Download Medroxyprogesterone acetate prevents the cardioprotective and anti

Am J Physiol Heart Circ Physiol 293: H1408–H1415, 2007.
First published April 13, 2007; doi:10.1152/ajpheart.00993.2006.
Medroxyprogesterone acetate prevents the cardioprotective and anti-inflammatory
effects of 17␤-estradiol in an in vivo model of myocardial ischemia
and reperfusion
Erin A. Booth and Benedict R. Lucchesi
Department of Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan
Submitted 11 September 2006; accepted in final form 11 April 2007
medroxyprogesterone acetate; membrane attack complex; C-reactive
protein; complement; lipid peroxidation
PREVIOUS PUBLICATIONS HAVE reported the protective effects of
estrogens against ischemia-reperfusion injury, atherosclerosis,
and arrhythmogenesis (4, 9, 37). However, the premature
termination of two large clinical trials has raised serious
questions regarding the cardioprotective effects of hormone
replacement therapy after menopause (12, 18, 19). One possible explanation for the negative effects of estrogens in the
clinical trials is the addition of progesterone to the hormone
regimen. Combination therapy is an accepted treatment for
women who have not undergone a hysterectomy to reduce the
risk of endometrial hyperplasia and uterine cancer (11).
Although unopposed estrogen is the optimal regimen for elevation of HDL cholesterol, the high rate of endometrial hyperplasia restricts the use of unopposed estrogen to women without a uterus. In women with a uterus, conjugated equine
estrogen with cyclic medroxyprogesterone acetate (MPA) has
the most favorable effect on HDL cholesterol without excess
Address for reprint requests and other correspondence: B. R. Lucchesi,
Dept. of Pharmacology, Univ. of Michigan Medical School, 1301C Medical
Science Research Bldg. III, 1150 West Medical Center Dr., Ann Arbor, MI
48109-0632 (e-mail: [email protected]).
H1408
risk of endometrial hyperplasia (40). The published trials
focused on the ability of the treatment regimen to prevent
primary or secondary coronary events; however, the extent of
myocardial damage after an event was not addressed. Several
studies suggest that 17␤-estradiol (E2) can protect against
ischemia-reperfusion injury in the heart and brain and that
combining different estrogen and progestin treatments may
result in different outcomes (6, 20, 33, 34).
Although tissue ischemia of sufficient duration produces
irreversible tissue injury and cell death, the extent of conversion from a viable to a nonviable state is increased significantly
when the previously ischemic tissue is reperfused. Irreversible
tissue injury associated with the restoration of blood flow is
referred to as reperfusion injury. Among the multiple factors
associated with ischemia-reperfusion injury are activation of
the complement cascade (16), generation of reactive oxygen
species (21), and emigration of neutrophils to the area at risk
(AAR) followed by release of cytotoxic constituents (10).
C-reactive protein (CRP), an acute-phase protein, is associated
with the pathogenesis of irreversible myocardial injury (8, 42).
The proposed mechanism of CRP involvement is through local
activation of the complement cascade (39), and increased
levels of CRP are reported to correlate with an increase in
tissue injury after ischemia and reperfusion (3, 13). Pharmacological inhibition of one or more of the multiple pathways of
the inflammatory response has been demonstrated to reduce the
extent of irreversible tissue injury after ischemia and reperfusion (15, 36). Previous studies have shown that estrogen has
the ability to reduce infarct size in the reperfused myocardium,
possibly through modulation of the complement cascade, by
reduction of CRP (5), or by an estrogen receptor-mediated
anti-inflammatory mechanism (25, 38).
The effects of progestins and progesterone on the cardiovascular system are controversial. The addition of progestin to the
hormone treatment regimen has been reported to inhibit estrogen’s protective effects in some, but not all, models of cardiovascular injury (27, 33, 41). Some of the negative effects may
be due to a proinflammatory effect of progestin or the ability of
progestin to antagonize estrogen’s anti-inflammatory effects,
endothelial function, and/or serum lipids (14, 40, 41). Oral
estrogen administration increases plasma levels of the acutephase CRP, whereas the progestin MPA attenuates this effect.
Because it is possible that elevated CRP is associated with
plaque destabilization and rupture, it has been suggested that
the proinflammatory effect of estrogen may account for the
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
0363-6135/07 $8.00 Copyright © 2007 the American Physiological Society
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Booth EA, Lucchesi BR. Medroxyprogesterone acetate prevents
the cardioprotective and anti-inflammatory effects of 17␤-estradiol in
an in vivo model of myocardial ischemia and reperfusion. Am J
Physiol Heart Circ Physiol 293: H1408–H1415, 2007. First published
April 13, 2007; doi:10.1152/ajpheart.00993.2006.—Previous studies
demonstrated the protective effects of estrogen administration in
models of cardiovascular disease. However, there is a discrepancy
between these data and those from the recent clinical trials with
hormone replacement therapy in menopausal women. One possible
explanation for the divergent results is the addition of progestin to the
hormone regimen in the Women’s Health Initiative and the Heart and
Estrogen/Progestin Replacement Study trials. The aim of the present
study was to examine the effects of a combination of 17␤-estradiol
(E2, 20 ␮g) and medroxyprogesterone acetate (MPA, 80 ␮g) on
infarct size in New Zealand White rabbits. Infarct size as a percentage
of the area at risk was significantly reduced by administration of E2 30
min before induction of myocardial ischemia compared with vehicle
(19.5 ⫾ 3.1 vs. 55.7 ⫾ 2.6%, P ⬍ 0.001). However, E2 ⫹ MPA failed
to elicit a reduction in infarct size (52.5 ⫾ 4.6%), and MPA had no
effect (50.8 ⫾ 2.6%). E2 also reduced serum levels of cardiac troponin
I, immune complex deposition in myocardial tissue, activation of the
complement system, and lipid peroxidation. All these effects were reversed by MPA. The results suggest that MPA antagonizes the infarctsparing effects of E2, possibly through modulation of the immune
response after ischemia and reperfusion.
ESTROGEN, PROGESTIN, AND ISCHEMIA-REPERFUSION INJURY
MATERIALS AND METHODS
Guidelines for animal research. The procedures used in this study
are in agreement with the guidelines of the University of Michigan
Committee on the Use and Care of Animals. The University of
Michigan Unit for Laboratory Animal Medicine provides veterinary
care. The University of Michigan is accredited by the American
Association of Accreditation of Laboratory Animal Health Care, and
the animal care use program conforms to the standards in the National
Institutes of Health Guide for the Care and Use of Laboratory
Animals [DHHS Publication No. (NIH) 86-23]. Animal protocols
were approved by the University of Michigan University Committee
on Use and Care of Animals.
Surgical preparation. Male New Zealand White rabbits (2.6 –3.2
kg body wt; Covance, Kalamazoo, MI) were anesthetized intramuscularly with xylazine (3.0 mg/kg) ⫹ ketamine (35 mg/kg) and then
intravenously with pentobarbital sodium (15 mg/kg). After insertion
of a cuffed endotracheal tube, the animals were placed on positivepressure ventilation using room air. The left jugular vein was isolated
and cannulated for drug administration. The left carotid artery was
isolated and instrumented with a Millar catheter micro-tip pressure
transducer (Millar Instrument, Houston, TX) positioned immediately
above the aortic valve to monitor aortic blood pressure. A lead II
electrocardiogram was monitored throughout the experiment. After a
left thoracotomy and pericardiotomy, the left anterior descending
coronary artery was identified. A 3-0 silk suture (Genzyme Biosurgery, Cambridge, MA) was passed under the artery and around a short
length of polyethylene tubing. Simultaneous downward displacement
of the polyethylene tubing during application of upward traction on
the suture resulted in occlusion of the coronary artery and cessation of
regional myocardial blood flow. Coronary artery occlusion was maintained for 30 min; then reperfusion was initiated by withdrawal of the
polyethylene tubing. Regional myocardial ischemia was verified by
the presence of a zone of cyanosis in the area of distribution of the
occluded vessel and changes in the electrocardiogram consistent with
the presence of transmural regional myocardial ischemia (ST segment
elevation).
Experimental protocol. Animals were allowed to stabilize for 15
min before beginning the protocol. Rabbits were randomized equally
among four treatment groups: E2 (20 ␮g), E2 ⫹ MPA (20 and 80 ␮g,
respectively), MPA (80 ␮g), and vehicle (1 ml of 20% DMSO-80%
polyethylene glycol). Thirty minutes after drug/vehicle administration, the left anterior descending coronary artery was subjected to 30
min of occlusion followed by 4 h of reperfusion. The hearts from eight
animals in each group were stained for determination of infarct size.
Tissue from the remaining five hearts in each group was used for
Western blot analysis and lipid peroxidation assays.
Determination of infarct size. After 4 h of reperfusion, the heart
was removed, the aorta was cannulated, and the coronary vascular bed
was perfused on a Langendorff apparatus with oxygenated KrebsHenseleit buffer at a constant flow of 22–24 ml/min for 10 min to
AJP-Heart Circ Physiol • VOL
clear the vascular compartment of plasma and blood cellular elements.
A 1% solution of triphenyltetrazolium chloride (TTC) in phosphate
buffer (pH 7.4, 37°C, 45 ml) was perfused through the heart. TTC
demarcates the noninfarcted myocardium within the AAR with a
brick-red color, indicating the presence of a formazan precipitate
resulting from the reduction of TTC by dehydrogenases in viable
myocardial tissue. Irreversibly injured tissue, lacking cytosolic dehydrogenases, is unable to form the formazan precipitate and appears
pale yellow. After TTC infusion, the left circumflex coronary artery
was ligated at the same site that was ligated during the induction of
regional myocardial ischemia. The perfusion pump was stopped, and
3 ml of a 0.25% solution of Evans blue dye was injected slowly
through a sidearm port connected to the aortic cannula. The dye
was passed through the heart for 10 s to ensure its uniform tissue
distribution. The presence of Evans blue dye was used to demarcate the left ventricular tissue that was not subjected to regional
ischemia (NLV), as opposed to the risk region (AAR). The heart
was removed from the perfusion apparatus and cut into three
transverse sections at right angles to the vertical axis. The right
ventricle, apex, and atrial tissue were discarded. The unblinded
investigator traced both surfaces of each transverse section onto
clear acetate sheets. The images were scanned and downloaded
into Adobe PhotoShop (Adobe Systems, Seattle, WA). For determination of the NLV, AAR, and infarct region, the number of
pixels occupying each area was calculated using Adobe PhotoShop
software. Total AAR is expressed as a percentage of the left
ventricle and infarct size as a percentage of the AAR.
Biochemical marker of irreversible myocardial injury. Plasma
concentrations of cardiac-specific troponin I (cTnI) were determined
by ELISA (Life Diagnostics, West Chester, PA). Briefly, serum was
prepared from whole blood drawn at baseline and 2 and 4 h after the
start of reperfusion: E2 (n ⫽ 9), E2 ⫹ MPA (n ⫽ 9), MPA (n ⫽ 9),
and vehicle (n ⫽ 9). Samples were frozen immediately in liquid
nitrogen and stored at ⫺80°C. On the day of the assay, samples were
thawed over ice and diluted appropriately with PBS. Protein concentrations were determined by comparison of the optical density of each
sample with a standard curve.
Immunofluorescent detection of MAC and CRP. MAC and CRP
levels in left ventricular tissue were determined as described previously (5). Briefly, tissue samples used for infarct size determination
were fixed in 10% buffered formalin immediately after completion of
the experimental protocol. The tissue samples were embedded in
paraffin blocks and cut into 2-␮m-thick sections, which were mounted
on glass slides. Two consecutive sections (mirror images) from a
single heart slice were mounted on each slide. The slides were washed
three times in xylene to remove the paraffin and rehydrated in an
ethanol gradient; they were placed in a boiling solution of a diluted
unmasking agent (Vector Laboratories, Burlingame, CA) and then
blocked with 5% milk for 45 min. Primary antibodies were incubated
at room temperature in a humidity chamber for 45 min. One section
per slide was incubated with a chicken anti-rabbit CRP antibody (5
␮g/ml final concentration; Immunology Consultants Laboratory,
Newberg, OR). The opposing transverse heart section was incubated
with a chicken anti-rabbit MAC antibody (1:2,500 final dilution;
developed in conjunction with Lampire Biological Laboratories, Pipersville, PA). After three consecutive washes in PBS-1% milk, each
section was incubated with a goat anti-chicken biotinylated secondary
antibody (1.5 ␮g/ml final concentration; Vector Laboratories) for 30
min. The slides were washed three times with PBS and then incubated
with fluorescein (Fluorescent Streptavidin Kit, Vector Laboratories)
for visualization of the proteins. For comparison, digital images were
captured using a digital camera (model DKC5000, Sony of America,
New York, NY) connected to a fluorescent stereoscope (model MX
FLIII, Leica, Wetzlar, Germany) and the accompanying software
(Leica). Images were analyzed using IP Lab software (Scanalytics,
Fairfax, VA) to determine mean fluorescence intensity per heart
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increased number of cardiovascular events during the first
years of the Heart and Estrogen/Progestin Replacement Study
trial. The increase in CRP was not observed when estrogen was
administered transdermally (17), suggesting that this increase
may not be an indicator of inflammation but, rather, an effect
of first-pass metabolism.
We previously demonstrated that acute estrogen administration alone significantly reduced infarct size and tissue
deposition of CRP and the membrane attack complex
(MAC) (5). The aim of the present study was to investigate
the anti-inflammatory effects of estrogen after myocardial
ischemia and reperfusion and determine whether treatment
with the combination of estrogen and the synthetic progesterone MPA reverses estrogen’s protective effects.
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ESTROGEN, PROGESTIN, AND ISCHEMIA-REPERFUSION INJURY
AJP-Heart Circ Physiol • VOL
RESULTS
Hemodynamic effects. Hemodynamic variables were obtained to determine the effects of E2 on arterial blood pressure
and heart rate. An insignificant decrease in arterial blood
pressure (5– 8 mmHg) followed by an immediate return to the
baseline values was observed after intravenous administration
of E2, MPA, E2 ⫹ MPA, and vehicle control. The rate-pressure
product [(mean arterial blood pressure ⫻ heart rate) ⫼ 100]
was used as an indicator of myocardial oxygen consumption.
Rate-pressure product decreased in each of the groups (E2,
E2 ⫹ MPA, MPA, and vehicle) from equilibration to 30 min
after treatment and then remained stable throughout the protocol, with no differences among the groups (Table 1).
Infarct size. E2, but not E2 ⫹ MPA, reduced infarct size.
Rabbits were randomized to three treatment groups: 20 ␮g of
E2, 20 ␮g of E2 ⫹ 80 ␮g of MPA, 80 ␮g of MPA, and vehicle
administered intravenously 30 min before myocardial ischemia
and 4 h of reperfusion. Infarcts, expressed as a percentage of
the AAR, were significantly smaller in E2- than in vehicletreated rabbits: 19.5 ⫾ 3.1 vs. 55.7 ⫾ 2.6% (P ⬍ 0.001). As
shown in Fig. 1A, addition of the progestin MPA to the
treatment regimen attenuated the infarct-sparing effect of E2
(52.5 ⫾ 4.6%), and MPA alone had no effect (50.8 ⫾ 2.6%).
The size of the AAR, expressed as a percentage of the total left
ventricle, was similar in each of the treatment groups: 57.1 ⫾
3.3, 57.8 ⫾ 3.0, 61.9 ⫾ 2.6, and 56.1 ⫾ 2.7% with vehicle, E2,
E2 ⫹ MPA, and MPA, respectively (Fig. 1B).
cTnI. Plasma concentrations of a biochemical marker of
irreversible injury are reduced after treatment with E2, but not
E2 ⫹ MPA. Plasma concentrations of cTnI were similar in all
groups at baseline. Plasma cTnI levels were significantly lower
in E2- than in vehicle-treated animals at 2 h (16.6 ⫾ 4.7 vs.
91.2 ⫾ 9.9 ng/ml, P ⬍ 0.05) and 4 h (28.8 ⫾ 6.7 vs. 103.5 ⫾
8.2 ng/ml, P ⬍ 0.05) after the onset of reperfusion. Plasma
cTnI concentration was reduced in E2 ⫹ MPA- and MAP-
Table 1. Effect of treatment on heart rate, mean arterial
blood pressure, and rate-pressure product in anesthetized
rabbits
HR, beats/min
Vehicle
E2
E2 ⫹ MPA
MPA
MABP, mmHg
Vehicle
E2
E2 ⫹ MPA
MPA
RPP, [(beats/min) ⫻
mmHg] ⫼ 100
Vehicle
E2
E2 ⫹ MPA
MPA
Baseline
30 min
After
Treatment
2h
4h
218⫾7.2
229⫾13.5
230⫾8.2
241⫾10.5
210⫾8.3
210⫾12.9
215⫾11.1
233⫾8.6
184⫾6.4
190⫾11.1
188⫾3.8
230⫾9.7
182⫾11.0
180⫾8.2
183⫾8.9
226⫾10.3
53.4⫾2.6
57.8⫾3.2
51.6⫾4.2
63.7⫾2.7
44.3⫾2.3
48.7⫾6.7
47.0⫾2.5
52.9⫾2.4
44.6⫾2.5
38.8⫾6.2
43.6⫾4.0
36.8⫾6.2
45.6⫾4.1
38.4⫾3.9
44.8⫾4.9
45.5⫾3.5
82.3⫾6.1
74.6⫾8.1
82.2⫾4.7
87.0⫾16.8
84.2⫾6.1
68.4⫾7.1
81.8⫾9.8
104⫾10.4
115.6⫾5.2
92.6⫾3.5
131.5⫾7.5
99.3⫾7.1
119.8⫾13.6 101.8⫾8.3
152⫾10.4
123⫾7.9
After Reperfusion
Values are means ⫾ SE. HR, heart rate; MABP, mean arterial blood
pressure; RPP, rate-pressure product; E2, 17␤-estradiol; MPA, medroxyprogesterone acetate.
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section. The sections were normalized to the amount of background
on each slide. The mean intensities for five hearts in each treatment
group were averaged and compared.
Assessment of complement inhibition. A red blood cell (RBC) lysis
assay was used to determine whether the pretreatment was able to
inhibit the rabbit complement system. The ex vivo analysis of complement activity is based on the C5b-9-dependent lysis of human
RBCs on exposure to rabbit plasma. Complement-mediated RBC
hemolysis was assessed by a turbidimetric method described previously (23). Rabbit serum was obtained from whole blood samples
drawn from rabbits that were treated with 20 ␮g of E2 (n ⫽ 11), 20
␮g of E2 ⫹ 80 ␮g of MPA (n ⫽ 11), 80 ␮g of MPA (n ⫽ 11), or
vehicle (1 ml of 20% DMSO-80% polyethylene glycol, n ⫽ 11). After
informed consent was obtained, human whole blood for the isolation
of RBCs was obtained by venipuncture of the forearm vein of a
healthy male donor who had not been exposed to any medication for
the past 7 days. The cells were washed three times in 10 ml of PBS
(pH 7.4) and diluted in PBS to achieve a final RBC concentration of
1 ⫻ 108 cells/ml. The assay was initiated by the addition of 15 ␮l of
diluted human RBCs to 185 ␮l of rabbit plasma, and the light
transmittance was monitored for 5 min. The final assay volume was
200 ␮l. Light transmittance (100%) was set with RBCs lysed with 1:1
rabbit plasma and deionized water.
Western blot analysis. Tissue samples from AAR and NLV were
homogenized in 1% SDS in Tris-buffered saline containing a protease
inhibitor cocktail (Complete Mini Protease Inhibitor, Boehringer
Mannheim). Homogenates were centrifuged at 7,200 g at 4°C for 15
min. The protein content of the supernatants was determined using a
bicinchoninic acid protein assay kit (Pierce, Rockford, IL). Forty
micrograms of protein were boiled for 10 min and loaded on a 10%
Tris-glycine gel (Bio-Rad, Hercules, CA). After 90 min of electrophoresis (130 V), the proteins were transferred to nitrocellulose
membranes at 40 V overnight at 4°C. Membranes were blocked in 5%
nonfat milk for 1 h before incubation with anti-rabbit C3 antibody
(1:500 dilution; ICN Biomedicals, Aurora, OH). Immunoblots were
then washed twice with TBS-0.05% Triton X-100 and then incubated
with anti-goat IgG (1:5,000 dilution; Sigma) labeled with horseradish
peroxidase. Immunoreactivity was visualized with the enhanced
chemiluminescence substrate kit (Amersham Biosciences, Piscataway, NJ). Images were captured using the EpiChem3 Darkroom
(UVP, Upland, CA), and the mean density of the bands was determined using Lab Works analysis software (UVP). The bands were
normalized to actin for determination of the intensity.
MDA measurements. The levels of malondialdehyde (MDA) in the
AAR and NLV were determined as an indicator of lipid peroxidation
(22). After 4 h of reperfusion, heart tissue was removed and immediately frozen until use. Frozen sections were homogenized in 1.15%
(wt/vol) KCl solution. A 100-␮l aliquot of the homogenate was added
to a reaction mixture containing 200 ␮l of 8.1% (wt/vol) SDS, 1,500
␮l of 20% (wt/vol) acetic acid (pH 3.5), 1,500 ␮l of 0.8% (wt/vol)
thiobarbituric acid, and 700 ␮l of distilled water. Samples were then
heated for 1 h at 95°C and centrifuged at 4,000 rpm for 10 min. The
absorbance of the supernatant was measured by spectrophotometry at
515–553 nm.
Materials. Unless otherwise noted, all materials were purchased
from Sigma Chemical (St. Louis, MO).
Statistical analysis. Values are means ⫾ SE. Differences between
control and experimental groups were determined using a one-way
ANOVA for multiple groups or repeated measures. Differences between groups were determined using Bonferroni’s post test. For cTnI,
differences within each time point were compared using Student’s
t-test for unpaired comparisons. P ⬍ 0.05 was considered to be
significant. Statistical analysis was performed using GraphPad Prism
(GraphPad Software, San Diego, CA).
ESTROGEN, PROGESTIN, AND ISCHEMIA-REPERFUSION INJURY
treated animals but was not significantly different from the
plasma cTnI concentration in vehicle-treated animals (Fig. 2).
Immunofluorescence. Immunofluorescence for MAC and
CRP was reduced in hearts from E2-, but not E2 ⫹ MPAtreated animals. Hearts from vehicle-treated animals demonstrated robust antibody staining with chicken anti-rabbit MAC
and chicken anti-rabbit CRP antibodies, indicating the deposition of both proteins in the myocardial region subjected
to ischemia and reperfusion. Conversely, in hearts from
E2-treated animals, fluorescence and deposition of CRP and
MAC were reduced. Fluorescence was similar in heart tissue
from E2 ⫹ MPA- and MPA-treated rabbits and vehicle-treated
rabbits (Fig. 3A). In tissue sections stained for MAC or CRP,
the mean intensity of fluorescence was significantly less (P ⬍
0.001) in E2- than in vehicle-treated animals (Fig. 3, B and C).
Mean fluorescence intensity of heart sections from animals
treated with E2 ⫹ MPA or MPA was similar to that from
vehicle-treated animals.
Assessment of complement inhibition. E2 treatment reduced
mean RBC hemolysis. The RBC hemolysis assay was used to
determine the ability of E2 to inhibit the activation of the
complement system. Hemolysis of human RBCs was reduced
in serum from rabbits pretreated with E2 compared with serum
from vehicle-treated animals: 41.6 ⫾ 8.6 vs. 72.3 ⫾ 6.1% (P ⬍
AJP-Heart Circ Physiol • VOL
0.05). As shown in Fig. 4, the inhibitory effect of E2 on RBC
lysis was prevented by addition of MPA (66.3 ⫾ 7.3%) or
E2 ⫹ MPA to the treatment regimen (76.9 ⫾ 5.9%).
Western blot analysis. Hearts from animals treated with E2,
but not E2 ⫹ MPA, demonstrated a reduction in complement
C3 protein. Myocardial tissue from the AAR and NLV of
animals treated with vehicle, E2, E2 ⫹ MPA, or MPA was
subjected to Western blot analysis. Western blot analysis
demonstrates bands for C3 in the soluble extract of the myocardial tissue from the AAR of hearts of animals treated with
vehicle, E2 ⫹ MPA, or MPA and subjected to ischemia and
reperfusion (Fig. 5A). In contrast, there was less detectable C3
in heart tissue from animals treated with E2, suggesting that E2
attenuated C3 protein expression. The C3 product displayed
bands at 80 kDa, as reported previously (36). The normalized
intensity of bands for samples from five animals is shown in
Fig. 5B.
MDA measurements. E2, but not E2 ⫹ MPA, reduced lipid
peroxidation, as assessed by the measurement of MDA. The
thiobarbituric acid reaction was used to quantitate the presence
of lipid peroxidation products in the AAR of hearts from
animals treated with E2, E2 ⫹ MPA, MPA, or vehicle and
subjected to ischemia and reperfusion. Acute treatment with E2
significantly decreased the amount of lipid peroxidation in
hearts compared with vehicle treatment, as determined by
MDA levels: 257.8 ⫾ 83.4 vs. 69.9 ⫾ 18.2 ␮M MDA/g tissue
(P ⬍ 0.05). E2 ⫹ MPA or MPA decreased the level of lipid
peroxidation compared with vehicle: 186.4 ⫾ 42.0 and
158.4 ⫾ 54.8 ␮M MDA/g tissue, respectively [P ⬎ 0.05 (not
significant)]. MDA levels significantly increased in animals
treated with vehicle, E2 ⫹ MPA, or MPA in the AAR compared with the NLV. There was no significant increase in the
AAR of E2-treated animals (Fig. 6).
DISCUSSION
Inflammation serves an important role in the pathogenesis
of many forms of disease, including myocardial ischemiareperfusion injury. Treatment with E2 results in anti-inflammatory effects that may be protective in the setting of ischemiareperfusion injury (7, 26, 31). The most notable finding of the
Fig. 2. Serum levels of cardiac-specific troponin-I (cTnI) as determined by
ELISA. E2 significantly reduced serum concentrations of cTnI at 2 and 4 h of
reperfusion compared with vehicle. Serum cTnI levels were reduced in animals
treated with 20 ␮g of E2 ⫹ 80 ␮g of MPA and 80 ␮g of MPA but were not
significantly lower than control. Values are means ⫾ SE (n ⫽ 9). ***P ⬍
0.001.
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Fig. 1. Effects of 17␤-estradiol (E2) and E2 ⫹ medroxyprogesterone acetate
(MPA) on infarct size in New Zealand White rabbits. A: E2 (20 ␮g) significantly decreased infarct size, expressed as percentage of area at risk (AAR),
whereas effect of E2 (20 ␮g) ⫹ MPA (80 ␮g) was similar to vehicle. MPA (80
␮g) had no effect on infarct size. B: risk regions were similar between groups.
LV, left ventricle. Values are means ⫾ SE (n ⫽ 8). ***P ⬍ 0.001.
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ESTROGEN, PROGESTIN, AND ISCHEMIA-REPERFUSION INJURY
present study is that E2 has a significant effect on CRP
expression and complement-mediated injury associated with
ischemia and reperfusion. In contrast to other studies, progesterone alone did not demonstrate a robust proinflammatory
effect; however, treatment with a combination of progesterone
and estrogen reversed the protective effects of E2 after ischemia.
Previous studies demonstrated the anti-inflammatory capacity of E2, and our laboratory showed that E2 modulates the
activation of the tissue-associated complement system in the
myocardium in response to reperfusion injury (5). The present
Fig. 4. Complement-mediated RBC hemolysis assay conducted after administration of vehicle, 20 ␮g of E2, 20 ␮g of E2 ⫹ 80 ␮g of MPA, or 80 ␮g of
MPA. Human RBCs were used as the target cell and rabbit serum drawn after
treatment as the source of complement proteins. Hemolytic response was
followed for 5 min. Values are means ⫾ SE (n ⫽ 11). *P ⬍ 0.05.
AJP-Heart Circ Physiol • VOL
study expands on these findings by demonstrating that E2
inhibits complement activation, as determined by a reduction
in complement-mediated RBC lysis and C3 protein expression.
C3 protein plays a central role in the classical and alternative
pathways of the complement cascade. The basal tissue and
serum concentrations of C3 are greater than other complement
components, and C3 concentration is amplified on activation of
the complement system. Although not statistically significant,
E2 was associated with a trend toward a decrease in tissue
protein levels of C3. Complement activity was assessed via the
ex vivo RBC lysis assay. In the present study, there was a
significant decrease in RBC lysis in the presence of E2 that was
negated when E2 was administered in combination with MPA.
Although activation of the complement cascade contributes
to the pathogenesis of ischemia-reperfusion injury, individual
complement components can trigger signaling pathways that
alter cellular responses without causing cell death. Although
the effects of C5a can be detrimental to the tissue, it has been
shown to elicit neuroprotective and cardioprotective effects
in vivo (24, 35). In vitro pretreatment with sublytic concentrations of complement has been reported to prevent cellular
damage from a subsequent exposure to a normally lytic concentration of complement (28, 29). In an in vivo experiment,
Tanhehco et al. (35) demonstrated the ability of sublytic levels
of complement to reduce infarct size after ischemia and reperfusion. It is possible that although estrogen does not completely inhibit the activation of complement after ischemia and
reperfusion, the level of activation is decreased, and the function of the sublytic complement is to exhibit protective cellular
responses without precipitating cell death. The addition of
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Fig. 3. A: representative immunofluorescent
images of hearts from animals treated with 20
␮g of E2, 20 ␮g of E2 ⫹ 80 ␮g of MPA, 80
␮g of MPA, or vehicle and stained for membrane attack complex (MAC) and C-reactive
protein (CRP). Hearts from 5 animals per
group were analyzed. Areas of bright deposits
indicate areas of MAC formation and CRP
deposition. The most intense staining is localized to the risk region of vehicle-, E2 ⫹
MPA-, and MPA-treated hearts. B and
C: mean intensity of staining for MAC and
CRP was significantly reduced in hearts from
animals treated with E2. Values are means ⫾
SE. ***P ⬍ 0.001.
ESTROGEN, PROGESTIN, AND ISCHEMIA-REPERFUSION INJURY
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progestin reverses this effect and, thereby, increases complement to levels that cause irreversible tissue injury.
Free radicals are involved in mediating the tissue injury
associated with the inflammatory process, including the activation of the complement cascade and tissue expression of
CRP (21). Lipid peroxidation represents a direct manifestation
of the deleterious effects of free radicals on cell membranes,
and initiation of intracellular signaling cascades by free radicals may propagate tissue injury (1, 43). In the present study,
we demonstrated a reduction in lipid peroxidation after E2
treatment that was not observed when MPA was added to the
treatment protocol. MPA alone caused a slight, but nonsignificant, decrease in lipid peroxidation. A reduction in free radical
activity, as demonstrated by reduced lipid peroxidation, may
contribute to the reduction in MAC and CRP activation in
hearts from E2-treated animals.
The present study clearly demonstrates that E2 protects the
heart from ischemia-reperfusion injury, most likely through
modulation of the inflammatory response to injury. The results
with the synthetic progesterone MPA are not as clear. The
addition of MPA to the treatment regimen significantly re-
Fig. 6. Malondialdehyde (MDA) levels in AAR and NLV of hearts from
animals treated with vehicle, E2, E2 ⫹ MPA, or MPA. Ischemia and reperfusion of the myocardium leads to a considerable increase in MDA levels in the
AAR, which is inhibited by E2, but not E2 ⫹ MPA or MPA. Values are
means ⫾ SE. *P ⬍ 0.05 vs. vehicle. ^^P ⬍ 0.01 vs. NLV. ^P ⬍ 0.05 vs. NLV.
AJP-Heart Circ Physiol • VOL
verses the protective effects of estrogen, as evaluated by infarct
size, serum cTnI levels, complement deposition and activation,
and lipid peroxidation. This reversal of estrogen’s action with
the addition of progesterone is consistent with previous observations that addition of the progestin MPA, but not norethindrone acetate, inhibited ethinyl estradiol’s infarct-sparing effects. Progesterone has also been shown to counteract estrogen’s protective effects after experimental stroke and
cerebrovascular inflammation (34). Treatment with MPA alone
caused a nonsignificant reduction in infarct size and cTnI
release. The MDA levels suggest a trend toward a reduction in
lipid peroxidation with MPA alone, which may suggest a
mechanism for the reduction of infarct size and the corresponding reduction of troponin levels. The trend toward an increase
in complement-mediated activation after treatment with MPA,
although not significant, agrees with the results of Sunday et al.
(34) that progesterone exacerbated the inflammatory response
in the cerebrovasculature and that this exacerbation opposed
the protective anti-inflammatory effects of E2. These proinflammatory effects of MPA may counteract the protective
effects on lipid metabolism, vascular reactivity, and atherosclerotic progression. The exact mechanism by which MPA modulates the inflammatory response to injury and negates estrogen’s infarct-reducing effects remains to be determined.
Treatment with the progestin MPA does not fully account
for the negative effects observed in the clinical trials. In the
Women’s Health Initiative, unopposed estrogen treatment did
not increase or decrease the risk of coronary heart disease but
did result in an increase in stroke (2, 19). There was an increase
in cardiovascular events in the first year of the Women’s
Health Initiative, which is possibly due to the associated
increase in CRP (30). Increases in CRP may be harmful,
because CRP has a proatherogenic role: it binds to and activates complement, facilitates LDL uptake by macrophages,
and increases plasminogen activator inhibitor-I expression and
activity, thereby inhibiting fibrinolysis. This increase in CRP,
which is not seen with transdermal estrogen, may partially be
a result of the route of administration.
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Fig. 5. Western blot analysis of cytosolic
extracts from the AAR (A) and normal left
ventricular region (NLV, C) of hearts from
rabbits subjected to ischemia and reperfusion. Positions of the molecular mass markers are shown at left. Bands for C3 (at ⬃80
kDa) appear in the AAR of rabbit hearts
treated with vehicle, E2 ⫹ MPA, and MPA
but are less distinct in the AAR of hearts
treated with E2. B and D: relative intensity
for AAR and NLV regions in bands normalized to actin.
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ESTROGEN, PROGESTIN, AND ISCHEMIA-REPERFUSION INJURY
GRANTS
This study was funded by the Cardiovascular Research Fund of the
University of Michigan Medical School.
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