides increased
insulin sensitivity (Villareal & Holloszy, 2004). Moreover,
in a double-blind study, DHEA-treated postmenopausal
women for 12 weeks showed an enhancement of end-
othelial function (Williams et al. 2004). Considering that
the adrenal steroid DHEA is the unique sexual steroid
hormone source in postmenopausal women (Labrie,
2010) and the risks associated with ORT (Prentice et al.
2009), the data presented here concerning the mechanism
of DHEA action suggest that DHEA could be used as a
successful alternative hormone replacement therapy for
postmenopausal women.
Indeed, some studies using animal models
demonstrated that DHEA treatment induces several
beneficial effects. It may reduce cardiac fibrosis in diabetic
rats (Aragno et al. 2008), delay atherosclerotic plaque
formation in rabbits (Hayashi et al. 2000), reduce weight
gain and fat deposition in rats (Han et al. 1998), increase
insulin sensitivity (Campbell et al. 2004) and insulin
secretion in rats (Medina et al. 2006), and reduce blood
pressure in ovariectomized rats (Bhuiyan & Fukunaga,
2010). In addition, DHEA increases in vitro endothelial
proliferation (Williams e t al. 2004), and protects end-
othelial cells against apoptosis (Liu et al. 2007), both
effects independently of oestrogen receptors. Besides, the
present study has for the first time shown that DHEA
treatment in vivo corrects both contractile and relaxation
responses to PHE and ACh in OVX rats. Therefore, DHEA
has several effects on the cardiovascular system, which
Figure 2. Reactive oxygen species in aorta
Representative fluorescence photomicrographs of microscopic sections of thoracic aorta from SHAM, OVX and
DHEA-treated rats. Vessels were labelled with the oxidative dye hydroethidine, which produces a red fluorescence
when oxidized to ethidium bromide by superoxide anion. Quantitative analysis for fluorescence photomicrographs
of microscopic sections of thoracic aorta from SHAM, OVX and DHEA-treated rats. Results are expressed as
means ± SEM. Two-way ANOVA:
P < 0.01 in comparison to SHAM and #P < 0.01 in comparison to OVX (n = 7).
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could be direct or indirect, which reduce cardiovascular
risk factors such as body weight gain or insulin resistance.
The present results suggest that the correction of
increase in PHE-induced contraction and decrease
in ACh-induced relaxation observed in aortas f rom
ovariectomized rats by DHEA treatment in part are related
Figure 3. eNOS protein expression and phosphorylation in
aorta
Representative Western blots (top) and quantitative analysis
(bottom) for total eNOS protein expression (A) and ser1177 eNOS
phosphorylation (B) in thoracic aorta from SHAM, OVX and
DHEA-treated rats. Results are expressed as means ± SEM. Two-way
ANOVA:
P < 0.01 in comparison to SHAM and #P < 0.01 in
comparison to OVX (n = 9).
to an increment in eNOS phosphorylation, improving NO
production, since the eNOS phosphorylation on ser-1177
enhances the NO production by eNOS (Dimmeler et al.
1999). In line with the present results obtained in
aorta, Bhuiyan & Fukunaga (2010) showed a decrease
in eNOS phosphorylation in kidney from ovariectomized
Wistar rats. Moreover, Widder et al. (2003) demonstra ted
Figure 4. Cu/Zn-SOD and Mn-SOD protein expression in aorta
Representative Western blots (top) and quantitative analysis
(bottom) for Cu/Zn-SOD (A) and Mn-SOD (B) protein expression in
thoracic aorta from SHAM, OVX and DHEA-treated rats. Results are
expressed as means ± SEM. Two-way ANOVA:
P < 0.01 in
comparison to SHAM and #P < 0.01 in comparison to OVX (n = 8).
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J Physiol 589.10 Dehydroepiandrosterone reduces reactive oxygen species in aorta 2593
that ovar iectomy is related to a reduction of eNOS
protein expression in aorta from SHR rats, which was
not observed in our study using aorta from Wistar
rats. Recently, consistent with our results, Bhuiyan &
Fukunaga (2010) also showed that DHEA enhances eNOS
phosphorylation in kidney from ovariectomized rats,
leading to blood pressure reduction. In addition, there
are some in vitro studies which have demonstrated that
DHEA directly stimulates eNOS phosphorylation in end-
othelial cells (Liu & Dillon, 2004; Formoso et al. 2006).
Furthermore, our results exclude a possible role of DHEA
treatment on impaired ability of vascular smooth muscle
to relax in response to exogenous NO donor, since the
endothelium-independent relaxation induced by SNP was
similar in all groups and was not changed by DHEA
treatment.
Besides the reduction of NO production by eNOS,
the increased superoxide anion in vascular cells provokes
a decline in NO bioavailability, leading also to end-
othelial dysfunction (Cai & Harrison, 2000). The main
source of superoxide anion in the vascular system is
the enzyme NADPH oxidase (Cai & Harrison, 2000).
Similar to other studies (Wassmann et al. 2001; Tsuda
et al. 2005), here we observed that beyond decrease in
eNOS phosphorylation, oxidative stress is also involved
in the reduced NO bioavailability in aorta from OVX
rats. Therefore, endothelial dysfunction in OVX rats is
due to both reduced eNOS phosphorylation and oxidative
stress. In fact, PHE and ACh response of aortic rings
from OVX rats were also corrected by scavenging super-
oxide anion and inhibiting NADPH oxidase. Reinforcing
our results, aorta from OVX rats displayed an increase
in ROS production. Oxidative stress-induced endothelial
dysfunction by oestrogen withdrawal has already been
shown by several studies. Virdis et al. (2000) observed that
endothelial dysfunction resulting from acute oestrogen
deprivation in women was caused by oxidative stress.
In SHR OVX rats reduction in relaxation response to
carbachol in aortic rings associated with increased super-
oxide anion production was demonstr a ted (Wassmann
et al. 2001). Moreover, oestrogen protects female OVX
mice against increase in ROS production in aorta, and
angiotensin II-induced intr a cellular ROS production in
vascular smooth muscle (Strehlow et al. 2003).
Several studies have shown a potential antioxidant
effect of DHEA. The present s tudy also demonstrates
a role of DHEA in reducing superoxide anion content
in aorta from OVX rats. This effect m ay represent
the mechanism by which DHEA treatment corrects the
vascular response to PHE a nd ACh in aorta from OVX
rats. Yorek et al. (2002) observed that DHEA reduced
plasma thiobarbituric acid-reactive substances (TBARS)
and superoxide anion production in arterioles from
diabetic rats, and Aragno et al. (2006) also demonstrated
reductioninROScontentinheartsfromdiabeticrats
treated with DHEA. It seems that DHEA has an anti-
oxidant effect that is not only in the cardiovascular system,
since it reduced oxidative stress-induced skeletal muscle
damage in diabetic rats (Aragno et al. 2004). Furthermore,
oxidative stress parameters in plasma and in peri-
pheral blood mononuclear cells in diabetic subjec ts were
significantly decreased by DHEA treatment (Brignardello
et al. 2007). Besides, in vitro studies have shown direct
Figure 5. NADPH oxidase subunits protein expression in aorta
Representative Western blots (top) and quantitative analysis
(bottom) for gp91phox (A) and p22phox (B) protein expression in
thoracic aorta from SHAM, OVX and DHEA-treated rats. Results are
expressed as means ± SEM. Two-way ANOVA:
P < 0.01 in
comparison to SHAM and #P < 0.01 in comparison to OVX (n = 8).
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2594 J. P. G. Camporez and others J Physiol 589.10
effects of DHEA on oxidative stress parameters. DHEA
reduced ROS production and NF-κB activation in end-
othelial cells treated with TNF-α (Guti
´
errez et al. 2007),
and protected endothelial cells against apoptosis (Liu et al.
2007) and stimulated their proliferation (Williams et al.
2004).
As discussed above and demonstrated in the pre-
sent study, menopause and ovariectomy induce oxidative
stress, mainly in the cardiovascular system. One reason
for this outcome could be the modulation of the activity
or expression of antioxidant enzymes. Tsuda et al. (2005)
demonstrated that aorta from female mice displayed less
NADPH oxidase activity and expression than aorta from
male mice, w hile ovariectomy showed an increase in
NADPH oxidase activity and expression in females. This
outcome has an important role in endothelial dysfunction
observed in OVX rats, since NADPH oxidase is the
main source of superoxide anion associated with end-
othelial dysfunction (Cai & Harrison, 2000). In addition,
antioxidant systems are also depressed by oestrogen
withdr awal, which contributes to oxidative stress-induced
endothelial dysfunction in OVX rats. Oestrogen deficiency
decreased SOD mRNA expression in aorta f rom OVX
mice, which was related to an increase in the presence
of the superoxide anion, and oestrogen replacement
therapy prevented this downregulation (Strehlow et al.
2003).
In our study we also observed a modulation of
SOD and NADPH oxidase protein expression following
ovariectomy, displaying reduced Cu/Zn-SOD protein
expression and increased gp91phox and p22phox (sub-
units of NADPH oxidase) protein expression, which
would explain the oxidative stress-induced endothelial
dysfunction. B esides, we observed a correction of
Cu/Zn-SOD downregulation and gp91phox and p22phox
increment by DHEA treatment, which could be the
main result of DHEA treatment, leading to reduced
ROS production and consequently enhanced vascular
function in OVX rats. Besides the vascular antioxidant
effect by modulating expression of enzymes involved
in oxidant/antioxidant activity, the present work shows
that DHEA treatment also enhances SOD activity in
aortafromagedrats(Wuet al. 2007), prevents oxidative
injury in obstructive jaundice in rats by increasing SOD
activity in liver (Celebi et al. 2004), and also raises SOD
activity in liver from diabetic rats (Aragno et al. 1999).
However, the present study is the first to demonstrate
that DHEA treatment can modulate Cu/Zn-SOD,
gp91phox and p22phox (NADPH oxidase) protein
expression.
In summary, this study confirms the pivotal role
of oxidative stress-induced endothelial dysfunction in
OVX rats. Furthermore, it indicates the potential role of
DHEA therapy, instead of oestrogen replacement therapy,
as an antioxidant, improving endothelial function,
reducing systolic and diastolic blood pressure, and
modulating eNOS phosphorylation, and Cu/Zn-SOD,
gp91phox and p22phox protein expression in aorta from
OVX rats.
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Author contributions
J.P.G.C., E.H.A. a nd C.R.O.C. contributed to the conception
and design of the experiments. J.P.G.C. performed and analysed
most of the experiments in this study, with technical assistance
from E.H.A., A.P.D., C.R.F. and L.V.R. All authors analysed and
interpreted data. J.P.G.C., L.V.R. and C.R.O.C. wrote the article.
Each of the authors approved the final version of the manuscript.
There were no conflicts of interest.
Acknowledgements
WearegratefultoDrRonaldD.P.K.Ranvaudforhelpful
suggestions on the manuscript. This work was funded by
research grants from FAPESP and CNPq, Brazil.
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    • "Its improved activity could be in part explained by an up-regulation of gene expression and protein level of MnSOD. Indeed, in studies which focused on antioxidant effects of steroids in ovariectomized female rats, an increase of MnSOD protein level has been observed after treatment with estradiol or progesterone [32] , whereas DHEA preferentially upregulated the expression of Cu/ZnSOD [31]. In orchiectomized male rats, testosterone was also able to increase MnSOD protein level compared to the control (sham operated) [62]. "
    [Show abstract] [Hide abstract] ABSTRACT: The brain has high energy requirements to maintain neuronal activity. Consequently impaired mitochondrial function will lead to disease. Normal aging is associated with several alterations in neurosteroid production and secretion. Decreases in neurosteroid levels might contribute to brain aging and loss of important nervous functions, such as memory. Up to now, extensive studies only focused on estradiol as a promising neurosteroid compound that is able to ameliorate cellular bioenergetics, while the effects of other steroids on brain mitochondria are poorly understood or not investigated at all. Thus, we aimed to characterize the bioenergetic modulating profile of a panel of seven structurally diverse neurosteroids (progesterone, estradiol, estrone, testosterone, 3α-androstanediol, DHEA and allopregnanolone), known to be involved in brain function regulation. Of note, most of the steroids tested were able to improve bioenergetic activity in neuronal cells by increasing ATP levels, mitochondrial membrane potential and basal mitochondrial respiration. In parallel, they modulated redox homeostasis by increasing antioxidant activity, probably as a compensatory mechanism to a slight enhancement of ROS which might result from the rise in oxygen consumption. Thereby, neurosteroids appeared to act via their corresponding receptors and exhibited specific bioenergetic profiles. Taken together, our results indicate that the ability to boost mitochondria is not unique to estradiol, but seems to be a rather common mechanism of different steroids in the brain. Thus, neurosteroids may act upon neuronal bioenergetics in a delicate balance and an age-related steroid disturbance might be involved in mitochondrial dysfunction underlying neurodegenerative disorders.
    Full-text · Article · Sep 2014
    • "However, Jacob et al. (2011), verified that 5-week DHEA treatment was able to increase glutathione peroxidase protein levels in liver of young and aged rats. Moreover, DHEA was an efficient antioxidant agent in the aorta of ovariectomized rats (CAMPOREZ et al., 2011). Finally, DHEA administration is associated with an increased cellular proliferation. "
    [Show abstract] [Hide abstract] ABSTRACT: Dehydroespiandrosterone (DHEA) is associated with improvements in chronic degenerative diseases, including obesity, insulin resistance, and cardiovascular diseases. Nevertheless, it is observed an increase in its concentration in individuals with liver lipid infiltration, but it is not precise if this condition emerges as a cause or a consequence. In this way, we aimed to identify gene expression alterations in lipid and glucose liver metabolism markers, as well as oxidative stress markers. For this purpose, male Wistar rats, 12-14 months old were treated with subcutaneous injections of DHEA (only dose of 10 mg kg-1); and after 7 days, hepatic gene expression by PCR real time were performed for the following genes: G6Pase, PEPCK, FAS, PPARγ, malic enzyme, ChREBP, LXR, catalase, GPx, iNOS, NADPH oxidase subunits and PCNA. We observed a tendency of reduction in G6Pase gene expression in treated group (p = 0.08). In addition, it was identified an increase in liver PPARγ and FAS gene expressions, two markers of increased activity of lipogenic pathway. We also observed an increase in iNOS gene expression, a known inductor of systemic and hepatic insulin resistance. In conclusion, our data indicates that the treatment with DHEA can be associated with the development of liver lipid infiltration and hepatic insulin resistance.
    Full-text · Article · Jun 2014
    • "One of these substances is dehydroepiandrosterone (DHEA), which is together with its sulfate ester (DHEAS) one of the most abundant steroids in the human body. Both DHEA and DHEAS have antioxidant effects (Camporez et al., 2011; Gao et al., 2005; Maninger et al., 2009). Evidence also indicates that DHEA and DHEAS are synthesized in the brain (Maninger et al., 2009). "
    [Show abstract] [Hide abstract] ABSTRACT: Tardive dyskinesia (TD) is a potentially irreversible consequence of long term treatment with antipsychotic drugs which is according to a well-known theory believed to be related to oxidative stress induced neurotoxicity. Dehydroepiandrosterone (DHEA) is an endogenous antioxidant with neuroprotective activity. The biosynthesis of DHEA depends upon the activity of cytochrome P450c17α (CYP17). The gene that encodes for CYP17 has a (T34C) single nucleotide polymorphism which enhances CYP17 transcription and expression. To test the hypothesis that carriership of a more active CYP17 variant would result in higher DHEA(S) levels and protect against neurotoxicity which results in orofaciolingual TD (TDof), limb-truncal TD (TDlt) or both (TDsum)? Tardive dyskinesia was assessed cross-sectionally in 146 Caucasian psychiatric inpatients from Siberia. Patients who are carriers of the Cyp17 genotype CC have less chance of developing TD compared to patients who are carriers of the Cyp17 genotypes TC or TT (p<0.05). However, these carriers have significant lower circulating DHEAS levels compared to carriers of the Cyp17 genotypes TC and TT (p<0.05). Conversely, carriers of the CYP17 T-allele have significant elevated DHEAS levels. After correcting for gender and age no significant relationship between Cyp17 genotype CC, the T-allelle and the C-allele and the DHEAS concentration of patients was observed. Conclusions: Although an association between the CYP17 CC genotype and TD is indicated, our findings do not support the hypothesis that this is mediated through increased DHEA(S) levels. We believe that the relationship between this polymorphism and neuroprotective effects of steroids is more complex and cannot be elucidated without taking the posttranslational regulation of the enzyme into account.
    Full-text · Article · Jan 2014
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