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© 1995 American Heart Association, Inc.
Articles |
Effect of 17
-Dihydroequilin Sulfate, a Conjugated Equine Estrogen, and Ethynylestradiol on Atherosclerosis in Cholesterol-Fed Rabbits
From the Department of Pathology (S., J.J., R.W.S.C.), The Bowman Gray School of Medicine of Wake Forest University, Winston-Salem, NC, and the Cardiovascular Diseases and Drug Safety and Metabolism Divisions (S.J.A., A.C.), Wyeth-Ayerst Research, Princeton, NJ.
Correspondence to Richard W. St. Clair, PhD, The Bowman Gray School of Medicine of Wake Forest University, Department of Pathology, Medical Center Blvd, Winston-Salem, NC 27157-1086.
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Abstract |
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Abstract The effect of 17
-dihydroequilin sulfate (DHES), a
water-soluble estrogen of conjugated estrogens (Premarin), and
ethynylestradiol (EE), a commonly used estrogen found in many oral
contraceptives, on the development of atherosclerosis was studied in
rabbits fed an atherogenic diet (0.2% cholesterol) for 24 weeks. Ten
animals were given 15
µg ·kg-1 · d-1 EE, 10 received
3.8 mg · kg-1 · d-1 of DHES, and the
remaining 10 sham-ovariectomized and 10 ovariectomized animals served
as cholesterol-fed controls. These doses were chosen to have similar
estrogenic potency. Plasma cholesterol concentrations increased to
about 900 mg/dL and did not differ among the experimental groups. After
24 weeks, plasma ß-VLDL and HDL cholesterol concentrations were the
same for all cholesterol-fed groups, while LDL cholesterol was
significantly higher in the two estrogen-treated groups. In spite of
this, both EE and DHES significantly reduced atherosclerosis by 35% in
the aortic arch and 75% to 80% in the thoracic and abdominal aorta.
The reduction in atherosclerosis was seen in animals with a wide range
(400 to 1400 mg/dL) of plasma cholesterol concentrations and was
independent of lipoprotein profile. ß-VLDL isolated from
estrogen-treated animals was not significantly different from control
ß-VLDL in its ability to stimulate cholesterol accumulation in THP-1
macrophages in culture. This suggests that the protective effect of
estrogens on the development of atherosclerosis is not mediated by
qualitative differences in ß-VLDL that affect uptake by macrophages.
The results of this study extend our knowledge of the range of
estrogens that reduce atherosclerosis. Given the lack of effect on
plasma lipid and lipoprotein concentrations, these data are consistent
with the conclusion that estrogens exert some of this beneficial effect
directly at the level of the arterial wall by influencing certain key
components in the pathogenesis of atherosclerosis.
Key Words: estrogens • lipoproteins • macrophages • ß-VLDL • HDL
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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There is considerable evidence that estrogens protect against the development of coronary heart disease in women.1 2 Although under certain conditions estrogens lower the LDL and increase the HDL concentrations in the blood, only about 25% of the beneficial effect of estrogens on the risk of coronary heart disease in women can be explained by changes in plasma lipoprotein profile.1 This suggests that estrogens may influence the development of atherosclerosis by other mechanisms, such as a direct effect on the artery wall. This conclusion is supported by results from several studies with cholesterol-fed rabbits in which estrogen treatment was associated with a significant reduction in aortic atherosclerosis that was independent of changes in plasma cholesterol or lipoprotein concentrations.3 4 5 Studies with nonhuman primates have also shown that estrogens and estrogen/progestin-containing oral contraceptives reduce atherosclerosis even though the changes in plasma lipoprotein profile and lipoprotein composition would have predicted greater atherosclerosis.6 The mechanism by which estrogens exert this beneficial effect on atherosclerosis is unknown, but it may involve one or more aspects of arterial wall lipoprotein metabolism.4 7
To our knowledge, the only estrogens that have been used in published
studies on experimental atherosclerosis in rabbits and nonhuman
primates are 17ß-estradiol and ethynylestradiol (EE). Although
administered by different routes at different doses with or without
combination with progestins (EE has been used only in combination with
progestins), both of these estrogens reduce
atherosclerosis.5 6 Premarin, a complex mixture of
water-soluble conjugated estrogens extracted from pregnant mares'
urine, is the most widely used drug for estrogen replacement therapy in
postmenopausal women in the United States.8 Nevertheless,
neither conjugated estrogens nor any of their individual components
have been tested for their effects on experimental atherosclerosis. Of
the 10 estrogens in conjugated estrogens that have been identified,
17-dihydroequilin sulfate (DHES) is the third most abundant, making
up about 15% of the total.9 10 DHES, similar to other
steroid sulfates, is deconjugated by the enzyme aryl sulfatase in a
variety of tissues to produce the active form of
17-dihydroequilin.9 10 As a part of other studies on
the cardioprotective effects of conjugated estrogens, sufficient
quantities of DHES were isolated and available to test its effects on
atherosclerosis. Thus, the purpose of the present study was to test
the effect of DHES on the development of aortic atherosclerosis in
ovariectomized rabbits fed an atherogenic diet. This is also the first
study of which we are aware that considers the effect of EE alone on
atherosclerosis. The doses of EE and DHES were chosen to provide
approximately equivalent estrogenic potency.
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Animals and Experimental Design
Adult female New Zealand White rabbits (n=45; weight, 3 to 4 kg) were obtained from the Franklin Rabbitry. They were individually housed in stainless steel cages in a room with a 12-hour light cycle and initially were fed a diet of commercial rabbit chow (Agway PROLAB rabbit formula). Thirty animals were ovariectomized (Ovx) and 15 were sham ovariectomized (Sham Ovx). Ovariectomy was carried out by using sterile procedures in animals anesthetized with ketamine (20 to 40 mg/kg IM) and xylazine (5 mg/kg IM). This procedure and the overall experimental protocol were approved by the Wake Forest University Animal Care and Use Committee. The animals were allowed to recover from surgery for 14 days and were then randomly allocated into five groups according to the experimental design shown in Fig 1
. The
Sham-Ovx animals were divided into two groups. Five animals
received cholesterol-free rabbit chow containing 5% (wt/wt) corn
oil, and the remaining 10 animals were fed the same rabbit chow to
which cholesterol was added at a final concentration of 0.2%
cholesterol. The cholesterol was dissolved in the warm corn oil before
adding it to the rabbit chow. This diet will be referred to as the
"cholesterol diet." The 30 Ovx animals were divided into groups
of 10 animals each and were fed the cholesterol diet alone or the
cholesterol diet plus EE (Sigma) or DHES (Wyeth-Ayerst). EE was added
to the diet by dissolving it in the warm corn oil. DHES was dissolved
in a few milliliters of water and added while the diet was being mixed
in a mechanical mixer. These diets were fed for the first 7 weeks at 35
g · kg-1 · d-1. During this time the
actual food consumption of each rabbit was recorded daily. From this we
found that animals receiving EE ate approximately 30% less than the
other animals. As a result, for the remaining 16 weeks of the study all
animals were fed at the same rate of 25
g · kg-1 · d-1 so that they would all
consume their entire diet daily. Since the estrogens were incorporated
into the diets, EE administered for the first 7 weeks was at a dose of
21 µg · kg-1 · d-1, and for
DHES the dose was 5.0
mg · kg-1 · d-1, both based on
a maximum food intake of 35
g · kg-1 · d-1. For the final 17
weeks of the experiment the diet intake was reduced to 25
g · kg-1 · d-1 for all animals. This
reduced the estrogen dosages to 15
µg · kg-1 · d-1 for EE and 3.8
mg · kg-1 · d-1 for DHES. The animals
had free access to water and the indicated diets for the 24-week
experimental period.
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Plasma Lipids and Lipoproteins
Body weight, total plasma cholesterol,
triglycerides, and HDL cholesterol (HDL-C) were determined
at baseline (after ovariectomy but before starting the experimental
diets) and after 4, 9, 14, 19, and 24 weeks of consuming the
experimental diets. At the time of necropsy (24 weeks), plasma
lipoprotein cholesterol distribution was measured as described below.
Animals were fasted overnight and were bled from the central artery of
the ear. Blood was collected in tubes containing 1 mg/mL EDTA. Total
plasma cholesterol and triglycerides were measured by using
automated enzymatic procedures. The cholesterol procedure is based on
the method of Allain et al11 ; the triglyceride
procedure is based on the method of Fossati and
Prencipe.12 HDL-C was determined by the
heparinmanganese precipitation method.13 At the time
of necropsy, lipoprotein cholesterol distribution was determined on
blood samples taken from the ear. In addition, 30 to 50 mL blood was
collected in tubes containing EDTA from the vena cava and used for
isolation of ß-VLDL.14 Total cholesterol, HDL-C, and
ß-VLDL cholesterol were measured. LDL cholesterol (LDL-C) was
calculated from the following formula: LDL-C=(total
cholesterol)-(HDL-C)-(ß-VLDL cholesterol). The isolated ß-VLDL
was also used for the cell-culture study described below.
Necropsy and Evaluation of Aortic Atherosclerosis
After 24 weeks the animals were anesthetized with ketamine
hydrochloride (20 to 40 mg/kg IM) and killed with an overdose of sodium
pentobarbital (100 mg/kg IV). The entire aorta, from the heart to the
iliac bifurcation, was removed and dissected free from adventitia. The
freshly removed aorta was opened longitudinally for its entire length.
The aorta was divided into arch, thoracic, and abdominal segments at
the level of the ductus arteriosus scar, the celiac artery, and the
iliac arteries, respectively, and each segment was photographed in
black and white. From these photographs, the areas covered with
atherosclerotic plaques in the arch, thoracic aorta, and abdominal
aorta were measured by using a computer digitizer15 ; the
operator was blinded to the treatment group. The percent of the total
intimal surface of each aortic segment covered with grossly visible
atherosclerosis was calculated. The aortic segments were kept on ice
and moist with saline during the entire procedure. Subsequently, all
segments were weighed and stored frozen (-20°C) until analyzed for
cholesterol content.
For measurement of aortic cholesterol content, the aortic segments were first minced with scissors and homogenized in chloroform/methanol (2:1) by using a glass homogenizer. Lipids were extracted according to the method of Folch et al.16 Total cholesterol was measured by the method of Rudel and Morris17 on a known volume of the chloroform extract. Another aliquot was taken for the separation of free and esterified cholesterol by thin-layer chromatography (TLC) on precoated glass plates of silica gel 60 (Merck) by using a solvent system of hexane/diethyl ether/acetic acid (146:50:4, vol/vol/vol), and cholesterol in the free and esterified fractions was measured.18 Recovery was corrected by using a [3H]cholesterol internal standard added to the initial Folch extract. Following extraction of lipids, the lipid-free dry weight of the tissue was determined by using gravimetric methods in tubes that had been tare-weighed prior to adding the tissue homogenate.
At necropsy the major reproductive organs were removed, and the vagina and uterus were weighed to determine if their weights were affected by estrogen treatment.
Plasma Concentrations of 17-Dihydroequilin and EE
Plasma concentrations of total (conjugated plus unconjugated)
17-dihydroequilin were measured by high-performance liquid
chromatography (HPLC). To 1 mL of plasma from treated animals or spiked
control plasma was added 1 mL 0.2 mol/L sodium acetate buffer, pH 5.0,
100 ng internal standard (
6,7-dehydroestrone), and 0.2
mL ß-glycuronidase/sulfatase (Sigma). The samples were incubated at
37°C for 1 hour. The samples were cooled to room temperature and
extracted with 15 mL diethyl ether. The organic phase was evaporated to
dryness, and the residue was reconstituted in 1 mL mobile phase. An
aliquot of the extract was injected onto a C-6, 4.6x250-mm, 5-µ
column (Alltech) and eluted with a mobile phase consisting of water,
methanol, butanol, and phosphoric acid (56:40:3:1). The mobile phase
was pumped at a flow rate of 0.75 mL/min. 17-Dihydroequilin had a
retention time of 41 minutes and was detected by a dual-electrode
coulometric detector operated in the oxidative-screen mode. The
electrochemical detector potentials were set as follows on an ESA
Coulochem II model 5200 detector: guard cell, 750 mV; electrode
1, 350 mV; and electrode 2, 700 mV. The signals from electrode 2 were
captured on a Spectra-Physics Integrator.
The recovery of 17-dihydroequilin from rabbit plasma was 76%. The
specificity of the HPLC method was assessed for the absence of
significant interfering peaks in the scans of control rabbit plasma
extracts and for interference from potential metabolites; under the
conditions described no interfering peaks were observed. The assay was
linear over a range of 100 to 1000 ng/mL. Typical correlation
coefficients of the standard curves were greater than .99. The intraday
precision of the method, described as the coefficient of variation for
replicate samples, ranged from 1.8% to 6.3%. The accuracy of the
assay, estimated as the percent difference between mean
17-dihydroequilin found and the theoretical value, varied between
-1.9% and 3.6% and was independent of the concentration measured.
Interday precision and accuracy varied between 3.5% and 8.7% and
-6.3% to 2.5%, respectively, and were independent of the
concentration.
Plasma concentrations of EE were determined by a radioimmunoassay. Based on a 1-mL plasma sample, the lower limit of quantification was 40 pg/mL.
Incubation of Rabbit ß-VLDL With THP-1 Macrophages
The THP-1 monocyte-like cell line19 was obtained
from the American Type Culture Collection and was maintained in
suspension in RPMI-1640 medium containing 10% heatinactivated
fetal bovine serum, 2-mercaptoethanol (5x10-5 mol/L),
glutamine (200 mmol/L), penicillin (100 IU/mL), streptomycin (100
µg/mL), and Eagle's vitamins. To differentiate THP-1 cells into
macrophages, cells were seeded into 35-mm culture dishes
(1x106 cells/dish) in RPMI-1640 medium containing
12-O-tetradecanoylphorbol 13-acetate (TPA; 50 ng/mL) and
were incubated for 72 hours. Following this incubation, cells were
washed with phosphate-buffered saline20 to remove
unattached cells, and fresh medium containing lipoprotein-deficient
serum, TPA, and rabbit ß-VLDL, isolated individually from each animal
at the time of necropsy, was added to the cells and incubated for an
additional 48 hours. The ß-VLDL was added at a final concentration of
100 µg protein/mL. The ß-VLDL could be added at equivalent protein
concentrations since the total cholesterol/protein ratio was similar
for ß-VLDL from all cholesterol-fed treatment groups (Ovx 8.18±0.22,
n=7; Sham Ovx 7.36±0.60, n=9; EE 7.53±0.39, n=9; DHES 8.13±0.72,
n=9). Cholesterol esterification was measured after 48 hours of
incubation with ß-VLDL by determining the incorporation of
[3H]oleate into cholesteryl esters.21 In
parallel incubations done in separate dishes, cellular free and total
cholesterol mass were measured by gas-liquid
chromatography,18 and esterified cholesterol mass was
calculated as the difference between total and free cholesterol. All
incubations were carried out in triplicate. Cell protein was determined
by the method of Lowry et al22 using bovine serum albumin
as the standard.
Statistics
Unless indicated otherwise, results are expressed as mean±SEM.
On data that satisfied the assumptions for a parametric test, either a
paired t test, one-way ANOVA, or two-way ANOVA was used for
analysis to identify the presence of statistically significant
differences among groups, with comparisons between groups performed by
using the Student-Newman-Keuls test. For data that did not satisfy
assumptions for parametric tests, the nonparametric Kruskal-Wallis one-
or two-way ANOVA on ranks was used. All analyses were performed by
using the SIGMASTAT software program (Jandel Scientific).
Values were considered statistically significantly different if
P<.05.
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Plasma Lipids and Lipoproteins
Baseline values of body weight and plasma total cholesterol, HDL-C, and triglyceride concentrations for the five experimental groups are shown in Table 1
. All animals
were consuming commercial rabbit chow at the time of these analyses.
Baseline values were obtained 7 to 10 days after ovariectomy or sham
ovariectomy. There were no significant differences between groups.
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After initiation of cholesterol feeding, there was a rapid increase in
plasma total cholesterol concentrations in all groups that began to
plateau in the non–estrogen-treated animals by about week 9 (Fig 2). The plateau in plasma cholesterol concentration was
reached slightly later in the estrogen-treated groups. As a result, the
mean plasma cholesterol concentration over time was calculated by
measuring the area under the plasma cholesterol curve over the full 24
weeks of the study. These values represent a more realistic
average of the plasma cholesterol concentration to which the arteries
were exposed over the duration of the study and will correct for the
differences in the shape of the plasma cholesterol curves over time
between the estrogen- and non–estrogen-treated groups. A similar
procedure has been used by other investigators.5 23 This
will be referred to as the "weighted" plasma cholesterol
concentration. A weighted average was also calculated for plasma HDL-C
and triglyceride concentrations. The weighted mean plasma
cholesterol concentrations in the four cholesterol-fed groups ranged
from 833 to 993 mg/dL (P=NS) compared with 72 mg/dL for
chow-fed control animals (Table 2). Differences in
plasma HDL-C concentrations among the four cholesterol-fed groups were
not significant, and there was a nonsignificant trend for
triglyceride concentrations to be lower in the two groups
receiving estrogens. Table 3 shows the distribution of
plasma lipoprotein cholesterol measured at the time of necropsy, when
total plasma cholesterol concentrations were nonsignificantly higher in
the estrogen-treated animals. ß-VLDL cholesterol concentrations also
were not different among the groups, with concentrations ranging from
468 to 600 mg/dL. In contrast, LDL-C concentrations were significantly
higher by approximately 200 mg/dL in the estrogen-treated groups.
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), 9 sham-ovariectomized, cholesterol-fed (
), 9
ovariectomized, cholesterol-fed (
), 9 ovariectomized,
cholesterol-fed, ethynylestradiol-treated (
), and 7
ovariectomized, cholesterol-fed, 17
) animals, respectively. P<.001 control vs all other
groups.
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Plasma Estrogen Concentrations and Their Effect on Reproductive
Organ Weights
The concentration of 17-dihydroequilin in the plasma of rabbits
at different times throughout the 24 weeks is shown in Fig 3. As indicated in "Methods," during the initial 7
weeks of the experiment all animals received a higher dose of the drug
than during the remaining 17 weeks. This is reflected by the drop in
17-dihydroequilin concentrations in the plasma at week 8. EE could
not be detected in the plasma of any of the animals receiving this
hormone, which implies that the concentration was less than the lower
limit of sensitivity (40 pg/mL) of the method at the time of
analysis. This is due, most likely, to the fact that EE has a
relatively short half-life in plasma,9 24 and by fasting
the animals for 24 hours prior to blood collection there was
insufficient EE remaining to detect. In contrast, plasma levels of
17-dihydroequilin were readily detected. With DHES at 3.8
mg · kg-1 · d-1 (PO), the plasma
levels of 17-dihydroequilin averaged 274±125 ng/mL (weeks 8 through
24) in animals fasted for 24 hours. The higher concentrations of
17-dihydroequilin in the plasma after a 24-hour fast relative to EE
are due to the much higher dose of DHES used to achieve an equivalent
estrogenic potency; additionally, its half-life in the plasma is much
longer.9
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The weights of the uterus and vagina at necropsy for all animals are
shown in Fig 4. Ovx animals without hormone treatment
had the lowest weights, followed by Sham-Ovx animals. Both groups of
estrogen-treated animals had substantial and equivalent increases in
organ weights, suggesting that both hormones were present in
sufficient concentration to have a similar or maximum effect on the
weight of these estrogen-responsive organs.
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Aortic Atherosclerosis
Atherosclerosis was evaluated by two methods: percent surface area
involvement with grossly visible atherosclerotic lesions and
cholesterol content. The aortic arch, thoracic aorta, and abdominal
aorta were evaluated separately. Results are expressed per milligram
wet weight of aortic tissue since the estrogens had no effect on total
lipid-free dry weight or the wet weight–to–lipid-free dry weight
ratio (data not shown). Free, esterified, and total cholesterol content
and percent surface area involvement were used for evaluation of
atherosclerosis. In the chow-fed Sham-Ovx control animals there was no
grossly visible atherosclerosis, and aortic cholesterol content
averaged about 1 mg/g wet weight with less than 10% as esterified
cholesterol (Table 4). Cholesterol feeding induced
atherosclerosis in both Sham-Ovx and Ovx animals, but there was no
significant difference in the severity of atherosclerosis between
Sham-Ovx and Ovx animals regardless of the lesion parameters measured
(Table 4). As a result, we combined the data from the cholesterol-fed
Sham-Ovx and Ovx groups into a single non–estrogen-treated control
group containing 18 animals, against which the two estrogen treatment
groups were compared for statistical analysis. The effect of
estrogen treatment on atherosclerosis in the three aortic sites is
shown in Table 5. Significant (P<.001)
differences in atherosclerosis were seen in the different aortic sites
and with estrogen treatment. The greatest amount of atherosclerosis was
seen in the aortic arch, followed by the thoracic and abdominal aorta.
In the cholesterol-fed control animals, atherosclerotic lesions covered
an average of 67.0% of the surface area of the aortic arch, compared
with 25.2% for the thoracic aorta and 17.9% for the abdominal aorta.
These differences among the aortic segments were highly significant
(P<.001), with significant differences between the aortic
arch and the abdominal and thoracic aorta but not between the thoracic
and the abdominal aorta (Table 5). A similar difference in distribution
of atherosclerosis among the arterial segments was seen with arterial
cholesterol content. EE and DHES reduced aortic atherosclerosis
significantly (P<.001) and to about the same extent. This
beneficial effect of estrogens was seen in all three arterial segments
but was greatest in the thoracic and abdominal aorta, where there was a
greater than 70% reduction in atherosclerosis as measured by
percent surface area, compared with only about a 35% reduction in
atherosclerosis in the aortic arch. As seen from the probability values
for the interaction term (segmentxtreatment) in Table 5, this
difference in the effect of estrogens on specific arterial segments
approached significance only for percent surface area and
esterified cholesterol content. When animals treated with individual
estrogens were compared with controls the reduction in atherosclerosis
was significant for both EE and DHES for all measures except esterified
cholesterol. At the doses used EE and DHES did not differ from
one another in their ability to reduce atherosclerosis.
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Total aortic cholesterol content and percent surface area involvement
were highly correlated in all three aortic sites (Fig 5). The correlation was lower in the aortic arch,
perhaps because with the high percentage of the surface area covered by
atherosclerotic plaques in this arterial site some of these plaques
became thicker by accumulating more cholesterol rather than further
expanding the surface area involved. For this reason, in subsequent
analyses we have used aortic cholesterol content as the primary measure
of atherosclerosis. Inspection of the data in Fig 5 for individual
animals readily reveals the magnitude of the effect of estrogens in
reducing atherosclerosis, particularly for the thoracic and abdominal
aorta. In the aortic arch the difference in atherosclerosis between the
estrogen-treated and non–estrogen-treated animals was much less.
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Significant positive correlations were found between the weighted mean
plasma cholesterol concentration and the total cholesterol content of
the three aortic segments in the cholesterol-fed control animals (solid
regression lines in Fig 6). In contrast, there were no
significant correlations between plasma cholesterol concentration and
atherosclerosis in the estrogen-treated animals in any of the aortic
segments. Thus, the protective effect of these estrogens on
atherosclerosis development was seen over the full range of plasma
cholesterol and lipoprotein concentrations.
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)
there was a significant positive correlation of atherosclerosis (as
measured by aortic total cholesterol content) with the weighted mean
TPC concentration for each aortic site (solid regression lines).
Correlation coefficients were obtained as described in the legend to
Fig 5
To determine if there were qualitative differences in the ß-VLDL from
estrogen-treated animals that might influence its metabolism by foam
cells of the atherosclerotic lesion, ß-VLDL isolated from individual
animals was incubated with THP-1 macrophages, and its effect on
cellular cholesteryl ester accumulation was measured (Table 6). Cells incubated with ß-VLDL had a total
cholesterol content that was increased by more than 10-fold over
control cells. The greatest increase was in the esterified cholesterol
fraction. There was no significant difference, however, in the ability
of ß-VLDL from estrogen-treated or control animals to stimulate
cellular cholesterol accumulation. This was true whether the Ovx and
Sham-Ovx control groups were treated separately or combined for
statistical comparisons with the EE and DHES groups. Cholesterol
esterification was measured in the same cells by using
[1-14C]oleate as a substrate (data not shown), and it
paralleled the mass changes shown in Table 6. These differences
remained insignificant when the sources of the ß-VLDL
(estrogen-treated or control animals) were compared.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Oral administration of DHES and EE significantly reduced the development of aortic atherosclerosis in ovariectomized cholesterol-fed rabbits. At the doses used there was a similar degree of reduction in atherosclerosis with either EE or DHES. This is the first study we know of in which a conjugated equine estrogen (DHES) or EE alone has been tested for its effect on experimental atherosclerosis. Other studies with EE have used it in combination with different progestins25 26 or with ß-estradiol 17-cypionate.3 These combinations and/or the generally higher concentrations of estrogens used in other studies may explain why we did not see an effect of EE on plasma lipid or lipoprotein concentrations.
As is typical of rabbits, atherosclerosis was greatest in the aortic arch followed by the thoracic and abdominal aorta. Ovariectomy alone had no effect on the development of atherosclerosis. At the doses used EE and DHES reduced atherosclerosis to a similar extent in all regions of the aorta. Atherosclerosis was reduced by 35% in the arch and by 75% to 80% in the thoracic and abdominal aorta. The reason for this site-specific difference is unclear, although an analogous effect has been reported in women and nonhuman primates, in which estrogens appear to have their greatest effect on the coronary arteries.7 25 Although we cannot exclude the possibility that the pathogenesis of atherosclerosis may be different in the aortic arch, the more likely possibility is that the rate of development of atherosclerosis in the aortic arch was simply so rapid that the mechanism by which estrogens exert their protective effects was overwhelmed. If this is true then estrogens might be expected to have a greater effect in the arch at lower plasma cholesterol concentrations.
Neither EE nor DHES caused a significant change in total plasma cholesterol or HDL-C concentrations. The only significant change in plasma lipoprotein levels was at week 24, when the estrogen-treated animals actually had higher LDL-C concentrations. It is difficult to imagine how this difference could result in less atherosclerosis, particularly since there was no significant change in the concentration of ß-VLDL, which is generally considered to be the more atherogenic lipoprotein in cholesterol-fed rabbits.27 This result differs from that of Haarbo et al,5 who report that 17ß-estradiol lowered serum total cholesterol concentrations in rabbits and that the effect was almost entirely on ß-VLDL. In their study, however, in animals selected for equivalent serum cholesterol or ß-VLDL concentrations, the amount of atherosclerosis was less with estrogen treatment, suggesting an additional effect of estrogens on the development of atherosclerosis beyond that due to the lowering of plasma cholesterol or ß-VLDL concentrations. This is consistent with our data, which show a protective effect of estrogens against atherosclerosis at all plasma cholesterol concentrations. These observations support the conclusion that estrogens reduce the development of atherosclerosis by mechanisms that are independent of plasma lipoprotein concentrations, perhaps by some direct effect on the arterial wall. This is consistent with the report that in women less than 25% of the reduction in coronary heart disease by estrogens can be accounted for by changes in known lipid risk factors1 and in other studies with rabbits4 5 and nonhuman primates6 in which a reduction in atherosclerosis was seen with little to no effect on plasma lipoproteins or other risk factors.
How estrogens influence the development of atherosclerosis is unclear.
One possibility is that estrogens may reduce the production of abnormal
lipoproteins by the artery wall, possibly through an antioxidant
effect. High concentrations of estrogens in vitro have been shown to
protect LDL against copper-induced oxidation and cell-mediated
modification and subsequently to inhibit lipoprotein uptake and
degradation by cultured macrophages.28 29 Whether
estrogens ever achieve concentrations in the artery wall sufficient to
inhibit LDL oxidation is unclear. Alternatively, estrogen could also
produce qualitative changes in plasma lipoproteins such that their
uptake by cells of the arterial wall is reduced. We addressed this
possibility by studying the ability of ß-VLDL isolated from
individual animals to load THP-1 macrophages in vitro with cholesteryl
esters (Table 6
) and to stimulate cholesterol esterification. The
results showed that ß-VLDL from estrogen-treated rabbits did not
significantly reduce cholesterol accumulation and esterification. This
is in agreement with the study by Henriksson et al,3 who
used ß-VLDL from ß-estradiol 17-cypionate– plus EE-treated
rabbits. This does not eliminate the possibility, however, that
lipoproteins from estrogen-treated animals may be protected in the
artery wall from conversion to abnormal species that in turn can be
taken up by macrophages via the scavenger receptor or other
mechanisms.
Estrogens could also reduce the development of atherosclerosis by a direct effect on the cells of the atherosclerotic plaque. Estrogens do not appear to affect aortic permeability to lipoproteins such as LDL30 ; instead they probably influence the metabolism or accumulation of lipoproteins after the lipoproteins have entered the arterial wall. This conclusion is supported by studies in cynomolgus monkeys, in which there is a significant reduction in LDL accumulation and degradation in the aorta and coronary arteries in animals treated with 17ß-estradiol and cyclic progesterone.7 A similar reduction in cholesteryl ester accumulation in 17ß-estradiol–treated rabbits is reported by Hough and Zilversmit.4 The mechanism of this effect is unknown. One possibility is that estrogens could act directly on macrophages to reduce lipoprotein uptake and subsequent foam cell development. Macrophages are a likely candidate cell since in cholesterol-fed rabbits they are the major cell type of the atherosclerotic lesion.31 The mechanism by which macrophage lipoprotein metabolism is affected by estrogens is unknown, but it could involve a direct effect on lipoprotein receptors or some aspects of cellular cholesterol trafficking or efflux. Whether any of these effects of estrogens are mediated via estrogen receptors is not clear.
From the present study it is not possible to determine the extent
to which the protective effects of estrogens against atherosclerosis
are related to the relative estrogenic potency of the individual
estrogens, since both EE and DHES were administered at levels selected
to have approximately equal estrogenic activity. Although the similar
increases in uterine weights are consistent with equivalent estrogenic
potency, it is possible that both estrogens were present at
concentrations sufficient to give a maximum uterotrophic response.
Dose-response studies will be necessary to determine the minimum
concentration of these estrogens needed to reduce atherosclerosis
development and its relation to overall estrogenic activity. It also is
difficult to meaningfully compare the concentrations of the individual
estrogens used in this study with physiological levels or levels
achieved in women taking low-dose estrogen therapy. This is
particularly true for Premarin, the most widely used estrogen for
postmenopausal treatment, since it is composed of 10 conjugated
estrogens, each with different estrogenic potency, interconvertibility,
and turnover rates.9 10 Although the concentrations used
in this study are generally less than in other studies, they still
represent pharmacological levels, and it is clear that the
protective effect of both EE and the conjugated equine estrogen DHES on
the development of atherosclerosis is independent of plasma lipid or
lipoprotein concentrations. This is seen most clearly in the data in
Fig 6. A better understanding of the mechanism(s) by which estrogens
exert this effect should provide new insight into the pathogenesis of
atherosclerosis as well as suggesting new approaches for drug
therapy.
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Acknowledgments |
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This study was supported by a grant from the Wyeth-Ayerst Co. From the Bowman Gray School of Medicine the authors wish to thank S.H. Hester and M.A. Leight for their technical assistance, J. Gardin, M. Post, and B.C. Bullock for their assistance with necropsies and atherosclerosis evaluation, and V. Wolfkamp for the preparation of this manuscript. At Wyeth-Ayerst Research we thank F. Hadely for the EE assays and M. Day for useful discussions and input. This work was done in partial fulfillment of the requirements for the PhD degree for Sulistiyani from the Bowman Gray School of Medicine of Wake Forest University.
Received April 14, 1994; accepted March 24, 1995.
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