Download Estrogen, Progesterone, and Aldosterone Interactions

Estrogen, Progesterone, and Aldosterone Interactions
Medroxyprogesterone Acetate But Not Drospirenone Ablates
the Protective Function of 17␤-Estradiol in Aldosterone
Salt–Treated Rats
Paula Anahi Arias-Loza, Kai Hu, Andreas Schäfer, Johann Bauersachs, Thomas Quaschning, Jan Galle,
Virginija Jazbutyte, Ludwig Neyses, Georg Ertl, Karl-Heinrich Fritzemeier,
Christa Hegele-Hartung, Theo Pelzer
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Abstract—Controversial results obtained from human and animal studies on the prevention of heart disease by estrogens
and progestins warrant a better understanding of nuclear hormone receptor function and interaction. To address this issue
and taking into account that effects of synthetic progestins are not only referable to action through the progesterone
receptor but may also be mediated by other steroid receptors, we characterized cardiovascular function and
inflammatory gene expression in aldosterone salt–treated rats on long-term administration of 17␤-estradiol, medroxyprogesterone acetate, and drospirenone, a new progestogen exhibiting antimineralocorticoid activity. The complex
pattern of cardiovascular injury in ovariectomized Wistar rats induced by chronic aldosterone infusion plus a high-salt
diet was significantly attenuated in sham-ovariectomized rats and by coadministration of 17␤-estradiol in ovariectomized animals after 8 weeks of continuous treatment. The beneficial role of 17␤-estradiol on blood pressure, cardiac
hypertrophy, vascular osteopontin expression, perivascular fibrosis, and impaired NO-dependent relaxation of isolated
aortic rings was completely abrogated by coadministration of medroxyprogesterone acetate. In contrast, drospirenone
was either neutral or additive to 17␤-estradiol in protecting against aldosterone salt–induced cardiovascular injury and
inflammation. The current results support the hypothesis of complex interactions among estrogen, progesterone,
glucocorticoid, androgen, and mineralocorticoid receptor signaling in cardiovascular injury and inflammation. Novel
progestins, such as drospirenone, confer superior effects compared with medroxyprogesterone acetate in a model of
aldosterone-induced heart disease because of its antimineralocorticoid properties. (Hypertension. 2006;48:994-1001.)
Key Words: aldosterone 䡲 estrogen 䡲 medroxyprogesterone-acetate 䡲 cardiac hypertrophy
E
xperimental studies provide ample and solid evidence for
a protective effect of unopposed estrogens in animal
models of human heart disease.1 These reports are in contrast
with the neutral or negative outcome of clinical trials on
hormone replacement therapy, which consists of combined
estrogen and progestin treatment, in the prevention of coronary
artery disease in postmenopausal women.2,3 In experimental
models, estrogens regulate cardiac and vascular gene expression
and function via 2 distinct nuclear estrogen receptors, estrogen
receptor-␣ and -␤ (ER␣ and ER␤), which are coexpressed and
may functionally interact with the mineralocorticoid receptor
(MR), the androgen receptor, and the progesterone receptor (PR)
in vascular smooth muscle cells, fibroblasts, endothelial cells,
and cardiac myocytes.4,5
In contrast to estrogens, which are highly selective ligands for
ER␣ and/or ER␤, progestogen effects are not only mediated by
PRs, but most synthetic progestins also interact with other
steroid receptors, such as the androgen, the glucocorticoid, and
the MR.6 Progestins might block not only estrogen activity in
reproductive tract organs but also in cardiac and vascular cells.7,8
Accordingly, several authors have speculated that such interactions may be responsible for the lack of protective effects in the
Women’s Health Initiative Trial.4 Of note, medroxyprogesterone
acetate (MPA), in addition to its progestogenic activity, also
exhibits androgenic and glucocorticoid activity.9,10 But the role
of progestins in cardiovascular disease remains poorly defined,
although synthetic progestogens, including MPA, may have
played a relevant role in important clinical trials on hormone
replacement therapy in cardiovascular disease. Drospirenone,
which is a new progestin derived from 17␣-spirolactone, closely
matches the pharmacological profile of natural progesterone but
is devoid of androgenic or glucocorticoid activity and has a
higher antimineralocorticoid potency.11–13
Mineralocorticoids play an important physiological role in
fluid and electrolyte balance. However, disproportionate elevation of aldosterone serum levels results not only in severe
Received March 28, 2006; first decision April 23, 2006; revision accepted August 11, 2006.
From the Medizinische Klinik I (P.A.A.-L., K.H., A.S., J.B., T.Q., J.G., V.J., G.E., T.P.), University of Würzburg, Würzburg, Germany; Schering AG
Berlin (K.-H.F., C.H.-H.), Berlin, Germany; and the Division of Cardiology (L.N.), University of Manchester, Manchester, United Kingdom.
Correspondence to Theo Pelzer, Medizinische Klinik I, University of Würzburg, Josef-Schneider Str 2, D-97080 Würzburg, Germany. E-mail
[email protected]
© 2006 American Heart Association, Inc.
Hypertension is available at http://www.hypertensionaha.org
DOI: 10.1161/01.HYP.0000242482.57186.e8
994
Arias-Loza et al
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vascular inflammation, fibrosis, hypertension, and cardiac
myocyte hypertrophy in rodents but may also play a causative
role in ⱕ10% of patients diagnosed previously with essential
hypertension.14 –16 Increased aldosterone serum levels have
also been shown to promote the development of congestive
heart failure, and MR antagonists, such as spironolactone or
eplerenone, improve clinical symptoms and prolong survival
in heart failure patients.17–19
Classical hormone replacement therapy may, thus, interfere with
mineralocorticoid functions. Accordingly, interactions among nuclear hormone receptors for estrogens, progestins, and mineralocorticoids are of specific interest in cardiovascular disease. To
address the issue, we studied the effect of 17␤-estradiol and
synthetic progestins on cardiovascular function, inflammatory
injury, and gene expression in aldosterone salt–treated rats as an
established model of disproportionate aldosterone activity.15
Specifically, we hypothesized1 that 17␤-estradiol would attenuate the detrimental effects of excessive MR activation,2 that
MPA would antagonize the protective function of estrogen,3 that
MPA would aggravate MR-mediated cardiovascular injury, and4
that drospirenone, as a progestin with antimineralocorticoid
activity, might confer more favorable effects than MPA on
cardiovascular function and inflammatory gene expression.
Methods
Animal Model and Treatment
Female Wistar rats were obtained from IFFA CREDO (Lyon,
France) at the age of 12 weeks. Animals in group 1 were sham
operated; animals in group 2 were ovariectomized (ovx) and uninephrectomized (npx) by right-sided nephrectomy. Groups 3 to 10
were subjected to ovariectomy and uninephrectomy plus aldosterone-salt treatment ([AST] rats; 0.75 ␮g aldosterone per hour via
Alzet pumps plus 1% NaCl added to tap). AST rats were treated as
follows: group 3: placebo; group 4: 17␤-estradiol (2 ␮g/kg per day);
group 5: 17␤-estradiol (E2) plus spironolactone (20 mg/kg per day);
group 6: E2 plus drospirenone low dose (3 mg/kg per day); group 7:
E2 plus drospirenone medium dose (9 mg/kg per day); group 8: E2
plus drospirenone high dose (30 mg/kg per day); group 9: E2 plus
MPA (3 mg/kg per day); and group 10: placebo plus MPA. Animals
in group 11 were sham-ovx, uni-npx, and received AST treatment as
described before. Steroids were dissolved in ethanol and administered by daily subcutaneous injections using either peanut oil
(17␤-estradiol) or castor oil (MPA, drospirenone) as a carrier;
spironolactone was supplied with a tap.7,13,20 All of the surgical
procedures were carried out under isoflurane anesthesia (isoflurane
1.5 vol% supplemented by 0.5 L of oxygen per minute) after
pretreatment with tribromoethanol/amylene hydrate (Avertin; 2.5%
weight:volume; 6 ␮L/g body weight IP). All of the protocols were
reviewed and accepted by the local ethics committee and performed
in accordance with the current National Institutes of Health (NIH)
Guide for the Care and Use of Laboratory Animals.
Hemodynamic Analysis
Hemodynamic measurements were performed as reported previously
under light isoflurane anesthesia and spontaneous respiration (isoflurane 1.5 vol%; 0.5 L of oxygen per minute).21 Left ventricular
pressure curves were recorded from the left ventricular cavity, and
systolic and diastolic blood pressure measurements were obtained on
catheter placement in the thoracic aorta. Measurements were performed by a single, trained observer blinded for treatment groups.
Morphometric Analysis
Body weight, heart weight, uterus weight, and tibia length were
measured after hemodynamic analysis. Relative heart weight was
calculated from absolute heart weight and tibia length to avoid a bias
Effects of E2, MPA, and Drospirenon in AST Rats
995
of the results by the adipose phenotype ovx rats. Serum E2 and
angiotensin II (Ang II) levels were measured by radioimmunoassays
(E2: DPC-Biermann; Ang II: Peninsula). Hearts were dissected into
apex, midventricle, and base. The midventricular section was embedded into TissueTec OCT (Sakura) and cut into 5-␮m sections for
hematoxylin and 6-␮m sections for picro sirius red staining. Cardiac
myocyte cross-sectional areas were calculated from manual tracings
of 80 randomly selected cardiac myocytes from each animal (total
measurements: 7.900 myocytes) using the “Image J” software
(NIH).22 Perivascular collagen accumulation was quantified around
6 randomly selected coronary arteries in 3 nonadjacent sections from
each animal (total measurements: 1.800 vessels) using the image
analysis system Scion (NIH).23 Media and intima areas and intima:media ratios were measured in 3 nonadjacent aortic sections from
each animal (total measurements: 295 sections) by manual tracing of
the intima–media border and media– externa border using the Image
J software. Uteri were fixed in buffered formaldehyde, cut into 4-␮m
transverse sections, and stained with hematoxylin/eosin for quantitative
evaluation of luminal epithelial cell height using an Axiophot 2
microscope and a KS 400 imaging system (Carl Zeiss Vision GmbH).
Vascular Reactivity Studies
Tissue harvest and organ chamber experiments were performed according to published protocols.24 The aorta was isolated after hemodynamic
analysis in a no-touch technique and placed into modified Krebs–Ringer
bicarbonate solution (4°C). Aortic rings were mounted on tungsten
stir-ups, placed in an organ bath containing 10 mL of modified
Krebs–Ringer bicarbonate solution (37°C [pH 7.4] and 95% O2/5%
CO2), connected to force transducers (Föhr Medical Instruments),
equilibrated, and stretched to optimum passive tension (2.5⫾0.2 g).
Rings were preconstricted with phenylephrine to ⬇70% of 100 mmol/L
KCl-induced tension and relaxations to acetylcholine ([ACH] 10⫺10 to
10⫺5 mol/L) or sodium nitroprusside ([SNP] 10⫺11 to 10⫺5 mol/L) were
obtained.
Immunohistochemistry
Aortic sections (2 ␮m) were mounted on glass slides, fixed with 4%
formaldehyde in 100 mmol/L of PBS (pH 7.4) before O/N incubation
at 4°C with primary antibodies directed against osteopontin (Abcam
rabbit polyclonal, 1:100).25 Osteopontin expression was visualized
using a commercial kit (Vectastain ABC Kit diaminobenzidine,
anti-rabbit IgG, Vector Labs) and quantified using the Scion software.26 Three randomly selected fields from 3 nonadjacent sections
per specimen were analyzed among 4 randomly selected animals per
group (total measurements: 1160 fields). Immediately adjacent
sections, in which osteopontin antibodies were omitted, served as
negative controls. Nuclei were stained with Hematoxylin QS
(Vector Labs).
Statistics
Statistical significance was calculated by 1-way ANOVA followed by
Student–Newman–Keuls post hoc testing. Values are mean⫾SEM, and
P values ⬍0.05 were considered significant.
Results
Global and Hemodynamic Measurements
Relative heart weight was significantly higher in estrogendepleted AST rats compared with sham-operated or ovx and npx
animals (⫹27.3⫾3.5% versus sham; P⬍0.01; Table). Cardiac
mass was lower in AST intact and in ovx AST rats receiving
17␤-estradiol substitution (⫺14⫾2.4% versus AST placebo;
P⬍0.01) compared with estrogen-depleted AST rats receiving
placebo. In contrast, cardiac mass remained elevated in rats
treated with E2 plus MPA (⫹24⫾5% versus AST⫹E2; P⬍0.01).
The combination of drospirenone and 17␤-estradiol reduced
relative heart weight to near-baseline levels. Very similar results
were obtained for absolute heart weight. Systolic and mean
996
Hypertension
November 2006
Global and Hemodynamic Measurements
Ovx
AST⫹E2
(n⫽10)
Ovx AST⫹
E2⫹spiro
(n⫽10)
Ovx AST⫹
E2⫹dro
3 mg
(n⫽10)
Ovx AST⫹
E2⫹dro
9 mg
(n⫽10)
Ovx AST⫹
E2⫹dro
30 mg
(n⫽10)
Sham
(n⫽10)
Ovx npx
(n⫽5)
Ovx AST
Placebo
(n⫽10)
Rel. heart weight,
mg/cm
171⫾2*
176⫾2*
259⫾10
219⫾6*
190⫾5*
217⫾14*
196⫾6*
Abs. heart weight, mg
650⫾7*
669⫾6*
997⫾40
858⫾24*
741⫾21*
837⫾55*
756⫾24*
Variable
Ovx AST⫹
E2⫹MPA
(n⫽10)
Ovx
AST⫹MPA
(n⫽5)
195⫾5*
272⫾11
270⫾14
221⫾6*
763⫾18*
1055⫾46
1028⫾54
863⫾34
Sham AST
(n⫽9)
Systolic BP, mm Hg
145⫾3*
143⫾6*
190⫾9
161⫾5*
143⫾5*
149⫾4*
150⫾6*
154⫾7*
191⫾12
189⫾10
160⫾3*
Mean BP, mm Hg
128⫾2*
132⫾6*
164⫾7
131⫾6*
116⫾6*
123⫾4*
120⫾6*
121⫾6*
155⫾8
152⫾12
139⫾3*
79⫾5*
63⫾6*
7⫾1
9⫾2
46⫾3*
29⫾6*
50⫾5*
43⫾8*
6⫾2
6⫾2
Serum Ang II,
pg/100 ␮L
Uterus weight, mg
699⫾88*†
101⫾6
107⫾6
447⫾47*
497⫾64*
275⫾19*†
283⫾20†
242⫾12†
533⫾23*
233⫾16†
Serum E2, pg/mL
25⫾8*
2⫾1
4⫾3
22⫾4*
19⫾3*
14⫾3*
19⫾3*
26⫾7*
18⫾7*
2⫾1
nd
nd
nd
26⫾1
nd
nd
nd
18⫾1†
17⫾1†
nd
Epithelium height, ␮m
4⫾1
660⫾113*†
16⫾5*
nd
Body weight, g
268⫾5*
297⫾8
326⫾8
287⫾5*
287⫾6*
295⫾8
286⫾5*
296⫾12
277⫾14*
298⫾5
257⫾8*
Heart rate, bpm
420⫾12
411⫾20
395⫾15
394⫾7
414⫾9
410⫾7
416⫾9
418⫾11
381⫾14
361⫾17
367⫾12
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nd indicates not done; dro, drospirenone; spiro, spironolactone; BP, blood pressure.
*P⬍0.05 vs ovx AST placebo; †P⬍0.05 vs ovx AST⫹E2.
blood pressure levels were significantly elevated in ovx, AST
rats compared with sham-operated or ovx/npx rats. Blood
pressure levels were lower in intact AST animals and in AST
rats receiving 17␤-estradiol (⫺15⫾2% and ⫺15⫾3% versus
placebo; both P⬍0.01). Combining drospirenone or spironolactone with E2 substitution caused a further reduction of blood
pressure, whereas MPA treatment resulted in increased blood
pressure in E2-substituted rats. Serum Ang II levels were lower
in ovx AST rats compared with sham-operated or ovx/npx
animals (⫺90⫾2.5% versus sham; P⬍0.001) and not affected by E2 or MPA treatment, whereas drospirenone and
spironolactone prevented the suppression of serum Ang II
levels. Uterus atrophy was observed in ovx rats but not in
sham-operated or E2-substituted animals. Uterus weight was
lower in estrogen-substituted rats receiving drospirenone,
unaltered by spironolactone, and increased in MPA-treated
AST rats. Estrogen serum levels were low in ovx and
increased to physiological levels in E2-substituted rats. Endometrial epithelium height was significantly lower in rats
receiving E2 plus MPA or drospirenone compared with
unopposed estrogen substitution. Body weight was higher in
estrogen-depleted animals and decreased on E2 substitution.
Heart rate was similar and within physiological limits among
all of the animals.
Perivascular Collagen Accumulation
AST resulted in significantly higher levels of perivascular
collagen deposition compared with sham treatment or uninephrectomy ovariectomy (⫹300⫾33% versus sham; P⬍0.001;
Cardiac Myocyte Cross-Sectional Area
Cardiac hypertrophy and a patchy pattern of perivascular fibrosis
were evident in AST rats from the gross morphological appearance of cardiac cross-sections (Figure 1A and 1B). The average
cardiac myocyte cross-sectional area was elevated in AST
compared with sham-operated rats (⫹128⫾12% versus sham;
P⬍0.001; Figure 2) and decreased substantially with E2 substitution (⫺57⫾1.3% versus AST placebo; P⬍0.001) and, to a
lesser extent in intact, non-ovx animals. No additional effect was
observed in groups receiving E2 plus drospirenone or spironolactone. MPA blocked the decrease of the cardiac myocyte crosssectional area in estrogen-substituted rats (⫹45⫾5% versus ASTE2) but, by itself, did not aggravate myocyte hypertrophy.
Figure 1. A, Cardiac morphology. Hematoxylin/eosin-stained
cross-sections illustrate cardiac hypertrophy in ovx AST rats vs
sham-operated or ovx-npx rats. Cardiac mass was lower in intact
AST rats and in ovx animals treated with 17␤-estradiol (E2), E2
plus spironolactone, E2 plus drospirenone (3, 9, and 30 mg/kg per
day) but not in AST rats treated with MPA or E2 plus MPA. B, Cardiac fibrosis. Cardiac cross-sections stained with picro sirius red
illustrate increased perivascular fibrosis in ovx AST rats that was
attenuated in intact animals and in ovx rats receiving E2, E2 plus
spironolactone, or E2 plus drospirenone, whereas cardiac collagen
deposition was highest in rats receiving MPA.
Arias-Loza et al
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Figure 2. Cardiac myocyte cross-sectional area. Increased cardiac myocyte cross-sectional areas in ovx AST rats vs control
were normalized by 17␤-estradiol, E2 plus spironolactone, and
E2 plus drospirenone and partially normalized in intact rats but
remained elevated in MPA or MPA plus E2-treated rats. Photomicrographs illustrate representative hematoxylin-stained
myocardial cross-sections. n⫽5 to 10 animals per group;
*P⬍0.05 s sham.
Figure 3). E2 treatment completely blocked perivascular fibrosis
in AST rats (⫺277⫾31% versus AST-placebo; P⬍0.001), and
no further reduction of collagen deposition was observed in
animals cotreated with E2 plus drospirenone or spironolactone. No significant perivascular fibrosis was observed in
intact rats receiving AST treatment. MPA aggravated perivascular fibrotic lesions and blocked the inhibitory effect of
17␤-estradiol on perivascular fibrosis.
Aortic Intima:Media Ratios
Aortic intima:media ratios increased in placebo-treated AST
rats compared with sham-operated or ovx and uni-npx animals (Figure 4). Combining estrogen substitution with AST
treatment resulted in reduced intima:media ratios that nearly
reached physiological levels. Similar observations were made
in intact, non-ovx animals receiving AST treatment. No
additional effect on vascular remodeling was observed in
animals receiving 17␤-estradiol plus drospirenone or spironolactone. The highest intima:media ratios were observed in
AST rats receiving E2 plus MPA.
Effects of E2, MPA, and Drospirenon in AST Rats
997
Figure 3. Perivascular collagen. Increased perivascular collagen
accumulation in ovx AST rats vs control was attenuated in intact
animals and by treatment of estrogen-depleted rats with 17␤estradiol, E2 plus spironolactone, and E2 plus drospirenone,
whereas MPA resulted in increased perivascular fibrosis. Photomicrographs illustrate representative vascular cross-sections
(picro sirius red stain). n⫽5 to 10 animals per group; *P⬍0.05 vs
sham; §P⬍0.05 vs ovx AST.
Vascular Osteopontin Expression
Osteopontin expression was higher in aortic specimens of ovx
AST rats compared with sham-operated or ovx and uni-npx
animals (Figure 5). 17␤-Estradiol effectively blocked the local
accumulation of osteopontin in the aortic media and intima layer.
Similar results were obtained in intact animals and in ovx
animals receiving E2 substitution combined with drospirenone
or spironolactone. In contrast, MPA blocked the reduction of
vascular osteopontin expression in estrogen-substituted AST rats
but, by itself, did not increase aortic osteopontin content.
Vasomotor Studies in Isolated Aortic Rings
The contractile response of isolated aortic rings toward norepinephrine treatment was increased, and ACH-induced vascular
relaxation was impaired in AST compared with sham-operated
rats (Figure 6A and 6B). MPA strikingly aggravated NOdependent vascular dysfunction as judged by impaired relaxation
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Figure 5. Vascular osteopontin expression. The photomicrographs illustrate representative immunohistochemistry stainings
for aortic osteopontin expression from all treatment groups
including negative control (opn antibody omitted). Increased
vascular osteopontin expression in ovx AST rats was attenuated
in intact animals and by treatment of ovx rats with 17␤estradiol, E2 plus spironolactone, and E2 plus drospirenone but
remained elevated with administration of MPA.
Figure 4. Aortic intima:media ratios. Increased aortic intima:
media ratios in ovx AST rats vs control were decreased in intact
animals and by treatment of ovx rats with 17␤-estradiol, E2 plus
spironolactone, and E2 plus drospirenone, whereas cotreatment
with MPA resulted in media hypertrophy and increased intima:
media ratios. Photomicrographs illustrate representative
hematoxylin/eosin-stained aortic cross-sections. n⫽5 to 10
animals per group; *P⬍0.05 vs sham; §P⬍0.05 vs ovx AST.
to ACH treatment of aortic rings (Figure 6B). Combined
treatment with 17␤-estradiol plus drospirenone markedly improved endothelium-dependent relaxation to ACH compared
with rats substituted with 17␤-estradiol (P⬍0.05 versus
AST⫹E2; Figure 6C). In contrast, MPA treatment resulted in
impaired endothelium-dependent relaxation (P⬍0.05 versus
AST ⫹E2 and P⬍0.01 versus all other groups; Figure 6C).
Relaxation to ACH was blunted in all of the groups after
preincubation with N G-nitro-L-arginine methyl ester (data not
shown). In contrast to endothelium-dependent relaxation, maximum endothelium-independent relaxations to the NO donor
SNP were comparable in all of the groups apart from MPAtreated animals in which even endothelium-independent relaxation was significantly impaired (Figure 6D).
Discussion
The major findings of this study can be summarized as follows:
(1) 17␤-estradiol substitution similar to sham ovariectomy attenuates aldosterone salt–induced hypertension, cardiac hypertrophy, vascular fibrosis, and osteopontin expression, as well as
endothelial dysfunction in estrogen-depleted rats; (2) the protec-
tive effects of 17␤-estradiol are abrogated by MPA; (3) MPA
aggravates aldosterone salt–mediated vascular fibrosis and dysfunction; and (4) drospirenone exhibits favorable effects on
cardiovascular morphology, function, and vascular osteopontin
expression.
The effects of AST on cardiovascular pathology have so
far only been studied in male but not in female rats.
Therefore, it is interesting to note that signs of disproportionate MR activation, such as cardiac hypertrophy, hypertension,
perivascular collagen accumulation, and endothelial dysfunction, as well as increased vascular osteopontin expression,
were observed predominantly in ovx AST rats, whereas their
severity was significantly reduced in AST rats receiving
either sham ovariectomy or ovariectomy plus 17␤-estradiol
substitution. One important finding reported here is, therefore, the observation that endogenous estrogens, as well as
substitution with physiological doses of 17␤-estradiol, completely blocked or significantly improved several key features
of cardiovascular injury under the condition of disproportionate MR activity.
Although the complex phenotype of AST rats suggests
multifactorial mechanisms through which estrogens might attenuate cardiovascular injury and dysfunction, 17␤-estradiol could
be discussed to interfere directly with MR activation. However,
this hypothesis is neither supported by the current literature nor
by measurements of Ang II serum levels, which were suppressed
in AST rats irrespective of E2 substitution, whereas MR antagonist treatment with spironolactone and drospirenone resulted in
increased serum Ang II levels. Therefore, it appears more likely
that 17␤-estradiol interacts with downstream signal transduction
pathways that become activated on AST in different organ
systems. Estrogen effects are mediated by 2 different receptors,
Arias-Loza et al
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Figure 6. Vascular reactivity studies. A, Phenylephrine-induced vasoconstriction was significantly increased in ovx AST rats and aggravated further by MPA. B, Administration of ACH in cumulative doses induced endothelium-dependent vasorelaxation, which was
impaired in ovx AST rats and further decreased in AST rats on MPA treatment. C, Spironolactone or drosperinone in all dosages significantly improved endothelium-dependent relaxation. D, NO donor SNP normalized endothelium-dependent relaxation in all groups but
not in ovx AST rats treated with MPA. n⫽5 to 10 animals per group; *P⬍0.05 vs sham; §P⬍0.05 vs ovx AST; †P⬍0.05 vs ovx AST E2.
ER␣ and ER␤, which confer redundant, as well as specific,
effects in the cardiovascular system.27–29 The current study used
the nonselective estrogen receptor agonist 17␤-estradiol. Further
studies that are currently underway in our laboratory will, thus,
be required to determine whether selective ER␣ and/or ER␤
agonists also attenuate cardiovascular injury in AST rats.
The extent of vascular injury in AST rats was closely
linked to vascular osteopontin expression levels. Osteopontin
plays an important role in cardiac and vascular fibrosis,
because targeted deletion of osteopontin ameliorates cardiovascular collagen accumulation after myocardial infarction,
aortic banding, and chronic Ang II infusion.16,30 –32 Decreased
osteopontin expression in estrogen-substituted AST rats thus
appears as a likely mechanism to explain the lower perivascular collagen accumulation and fibrosis. Therefore, it is
interesting to note that estrogens increase osteopontin expression under in vitro conditions and that ER␣ directly binds and
transactivates the osteopontin promoter in rat osteosarcoma
cells.33,34 The differential expression pattern of osteopontin
under in vitro and in vivo conditions is most likely explained
by very different experimental conditions. Specific studies
will be required to characterize the mechanisms by which
mineralocorticoids and estrogens regulate vascular osteopontin expression in vivo.
In parallel to vascular osteopontin expression, impaired
ACH induced relaxation of isolated aortic rings from ovx
AST rats improved on estrogen substitution. These observations indicate that decreased NO bioavailability in ovx rats
improves with estrogen substitution. Similar observations
have been made previously in estrogen-depleted SHR rats
and have been linked to ER␣ expression in ERKO mice.35,36
Protective estrogen effects in AST rats were, however, not
limited to the vasculature but extended to cardiac muscle, as
well, because cardiac mass was lower, and cardiac myocyte
cross-sectional areas were smaller in estrogen-substituted
compared with placebo-treated AST rats. These findings
could primarily be explained by lower blood pressure levels
in AST rats receiving 17␤-estradiol substitution. Beyond that,
estrogens attenuate cardiac hypertrophy directly by regulation
of signal transduction pathways that promote or inhibit the
development of cardiac hypertrophy, such as ANP expression
or mitogen-activated protein kinase activation.21,37 Based on
the current results and experimental strategy, we cannot rule
out that similar mechanisms might be operative in AST rats
as well, although the reduction of blood pressure already
provides a direct mechanism for decreased cardiac mass in
E2-substituted rats.
The second and functionally important finding of this study
is the observation that MPA almost completely blocked the
protective function of 17␤-estradiol in AST rats. Unfavorable
effects of MPA in AST rats parallel previous reports in which
progestins abrogated the reduction of vascular injury after
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November 2006
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carotid balloon injury on estrogen substitution.7 Within this
context, it is important to note that MPA binds not only to the
PR but also acts as an androgen and glucocorticoid receptor
ligand.9 The additional increase in E2-stimulated uterine
weight by MPA is a clear sign of androgenic pharmacological
activity, and it may be argued that the androgenic activity of
MPA may blunt the beneficial effects of estrogens.38 Similar
observations have been made for the human situation in
which progestins with androgenic partial activities can reverse the beneficial lipid profile of estrogens.10 Collective
evidence, thus, strongly suggests that synthetic progestins act
differently because of different profiles in the activation of,
for example, androgen and glucocorticoid receptors, and it
seems likely that some progestins increase vascular injury,
whereas others exert rather beneficial effects.11,39,40
The observation that MPA attenuated the protective effect
of 17␤-estradiol in AST rats could be explained either by
enhanced mineralocorticoid activity or, alternatively, by inhibition of estrogen action. To discriminate between these
mechanisms, we analyzed the cardiovascular phenotype of
ovx AST rats on long-term administration of MPA. Aggravation of perivascular fibrosis and endothelium-dependent
vasorelaxation in MPA-treated AST rats strongly suggest an
enhancement of vascular remodeling and dysfunction. Because long-term androgen treatment in rats results in cardiac
damage and hypertension via an indirect mineralocorticoid
activity, we cannot exclude that the androgenic partial activity of MPA may have contributed to impaired vascular
relaxation and perivascular fibrosis.41
Drospirenone, which is a progestin with potent MR antagonist
properties and without additional androgenic and glucocorticoid
activity, might exhibit different and protective effects on cardiovascular injury in E2-substituted AST rats as compared with
MPA.11,42 If this hypothesis was true, cardiac and vascular
damage, vascular reactivity, and osteopontin expression should
either be unaffected or even improved in drospirenone-treated
AST rats. The current results strongly support this hypothesis,
because drospirenone at all of the tested dosages conferred more
favorable and protective effects on cardiac hypertrophy, vascular
inflammation, endothelial dysfunction, and osteopontin expression under conditions of disproportionate MR activation than
MPA. The observation, that drospirenone caused a further
reduction of blood pressure and cardiac mass in estrogensubstituted rats is in line with recent findings in postmenopausal
women. Clinical studies have confirmed the protective function
of drospirenone in hypertension and cardiac hypertrophy, although the magnitude of these effects was more moderate in
human studies. These differences are most likely explained by
the fact that we used a model of cardiovascular injury that is
strictly aldosterone dependent, whereas aldosterone levels might
not have been elevated among a significant proportion of
patients who were enrolled in clinical studies on drospirenone. In
addition, it should be noted that, in contrast to the human
situation, 17␤-estradiol already caused a significant reduction of
blood pressure and cardiac mass in AST rats.43– 45
Perspectives
We propose that novel strategies of hormone replacement
therapy, including progestins with antimineralocorticoid ac-
tivity, such as drospirenone, could be advantageous compared
with MPA.
Sources of Funding
T.P. has received support from the Interdisciplinary Center for Clinical
Research Würzburg (TP F-11); P.A.A.-L. and V.J. received support
from the German Academic Exchange Service; L.N. has received
support from the Medical Research Council (United Kingdom), the
British Heart Foundation, and from the EUMORPHIA consortium
(European Union framework program 5); and G.E. has received support
from the Deutsche Forschungsgemeinschaft (SFB 688).
Disclosures
T.P. and L.N. have received financial support from Schering AG
Berlin that relate to the evaluation of novel steroid hormone receptor
ligands. None of the remaining authors report any conflicts.
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Hypertension. 2006;48:994-1001; originally published online September 25, 2006;
doi: 10.1161/01.HYP.0000242482.57186.e8
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