(Circulation. 1999;100:1727-1733.)
© 1999 American Heart Association, Inc.
Basic Science Reports |
The Direct Antiatherogenic Effect of Estrogen Is Present, Absent, or Reversed, Depending on the State of the Arterial Endothelium
A Time Course Study in Cholesterol-Clamped Rabbits
From Novo Nordisk, Maaloev (P.H., E.E.); Center for Clinical and Basic Research, Ballerup (H.L.A.); and Clinical Institute, Odense University (M.R.A., 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 |
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Background—This study further investigated the relationship between estrogen, arterial endothelium, and nitric oxide (NO) in cholesterol-clamped rabbits.
Methods and Results—Rabbits were ovariectomized, balloon-injured in the thoracic aorta, and grouped to receive cholesterol-enriched chow together with either 17ß-estradiol or vehicle for 1, 2, 4, or 8 weeks. In the undamaged aorta, cholesterol accumulation of the placebo rabbits was significantly increased from week 4 to 8 (P<0.001). This increase was almost completely inhibited by estrogen (P<0.001). In the balloon-injured aorta, the estrogen and placebo rabbits accumulated similar amounts of cholesterol in the reendothelialized areas. In the deendothelialized areas, the estrogen group surprisingly accumulated significantly more cholesterol than the placebo group. This difference was apparent from week 2 and became significant at week 8 (P<0.01). Circulating nitrite/nitrate were significantly increased by estrogen at weeks 1, 2, and 4 but not at week 8. Similarly, in additional experiments, basal NO release was significantly higher in estrogen-treated than in placebo-treated rabbits after 4 (P<0.05) but not after 8 weeks. Stimulated NO release and endothelial NO synthase activity did not differ between groups. Mononuclear-endothelial cell binding was reduced by 50% by estrogen after 4 weeks (P<0.05). This difference, however, was abolished by coadministration of NG-nitro-L-arginine methyl ester, an inhibitor of NO production.
Conclusions—The direct antiatherogenic effect of estrogen was present, absent, or reversed, depending on the state of the arterial endothelium, and preceded by a transient increase in NO production followed by a reduced mononuclear-endothelial cell binding.
Key Words: atherosclerosis • balloon • endothelium • estrogen • nitric oxide
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Introduction |
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Atherosclerosis and its complications remain the most common cause of death in postmenopausal women of the West.1 Several epidemiological studies suggest that estrogen replacement therapy significantly attenuates the progression of this disease.1 2 Underlying mechanisms are not well understood but are thought to involve both effects of estrogen on circulating lipids and lipoproteins and, more importantly, direct effects of estrogen on cells/structures within the arterial wall.
A variety of animal studies support the suggestions of epidemiological studies through the demonstration of a significant inhibition of diet-induced or spontaneous atherosclerosis after estrogen treatment.3 4 5 However, in these experiments, studies of the direct antiatherogenic effect of estrogen are often hampered by differences in plasma cholesterol levels between groups. We have developed a special model for use of studies of the direct antiatherogenic effect of estrogen: the cholesterol-clamped rabbit. In this model the amount of cholesterol added to the chow of each rabbit is not constant but continuously adjusted according to weekly plasma cholesterol determinations. In this way, all rabbits are maintained at a similar plasma cholesterol concentration, resulting in all aortas being exposed to a similar average plasma cholesterol level. With this model, we have previously shown that the direct antiatherogenic effect of estrogen is abolished by balloon catheter injury,6 7 8 and significantly attenuated by long-term treatment with NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide (NO) synthase (eNOS).7 These findings suggest that the endothelium and endothelial NO are involved in the mechanism by which estrogen inhibits atherogenesis independently of changes in plasma lipids. In the previous experiments, however, the animals were euthanized 12 weeks after injury. At that time, the balloon-injured area had accumulated much more cholesterol than the surrounding undamaged aorta. Thus, the abolishment of the direct antiatherogenic effect of estrogen by balloon catheter injury could be due to the high accumulation of cholesterol rather than to the state of the arterial endothelium. Furthermore, because the balloon-injured area was not separated into reendothelialized and deendothelialized tissue, the above experiments did not discriminate between effects of estrogen on aorta with regenerating endothelium and effects of estrogen on aorta where the endothelium is completely absent.
The purpose of the current study was to further investigate the relationship between estrogen, arterial endothelium, and NO in cholesterol-clamped ovariectomized rabbits. In the main experiment (atherosclerosis experiment), the direct effect of estrogen on undamaged and balloon-injured aorta and on circulating plasma nitrite/nitrate levels was studied at different time points during initiation of atherosclerosis (1 to 8 weeks). Before euthanasia, the rabbits were injected with Evans blue dye, enabling separation of the balloon-injured area into reendothelialized tissue and still deendothelialized tissue. In 2 additional experiments (NO experiments), the direct effect of estrogen on (I) eNOS activity and basal and stimulated release of NO from the endothelium, and (II) mononuclear-endothelial cell binding in the presence and absence of L-NAME, was studied after 4 (I+II) and 8 (I) weeks of cholesterol feeding.
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Methods |
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Sexually mature female New Zealand White rabbits (n=254) were purchased from Interfauna (Huntingdon, England). The rabbits were housed in individual stainless steel cages at a room temperature of 18±2°C with a 12-hour light cycle and free access to drinking water. All experimental procedures were performed in accordance with the Danish regulations for experiments with animals.
Atherosclerosis Experiment
Experimental Design
One hundred fifty rabbits were anesthetized with
intravenous pentobarbital and underwent bilateral
ovariectomy and balloon catheter injury in the lower thoracic aorta as
described previously.7 8 The rabbits were allowed to
recover for 3 weeks before treatment was initiated.
The rabbits were then divided into 10 groups with similar baseline values of plasma cholesterol and body weight to receive intervention with (1) cholesterol-enriched chow together with either 17ß-estradiol (17ß-E2) cypionate (Sigma) or vehicle (corn oil; Nomeco) for 1, 2, 4, or 8 weeks (estrogen and placebo groups; n=15) or (2) regular (vehicle-enriched) chow together with vehicle for 0 (start of cholesterol feeding period) or 8 weeks (chow groups; n=15). 17 ß-E2 cypionate was given subcutaneously in a concentration of 50 µg/kg every 3rd day; vehicle was administered similarly. This dose of estrogen was chosen because it produces plasma estradiol levels in rabbits comparable to those seen in women receiving estrogen replacement therapy.9 Dietary cholesterol was given in individual amounts, aiming at a plasma concentration of about 25 mmol/L in all rabbits.6 7 8 Blood samples for plasma cholesterol determination were drawn 1 to 2 times weekly and at euthanasia. The final blood samples were additionally used for measurement of circulating nitrite/nitrate levels.
The animals were injected intravenously with 5 mL of Evans blue dye (5 mg/mL), which was allowed to circulate for 5 minutes before the rabbits were killed with an overdose of intravenous pentobarbital.6 7 8 9 The aorta was then removed from the level of the aortic valves to the level of the diaphragm, opened longitudinally, and divided into 4 parts: arch (valves to ductus arteriosus; undamaged), upper thoracic (ductus arteriosus to second intercostal arteries; undamaged), lower thoracic (fifth intercostal arteries to celiac artery; balloon-injured), and abdominal (celiac artery to renal arteries; undamaged) aorta. The balloon-injured lower thoracic aorta was further separated into white tissue, consisting of reendothelialized endothelium, and blue tissue, consisting of still deendothelialized endothelium. Each aortic part was fixed with pins on a cork board, and the intima/inner media was stripped from the outer media, weighed, and stored at -20 C° until analyzed for aortic cholesterol content.6 7 8
Measurement of Circulating Nitrite/Nitrate Levels
Plasma nitrite/nitrate levels were measured with the use of
Griess reagent.10 To reduce the lipid content, plasma
samples were initially centrifuged at 14 000 rpm for 2
minutes, and the infranate was carefully removed. This procedure was
repeated once. The samples (50 uL) were incubated in microtiter plate
wells for 45 minutes at room temperature with 10 uL of 30 uM NADPH and
40 uL of freshly prepared Master Mix (glucose-6-phosphate 15
mmol/L, G6P dehydrogenase 4800 U/L, nitrate reductase 2400 U/L, and
NaPi buffer 0.448 mol/L, PH 7.4) to convert nitrate to nitrite.
Standards of nitrite and nitrate (5 to 200 uL) were run in parallel.
Total nitrite (nitrite+nitrate) was analyzed by reacting the
samples with Griess reagent (0.1% N-(1-naphtyl) ethylenediamine HCl
and 1% sulfanilamide in 5% phosphoric acid). The absorbance of the
reacted samples was read at 540 nm after 10 minutes of
incubation at 20°C. The nitrate standards gave absorbance values that
were >90% of the corresponding nitrite standards. The interassay
variation coefficient for the standards with the same absorbance value
as the plasma samples were <2%.
NO Experiment I
Forty-four rabbits were ovariectomized, individually
cholesterol-fed, and treated with either 17ß-E2 cypionate
or vehicle as described above for 4 or 8 weeks (n=11). At euthanasia,
the thoracic aorta was removed and carefully dissected free of
connective tissue, for measurement of basal and stimulated NO release,
eNOS activity, and aortic cholesterol content (lower 3
cm).6 7 8
Basal and Stimulated NO Release
Rings from the upper thoracic aorta were suspended in organ
baths for the measurement of isometric tension as described
previously.11
In one ring, basal release of NO was determined indirectly by the magnitude of contraction evoked by 100 uM of L-NAME (Sigma). In another ring, a cumulative dose-response curve for acetylcholine (Sigma) was performed. Both rings were precontracted with phenylephrine to 30% (basal release) and 50% (acetylcholine-mediated relaxation) of the contractile response evoked by 122 mmol/L potassium. The degree of contractions/relaxations is expressed as a percent of this contraction.
eNOS Activity
The middle thoracic aorta was removed and transferred to
ice-cold, oxygenated saline. The aorta was opened
longitudinally, and endothelial cells were obtained by
a single scrape with a razor blade along the luminal surface. The cells
were placed in Eppendorf tubes containing 50 uL of 50 mmol/L TRIS
buffer (pH 7.4) and immediately frozen in liquid nitrogen. The activity
of eNOS was determined by conversion of
14C-L-arginine
to14C-L-citrulline by a modification
of methods previously described.12 In short, the cells
were homogenized by 5 cycles of freeze-thawing and
incubated for 30 minutes at 37°C in a reaction buffer containing
14C-L-arginine and
calmodulin, tetrahydrobiopterin, FAD, and ß-NADPH.
14C-L-citrulline was isolated by
column chromatography and quantified by liquid
scintillation counting. To express the activity per
endothelial cell, the number of cells in each sample
was determined by cell counting in a counting chamber at the light
microscopic level. Samples with endothelial cells from
pig aorta were used as control material. The interassay variation
coefficient was 17%.
NO Experiment II
Sixty rabbits were ovariectomized, individually
cholesterol-fed, and treated with 17ß-E2 or vehicle as
described above, either alone or together with 160 µg/mL L-NAME in
their drinking water for 4 weeks (n=15). At euthanasia, the thoracic
aorta was removed and carefully dissected free of connective tissue,
for measurement of mononuclear-endothelial cell binding
and aortic cholesterol content (lower 3
cm).6 7 8
Mononuclear Cell Adhesion Experiment
Mononuclear cell adhesion was determined as previously
described.13 Briefly, a 3-cm segment of the upper thoracic
aorta was opened longitudinally and, with the
endothelial side up, was incubated in a 35-mm culture
dish with Hanks' balanced salt solution (HBSS) on a rocking platform.
After 10 minutes, the HBSS medium was replaced by binding medium
containing fluorescently labeled human mononuclear cells
(5x105 cells/mL). The aortic segment was
incubated with the mononuclear cells for 30 minutes, after which the
medium was aspirated and the segment washed twice with fresh binding
medium. Adherent cells were counted blindly under fluorescence
microscopy from 9 predetermined, equally distributed sites.
Statistics
Differences between groups were evaluated by a 2-way ANOVA with
post hoc analysis using the Student–Newman-Keuls test or the
Student's unpaired t test (nitrite/nitrate data). All
statistical analyses were performed with the GraphPad Prism
software program. P
0.05 was considered the level of
statistical significance.
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Results |
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Atherosclerosis Experiment
Characteristics of the 10 Rabbit Groups
The rabbits were well matched with regard to initial levels of body weight and plasma cholesterol (Table
). None of the
rabbit groups lost weight during the experimental period. The plasma
cholesterol level of the chow rabbits was not significantly
different at weeks 0 and 8. The cholesterol-fed groups
showed a considerable rise in plasma cholesterol levels
compared with the chow groups: the mean concentration of plasma
cholesterol during the experimental period (AUC/number of
days) increased with time (P<0.001 by 2-way ANOVA), but was
without difference between the estrogen and placebo group at each time
point. The amount of dietary cholesterol, which was used to
adjust each rabbit's plasma cholesterol to 25 mmol/L,
increased with time, but was already 2 times higher in the estrogen
than in the placebo group after the first week of
cholesterol feeding (P<0.001 by 2-way
ANOVA).
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Aortic Atherosclerosis
The data for the undamaged aorta are shown in Figure 1. Aortic cholesterol
accumulation of the chow rabbits did not change from week 0 to week 8.
Aortic cholesterol accumulation of the
cholesterol-fed groups was at a similar low and constant
level for the first 4 weeks. From weeks 4 to 8, however, accumulation
more than doubled in the placebo group (P<0.001 by multiple
comparison). Estrogen almost completely inhibited this increase,
resulting in a time-dependent overall effect of estrogen treatment on
aortic cholesterol accumulation (P<0.001 by
2-way ANOVA).
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The data for the balloon-injured aorta are shown in Figure 2. Cholesterol accumulation
of the chow rabbits was similar at weeks 0 and 8 and not significantly
different from that in the undamaged aorta in either white (top) or
blue (bottom) tissue. Cholesterol accumulation of the
cholesterol-fed groups increased steadily during the
experiment (P<0.001 by 2-way ANOVA) and was higher at all
time points than that in the undamaged aorta in both white and blue
tissue. The white tissue with regenerated endothelium
displayed similar aortic cholesterol accumulation in the
estrogen and placebo group at all time points. The blue tissue denuded
of endothelium, however, displayed an overall
significantly higher aortic cholesterol accumulation in the
estrogen than in the placebo group (effect of estrogen treatment,
P=0.004 by 2-way ANOVA). This difference was present
from week 2, but did not become significant until week 8
(P<0.01 by multiple comparison). The contribution of blue
tissue to the total balloon-injured area varied from 30% to 50% (wet
weight/wet weight) at the different points and was not significantly
different between the estrogen and placebo group.
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Circulating Plasma Nitrite/Nitrate Levels
In the chow groups, plasma nitrite/nitrate levels were lower than
in the cholesterol-fed groups and showed significantly
higher values at week 0 than at week 8 (P=0.01) (Figure 3). In the cholesterol-fed
groups, plasma nitrite/nitrate were also significantly affected by time
(P=0.02 by 2-way ANOVA); showing an initial increase from
week 1 to 2, and a subsequent fall from week 2 to 8. Plasma
nitrite/nitrate was markedly increased by estrogen treatment
(P<0.001 by 2-way ANOVA). This effect reached statistical
significance at week 1, 2 (P<0.05), and 4
(P<0.001) but not at week 8 (Student's unpaired
t test).
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NO Experiment I
Three rabbits in the 4-week estrogen group were excluded due to
anorexia or resistance to diet-induced
hypercholesterolemia. That left 41 rabbits in
the experiment. The mean concentration of plasma
cholesterol during the experimental period was
significantly higher at week 8 than at week 4, but not significantly
different between the estrogen and placebo group at each time point
(data not shown). As in the above experiment, aortic
cholesterol was not significantly different between the 2
groups at week 4 but was significantly lower in the estrogen group than
in the placebo group at week 8 (P<0.001 by multiple
comparison) (Figure 4, top).
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eNOS Activity and Basal and Stimulated NO Release
eNOS activity was similar at week 4 and 8, and not significantly
different between the estrogen and placebo group (Figure 4
, middle). The magnitude of vasoconstriction induced by L-NAME (basal NO
release) was not significantly different at week 4 and 8 (Figure 4, bottom). At week 4, vasoconstriction was significantly
greater in the estrogen than in the placebo group (P<0.01
by multiple comparison). This difference, however, was no longer
present at week 8. Acetylcholine-induced relaxation (stimulated NO
release) was not significantly different at week 4 and 8, and not
significantly different between the estrogen and placebo group, despite
a trend to an estrogen-mediated improvement in dilatation response
after 8 weeks (Figure 5).
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NO Experiment II
Neither the mean concentration of plasma cholesterol
during the experimental period nor the aortic accumulation of
cholesterol was significantly different between the 2
groups after 4 weeks of individualized cholesterol feeding
(data not shown).
Mononuclear Cell Adhesion
In rabbits not administered with L-NAME, aortic segments from
estrogen-treated rabbits demonstrated a 50% decrease in cell binding
compared with placebo-treated rabbits (P<0.05 by multiple
comparison) (Figure 6). This difference,
however, was significantly reduced by L-NAME administration
(interaction, P=0.05 by 2-way ANOVA). This resulted in no
significant decrease in cell binding by treatment with estrogen in
combination with L-NAME. L-NAME administration alone did not
significantly change aortic cell binding.
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Discussion |
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Atherosclerosis Experiment
Cholesterol feeding, in contrast to oil-enriched chow, significantly increased plasma and aortic cholesterol. The higher amount of dietary cholesterol given to the estrogen than to the placebo group suggests that estrogen would have significantly reduced plasma cholesterol levels had the rabbits not been individually fed cholesterol. The high levels of circulating cholesterol did not accumulate significantly in the undamaged aorta of the 2 groups within the first 4 weeks. Within the following 4 weeks, however, aortic cholesterol more than doubled in the placebo-treated rabbits. The inhibition of aortic cholesterol accumulation by estrogen, mediated independently of plasma cholesterol levels, suggests that estrogen in some way increases the resistance of the artery wall to atherosclerosis. Alternatively, estrogen may inhibit atherosclerosis by favorably affecting plasma lipoprotein patterns. Such changes are not equalized by the cholesterol clamping technique and were not measured in the current study. A number of studies, however, suggest that the beneficial effect of estrogen is either independent of or only partially explained by changes in plasma lipoproteins.3 4 5 6 7 8
This present study confirms our previous finding that the direct antiatherogenic effect of estrogen is abolished by balloon catheter injury in cholesterol-fed rabbits.6 7 8 In previous experiments, however, the rabbits were euthanized 12 weeks after surgery. At that time, the balloon-injured area consists of areas denuded of endothelium as well as areas covered with regenerated endothelial cells that are irregularly shaped, lack alignment in the direction of the blood flow, and exhibit endothelial dysfunction.14 15 In this study, the effects of estrogen on the balloon-injured aorta were therefore separated into effects on reendothelialized and effects on deendothelialized tissue. The results suggest that the overall lack of effect of estrogen on balloon-injured tissue is the aggregate of a neutral effect of estrogen on reendothelialized tissue and a paradoxical atherogenic effect of estrogen on deendothelialized tissue. Furthermore, it demonstrates that these effects are also present at low levels of aortic cholesterol accumulation, adding further support to the idea that the different effects of estrogen on undamaged and balloon-injured aorta are explained by changes in the endothelial state rather than by a general attenuation of the antiatherogenic effect of estrogen at high levels of atherosclerosis.
NO Data
A paradoxical atherogenic effect as demonstrated in the
atherosclerosis experiment has previously been observed
in endothelium-denuded aorta of female compared with
male rabbits13 and in balloon-injured (nonseparated) aorta
of rabbits treated with estrogen+L-NAME.7 Thus, absence of
endothelial NO, either by endothelial
denudation or enzyme inhibition, may play a role for this previously
unnoticed effect of estrogen/female sex on vascular tissue. NO is
released from the endothelium both continuously (basal
release), and in response to different agents (stimulated release) and
acts as an endogenous vasodilator as well as an
antiatherogenic molecule.16 In the present study, estrogen
selectively increased basal release of NO. This is in accordance with
previous reports in animals and humans,17 18 although the
cause of this phenomenon is not known. It may not be explained by
changes in eNOS protein level, as this would affect stimulated release
of NO as well. Changes in eNOS protein level would also result in
altered aortic eNOS activity which could not be demonstrated in the
current experiment after 4 or 8 weeks. Thus, estrogen may affect basal
NO release via alternative mechanisms. It has been suggested that
estrogen via an interaction with its receptor induces a moderate
elevation of free cytosolic calcium in endothelial
cells, resulting in increased activity of the calcium-dependent
eNOS.19 Such a moderate increase in calcium level would be
masked by the more significant increase following stimulation with
acetylcholine. Similarly, the increased eNOS activity in vivo would be
masked in the current NOS assay, where calcium and all other known
cofactors are added in excess. Alternatively, it has been suggested
that estrogen inhibits superoxide anion production, a free
radical able to react rapidly with NO leading to its
inactivation.20
Estrogen increased basal NO release from aortic rings only before differences in aortic cholesterol became apparent. This is in accordance with a previous study, where an increase in basal NO release by female sex was present before cholesterol feeding was initiated but not after 10 to 15 weeks of cholesterol feeding.17 A transiency in the effect of estrogen on basal NO release is further supported by the data for circulating nitrite/nitrate, showing an increase in the estrogen-treated groups after 1,2, and 4 but not after 8 weeks of cholesterol feeding. Thus, a possible involvement of NO in the direct antiatherogenic effect of estrogen may take place at an early stage of atherogenesis.
We have recently shown that long-term inhibition of NO synthesis significantly attenuates the direct antiatherogenic effect of estrogen in cholesterol-fed rabbits.7 NO has been demonstrated to decrease vascular cell adhesion molecule-1 (VCAM-1) expression in cultured endothelial cells,21 and to inhibit monocyte-endothelial cell interaction.22 A similar regulatory effect on VCAM-1 expression has been demonstrated for estrogen.23 Thus, a likely mechanism by which NO could mediate estrogen's antiatherogenic effect is through a reduction in mononuclear-endothelial cell binding. Our data, showing a 50% decrease in cell binding of aortic segments from estrogen-treated rabbits compared with placebo-treated rabbits and an abolishment by this effect by simultaneous treatment with L-NAME, suggest (1) that the effect of estrogen on VCAM-1 expression is translated into a reduced mononuclear-endothelial cell binding in vivo and (2) that this effect of estrogen is mediated by its ability to increase NO production.
Conclusion
This study shows that the direct antiatherogenic effect of
estrogen is already present after 8 weeks in the
cholesterol-clamped rabbit model. The effect varied with
the state of the aortic endothelium, was paradoxically
reversed in denuded areas, and was preceded by a transient increase in
basal NO release and circulating nitrite/nitrate concentrations. This
transient increase in NO production may be involved in the
direct antiatherogenic effect of estrogen by in turn mediating a 50%
reduction in mononuclear-endothelial cell binding, one
of the earliest step in atherogenesis. Thus, these data add support to
the notion that the endothelium and
endothelial NO are involved in the mechanism by which
estrogen inhibits atherosclerosis independently of
plasma cholesterol levels.
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Acknowledgments |
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These studies were supported by grants from the Danish Heart Foundation.
Received March 15, 1999; revision received June 9, 1999; accepted June 16, 1999.
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Barrett-Connor E, Bush TL. Estrogen and coronary heart disease in women. JAMA. 1991;265:1861–1867.
2. 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]
3.
Adams MR, Kaplan JR, Manuck SB, Koritnik DR, Parks JS,
Wolfe MS, Clarkson TB. Inhibition of coronary artery
atherosclerosis by 17-beta estradiol in ovariectomized
monkeys. Arteriosclerosis. 1990;10:1051–1057.
4. 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.
5.
Bourassa PK, Milos PM, Gaynor BJ, Breslow JL, Aiello
RJ. Estrogen reduces atherosclerotic lesion development in
apolipoprotein E-deficient mice. Proc Natl Acad Sci
U S A. 1996;93:10022–10027.
6.
Holm P, Stender S, Andersen HØ, Hansen BF, Kjeldsen
K, Nordestgaard BG. Antiatherogenic effect of estrogen abolished by
balloon catheter injury in cholesterol-clamped rabbits.
Arterioscler Thromb Vasc Biol. 1997;17:1504–1511.
7. Holm P, Korsgaard N, Shalmi M, Andersen HL, Hougaard P, Skouby SO, Stender S. Significant reduction of the antiatherogenic effect of estrogen by long-term inhibition of nitric oxide synthesis in cholesterol-clamped rabbits. J Clin Invest. 1997;100:821–828.[Medline] [Order article via Infotrieve]
8.
Holm P, Shalmi M, Korsgaard N, Guldhammer B, Skouby
SO, Stender S. A partial estrogen receptor agonist with strong
antiatherogenic properties without noticeable effect on
reproductive tissue in cholesterol-fed female and male
rabbits. Arterioscler Thromb Vasc Biol. 1997;17:2264–2272.
9. Holm P, Andersen HØ, Nordestgaard BG, Hansen BF, Kjeldsen K, Stender S. Effect of estrogen 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]
10. Verdon CP, Burton BA, Prior RL. Sample pretreatment with nitrate reductase and glucose-6-phosphate dehydrogenase quantitatively reduces nitrate while avoiding interference by NADP+ when the Griess reaction is used to assay for nitrite. Anal Biochem. 1995;224:502–508.[Medline] [Order article via Infotrieve]
11. Furchgott RF, Zawadski JV. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature. 1980;288:373–376.[Medline] [Order article via Infotrieve]
12.
Fabricius M, Rubin I, Bundgaard M, Lauritzen M. NOS
activity in brain and endothelium: Relation to
hypercapnic rise of cerebral blood flow in rats. Am J
Physiol. 1996;271:H2035–H2044.
13.
Holm P, Andersen HL, Arrøe G, Stender S. Gender gap in
aortic cholesterol accumulation in
cholesterol-clamped rabbits: role of the
endothelium and mononuclear-endothelial
cell interaction. Circulation. 1998;98:2731–2737.
14.
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;81:1667–1679.
15.
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.
16.
Moncada S, Higgs A. The L-arginine-nitric oxide
pathway. N Engl J Med. 1993;329:2002–2012.
17. Hayashi T, Fukuto JM, Ignarro LJ, Chaudhuri G. Gender differences in atherosclerosis: possible role of nitric oxide. J Cardiovasc Pharmacol. 1995;26:792–802.[Medline] [Order article via Infotrieve]
18.
Sudhir K, Jennings GL, Funder JW, Komerasoff PA.
Estrogen enhances basal nitric oxide release in the forearm vasculature
in perimenopausal women. Hypertension. 1996;28:330–334.
19. Rubanyi GM, Freay AD, Kauser K, Sukovich D, Burton G, Lubahn DB, Couse FJ, Curtis SW, Korach KS. Vascular estrogen receptors and endothelium-derived nitric oxide production in the mouse aorta. Gender difference and effect of estrogen receptor disruption. J Clin Invest. 1997;99:2429–2437.[Medline] [Order article via Infotrieve]
20.
Arnal JF, Clamens S, Pechet C, Negre-Salvayres A,
Allera C, Girolami J-P, Salvayres R, Bayard F. Ethinylestradiol does
not enhance the expression of nitric oxide synthase in bovine
endothelial cells but increases the release of
bioactive nitric oxide by inhibiting superoxide anion
production. Proc Natl Acad Sci U S A. 1996;93:4108–4113.
21.
Khan BV, Harrison DG, Olbrych MT, Alexander RW, Medford
RM. Nitric oxide regulates vascular cell adhesion molecule-1 gene
expression and redox-sensitive transcriptional events in human vascular
endothelial cells. Proc Natl Acad Sci
U S A. 1996;93:9114–9119.
22.
Tsao PS, McEvoy LM, Drexler H, Butcher EC, Cooke JP.
Enhanced endothelial adhesiveness in
hypercholesterolemia is attenuated by
L-arginine. Circulation. 1994;89:2176–2182.
23.
Caulin-Glaser T, Farrell WJ, Pfau SE, Zaret B, Bunger
K, Setaro JF, Brennan JJ, Bender JR, Cleman MW, Cabin HS, Remetz MS.
Modulation of circulating cellular adhesion molecules in postmenopausal
women with coronary artery disease. J Am Coll
Cardiol. 1998;31:1555–1560.
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F. Grodstein, J. E. Manson, M. J. Stampfer, and W. C. Willett The Discrepancy between Observational Studies and Randomized Trials of Menopausal Hormone Therapy Ann Intern Med, May 4, 2004; 140(9): 764 - 765. [Full Text] [PDF]
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 E. Lokkegaard, Z. Jovanovic, B. L. Heitmann, N. Keiding, B. Ottesen, Y. A. Hundrup, E. B. Obel, and A. T. Pedersen Increased Risk of Stroke in Hypertensive Women Using Hormone Therapy: Analyses Based on the Danish Nurse Study Arch Neurol, October 1, 2003; 60(10): 1379 - 1384. [Abstract] [Full Text] [PDF]
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 C. Noel Bairey Merz, B. D. Johnson, B. L. Sharaf, V. Bittner, S. L. Berga, G. D. Braunstein, T. K. Hodgson, K. A. Matthews, C. J. Pepine, S. E. Reis, et al. Hypoestrogenemia of hypothalamic origin and coronary artery disease in premenopausal women: a report from the NHLBI-sponsored WISE study J. Am. Coll. Cardiol., February 5, 2003; 41(3): 413 - 419. [Abstract] [Full Text] [PDF]
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 K. L. Chambliss and P. W. Shaul Estrogen Modulation of Endothelial Nitric Oxide Synthase Endocr. Rev., October 1, 2002; 23(5): 665 - 686. [Abstract] [Full Text] [PDF]
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 R. P. Byington, C. D. Furberg, D. M. Herrington, J. A. Herd, D. Hunninghake, M. Lowery, W. Riley, T. Craven, L. Chaput, C. C. Ireland, et al. Effect of Estrogen Plus Progestin on Progression of Carotid Atherosclerosis in Postmenopausal Women With Heart Disease: HERS B-Mode Substudy Arterioscler Thromb Vasc Biol, October 1, 2002; 22(10): 1692 - 1697. [Abstract] [Full Text] [PDF]
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K. K. Koh, J. Y. Ahn, D. K. Jin, B.-K. Yoon, H. S. Kim, D. S. Kim, M.-S. Shin, J. W. Son, I. S. Choi, and E. K. Shin Effects of Continuous Combined Hormone Replacement Therapy on Inflammation in Hypertensive and/or Overweight Postmenopausal Women Arterioscler Thromb Vasc Biol, September 1, 2002; 22(9): 1459 - 1464. [Abstract] [Full Text] [PDF]
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 K. K. Koh Effects of estrogen on the vascular wall: vasomotor function and inflammation Cardiovasc Res, September 1, 2002; 55(4): 714 - 726. [Full Text] [PDF]
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H. Y. Chan, X. Yao, S. Y. Tsang, J.-P. Bourreau, F. L. Chan, and Y. Huang Isoproterenol amplifies 17{beta}-estradiol-mediated vasorelaxation: role of endothelium/nitric oxide and cyclic AMP Cardiovasc Res, February 15, 2002; 53(3): 627 - 633. [Abstract] [Full Text] [PDF]
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 J. B. Chang and T. A. Stein Ten-Year Outcome After Saphenous Vein Patch Angioplasty in Males and Females After Carotid Endarterectomy Vascular and Endovascular Surgery, January 1, 2002; 36(1): 21 - 27. [Abstract] [PDF]
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 C. Amant, P. Holm, S.-h. Xu, N. Tritman, M. Kearney, and D. W. Losordo Estrogen Receptor-Mediated, Nitric Oxide-Dependent Modulation of the Immunologic Barrier Function of the Endothelium: Regulation of Fas Ligand Expression by Estradiol Circulation, November 20, 2001; 104(21): 2576 - 2581. [Abstract] [Full Text] [PDF]
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D. W. Losordo and J. M. Isner Vascular endothelial growth factor-induced angiogenesis: crouching tiger or hidden dragon? J. Am. Coll. Cardiol., June 15, 2001; 37(8): 2131 - 2135. [Full Text] [PDF]
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 R. K. Dubey and E. K. Jackson Estrogen-induced cardiorenal protection: potential cellular, biochemical, and molecular mechanisms Am J Physiol Renal Physiol, March 1, 2001; 280(3): F365 - F388. [Abstract] [Full Text] [PDF]
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 D. M. Herrington, D. M. Reboussin, K. B. Brosnihan, P. C. Sharp, S. A. Shumaker, T. E. Snyder, C. D. Furberg, G. J. Kowalchuk, T. D. Stuckey, W. J. Rogers, et al. Effects of Estrogen Replacement on the Progression of Coronary-Artery Atherosclerosis N. Engl. J. Med., August 24, 2000; 343(8): 522 - 529. [Abstract] [Full Text] [PDF]
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M. R Andersen and S. Stender Endothelial nitric oxide synthase activity in aorta of normocholesterolemic rabbits: regional variation and the effect of estrogen Cardiovasc Res, July 1, 2000; 47(1): 192 - 199. [Abstract] [Full Text] [PDF]
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