© 1997 American Heart Association, Inc.
Articles |
Antiatherogenic Effect of Estrogen Abolished by Balloon Catheter Injury in Cholesterol-Clamped Rabbits
From the Departments of Obstetrics and Gynecology (P.H.) and Thoracic Surgery (H.õ.A.), Rigshospitalet; the Department of Pathology, Hvidovre Hospital (B.F.H.); and the Department of Clinical Biochemistry, Herlev Hospital (B.G.N.), University of Copenhagen; and the Clinical Institute, University of Odense (S.S.) (Denmark).
Correspondence to Pernille Holm, MD, Department of Women's Healthcare Biology, Novo Nordisk Park, 2760 Måløv, Denmark. E-mail PHlm{at}novo.dk
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Abstract The purpose of this study was to investigate the importance of an intact endothelial cell layer on the direct antiatherogenic effect of estrogen on the arterial wall. Thirty rabbits were bilaterally ovariectomized and subjected to mechanical injury of the endothelium by balloon catheterization of the upper thoracic aorta. Immediately after the operation, treatment was initiated with either 17ß-estradiol or placebo given intramuscularly. All rabbits were clamped at a similar plasma cholesterol level from 1 week before the operation until the experiment was terminated 13 weeks later. In the undamaged aorta, ie, the aortic arch, the lower thoracic aorta, and the upper abdominal aorta, the estrogen-treated rabbits had one third (P=.06), one sixth (P=.002), and one seventh (P=.001), respectively, the accumulation of cholesterol of the placebo-treated rabbits. In the upper thoracic aorta that had been subjected to mechanical injury of the endothelium, however, aortic cholesterol accumulation was not significantly different between the two groups. Similar results were obtained by histological evaluation of the aortic tissues. Immunohistochemical staining with antibodies against macrophages, smooth muscle cells, and T lymphocytes revealed no significant differences in the intimal distribution of cells between estrogen- and placebo-treated rabbits, except for a higher number of T lymphocytes per unit intimal area of the undamaged aortic arch (P<.0005) in the estrogen-treated-rabbits than the placebo-treated rabbits. This is the first study to demonstrate that the antiatherogenic effect of estrogen is abolished by balloon catheter injury in cholesterol-clamped rabbits. These results may indicate that an intact endothelial cell layer is crucial for the direct antiatherogenic effect of estrogen on the arterial wall.
Key Words: atherosclerosis • arterial wall • cholesterol • endothelium • 17ß-estradiol
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Introduction |
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Several human epidemiological studies have shown an association between estrogen replacement therapy and a reduced risk of cardiovascular disease in postmenopausal women.1 2 3 4 5 Underlying mechanisms, however, have not been completely defined. Although estrogen is able to favorably alter the plasma lipid profile,6 7 8 this effect does not account for the total extent of cardiovascular protection associated with estrogen replacement therapy.1 9 Estrogen may possess additional beneficial effects on the cardiovascular system, most likely at the level of the arterial wall.
Experimental animal studies have revealed two major direct actions of estrogen on the arterial wall. First, estrogen seems able to inhibit cholesterol accumulation and intimal hyperplasia in the aorta as well as in the coronary arteries of cholesterol-fed rabbits and monkeys.10 11 12 This may be partly explained by suppression of the arterial uptake and/or degradation of LDL.13 However, the target cells or structures in the arterial wall that mediate the antiatherogenic effect of estrogen are not known at present. Second, estrogen seems capable of normalizing abnormal vasomotor response in atherosclerotic coronary arteries of cholesterol-fed monkeys.14 15 This observation has recently been extended to humans,16 17 and the effect is believed to be mediated through an action of estrogen on the vascular endothelium.
Abnormal vasomotor response is one of the earliest signs of endothelial dysfunction, reflecting an inadequacy of endothelial cells (ECs) to release the correct balance of relaxation and constriction factors.18 The endothelium, however, is not only important for the dynamic properties of the arterial wall but also constitutes a barrier between plasma and the intima for entry of cells and plasma macromolecules such as lipoprotein particles.19 For this reason, one might speculate that the endothelium is the target not only for the beneficial effect of estrogen on vasomotor response but also for the direct antiatherogenic effect of estrogen on the arterial wall.
We hypothesized that the direct antiatherogenic effect of estrogen on the arterial wall was dependent on the presence of an intact EC layer. The present study was designed to investigate whether the direct effect of estrogen on atherogenesis, ie, the effect not mediated through reduced plasma cholesterol levels, was different in undamaged and balloon-injured aortic tissue within the same cholesterol-clamped rabbit.
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Animals
Thirty-eight sexually mature female white rabbits of the Danish Country Strain were purchased from Statens Seruminstitut, Copenhagen, Denmark. Initial body weight before the study was 2.5 to 3.5 kg. All experimental procedures were conducted in compliance with Danish regulations for experiments on animals.
Surgery
Ovariectomy and balloon catheter injury were performed on 30
animals as follows. Anesthesia was induced and maintained
with repeated small doses of intravenous pentobarbital. An
average total dose of 
50 mg/kg body weight was given to each
rabbit. The abdomen was opened through a midline incision and both
ovaries removed. A segment of the abdominal aorta just above the origin
of the inferior mesenteric artery was dissected free, and
the animal was heparinized with 100 IU heparin/kg body weight. The
aorta was then cross-clamped over a small segment and incised
longitudinally, and a 4F embolectomy catheter (Baxter Healthcare Corp)
was inserted and advanced as far as the beginning of the aortic arch.
The balloon was inflated with 0.6 mL of saline (distension, 9.0
mm) and the catheter retracted 3 cm. Finally, the balloon was deflated
and the catheter withdrawn. The aorta was sutured with polypropylene
7-0 and the caudal blood supply reestablished. The duration of each
operation was 30 to 45 minutes.
Estrogen Treatment
Immediately after the operation, treatment was initiated with
either 17ß-estradiol cypionate (estrogen group) or vehicle (placebo
group). The solution of 17ß-estradiol cypionate (Sigma Chemical
Co) was prepared as described previously.20 Rabbits in the
estrogen group were injected intramuscularly with 50 µg
17ß-estradiol cypionate/kg body weight every third day, and the
rabbits in the placebo group were injected intramuscularly with a
corresponding volume of corn oil.
At the end of the experiment, the plasma trough concentrations of estradiol, estrone, and estrone sulfate were measured by specific radioimmunoassay after extraction and chromatography.21 The detection limit of the estradiol and estrone assay was 100 pmol/L and that of the estrone sulfate assay 200 pmol/L.
Plasma and Dietary Cholesterol
In an attempt to minimize the effect of estrogen on plasma
cholesterol levels, cholesterol feeding was
commenced 1 week before hormone treatment and operation. To ensure that
an average plasma cholesterol level of 25
mmol/L was maintained in all rabbits, the amount of
cholesterol added to the chow of each animal was adjusted
individually according to regularly assayed blood
cholesterol levels as determined by enzymatic cholesteryl
ester hydrolysis and cholesterol oxidation followed by a
color reaction (CHOD-PAP, Boehringer Mannheim). Each rabbit
received 100 g of chow daily, consisting of 89 to 90 g of
standard rabbit pellets (Altromin 2113) enriched with 0 to 1 g of
cholesterol (CH-UPS, Sigma) dissolved in 10 g of corn
oil (BP80, Mecobenzon). The distribution of cholesterol
between VLDL (d<1.006 g/mL), IDL
(1.006<d<1.019 g/mL), LDL
(1.019<d<1.063 g/mL), and HDL (d>1.063
g/mL) lipoproteins was determined 6 and 12 weeks after
cholesterol feeding was commenced by
ultracentrifugation as described
previously.20 All blood samples were drawn from the
lateral ear vein into tubes containing Na2EDTA (2 mL for
plasma cholesterol determinations and 10 mL for
ultracentrifugation).
Preparation of Aortic Tissue
At the end of the experiment after 13 weeks of
cholesterol feeding (ie, 12 weeks after the operation and
initiation of estrogen or vehicle treatment), the rabbits were injected
intravenously with 5 mL of Evans blue dye (5 mg/mL).
Evans blue dye binds with circulating albumin, and the
uninjured endothelium serves as a barrier to this
dye-protein complex, whereas areas denuded of
endothelium stain royal blue. The dye was allowed to
circulate for 5 minutes before the rabbits were killed with
intravenous pentobarbital (50 to 100 mg/kg body
weight). The cardiovascular system was flushed with 500
mL of saline via the left ventricle of the heart; blood and
perfusate left through an incision in the inferior
vena cava. After it was flushed, the entire aorta was dissected free
and carefully freed of adventitia. The balloon-injured area was easily
identified by its blue staining. It had a patchy appearance with blue
(still deendothelialized) and white
(reendothelialized) regions. The
reendothelialized regions appeared as "islands"
around the branch orifices and at the proximal and distal edges of
the previously injured aorta. The proportion of white tissue determined
by visual examination was almost similar in estrogen-treated and
untreated rabbits (30±6% versus 24±6%), suggesting that the extent
of endothelial regeneration was similar in the two
groups. The blue-stained segment was removed together with three
undamaged segments, one each from the aortic arch, the lower thoracic
aorta, and the upper abdominal aorta (Fig 1
). The undamaged segment from the upper
abdominal aorta included the celiac and superior mesenteric orifices
but not the renal orifices or the site of cross-clamping and incision.
One specimen of unopened aorta 3 mm long was taken from each of
these four segments for histological and
immunohistochemical evaluation; the rings were formalin fixed, paraffin
embedded, and cut into 3-µm sections. The remaining tissue was used
to determine cholesterol content: to outline the luminal
surface area, the aortic segments were cut open and pinned to paper on
a cork board. The aortic tissue was then stripped into an inner layer
comprising the intima and inner media and an outer layer comprising the
remaining media. All parts were weighed and stored at -20°C until
chemical analysis.
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Quantification of Atherosclerosis Development
Cholesterol content was determined in the
intima–inner media and the outer media layers; aortic tissue was
extracted with at least 20 volumes of chloroform/methanol (1:1,
vol/vol). After addition of chloroform/methanol, the extract was
washed by the Folch procedure.22 Subsequent to
evaporation, the extracted lipids were redissolved in isopropanol and
the cholesterol content determined by the same enzymatic
kit (CHOD-PAP) used for plasma cholesterol determinations.
The validity of this procedure has been tested
previously.23
Intimal hyperplasia was measured as described earlier.20 In brief, 3-µm sections of the four aortic segments were stained with elastic–van Gieson's and elastic–hematoxylin/eosin stains. The aortic cross sections were projected onto a white point grid, and the number of points over the intima and media was counted twice. The recorded value was the mean of the two counts. All histological measurements were performed by the same observer (P.H.) who was blinded to the treatment groups.
Immunohistochemistry
For definition of cell populations in the intima, 3-µm
sections of undamaged (aortic arch) and balloon-injured (upper
thoracic) aorta were stained with monoclonal antibodies to
macrophages, smooth muscle cells (SMCs), and T lymphocytes.
Sections from the lower thoracic and upper abdominal aortas were not
included in these studies because intimal thickening was present in
only one rabbit in the estrogen group. Macrophages were
identified by use of RAM11 antibody (anti-rabbit macrophage,
DAKO Corp), SMCs with HHF35 antibody (anti–smooth muscle
-actin, DAKO A/S), and T lymph-ocytes with L11/135 antibody
(anti-rabbit CD43, Serotec). An avidin-biotin method was used with
these antibodies. Mast cells were visualized by enzymatic stain
(LEDER, naphthol AS–D-chloracetate).
The quantification of macrophages and SMCs present within the intima was performed in the following way. In a representative field of view of the intima (magnification x400), the number of stained cells as well as the number of other cells were counted, and stained cells were expressed as a percentage of the total number of cells. Because of their relatively low numbers, all T lymphocytes and mast cells in the intima were counted and related to the intimal cross-sectional area (number of points).
For determination of estrogen receptor content within the rabbit aorta, 3-µm sections of the four aortic segments were stained with Dako 1D5 (DAKO A/S), a recently developed, commercially available monoclonal anti-human estrogen receptor antibody, which can be used on routinely processed, formalin-fixed tissue.24 The antibody reacts with the N-terminal domain of the receptor, and the staining is predominantly localized to the nucleus, with no cytoplasmic staining. To improve the staining pattern, a microwave antigen retrieval technique was used. The tissue sections were microwaved in 5 L of 0.01 mol/L citrate buffer at pH 6.0 for 2x 5 minutes. The tissue sections were then allowed to cool before being rinsed in Tris-buffered saline and stained as above. Positive control tissue, consisting of rabbit mammary tissue, was used in all staining runs. All immunohistochemical evaluations were performed by the same observer (P.H.) who was blinded to the treatment groups.
Extent of Arterial Damage Following Balloon
Catheterization
Eight rabbits were injured by balloon catheter in the upper
thoracic aorta according to the same procedure as described above. Two
of these rabbits were killed immediately after surgery to determine how
much of the original endothelium had been removed by
the denuding maneuver. Five minutes before the two rabbits were killed,
5 mL of Evans blue dye was injected intravenously. The
aortas were removed and opened longitudinally, and the extent of
endothelial denudation as assessed by the percentage of
blue staining was determined in the balloon-injured area.
The remaining six rabbits were killed 5 (n=3) or 10 (n=3) days after surgery to investigate whether the deeper arterial layers (ie, internal elastic lamina or SMCs of the media) had suffered from the balloon catheterization. After injection with Evans blue dye, the aorta was removed and aortic rings were taken from within, proximal to, and distal to the balloon-injured area. The rings were formalin fixed and paraffin embedded, and 3-µm sections were cut and stained with elastic–van Gieson's and elastic–hematoxylin/eosin stains. All histological evaluations were performed by the same observer (P.H.) who was blinded to the treatment groups.
Statistical Methods
All results are given as mean±SEM. The Mann-Whitney
U test (two tailed) was used for comparison between
groups.
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Results |
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The two groups of rabbits were well matched with regard to age and initial values of plasma cholesterol and body weight. At the time of balloon injury, the mean body weight of the estrogen group was 3.1±0.04 kg (range, 3.0 to 3.3) and that of the placebo group 3.1±0.04 kg (range, 2.9 to 3.5). One rabbit in the estrogen group died during the operation due to an overdose of anesthesia. Another rabbit in the placebo group had to be killed during week 5 because of sudden paralysis of the lower body. That left 28 rabbits in the main study. All rabbits thrived without visible side effects of the operation or estrogen therapy. The weight gain during the 13-week experimental period was 0.7±0.1 kg in the estrogen group and 0.6±0.1 kg in the placebo group.
Estrogen Levels
At the end of the experiment, the mean trough level of estradiol
in the estrogen group of rabbits was 601±26 pmol/L, and the
mean trough level of estrone was 389±28 pmol/L. Neither
estradiol nor estrone was detectable in the placebo group. The mean
trough concentration of estrone sulfate was 1.52±0.08 nmol/L in
the estrogen group and 0.57±0.03 nmol/L in the placebo group. A
difference in hormone levels between the two groups was confirmed at
necropsy by the observation that the uteruses of estrogen-treated
rabbits were 5- to 10-fold larger than those of the placebo-treated
rabbits.
Cholesterol Feeding
We succeeded in maintaining a similar mean concentration of plasma
cholesterol in the two groups of rabbits throughout the
experimental period (Fig 2, upper panel).
Calculated as the area under the curve, the mean plasma
cholesterol concentration was 23.5±0.4 mmol/L
in the estrogen group and 23.8±0.3 mmol/L in the placebo
group. However, this similarity was achieved at the expense of the
total amount of dietary cholesterol, which was
significantly higher for rabbits in the estrogen group than the placebo
group (31.8±2.6 versus 21.1±1.1 g, P=.001).
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The distribution of plasma cholesterol between lipoprotein
fractions after 6 and 12 weeks of cholesterol feeding is
shown in Fig 2
(middle and lower panels). After 6 weeks, the rabbits in
the estrogen group had a significantly higher concentration of VLDL
cholesterol (P=.02) and HDL
cholesterol (P=.002) than those in the placebo
group, which was balanced by a nonsignificantly lower level of LDL
cholesterol. After 12 weeks, however, these differences had
disappeared.
Extent of Aortic Atherosclerosis
Representative tissue samples from undamaged and
balloon-injured aortas of the two groups are shown in Fig 3. In the undamaged aorta (ie, the aortic
arch, the lower thoracic aorta, and the upper abdominal aorta), the
estrogen-treated rabbits had one third (P=.06), one sixth
(P=.002), and one seventh (P=.001), respectively,
the amount of cholesterol accumulation in the placebo
rabbits (Fig 4, upper panel). However, in
the balloon-injured part, ie, the upper thoracic aorta, the rabbits
treated with estrogen had accumulated an amount of
cholesterol similar to that of the rabbits treated with
placebo. The data, when expressed on a per milligram wet weight basis,
were similar to the results of cholesterol accumulation
when they were expressed per area or per milligram of protein. The
cholesterol accumulation in the outer media was not
significantly different between the two groups in any of the four parts
of the aorta (data not shown).
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The ratio of intimal area to intimal-plus-medial area in
histological cross sections of the undamaged lower
thoracic and upper abdominal aortas was significantly reduced in
rabbits receiving estrogen compared with those receiving placebo
(P=.004 and P=.006, respectively; Fig 4, lower
panel). In the aortic arch this reduction in area ratio did not reach
statistical significance (P=.13). This finding is consonant
with the aortic cholesterol data, which showed that the
reduction by estrogen was also most pronounced in the distal parts of
the aorta. In the balloon-injured part of the aorta, the ratio of
intimal area to intimal-plus-medial area was not statistically
different between the two groups. The medial cross-sectional area
decreased with increasing distance from the aortic arch, with no
difference between the estrogen group and the placebo group.
The extent and severity of atherosclerosis in the
undamaged parts of the aorta in all rabbits were most severe in the
aortic arch, with lesser involvement in the more distal parts of the
aorta (Fig 4, upper and lower panels). This regional variation
throughout the length of the aorta in rabbits is already well
recognized.
Immunohistochemistry
Aortic sections stained with RAM11, HHF35, and L11/135 are shown
in Fig 5. RAM11-positive,
macrophage-derived foam cells were abundant in the intima
of the undamaged aortic arch, whereas HHF35-positive SMCs were the
predominant cell type in the intima of the balloon-injured upper
thoracic aorta (Table). CD43-positive T
lymphocytes were occasionally observed in the intima, but no mast cells
were seen. There were no significant differences in the distribution of
cells between estrogen- and placebo-treated rabbits except for the
number of T lymphocytes per unit of intimal area in the undamaged
aortic arch, which was only one tenth the amount in the estrogen group
than in the placebo group (P<.0005).
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No evidence of estrogen receptor expression within the rabbit aorta was
found when assessed immunohistochemically with the antibody Dako 1D5
(Fig 6). From a total of 120 aortic
sections, no positive immunoreactivity was found. The sample of rabbit
mammary tissue, which served as an internal positive control, however,
showed positive nuclear staining in all staining runs (Fig 6),
suggesting that the antibody does cross-react with rabbit tissues.
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Extent of Arterial Damage Following Balloon
Catherization
In both animals killed immediately after surgery, the
balloon-injured area stained a uniform blue with no visible islands of
white tissue, suggesting that all ECs had been removed. In the six
animals killed 5 and 10 days after surgery, the balloon-injured area
was still primarily blue but with a few minor white spots, indicating
the start of regeneration of ECs. The cross sections from the upper
thoracic aorta exposed to balloon injury were indistinguishable from
those of the undamaged aortic arch and the undamaged lower thoracic
aorta: in neither tissue were there any ruptures of the internal
elastic lamina or necrosis of SMCs of the media (Fig 7).
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Discussion |
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The principal finding of this study is that the antiatherogenic effect of estrogen is abolished by balloon catheter injury in cholesterol-clamped rabbits. This finding suggests that an intact EC layer is a prerequisite for the direct antiatherogenic effect of estrogen on the arterial wall. The estradiol dose given intramuscularly to the rabbits resulted in plasma trough concentrations comparable to those seen in women receiving estrogen replacement therapy.25 Also, the plasma levels of estrone and estrone sulfate were within the human physiological range.26 Enlargement of the uterus in the estrogen-treated rabbits implies that the dose of estradiol given produced a pharmacological effect. No difference between the two groups was seen in total body weight gain, suggesting that estrogen treatment did not affect the general well-being of the rabbits.
Estrogen replacement therapy in rabbits has previously been shown to
either reduce or have no effect on plasma cholesterol
levels.10 23 To study the direct effect of estrogen on the
arterial wall, we maintained all rabbits at the same plasma
cholesterol concentration of 25 mmol/L. In
the present study, the estrogen-treated rabbits probably would have
had a lower plasma cholesterol level if they had not been
fed a higher amount of cholesterol than the placebo-treated
rabbits. The cholesterol-lowering effect of estrogen may be
due to induction of LDL receptors in the liver.27 28 The
distribution of cholesterol between lipoprotein fractions
was not significantly affected by estrogen therapy, apart from a minor
elevation of VLDL and HDL cholesterol in the first half of
the study. However, the possibility of subtle changes in lipoprotein
composition mediated by estrogen cannot be excluded; eg, several
studies have suggested that estrogen either alone or combined with a
progestin reduces LDL particle size in monkeys12 29 30 and
postmenopausal women.31 32
Previous animal studies have shown that estrogen is able to afford protection against atherosclerosis independent of beneficial changes in plasma lipid levels.10 11 12 23 The results of our study strongly support this finding: although all rabbits were clamped at a similar plasma cholesterol level and the estrogen group subsequently was given a larger amount of dietary cholesterol than the placebo group, estrogen nevertheless reduced atherogenesis in the undamaged aorta. This antiatherogenic effect of estrogen, mediated independently of changes in plasma lipids, may be due to a direct effect of estrogen on the arterial wall.
Our conclusion that the direct antiatherogenic effect of estrogen is abolished by balloon catheter injury is based on the assumption that estrogen would have had a beneficial effect on the upper thoracic aorta if this area had not been balloon injured. We have tested this assumption as part of another experiment (to be published elsewhere), in which two groups of female rabbits were subjected to the same experimental protocol as that described above, except that the rabbits were not balloon injured in the upper thoracic aorta. The cholesterol accumulation in the upper thoracic aorta was reduced to one third in the estrogen group compared with the level of the placebo group (P<.0001), suggesting that estrogen has the same antiatherogenic effect at the level of the upper thoracic aorta as in other undamaged parts of the aorta. There was a regional variation in the direct antiatherogenic effect of estrogen within the rabbit aorta, with the protection being more pronounced in the upper abdominal and lower thoracic aortas than the aortic arch. This observation is in accordance with previous findings in cholesterol-fed rabbits treated with estrogen.33
It has previously been shown that balloon injury of the rabbit aorta of the same severity as in the present study removes >90% of the endothelium, while the internal elastic membrane and SMCs of the media are left intact.34 This finding is consistent with ours, which showed complete endothelial denudation of the balloon-injured segment immediately after surgery, as assessed by Evans blue staining, and no signs of damage to the deeper arterial cell layers 5 and 10 days after surgery. At the time of necropsy (12 weeks after balloon injury), islands of regenerated endothelium were present in the balloon-injured area of all animals. However, ECs that regenerate after balloon injury may differ from native ones: they are irregularly shaped, lack alignment in the direction of blood flow, and exhibit endothelial dysfunction.35 36 Thus, our results suggest that an intact endothelium is crucial for the direct antiatherogenic effect of estrogen on the arterial wall simply because this benefit is abolished in aortic tissue in which the endothelium is either absent or aberrant.
Despite the fact that vascular smooth muscle is an estrogen-sensitive tissue,37 38 we were unable to demonstrate estrogen receptor expression in the rabbit aorta by the present technique. This finding reinforces our hypothesis about an SMC-independent, endothelium-based antiatherogenic effect of estrogen. The importance of an intact EC layer is further supported by the observation that estrogen therapy significantly inhibits transplant arteriosclerosis in cholesterol-fed rabbits treated with cyclosporin,39 whereas cholesterol-fed rabbits receiving estrogen therapy without cyclosporin are not protected against transplant arteriosclerosis.20 Cyclosporin is an immunosuppressive agent that has been shown to attenuate immunological damage to the endothelium.40 However, the above-mentioned results are not consistent with those of a recent study, which showed that estrogen treatment of rabbits undergoing balloon injury may inhibit SMC proliferation and thereby myointimal thickening.38 The fact that those rabbits38 were not cholesterol fed may explain the discrepancy.
Interest has lately focused on the ability of estrogen to increase the production of NO in ECs by inducing NO synthase enzymes.41 NO is an endogenous vasodilator responsible for the vascular relaxation induced by acetylcholine and other endothelium-dependent vasodilators.42 Decreased synthesis and/or increased degradation of NO are most probably the cause of the abnormal vascular responses seen in atherosclerotic coronary arteries. The ability of estrogen to increase NO production in the endothelium may therefore be the mechanism behind the beneficial effect of estrogen on abnormal vasomotion in atherosclerotic coronary arteries.
NO not only is the most potent endogenous vasodilator but also inhibits other key events in the atherosclerotic process. NO has been shown to inhibit platelet adhesion and aggregation, reduce monocyte adherence, decrease oxidation of LDL particles, and inhibit proliferation of SMCs.43 The antiatherogenic effect of NO is supported by recent findings showing that sustained enhancement of vascular NO activity is associated with an inhibition of intimal lesion formation,44 whereas chronic inhibition of NO synthase accelerates atherogenesis in hypercholesterolemic rabbits.45 46 The estrogen-mediated increase in production and release of NO from the endothelium may therefore also be involved in the direct antiatherogenic effect of estrogen on the arterial wall.
The accelerated atherogenesis following balloon injury appears to be a direct consequence of the tissue-injury-and-repair response mediated by complex interactions among blood-borne platelets, leukocytes, lipids, cytokines, and vascular cells.47 This process seems to be unaffected by estrogen treatment. The difference in pathogenesis of conventional atherogenesis versus that following balloon injury is reflected by the fact that SMCs were abundant in the intima of balloon-injured aortas, whereas foam cell–derived macrophages were the predominant cell type in the intima of undamaged aortas. Estrogen did not seem to have an effect on the distribution of these cells. Whether the difference between the two groups in terms of the relative amounts of T lymphocytes in the undamaged aorta is important for the antiatherogenic effect of estrogen is not known, although that could be a possibility. However, our findings need to be confirmed by other studies.
In conclusion, this study in cholesterol-fed rabbits suggests that the endothelium is the target not only for the beneficial effect of estrogen on vasomotor response but also for the direct antiatherogenic effect of estrogen on the arterial wall. Perhaps these effects of estrogen are not due to separate mechanisms but are mediated, at least in part, through a common pathway, such as a stimulatory effect of estrogen on NO synthesis from the endothelium.
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Acknowledgments |
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This study was supported by grants from the Danish Heart Foundation. We acknowledge the excellent technical help of Hanne Damm and Lone Christensen and thank Birgit Svenstrup, Seruminstituttet, for assistance in measuring plasma hormone levels.
Received December 19, 1995; accepted September 4, 1996.
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References |
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1. Bush TL, Barrett-Connor E, Cowan LD, Criqui MH, Wallace RB, Suchindran CM, Tyroler HA, Rifkind BM. Cardiovascular mortality and noncontraceptive use of estrogen in women: results from the Lipid Research Clinics Program Follow-up Study. Circulation. 1987;75:1102-1109.
2. Stampfer MJ, Colditz GA, Willet WC, Manson JE, Rosner B, Speizer FE, Hennekens CH. Postmenopausal estrogen therapy and cardiovascular disease: ten-year follow-up from the Nurses' Health Study. N Engl J Med. 1991;325:756-762.[Abstract]
3.
Henderson BE, Paganini-Hill A, Ross RK.
Decreased mortality in users of estrogen replacement therapy.
Arch Intern Med. 1991;151:75-78.
4. Stampfer MJ, Colditz GA. Estrogen replacement therapy and coronary heart disease: a quantitative assessment of the epidemiologic evidence. Prev Med. 1991;20:47-63.[Medline] [Order article via Infotrieve]
5.
Sullivan JM, Vander Zwaag R, Hughes JP, Maddock V,
Kroetz FW, Ramanathan KB, Mirvis DM. Estrogen replacement and
coronary artery disease: effect on survival in postmenopausal
women. Arch Intern Med. 1990;150:2557-2562.
6.
Lobo RA. Effects of hormonal replacement on
lipids and lipoproteins in postmenopausal women. J
Clin Endocrinol Metab. 1991;73:925-930.
7. Crook D. Postmenopausal oestrogens and progestogens: effects on plasma lipoprotein risk markers for coronary heart disease. In: Asch R, Studd JWW, ed. Progress in Reproductive Medicine. Lancashire, England: Parthenon; 1993:261-272.
8. Seed M, Crook D. Post-menopausal hormone replacement therapy, coronary heart disease and plasma lipoproteins. Curr Opin Lipidol. 1994;5:48-58.[Medline] [Order article via Infotrieve]
9.
Barrett-Connor E, Bush TL. Estrogen and
coronary heart disease in women. JAMA. 1991;265:1861-1867.
10. Haarbo J, Leth-Espensen P, Stender S, Christiansen C. Estrogen monotherapy and combined estrogen-progestogen replacement therapy attenuate aortic accumulation of cholesterol in ovariectomized cholesterol-fed rabbits. J Clin Invest. 1991;87:1274-1279.
11. Haarbo J, Hansen BF, Christiansen C. Hormone replacement therapy prevents coronary artery disease in ovariectomized cholesterol-fed rabbits. APMIS. 1991;99:721-727.[Medline] [Order article via Infotrieve]
12.
Adams MR, Kaplan JR, Manuck SB, Koritnik DR, Parks JS,
Wolfe MS, Clarkson TB. Inhibition of coronary artery
atherosclerosis by 17-ß estradiol in ovariectomized
monkeys. Arteriosclerosis. 1990;10:1051-1057.
13. Wagner JD, Clarkson TB, St Clair RW, Schwenke DC, Shively CA, Adams MR. Estrogen and progesterone replacement therapy reduces low density lipoprotein accumulation in the coronary arteries of surgically postmenopausal cynomolgus monkeys. J Clin Invest. 1991;88:1995-2002.
14.
Williams JK, Adams MR, Klopfenstein HS. Estrogen
modulates responses of atherosclerotic coronary
arteries. Circulation. 1990;81:1680-1687.
15. Williams JK, Adams MR, Herrington DM, Clarkson TB. Short-term administration of estrogen and vascular responses of atherosclerotic coronary arteries. J Am Coll Cardiol. 1992;20:452-457.[Abstract]
16.
Reis SE, Gloth ST, Blumenthal RS, Resar JR, Zacur HA,
Gerstenblith G, Brinker JA. Ethinyl estradiol acutely attenuates
abnormal coronary vasomotor responses to acetylcholine in
postmenopausal women. Circulation. 1994;89:52-60.
17. Rosano GMC, Sarrel PM, Poole-Wilson PA, Collins P. Beneficial effect of oestrogen on exercise-induced myocardial ischaemia in women with coronary artery disease. Lancet. 1993;342:133-136.[Medline] [Order article via Infotrieve]
18. Luscher TF. The endothelium as a target and mediator of cardiovascular disease. Eur J Clin Invest. 1993;23:670-685.[Medline] [Order article via Infotrieve]
19. Schwenke DC, Zilversmit DB. The arterial barrier to lipoprotein influx in the hypercholesterolemic rabbit, 2: long-term studies in deendothelialized and reendothelialized aortas. Atherosclerosis. 1989;77:105-115.[Medline] [Order article via Infotrieve]
20. Holm P, Andersen HO, Nordestgaard BG, Hansen BF, Kjeldsen K, Stender S. Effect of oestrogen replacement therapy on development of experimental arteriosclerosis: a study in transplanted and balloon-injured rabbit aortas. Atherosclerosis. 1995;115:191-200.[Medline] [Order article via Infotrieve]
21. Lykkesfeldt G, Bennet P, Lykkesfeldt AE, Micic S, Mrller S, Svenstrup B. Abnormal androgen and oestrogen metabolism in men with steroid sulphatase deficiency and recessive X-linked ichthyosis. Clinl Endocrinol. 1985;23:385-393.
22.
Folch J, Lees M, Sloane Stanley GH. A simple
method for the isolation and purification of total lipids from animals
tissue. J Biol Chem. 1957;226:497-509.
23.
Haarbo J, Svendsen OL, Christiansen C.
Progestogens do not affect aortic accumulation of
cholesterol in ovariectomized cholesterol-fed
rabbits. Circ Res. 1992;70:1198-1202.
24. Goulding H, Pinder S, Cannon P, Pearson D, Nicholson R, Snead D, Bell J, Elston CWE, Robertson JF, Blamey RW, Ellis IO. A new immunohistochemical antibody for the assessment of estrogen receptor status on routine formalin-fixed tissue samples. Hum Pathol. 1995;26:291-294.[Medline] [Order article via Infotrieve]
25. Levrant SG, Barnes RG. Pharmacology of estrogens. In: Lobo RA, ed. Treatment of the Postmenopausal Woman: Basic and Clinical Aspects. New York, NY: Raven Press; 1994:57-68.
26. Statens Seruminstitut. HDndbog 93 fra Statens Seruminstitut. Copenhagen, Denmark, 348 pp.
27.
Ma PTS, Yamamoto T, Goldstein JL, Brown MS.
Increased mRNA for low density lipoprotein receptor in livers of
rabbits treated with 17±-ethinyl estradiol. Proc Natl
Acad Sci U S A. 1986;83:792-796.
28. Henriksson P, Stamberger M, Eriksson M, Rudling M, Diczfalusy U, Berglund L, Angelin B. Oestrogen-induced changes in lipoprotein metabolism: role in prevention of atherosclerosis in the cholesterol-fed rabbit. Eur J Clin Invest. 1989;19:395-403.[Medline] [Order article via Infotrieve]
29.
Parks JS, Pelkey SJ, Babiak J, Clarkson TB.
Contraceptive steroid effects on lipids and lipoproteins in cynomolgus
monkeys. Arteriosclerosis. 1989;9:261-268.
30.
Wagner JD, St Clair RW, Schwenke DC, Shively CA, Adams
MR, Clarkson TB. Regional differences in arterial
low density lipoprotein metabolism in surgically
postmenopausal cynomolgus monkeys. Arterioscler
Thromb. 1992;12:717-726.
31. Campos H, Sacks FM, Walsh BW, Schiff I, O'Hanesian MA, Krauss RM. Differential effects of estrogen on low-density lipoprotein subclasses in healthy postmenopausal women. Metabolism. 1993;42:1153-1158.[Medline] [Order article via Infotrieve]
32. Van der Mooren MJ, de Graaf J, Demacker PNM, de Haan AFJ, Rolland R. Changes in the low-density lipoprotein profile during 17ß-estradiol-dydrogesterone therapy in postmenopausal women. Metabolism. 1994;43:799-802.[Medline] [Order article via Infotrieve]
33.
Sulistiyani SJ, Adelman A, Chandrasekaran J, Rayo J, St
Clair RW. Effect of 17±- dihydroequilin sulfate, a conjugated
equine estrogen, and ethynyl estradiol on
atherosclerosis in cholesterol-fed
rabbits. Arterioscler Thromb Vasc Biol. 1995;15:837-846.
34. Schwenke DC, Zilversmit DB. The arterial barrier to lipoprotein influx in the hypercholesterolemic rabbit, 1: studies during the first two days after mild aortic injury. Atherosclerosis. 1989;77:91-103.[Medline] [Order article via Infotrieve]
35.
Weidinger FF, McLenachan JM, Cybulsky MI, Gordon JB,
Rennke HG, Hollenberg NK, Fallon JT, Ganz P, Cooke JP.
Persistent dysfunction of regenerated endothelium after
balloon angioplasty of rabbit iliac artery.
Circulation. 1990;81:1667-1679NB.
36.
Saroyan RM, Roberts MP, Light JT, Chen I, Vaccarella
MY, Bang DJ, Kvamme P, Singh S, Scalia SV, Kerstein MD, Kadowitz PJ,
McNamara DB. Differential recovery of prostacyclin and
endothelium-derived relaxing factor after vascular
injury. Am J Physiol. 1992;262:H1449-H1457.
37. Fischer-Dzoga K, Wissler RW, Vesselinovitch D. The effect of estradiol on the proliferation of rabbit aortic medial tissue culture cells induced by hyperlipemic serum. Exp Mol Pathol. 1983;39:355-363.[Medline] [Order article via Infotrieve]
38. Foegh ML, Asotra S, Howel MH, Ramwell PW. Estradiol inhibition of arterial neointimal hyperplasia after balloon injury. J Vasc Surg. 1994;19:722-726.[Medline] [Order article via Infotrieve]
39. Cheng LP, Kuwahara M, Jacobsson J, Foegh ML. Inhibition of myointimal hyperplasia and macrophage infiltration by estradiol in aorta allografts. Transplantation. 1991;52:967-972.[Medline] [Order article via Infotrieve]
40.
Andersen HO, Madsen G, Nordestgaard BG, Hansen BF,
Kjeldsen K, Stender S. Cyclosporin suppresses transplant
arteriosclerosis in the aorta-allografted,
cholesterol-clamped rabbit: suppression preceded by
decrease in arterial lipoprotein permeability.
Arterioscler Thromb. 1994;14:944-950.
41.
Weiner CP, Lizasoain I, Baylis SA, Knowles RG, Charles
IG, Moncada S. Induction of calcium-dependent nitric oxide
synthases by sex hormones. Proc Natl Acad Sci
U S A. 1994;91:5212-5216.
42.
Moncada S, Higgs A. The L-arginine-nitric oxide
pathway. N Engl J Med. 1993;329:2002-2012.
43. Cooke JP, Tsao P. Cellular mechanisms of atherogenesis and the effects of nitric oxide. Curr Opin Cardiol. 1992;7:799-804.
44. Cooke JP, Singer AH, Tsao P, Zera P, Rowan RA, Billingham ME. Antiatherogenic effects of L-arginine in the hypercholesterolemic rabbit. J Clin Invest. 1992;90:1168-1172.
45.
Naruse K, Shimizu K, Muramatsu M, Toki Y, Miyazaki Y,
Okumura K, Hashimoto H, Ito T. Long-term inhibition of NO
synthesis promotes atherosclerosis in the
hypercholesterolemic rabbit thoracic aorta.
Arterioscler Thromb. 1994;14:746-752.
46.
Cayatte AJ, Palacino JJ, Horten K, Cohen RA.
Chronic inhibition of nitric oxide production accelerates
neointima formation and impairs endothelial
function in hypercholesterolemic rabbits.
Arterioscler Thromb. 1994;14:753-759.
47. Ferns GAA, Stewart-Lee AL, ?nggDrd EE. Arterial response to mechanical injury: balloon catheter de-endothelialization. Atherosclerosis. 1992;92:89-104.[Medline] [Order article via Infotrieve]
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