© 2000 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Physiological Concentration of 17ß-Estradiol Retards the Progression of Severe Atherosclerosis Induced by a High-Cholesterol Diet Plus Balloon Catheter Injury
Role of NO
From the Department of Geriatrics, Nagoya University School of Medicine, Nagoya, Japan.
Correspondence to Toshio Hayashi, MD, Department of Geriatrics, Nagoya University School of Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya 466, Japan. E-mail hayashi{at}med.nagoya-u.ac.jp
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Abstract—The molecular mechanisms of the antiatherosclerotic effects of estrogen are not yet known. We evaluated the effects of 17ß-estradiol (E2) on high cholesterol diet– (HCD; standard diet and 1% cholesterol) and balloon injury–induced atherosclerosis in female New Zealand White rabbits. The abdominal aortas of 40 oophorectomized (Groups 1 through 5) and 8 nonoophorectomized (Group 6) rabbits were injured by balloon catheter, and the animals were then divided into the following groups and treated for 10 weeks: Group 1, standard diet; Group 2, standard diet plus a moderate dose of E2 (100 µg · kg-1 · d-1); Group 3, HCD; Group 4, HCD plus a moderate dose of E2; Group 5, HCD plus a low dose of E2 (20 µg · kg-1 · d-1); and Group 6, HCD in nonoophorectomized rabbits. After the treatment phase, plasma E2 was increased up to 282.2±45.5 pg/mL in Group 2, 263.0±41.5 pg/mL in Group 4, 87.9±18.8 pg/mL in Group 5, and 45.6±7.3 pg/mL in Group 6. HCD-mediated increases in plasma lipid levels were not changed by E2 treatment, whereas E2 decreased the aortic intimal thickening in Group 2 animals compared with those in Group 1 and reduced atherosclerosis in the thoracic and abdominal aortas of Group 4, 5, and 6 rabbits compared with those in Group 3. E2 restored the impaired abdominal aortic endothelium–dependent relaxation of balloon-injured and HCD-supplemented rabbits, and E2 increased basal nitric oxide (NO) release. The basal NO–releasing effect showed a significant, inverse relation with the severity of atherosclerosis. Plasma E2 concentration also showed a significant, inverse relation with atherosclerotic area. In conclusion, physiological concentrations of E2 can retard the progression of severe atherosclerosis and stabilize atheromas induced by HCD and balloon injury. The retardation may be partially mediated by endothelial NO function in vessels treated with E2.
Key Words: nitric oxide • estradiol • arteriosclerosis • balloon injury • endothelium
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Introduction |
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It is well known that estrogen retards the development of atherosclerosis,1 but the mechanisms of action are not clearly established. Estrogen replacement therapy suppresses the incidence of cardiovascular disease in postmenopausal women,2 and it reduces plasma LDL cholesterol and increases HDL cholesterol levels.3 However, the alterations in lipid profiles reportedly account for only a limited portion of the protective effects of estrogen against cardiovascular disease.4 Estrogen exerts a direct action on the vessel wall via incompletely understood mechanisms.5 Recently, estradiol was found to inhibit leukocyte adhesion and transendothelial migration in rabbits, which suggests one of the mechanisms of a direct antiatherosclerotic effect by estrogen on the vessel wall.6 Nitric oxide (NO), released from endothelial nitric oxide synthase (endothelial NOS), has been demonstrated to offer protection against the development of atherosclerosis by producing vascular dilatation, inhibiting monocyte adhesion to the endothelium, and other modes of action.7 8 9 We have found that estrogen acts via an NO-mediated system in vivo and in vitro. The aortas of intact (nonoophorectomized) female rabbits release a greater amount of NO than do the aortas of oophorectomized female or of male rabbits, and thus, NO might effectively protect against atherosclerosis in female versus male rabbits.10 11 17ß-Estradiol (E2) increases endothelial NOS activity via a receptor-mediated system in cultured endothelial cells.12 13 This information led us to investigate the effects of E2 on atherosclerosis and endothelial dysfunction induced by a high-cholesterol diet (HCD) and balloon injury.
Our preliminary experiment had shown that severe atherosclerosis (almost 75% stenosis) developed in the abdominal aorta in response to cholesterol feeding after balloon injury. We had also shown that an HCD induces atherosclerosis more slowly in female rabbits than in males.11 The controversy remained as to whether balloon injury diminishes the acetylcholine (ACh)-induced, NO-mediated relaxation and whether the distance between the endothelium and medial smooth muscle cells inhibits the effects of NO.14 15 It is not known whether E2 can prevent the impairment of endothelium-dependent relaxation or basal NO release in aortas with severe atherosclerosis induced by balloon catheter injury and an atherogenic diet. Therefore, in the current study, we evaluated how E2 treatment affects basal NO release, as evaluated by contractions in response to NG-monomethyl-L-arginine acetate (L-NMA) and aortic cGMP levels in atherosclerotic vessels. This report shows the antiatherosclerotic effect of E2 in the aortas of oophorectomized female rabbits treated with an HCD and balloon injury.
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Methods |
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Chemicals and Solutions
ACh, prostaglandin F2
(PGF2), calcium ionophore A-23187,
indomethacin, and L-NMA were all purchased from Sigma
Chemical Co. Monoclonal antibodies against estrogen receptors- and
-ß were purchased from Transduction Laboratories.
Nitroglycerin (NTG; 10% wt/wt triturate in lactose)
was from Nihon Kayaku Co, Ltd. Krebs-Henseleit solution (118
mmol/L NaCl, 4.7 mmol/L KCl, 1.5 mmol/L
CaCl2, 1.2 mmol/L
MgSO4, 1.2 mmol/L
KH2PO4, 25 mmol/L
NaHCO3, 11 mmol/L glucose, and 0.002
mmol/L EDTA; pH 7.4) was saturated with 95%
O2/5% CO2. The composition
of the depolarizing KCl solution was similar to that of Krebs-Henseleit
buffer, except for the replacement of NaCl by an equimolar amount of
KCl. All concentrations shown are those in the final bath.
Animals
A total of 48 female New Zealand White rabbits, 3 to 4 months
old and weighing 2.0 to 2.4 kg, were obtained from Kitayama Rabbits
(Ina, Japan). The rabbits were housed individually in stainless steel
cages at 20±3°C with a 12-hour light/dark cycle and with free access
to water. All animals were initially fed standard rabbit chow (Oriental
Yeast Co, Ltd) for 2 weeks. After 2 weeks of the standard chow diet, 40
rabbits, which were bilaterally oophorectomized, and 8
nonoophorectomized rabbits were used in this study. Six weeks after
oophorectomy, the abdominal aortas of all rabbits were injured by
balloon as described previously, with slight
modification.14 In brief, a 3F Fogarty catheter was
inserted from the right femoral artery and advanced to a position just
below the diaphragm. The balloon was inflated with 0.6 mL of
saline (distension, 8.0 mm), and the catheter was pulled 3 times
until the bifurcation of the iliac arteries was reached. Finally, the
balloon was deflated and the catheter withdrawn. Before balloon injury,
we confirmed that plasma concentrations of estrogen were <5
pg/mL.
The oophorectomized (Groups 1 through 5) and nonoophorectomized (Group 6) balloon-injured rabbits were then subdivided into the following groups, depending on diet and E2 treatment given for the next 10 weeks. Group 1 received a standard chow diet plus 1% ethanol (the solvent for E2); Group 2 received a standard diet and E2 at 100 µg · kg-1 · d-1; Group 3 received an HCD (standard diet with 1% cholesterol) plus 1% ethanol; Group 4 received an HCD plus 100 µg · kg-1 · d-1 of E2; Group 5 received an HCD plus 20 µg · kg-1 · d-1 of E2; and Group 6, the nonoophorectomized rabbits, received an HCD only. Each group consisted of 8 rabbits. E2 was diluted in 1% ethanol (100 µg/mL) and administered daily at 9 AM by subcutaneous injection. Feeding was restricted to 100 g/d. Blood was sampled 4 and 24 hours after the last feeding for the measurement of plasma E2 concentrations. The general appearance of the rabbits was observed daily, and they were weighed every 4 weeks. All of the rabbits appeared healthy throughout the course of study. All experiments were conducted in accordance with institutional guidelines for animal research.
Determinations of Plasma Lipid and E2
Concentrations
Total cholesterol and triglyceride
levels were measured by enzymatic assays as described
previously.16 17 The HDL cholesterol was
determined after precipitation with
phosphotungstate-MgCl2.17 The plasma
concentration of E2 was quantified as described
previously.18
Histological Evaluation of Aortic
Atherosclerosis
After 10 weeks of treatment, the rabbits were killed by
exsanguination after being anesthetized with pentobarbital (50
mg/kg IV). Cross sections of the descending thoracic aorta and
abdominal aorta, adjacent to each segment taken for evaluation of
endothelium-dependent responses, were stained with
hematoxylin-eosin to examine the endothelial lining and
with van Gieson’s elastic stain to determine the thickness of the
intima. Morphometric analysis was performed as described by
Weiner et al.19 In brief, the complete section in each
block was projected onto a vertical surface with a projecting
microscope. Six samples from each rabbit aorta were analyzed
with the objective lens. The contours of the lumen and internal elastic
lamina were traced, and the tracings were digitized (PC-9801 ES, NEC)
by using a graphics tablet. The mean surface involvement by
atherosclerotic lesion per vessel per animal was calculated by summing
all results obtained after dividing the lesion circumference by the
circumference of the internal elastic lamina and then dividing the sum
by the number of sections studied. Circumferences of lesion and normal
vessel were defined as circumferences of each part of internal elastic
lamina. The area occupied by atherosclerotic lesions was defined as the
percent area bounded by the lumen and the internal elastic lamina for
the ideal luminal area. The ideal luminal area was calculated from the
perimeter of the internal elastic lamina on the assumption that the
true shape of the vessel was circular, to exclude the artificial effect
due to fixation in 10% formalin solution. The mean area occupied by
the lesions per vessel per animal was calculated by summing the areas
occupied by lesion of all sections and dividing the sum by the number
of sections per vessel (n=6 for 1 vessel). Data were transferred to a
minicomputer (Macintosh Quadra 700, Apple, Ltd) for further
analysis.
Assays for Tissue Cholesterol Content
To assay for free and esterified cholesterol
content, the 2-cm-long segment of the abdominal aorta, from the portion
enclosed by the diaphragm (9 cm distal to the aortic valve) to the
portion 11 cm distal to the aortic valve, was weighed, minced, and
homogenized in 20 volumes of chloroform/methanol (2:1,
vol/vol) containing 0.001% BHT as an antioxidant in a motor-driven,
glass-glass homogenizer at 0°C to 2°C. Total lipid
extraction was carried out in the homogenate by using the
Folch procedure. The lipid-containing fraction was dried under
N2 and resuspended in isopropyl alcohol. The
total cholesterol and free cholesterol levels
were measured in the extract by the specific enzymatic assays mentioned
in plasma lipid analysis. The esterified
cholesterol was calculated as the difference between total
and free cholesterol (Wako Pure Chemical Industries,
Ltd).20
Immunocytochemical Analysis
Cross sections of the descending thoracic aorta, adjacent to
each segment taken for the evaluation of
endothelium-dependent responses (1 of continuous
sections used for histological evaluation of aortic
atherosclerosis), were deparaffinized with xylene and dehydrated with a
graded series of alcohol. The specimens were preincubated for 30
minutes with methanol containing 0.3%
H2O2 and washed for 10
minutes with PBS. The specimens were permeabilized with
0.1% Triton X-100 in PBS for 20 minutes; washed with PBS; blocked with
normal horse serum for 1 hour; and then incubated with primary
monoclonal antibody diluted in PBS for estrogen receptor- or -ß,
anti-rabbit macrophages (RAM11), anti–smooth muscle sells
(HHF35), or anti–endothelial NOS for 60 minutes. The
sections were then washed again with PBS. A biotynylated rabbit
anti-mouse IgG (1:500 dilution) was added, and the specimens were
incubated for another 30 minutes and washed with PBS, followed by
incubation for 30 minutes with avidin-biotin peroxidase complex reagent
(ABC kit, Vector Laboratories). The result was a brown peroxidase
reaction product of diaminobenzidine. Cell nuclei were
counterstained with methyl green.21 Negative controls
included substitution of the primary antiserum with either PBS or
irrelevant antibodies. Each field on the slide was scored for the
number of macrophage antibody–positive cells and
analyzed statistically as described in a previous
report.21 Five samples were prepared from each rabbit.
Isometric Tension Measurement
The thoracic and abdominal aortas of the rabbits were removed
carefully to protect the endothelial lining, cleared of
adhering fat and connective tissue, and cut into 2-mm-wide transverse
rings. The thoracic aorta was taken from the orifice of the left first
costal artery (
4 cm distal to the aortic valve) to the 3 cm above
the portion enclosed by the diaphragm (7 cm distal to the aortic
valve). The abdominal aorta was taken from 4 cm below the portion
enclosed by the diaphragm (13 cm distal to the aortic valve) to 3 cm
distal to the bifurcation of the internal iliac arteries. The
optimal passive load for both control and atherosclerotic aortas was
determined as the contractile response to 122 mmol/L KCl. Before
starting the experiment, the rings were stretched to their
predetermined optimal force, mounted on stainless steel hooks in
20-mL-capacity muscle chambers, and bathed in Krebs-Henseleit solution,
pH 7.4 at 37°C, for 1 hour. Force was measured isometrically with a
force displacement transducer (model DSA-603, Minebea Co, Ltd) and
displayed on a multipen recorder (model R-60, Rika Denki Co, Ltd).
Experiments were conducted to determine the responsiveness of the
endothelium-intact aortic rings to an
endothelium-dependent vasodilator, ACh. The
responsiveness of the endothelium-denuded aortic rings
to the endothelium-independent vasodilator NTG was also
determined. In these experiments, PGF2
(2.6x10-6 mol/L) initially induced the
submaximal force. To investigate the tone-related release of NO from
the endothelium-intact aortic rings, moderate vascular
tone (35% to 50% of the contractile response obtained with 122
mmol/L KCl) was induced by low PGF2
concentrations (0.8x10-6 mol/L).
Concentration-related contractile responses to L-NMA
(10-6 to 10-4 mol/L) were
assessed.10 In some experiments,
indomethacin (5x10-6 mol/L) was
added to the muscle chambers for 60 minutes before the start of the
experiment to rule out the contribution of prostanoids.
Measurement of Nitrite and Nitrate
(NO2-/NO3-)
The plasma concentrations of
NO2-/NO3-
(NOx) were measured with an automated NO
detector–high-performance liquid
chromatography system (ENO10, Eicom Co) as described
previously.22 In brief, samples were collected in an
automated sample injector connected to an automated NO detector.
NO2- and
NO3- in the dialysates were
separated by a reverse-phase separation column packed with polystyrene
polymer (NO-PAK, 4.6x50 mm; Eicom), and
NO3- was reduced to
NO2- in a reduction column
packed with copper-plated cadmium (NO-RED, Eicom).
NO2- was mixed with a Griess
reagent (0.5% sulfonamide, 0.025%
N-(1-naphthylethyl)ethylenediamine
dihydrochloride, and 1.25% HCl) to form a purple azo dye in a reaction
coil. The color of the product of the Griess reaction was measured
at 540 nm by a flow-through spectrophotometer (NOD-10, Eicom). The
mobile phase, which was delivered by a pump at a rate of 0.33 mL/min,
was 10% methanol containing 0.15 mol/L
NaCl/NH4Cl and 0.5g/L disodium EDTA.
Determination of cGMP
The cGMP concentration in the homogenates of aortas
was determined by a specific radioimmunoassay (RPN226,
Amersham).23 In brief, 4 aortic rings from the segment of
abdominal aorta from 2 cm distal to the portion enclosed by the
diaphragm (11 cm distal to the aortic valve) to 2 cm distal to
the bifurcation of the internal iliac arteries (wet weight each was
10±1 mg) were incubated for 30 minutes in test tubes containing
Krebs-Henseleit buffer saturated with 95% O2/5%
CO2. The rings were frozen in
LN2 and stored at -80°C. To determine cGMP
levels in aortic rings, they were homogenized in 1 mL of
6% trichloroacetic acid at 4°C and centrifuged at
12 000g for 5 minutes. The supernatant was washed 4 times
with 4 mL of water-saturated ethyl ether. Liquid samples were then
frozen at -80°C and lyophilized overnight. The lyophylate was
resolubilized in 1 mL of 0.05 mol/L sodium acetate buffer, and 50-µL
aliquots were placed in test tubes. Samples were assayed by a
radioimmunoassay for cGMP.23 Solids left from the
initial homogenization step were digested in 1 mL
of 0.1N NaOH overnight, after which total protein was determined.
Data Analysis
Relaxation was measured as the percentage of decrease in force
below that evoked by PGF2 in
arterial rings. Contraction in response to L-NMA was
measured as the percentage of increase in force above that evoked by
PGF2 in arterial rings. Results
were expressed as mean±SEM and represent unpaired data. Data
were compared by ANOVA with repeated measures. When a significant
F value was found, Scheffe’s test for multiple comparisons
was used to identify differences among groups. A level of
P<0.05 was considered statistically significant.
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Plasma Lipid and E2 Concentrations
There was no significant difference in serum HDL cholesterol, total serum protein, and body weight in all groups of rabbits over the course of study. In the animals receiving regular chow (Groups 1 and 2), total serum cholesterol or triglycerides did not change significantly over the course of the study. The addition of 1% cholesterol to the diet (Groups 3 through 6) significantly increased total cholesterol and triglyceride levels compared with their respective values in Group 1 animals (the Table
). Treatment with
E2 did not affect plasma lipid levels in this
study. The plasma concentration of E2 was
415.5±93.5 and 132.5±23.1 pg/mL, respectively, in Groups 4 and
5 after 4 hours of E2 administration.
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Histological Examination of
Atherosclerosis
Histological examination of the thoracic aortas
revealed more atheromatous lesions, as indicated by the
mean percentage of luminal encroachment and mean lesion area, in the
hypercholesterolemic (Group 3) group than in the
E2-treated groups (Group 4 or 5) or the
nonoophorectomized group (Group 6). The area of
atherosclerosis in the thoracic aorta was reduced to
20% by treatment with a moderate dose of E2 in
Group 4, and a 70% decrease was observed after low-dose
E2 treatment in Group 5 compared with the HCD
group (Group 3; Figure 1, left). An
50% decrease was observed in the nonoophorectomized rabbit aortas
(Group 6; Figure 1, left). Histologically, the
abdominal aortas of rabbits revealed more atheromatous
lesions than did the thoracic aortas. The abdominal aortas of HCD-fed
and balloon-injured rabbits (Group 3) showed severe
atherosclerosis with almost 75% stenosis,
although this was improved significantly in response to
E2 treatment in Groups 4 and 5.
E2 treatment in the rabbit group fed a regular
diet inhibited the intimal thickening after endothelium
denudation by balloon injury (Group 1 versus Group 2). The
atherosclerotic area of abdominal aorta was 60% diminished by a
moderate dose of E2 in Group 4, by 40% by a low
dose of E2 in Group 5, and by 25% in
nonoophorectomized rabbits (Group 6) compared with the HCD group (Group
3; Figure 1, right). These results suggest that
E2 can retard the progression of complicated
atherosclerosis induced by an HCD and balloon
injury.
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Aortic Cholesterol Content
The total and esterified cholesterol contents in the
vessels exhibited the same tendency as the atherosclerotic areas. Free
cholesterol levels increased as well in response to
cholesterol feeding, although the change was not
significant compared with the total and esterified
cholesterol content (Please see Figure
I online at
http://atvb.ahajournals.org).
Immunocytochemical Analysis
Atheroma in the abdominal aorta was composed of many
macrophage-derived foam cells and intimal smooth muscle
cell proliferation (Figure 2, top). A
significant antiatherosclerotic effect of estrogen was shown, and the
relative number of macrophages decreased in this study.
Estrogen treatment in Groups 4 and 5 not only reduced the area of
atherosclerosis but also decreased the area occupied by
macrophages (Figure 2, bottom).
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Endothelium-Dependent and -Independent
Relaxations
In all experimental groups, the
endothelium-dependent vasodilator ACh produced
concentration-dependent relaxations of the precontracted, thoracic
aortic rings with an intact endothelium (Figure 3, left). The magnitude of relaxation of
thoracic aortic rings from the hypercholesterolemic
animals (Group 3) was diminished. There was no significant difference
in endothelium-dependent relaxation observed among the
thoracic aortic rings obtained from the
normocholesterolemic groups (Groups 1 and 2) or from
the hypercholesterolemic animals that had been
administered moderate or low doses of E2 (Groups
4 and 5). Endothelium-dependent relaxation in
hypercholesterolemic, nonoophorectomized female rabbits
(Group 6) showed an intermediate response between HCD rabbits (Group 3)
and the treatment group given a low dose of E2
(Group 5).
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The endothelium-independent vasodilator NTG produced
concentration-dependent relaxations in precontracted,
endothelium-denuded thoracic aortic rings. No
significant difference in endothelium-independent
relaxation was observed in the aortic rings of all groups of rabbits
(Figure 3, middle). The inhibition of NOS by L-NMA
(10-7 to 10-4 mol/L) led
to a contractile response in aortic rings precontracted with
PGF2. The contractile response to L-NMA was
concentration dependent, and its magnitude was decreased in
endothelium-intact aortic rings of atherosclerotic
rabbits (Group 3) compared with rings from rabbits treated with the
normal diet (Groups 1 and 2; Figure 3, right). The L-NMA
contractile response was higher in E2-treated
rabbits (Groups 4, 5, and 6) than in untreated, atherosclerotic animals
(Group 3). Preincubation with indomethacin did not
affect the endothelium-dependent relaxation.
Balloon injury and an atherogenic diet diminished the ACh-induced,
NO-mediated relaxations in the abdominal aortas of rabbits (Figure 4, left). The ACh-mediated relaxation was
improved significantly by E2 treatment in rabbits
fed the regular diet (Group 1 versus Group 2). The ACh-mediated
relaxation was significantly abolished in the abdominal aortas of Group
3 rabbits (Figure 4, middle). E2
dose-dependently restored relaxation in the abdominal aortic rings of
Group 4, Group 5, and nonoophorectomized rabbits (Group 6). There was
no significant difference observed in
endothelium-independent relaxation in all groups of
rabbit aortic rings (Figure 4, middle). Inhibition of NOS by
L-NMA (10-7 to 10-4
mol/L) led to a contractile response in abdominal aortic rings
precontracted with PGF2, which was smaller
than that in the thoracic aorta. The L-NMA contractile response was
concentration dependent, and its magnitude was decreased in
endothelium-intact aortic rings of atherosclerotic
rabbits (Group 3) compared with rings from normal diet–treated rabbits
(Groups 1 and 2; Figure 4, right). The L-NMA contractile
response was higher in E2-treated rabbits (Groups
4, 5, and 6) than in untreated, atherosclerotic animals (Group 3).
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Plasma Nitrite and Nitrate
(NO2-/NO3-) and
Aortic cGMP Concentrations
The plasma concentration of NOx
(NO2-/NO3-)
was 42.9±5.7, 50.2±4.2, 48.3±4.7, 66.1±5.2, 57.9±4.1, and
54.6±2.9 µmol/L, respectively, in Groups 1 through 6.
E2 treatment or no oophorectomy (Groups 2, 4, 5,
and 6) tended to have increased concentrations of
NO2-/NO3- in plasma
compared with the other 2 groups of rabbits (Groups 1 and 3); however,
these differences did not achieve statistical significance
(P=0.061, Group 3 versus Group 4).
Hyperlipidemia did not affect the concentration of
NO2-/NO3-
in the present study. E2 treatment increased
the cGMP level in both the normal and the
hypercholesterolemic diet–supplemented rabbits. Thus,
cGMP levels were higher in Group 2 than in Group 1 animals and in Group
4 and 5 versus Group 3 animals (the Table). The cGMP level in
Group 6 rabbits was between the levels of Groups 3 and 4 (the
Table).
Relation of Estrogen Dose to Antiatherosclerotic and NO-Releasing
Effects in Rabbit Aortas
We used multiple regression analysis to investigate the
relation of plasma estrogen concentration, its antiatherosclerotic
effect in rabbit abdominal aortas, and the NO-releasing effect as
determined by aortic cGMP concentrations (Figure 5). Plasma estrogen levels showed a
dose-dependent antiatherosclerotic and NO-releasing effect, although
R2 was smaller than that for the
relation between estrogen concentration and atherosclerotic area. Thus,
E2 treatment might show an antiatherosclerotic
effect in these ways. The physiological and human
replacement dose of estrogen might act by increasing basal NO levels,
although it is possible that some antiatherosclerotic effects of
estrogen may be unrelated to the effects of NO in rabbit
atherosclerosis.
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Discussion |
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The aim of the present study was to shed light on the effect of E2on severe atherosclerosis and to investigate its possible mechanism(s) of action on atherosclerosis. Our experiments were specifically designed to evaluate the effects of E2 on the progression of atherosclerosis induced by balloon catheter injury and an atherogenic diet and on the atherosclerosis-associated impairment of endothelial NO function.
Plasma E2 concentrations were achieved by
treatment with 2 doses of E2: 1 was a moderate
dose (100 µg · kg-1 ·
d-1, yielding a mean E2
concentration of 263.0±41.5 pg/mL in Group 4) and the other was a low
dose (20 µg · kg-1 ·
d-1, yielding a mean E2
concentration of 87.9±18.8 pg/mL in Group 5) in HCD-induced
atherosclerotic rabbits. These plasma E2
concentrations are almost identical to those of the human menstrual
phase and are also seen in patients receiving E2
replacement therapy after menopause. As a control study, we examined
female rabbits without oophorectomy (Group 6); their plasma
E2 concentration (45.6±7.3 pg/mL) is the true
physiological level in rabbits.
E2 treatment in oophorectomized and
nonoophorectomized rabbits did not produce any significant variations
in plasma lipid levels (the Table). However, the present
study did show that E2 administration retarded
the progression of severe atherosclerosis induced by an
HCD plus balloon injury in the rabbit aorta. The mechanisms of the
antiatherosclerotic effects of E2 may be due to a
direct effect on the vessel wall. In fact, E2
treatment improved the HCD- and balloon injury–induced
atherosclerosis and restored the impaired
endothelial function in this study. We analyzed
these effects in the thoracic and abdominal aortas.
The objective of our study in the thoracic aorta was to evaluate the effect of estrogen on HCD-induced atherosclerosis. Our results support the finding of an antiatherosclerotic effect of estrogen described previously.24 25 However, the current study demonstrated that the antiatherosclerotic effect of E2 is dose dependent. E2 treatment increased NO-mediated vascular responses and basal NO release that had been decreased in atherosclerosis. We speculate from these results that protection of endothelial function, especially of basal NO release, may contribute to the protection against atherosclerosis.
The experiment in abdominal aortas of rabbits fed the HCD after balloon
injury was carried out to investigate the effects of estrogen on
atherosclerosis and endothelial
function recovery after endothelial injury. The
pathological findings in abdominal aortas were similar to the findings
in restenotic lesions after percutaneous
transluminal coronary angioplasty in human coronary
arteries, especially in plaques prone to rupture, which are rich in
macrophage-derived foam cells and contain large amount of
lipid.26 27 The findings in our study included intimal
smooth muscle cell proliferation and the appearance of some
macrophage-derived foam cells. The significant
antiatherosclerotic effect of estrogen in this study may be due to the
decreased number of macrophages in the atheromas of
rabbit aortas after E2 treatment. This might mean
that estrogen also plays a role in stabilizing the
atheroma; however, the effect of
physiological concentrations of
E2 on the abdominal aorta was observed to be weak
compared with that observed in the thoracic aorta. A significant,
inverse relation between basal NO and the severity of
atherosclerosis was observed statistically. The
expression of inducible NOS and peroxynitrite, a reaction product
of NO and O2-, has been
reported in atherosclerosis.28 However,
our study showed that inducible NOS–positive cells were very sparse in
the abdominal aortas of rabbits. In addition, areas positive for
nitrotyrosine, a marker of peroxynitrite, were not different in the
abdominal aortas of Group 3 and 5 rabbits (data not shown). Vascular
responses to the endothelium-independent vasodilator
NTG were also not different among the 6 groups of animals with
HCD-induced atherosclerosis in thoracic aortas and in
abdominal aortas subjected to balloon injury plus HCD-induced
atherosclerosis. On the other hand, NTG was more potent
in the thoracic aortas than in abdominal aortas (Figures 3 and 4). We speculate that severe and encroached whole-vessel
atherosclerosis in the abdominal aorta may affect not
only the initial precontraction force but also vessel elasticity in
response to NTG-induced relaxation.
Our present results contrast with previous reports describing
abolition of the antiatherosclerotic effect of estrogen on severe
atherosclerosis induced by an HCD and balloon
injury.29 30 The difference between our results and
previous reports may be due to the following reasons. We did not clamp
the plasma cholesterol level, which may have had some
additional effects, although these levels did not show any
statistically significant differences. Second, the dose and mode of
estrogen treatment were different. We selected 2 doses to achieve
moderate (ie, preovulation levels in young women) and
physiological (ie, replacement levels in
postmenopausal women) concentrations. Then we examined the
nonoophorectomized female rabbits to evaluate the effect of a truly
physiological concentration of estrogen in rabbits.
The plasma E2 concentration obtained by low-dose
E2 supplement (Group 5) was higher than that in
nonoophorectomized rabbits (Group 6). In other words, the replacement
level of plasma E2 for humans was higher than the
true E2 physiological
concentration for rabbits; thus, a stronger antiatherosclerotic effect
was observed in Group 5 than in Group 6. As a result, we could compare
the effects of various concentrations of E2,
which showed an antiatherosclerotic effect in a dose-dependent manner
(Figure 5). Our selection of subcutaneous injection as a route
of administration for E2 may have abolished the
adverse effect that oral supplementation with estrogen may produce on
triglycerides, which is a result of its passage through the
liver. Last, the beginning of HCD feeding that occurred 6 weeks after
oophorectomy was different from that of previous studies, in which HCD
feeding was started immediately after oophorectomy. It has been
suggested that an observation period of 4 weeks is necessary to achieve
a nonestrous status after oophorectomy and to obtain confirmation of no
significant estrogen release.31
It is important to determine the role of NO in relation to the antiatherosclerotic effect of estrogen. NO has antiatherosclerotic effects of its own, and the inhibition of intrinsic NO synthesis by NOS inhibitors is known to worsen atherosclerosis.8 The present study shows that balloon injury and an atherogenic diet (ie, endothelial denudation and hyperlipidemia) significantly diminished the ACh-induced, NO-mediated relaxation in the abdominal aortas of Group 3 rabbits. We also studied how E2 treatment affects basal NO release, as evaluated by contraction to L-NMA and aortic cGMP levels in atherosclerotic vessels. A significant, inverse relation between cGMP concentration and the severity of atherosclerosis was observed statistically. This may also imply that basal NO release plays an important role in the antiatherosclerotic effect of NO. E2 treatment increased basal NO release in a dose-dependent manner in atherosclerotic vessels, and our previous study showed an increased release of NO in cultured bovine and human endothelial cells in response to estrogen.12 We have also shown that basal NO release is greater in female rabbits than in male or oophorectomized female rabbits, and it is possible that a high concentration of E2 in female rabbits significantly retards atherosclerosis progression than in males due to a higher level of basal NO release in females.10 11 Estrogen has also been reported to be effective for neovascularization and endothelial repair in rabbit vessels by means of an NO-dependent mechanism.32 33 The increase in basal NO release by a low dose of extrinsic estrogen in this study may indicate that the antiatherosclerotic effect of estrogen occurs through NO.10 11 Furthermore, this assumption is supported by a recent study, which showed that inhibition of NOS reduced the antiatherosclerotic effect of estrogen in cholesterol diet–induced atherosclerosis in the rabbit aorta.34 However, we must consider another possibility that improved endothelial function due to estrogen treatment was not only the cause of the antiatherosclerotic effect but also the result of diminished atherosclerotic lesion formation through mechanisms other than NO. Some reports have described that the effects of estrogen are not through NO but through their dependence on another system and have concluded that NO does not play a role in some types of atherosclerosis.35 36 Additional study may be necessary to evaluate the antiatherosclerotic mechanisms of E2 on HCD- and balloon injury–induced atherosclerosis.
Regarding the mechanism for the antiatherosclerotic effects of
estrogen, we can rule out the role of estrogen receptors on the vessel
wall.37 Estrogen receptor-ß was recently identified, and
we have observed in preliminary studies that its distribution increases
in the atherosclerotic rabbit aorta (data not shown). Estrogen has been
shown to have a direct vascular effect in estrogen
receptor-–knockout mice, which suggests the important role of
estrogen receptor-ß. As these results are consistent with
ours, it may be important to clarify the mechanism of estrogen’s
effect on atherosclerosis with regard to estrogen
receptors.38
In conclusion, the restoration of NO release in response to E2 protects endothelial function in atherosclerotic vessels, and physiological concentrations of E2 can retard the progression of severe atherosclerosis and stabilize atheromas induced by an HCD and balloon injury. The present study indicates a favorable future for estrogen replacement therapy for the treatment of atherosclerosis.
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Acknowledgments |
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This study was supported in part by a grant-in-aid from the Ministry of Education, Science and Culture of Japan, No. 05670603.
Received January 12, 2000; accepted March 6, 2000.
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References |
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