(Circulation. 1998;98:2731-2737.)
© 1998 American Heart Association, Inc.
Basic Science Reports |
Gender Gap in Aortic Cholesterol Accumulation in Cholesterol-Clamped Rabbits
Role of the Endothelium and Mononuclear-Endothelial Cell Interaction
From Novo Nordisk A/S (P.H., H.L.A.), Måløv, Denmark, and Clinical Institute, Odense University (G.A., S.S.), Odense, 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—The purpose of the present study was to investigate plasma lipid–independent mechanisms for the sex difference in the development of atherosclerosis.
Methods and Results—In the first experiment, 20 male and 20
female rabbits were balloon-injured in the middle thoracic aorta and
maintained at the same plasma cholesterol level of
25 mmol/L by use of individualized cholesterol
feeding for 13 weeks. In the undamaged aorta, female rabbits had
accumulated less than half the amount of cholesterol found
in male rabbits (P<0.05). In the balloon-injured aorta,
cholesterol accumulation was 3- to 4-fold higher than in
the undamaged aorta, with no difference between groups. When
cholesterol accumulation data for the balloon-injured aorta
were separately assessed for blue
(deendothelialized) and white
(reendothelialized) tissue, blue tissue surprisingly
revealed a reverse gender gap, ie, a significantly higher accumulation
of cholesterol in females than in males
(P<0.05). White tissue, which constituted the majority
of the balloon-injured area, showed no difference in aortic
cholesterol accumulation between groups. In the second
experiment, 6 male and 6 female rabbits were fed standard rabbit
pellets and 6 male and 6 female rabbits were fed a 0.5%
cholesterol-enriched chow for 2 weeks. Mononuclear cell
binding was 5-fold higher in aortic segments from
hypercholesterolemic than from
normocholesterolemic rabbits (P<0.001).
In hypercholesterolemic rabbits, cell binding was
significantly lower in female than in male rabbits
(P<0.05) and showed higher values in
atherosclerosis-prone regions. These differences were
not found in normocholesterolemic animals.
Conclusions—The present results suggest that female atheroprotection is independent of sex differences in plasma cholesterol but vitally dependent on the state of the arterial endothelium and involves mononuclear-endothelial cell adhesion as an early step.
Key Words: aorta • atherosclerosis • balloon • endothelium • leukocytes
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Introduction |
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Coronary atherosclerosis is the leading cause of death in the western world.1 One of the most important risk factors for this disease is male sex, with a male-to-female ratio of death of coronary disease close to 4 before the age of 50.2 This sex difference is not eliminated by mathematical adjustments for sex differences in the prevalence of classic risk factors such as hyperlipidemia, hypertension, diabetes, smoking, and obesity.3 An inborn difference in arterial wall structure has been suggested, but results are controversial.4 5 6
The male-to-female ratio of death due to coronary disease declines gradually from the age of female menopause.2 Furthermore, a variety of epidemiological studies suggest that the risk of coronary disease is increased in ovariectomized and postmenopausal women compared with age-matched menstruating women7 8 and conversely is decreased in women taking estrogen replacement therapy compared with age-matched controls.9 10 Thus, a likely explanation for the sex difference in coronary disease prevalence is the presence of higher circulating blood levels of estrogen in women compared with men. On the other hand, evidence exists that men treated with estrogen, either as primary or secondary prophylaxis, experience an excess number of cardiovascular events compared with their nontreated counterparts.11 12 13 Furthermore, the endogenous levels of estrogen in men are only poorly correlated with coronary disease.2
In humans, the sex difference in atherosclerosis extent is present in the coronary arteries but not in the aorta.14 It has previously been shown that monkeys and rabbits fed a cholesterol-enriched diet also show a higher extent of atherosclerosis in males than in females.15 16 In the rabbit study, this difference was demonstrated in aortic tissue, which suggests that the rabbit aorta is a better model for coronary than for aortic atherosclerosis in humans. In that study, the male-female difference in atherosclerosis extent was present despite similar plasma cholesterol levels, which suggests a direct action of female sex at the level of the arterial wall. The purpose of the present study was to investigate whether a direct atheroprotective effect of female sex (1) depends on the state of the arterial endothelium (atherosclerosis experiment) and (2) is preceded by differences in endothelial adhesiveness for mononuclear cells (mononuclear cell adhesion experiment).
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Methods |
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All animals were sexually mature rabbits of the Danish Country Strain obtained from Statens Serum Institute, Copenhagen. The rabbits were housed in individual stainless steel cages at a room temperature of 20°C and with a 12-hour light cycle. The animals had free access to drinking water. All experimental procedures were performed in accordance with Danish regulations for experiments with animals.
Atherosclerosis Experiment
Experimental Design
Twenty male and 20 female rabbits were fed a
cholesterol-enriched diet of chow (Altromin 2113)
for 13 weeks (80 g/d). Cholesterol (Sigma Chemical Co) was
added to the chow in 4 different concentrations: 0%, 0.25%, 0.5%,
and 1% wt/wt.17 18 19 20 Guided by weekly plasma
cholesterol determinations (CHOD-PAP, Boehringer
Mannheim), we maintained all animals at a plasma
cholesterol level of 25 mmol/L using the 4
different chow preparations.17 18 19 20
After 1 week of cholesterol feeding, the rabbits were anesthetized with intravenous pentobarbital (50 mg/kg body weight) and underwent balloon catheter injury in the middle thoracic aorta as described previously.19 20 The postoperative mortality rate was 0%.
After 12 weeks of cholesterol feeding, blood samples were drawn for determination of the distribution of cholesterol among lipoprotein fractions.17 18 One week later, the rabbits were injected 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 (50 to 100 mg/kg body weight).17 18 19 20 The aorta was then removed from the level of the aortic valves to the level of the diaphragm.
Preparation of Aortic Tissue
The aorta was cleaned of adherent adventitial tissue, opened
longitudinally, and divided into 3 parts: 1 part extended from the
aortic valves to the second intercostal arteries (arch plus upper
thoracic; undamaged), 1 from the second to the fifth intercostal
arteries (middle thoracic; balloon injured), and 1 from the fifth
intercostal arteries to the celiac artery (lower thoracic; undamaged).
The balloon-injured middle thoracic aorta was further separated into
white tissue, consisting of reendothelialized areas,
and blue tissue, consisting of still
deendothelialized areas. Each aortic part was
fixed with pins on a cork board, and the inner layer containing the
intima and part of the media was stripped from the outer media. The
outer layer was discarded, and the inner layer was weighed and stored
within 1 hour at -20°C until analyzed.
Evaluation of Atherosclerosis Extent
Aortic cholesterol was determined as described
previously.18 19 20 In brief, the aortic tissue was
minced and extracted with 
20 vol of chloroform/methanol for 24 hours.
The extract was washed by the Folch procedure, and the
cholesterol content was determined enzymatically after
evaporation and redissolution in isopropanol. The validity of this
procedure has been tested previously.21
Mononuclear Cell Adhesion Experiment
Experimental Design
Six male and 6 female rabbits were fed standard rabbit pellets,
and 6 male and 6 female rabbits were fed a 0.5%
cholesterol-enriched chow. After 2 weeks, blood samples
were drawn for determination of plasma cholesterol levels,
after which the rabbits were killed by an overdose of
intravenous pentobarbital. The aorta was removed, placed in
cold PBS, and carefully freed of adventitia.
Isolation and Fluorescence Labeling of Mononuclear
Cells
Whole human blood was retrieved by venous puncture in sodium
citrate (10:1 vol/vol) and heparin (10 U/mL). The blood was carefully
layered onto a cushion of Lymphoprep (Nycomed Pharma A/S) and
centrifuged at 782g for 30 minutes at room
temperature. The buffy coat was removed and washed twice with RPMI 1640
medium (ATTC). The resulting cell pellet was allowed to incubate in
RPMI 1640 medium containing tetramethylrhodamine isothiocyanate 3
µg/mL (TRITC; CalBiochem) for 15 minutes at room temperature. To
separate labeled cells from the remaining dye, the cell suspension was
carefully underlaid with a layer of fetal calf serum and
centrifuged at 425g for 10 minutes at room
temperature. The cells were finally washed in RPMI 1640 medium and
resuspended in HBSS (Life Technologies) containing 2 mmol/L
Ca2+, 2 mmol/L Mg2+,
and 20 mmol/L HEPES for binding studies.
Binding Assay
The binding assay was performed as described previously by Tsao
et al.22 Segments (15 mm) were excised
from the level of the aortic arch (immediately below the carotid
arteries), the thoracic aorta (second to third intercostal arteries),
and the abdominal aorta (celiac to superior mesenteric artery). The
segments were opened longitudinally and, with the
endothelial side up, were affixed with needles to 35-mm
culture dishes containing 2 mL of HBSS. The dishes were then placed on
a rocking platform at room temperature.
After 10 minutes, the HBSS medium was replaced by binding medium containing mononuclear cells at a concentration of 5x105 cells/mL. The aortic segments were incubated with the mononuclear cells for 30 minutes, after which the medium was aspirated and the segments washed twice with fresh binding medium to remove nonadherent cells. After an additional 5 minutes on the rocking platform, the aortic segments were removed and placed on a glass slide with the endothelial side up. Adherent cells were counted under fluorescence microscopy from 9 predetermined, equally distributed sites on each segment. The identification of the segments was revealed after all segments were counted.
Statistics
In the atherosclerosis experiment, comparisons
between groups were made by use of the Wilcoxon U
test for unpaired samples. In the mononuclear cell adhesion experiment,
we evaluated differences between groups by a 2-way ANOVA with post hoc
analysis using the Newman-Keuls test. In Figure 5
, data were
analyzed by the Kruskal-Wallis test followed by a Dunn's
multiple comparison test. P<0.05 was considered
statistically significant. All analyses were performed with the
GraphPad Prism software program.
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) are combined with data from a previous study with a
similar design. Top, Undamaged thoracic aorta. Bottom, Balloon-injured
aorta. Comparisons between groups were made by use of Kruskal-Wallis
test followed by Dunn's multiple comparison test. Data are mean±SEM.
*P<0.05, ***P<0.001. OVX indicates
ovariectomized; E2, 17ß-estradiol. |
Results |
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Atherosclerosis Experiment
Characteristics of Female and Male Rabbits
Initial body weights were similar in the female and male rabbit groups. Both groups thrived and gained weight during the study period. The female group, however, had a significantly greater weight gain than the male group (Table
). The initial
plasma cholesterol concentration was significantly higher
in the female than in the male rabbits. This difference was eliminated
by cholesterol feeding, which elevated plasma
cholesterol to a similar level of 25 mmol/L in 31
rabbits. The remaining 9 animals (6 males and 3 females) had lower
plasma cholesterol concentrations throughout the study
either because of anorexia or because of a pronounced resistance to the
development of diet-induced
hypercholesterolemia. These rabbits were
excluded from the study. The amount of dietary cholesterol,
which was used to adjust each rabbit's plasma cholesterol
to 25 mmol/L, was not different between groups. The mean exposure
of plasma cholesterol to the aortic wall, which can be
calculated as the area under the time versus plasma
cholesterol curve, divided by the duration of the
experiment, was similar in the female and male rabbits at the end of
the experiment (Table). The female rabbits had a significantly higher
level of VLDL and a significantly lower level of LDL than the male
rabbits, but none of these parameters were, within the
observed values, statistically significantly related to aortic
cholesterol accumulation.
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Aortic Atherosclerosis
In undamaged aortas, ie, the aortic arch plus upper thoracic
aorta and the lower thoracic aorta, female rabbits had only one half
and one third, respectively, the accumulation of
cholesterol found in male rabbits (P<0.05)
(Figure 1). In the balloon-injured middle
thoracic aorta, cholesterol accumulation was 3- to 4-fold
higher than in the undamaged aorta and was not different between the 2
groups. When the cholesterol accumulation data for the
balloon-injured area were separately assessed for blue and white
tissue, the blue tissue, denuded of endothelium,
surprisingly revealed a reverse gender gap, ie, a significantly higher
accumulation of cholesterol in the female than in the male
group (P<0.05) (Figure 2).
The white tissue, with regenerated endothelium, showed
no difference in aortic cholesterol accumulation between
the female and the male groups. The majority of the balloon-injured
middle thoracic aorta consisted of reendothelialized
white tissue, whereas only a minor part consisted of
deendothelialized blue tissue (0.20±0.03 and
0.16±0.03 blue tissue mg wet wt /total tissue mg wet wt for females
and males, respectively).
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Mononuclear Cell Adhesion Experiment
At the end of the 2-week experiment, female rabbits had a
higher plasma cholesterol level than male rabbits in the
normocholesterolemic group (0.8±0.2 versus
0.4±0.1 mmol/L; P<0.05), whereas there was no
significant difference between female and male rabbits in the
hypercholesterolemic group (12.7±1.3 versus
10.0±1.1 mmol/L).
Figure 3 shows photomicrographs of
aortic segments from normocholesterolemic (A),
hypercholesterolemic male (B), and
hypercholesterolemic female (C) rabbits. Aortic
segments from normocholesterolemic rabbits demonstrated
only minimal binding of mononuclear cells to the
endothelial cells. There was no difference in cell
binding either between female and male rabbits or among the different
aortic segments within each group (Figure 4). Aortic segments from
hypercholesterolemic rabbits, however, showed a 5-fold
increase in cell binding compared with
normocholesterolemic rabbits (P<0.001)
(Figure 4). This increase was significantly attenuated in the female
compared with the male group as determined by a 2-way ANOVA
(P<0.05). There was also a regional variation in the
magnitude of cell binding: the aortic arch bound a higher amount of
mononuclear cells than the thoracic aorta (P<0.05), which
bound a higher amount of mononuclear cells than the abdominal aorta.
The latter difference, however, was only marginally significant
(P<0.10).
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Discussion |
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Atherosclerosis Experiment
It was possible to maintain plasma cholesterol at
25 mmol/L for 13 weeks in 31 of 40 animals. In these animals,
the amount of dietary cholesterol necessary to maintain the
elevated plasma cholesterol was without significant
difference between the 2 groups, which suggests a neutral effect of
female sex on total plasma cholesterol in
cholesterol-fed rabbits. Aortic accumulation of
cholesterol in the females was less than half that in the
males. These results are in accordance with previous
findings16 that showed an identical elevation of
plasma cholesterol in female and male rabbits fed a fixed
(2%) cholesterol-enriched diet for several weeks and
significantly less aortic atherosclerosis in female
than in male rabbits after 10 weeks. After 15 weeks, however, this sex
difference was no longer present. These findings suggest that the
direct, ie, plasma lipid–independent, atheroprotective effect of
female sex involves a retardation rather than an inhibition of the
development of atherosclerosis. To further explore this direct atheroprotection by female sex, we subjected all rabbits to balloon catheter injury in the middle thoracic aorta. We18 have previously shown that this procedure results in complete endothelial denudation, whereas the deeper arterial layers, ie, the internal elastic lamina and the smooth muscle of the media, are left intact. At necropsy 12 weeks after surgery, the balloon-injured area consists partly of areas denuded of endothelium and partly of areas covered with regenerated endothelial cells that are irregularly shaped, lack alignment in the direction of the blood flow, and exhibit endothelial dysfunction.23 The present findings, which show lower cholesterol accumulation in female than in male rabbits in the intact aorta, similar cholesterol accumulation in the 2 groups in the aorta with regenerated endothelium, and higher accumulation in the female than in the male group in the aorta denuded of endothelium, suggest that the direct atheroprotective effect of female sex is dependent on the state of the arterial endothelium. If such a dependence relies on upregulation of an endothelial antiatherogenic factor, the findings furthermore suggest that this occurs along with upregulation of other vascular, potentially atherogenic factors that eliminate or overrule protection in aortas with dysfunctioning or absent endothelium. To the best of our knowledge, effects of gender on cholesterol accumulation after balloon catheter injury have not previously been investigated in a hypercholesterolemic animal model.
Mononuclear Cell Adhesion Experiment
The finding of 5-fold higher binding of mononuclear cells to the
endothelium of vascular segments from
hypercholesterolemic rabbits compared with that of
normocholesterolemic rabbits is consistent with
previous observations.22 Adherence of mononuclear
cells to the vascular endothelium is one of the
earliest detectable abnormalities in the arteries of
hypercholesterolemic animals.24
The adherent cells infiltrate the subintima and transform into
lipid-engorging macrophages, resulting in the development of
foam cells and fatty streaks, the first gross pathological lesion of
atherosclerosis. The exact mechanism that initiates
this mononuclear-endothelial cell interaction is not
known, but expression of endothelial adhesion molecules
and chemotactic proteins induced by
hypercholesterolemia may be
involved.25 26
The long-term cholesterol-fed rabbit exhibits a characteristic regional pattern in the development of atherosclerosis: the lipid lesions are most pronounced in the aortic arch and involve smaller areas in the more distal parts of the aorta.17 18 19 20 27 28 The present findings showing a similar regional pattern in mononuclear-endothelial cell binding in hypercholesterolemic rabbits are supportive of a relation between an early (weeks) increase in endothelial adhesiveness and later (months) development of lipid lesions in these animals. Thus, the significantly lower mononuclear-endothelial cell binding found in the aortas of female compared with male hypercholesterolemic rabbits may play a role in the mediation of the endothelium-dependent sex difference in aortic cholesterol accumulation described in the atherosclerosis experiment. The mechanism by which female sex is able to affect endothelial adhesiveness is not known. It has previously been shown that basal release of endothelial nitric oxide (NO) is significantly higher in aortic rings from female than from male normocholesterolemic rabbits29 and that endothelial constitutive NO synthase (cNOS), the enzyme that catalyzes NO formation, is upregulated in pregnant and estrogen-treated guinea-pigs.30 In a recent study, mononuclear-endothelial cell interaction was increased by long-term administration of an NO synthase antagonist and conversely inhibited by long-term administration of the NO precursor L-arginine in cholesterol-fed rabbits, which suggests that NO is able to regulate endothelial adhesiveness for mononuclear cells.22 An endothelial factor that is regulated by female sex and that affects endothelial adhesiveness could thus be identical to NO. This relationship, however, was not addressed in the present study and warrants further investigation.
Importance of Estrogen
We20 have previously conducted a rabbit
study of the same duration and with the same plasma
cholesterol levels as in the
atherosclerosis experiment performed in the present
study; the previous study consisted of intact males, intact males
treated with estrogen, ovariectomized females, and ovariectomized
females treated with estrogen. Because the male rabbits of that
experiment accumulated nearly the same amount of
cholesterol in the thoracic aorta as the male rabbits of
the present experiment (26 and 21 nmol/mg wet weight,
respectively), we compared the results of the 2 experiments and found a
consistent pattern in the accumulation of
cholesterol. As shown in
Figure 5
(top), the gender gap in atherosclerosis
development is abolished when males are treated with estrogen. When
females are ovariectomized, which eliminates their
endogenous estrogen production, their protection is
lost, and when they are given estrogen replacement, their protection is
regained. Interestingly, the difference in
atherosclerosis extent between intact and
ovariectomized females may be more prominent when females are kept in
the same room as males, as in the present study, than when intact
and ovariectomized females are kept
apart.27 31 32 Probably, the presence of males
increases the endogenous production of estrogen in
females. The reason ovariectomized females tend to develop even more
atherosclerosis than male rabbits (P<0.10)
is not known, but the same trend is seen in monkeys fed an atherogenic
diet.15 One possibility is that males, with their
low endogenous estrogen production, are protected
relative to ovariectomized females that are completely devoid of
estrogen. As can be seen in Figure 5 (bottom), denudation of the aortic
endothelium by balloon catheter injury eliminates all
differences in aortic cholesterol accumulation between the
groups. Taken together, these results suggest that the direct
atheroprotection conferred by female sex, demonstrated above, is
explained exclusively by the presence of higher estrogen concentrations
in females than in males and that this effect of estrogen depends on an
intact endothelium.
Results from previous experimental animal studies investigating the effect of estrogen on balloon catheter–induced atherosclerosis are controversial. In normocholesterolemic rats, mice, and rabbits, estrogen protects against neointimal hyperplasia after balloon catheter injury.33 34 35 36 37 38 39 It has been suggested that this effect is mediated by a facilitation of the reendothelialization and thus the functional endothelial recovery of the damaged artery.38 39 In hypercholesterolemic rabbits and monkeys, estrogen has no effect neither on neointimal hyperplasia or on cholesterol accumulation after balloon catheter injury.17 18 19 20 40 One explanation for this discrepancy between effects of estrogen on balloon catheter–induced atherosclerosis in normocholesterolemic and hypercholesterolemic animals is that hypercholesterolemia impairs reendothelialization and thus counteracts or abolishes the beneficial effect of estrogen.
In the present study, we observed a surprisingly higher cholesterol accumulation in female than in male rabbits in the deendothelialized aorta. A paradoxical atherogenic effect of estrogen has previously been observed in balloon-injured aorta of rabbits treated with estrogen plus NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO production.19 Thus, absence of endothelial NO, either by enzyme inhibition or by endothelial denudation, may play a role in this previously unnoticed effect of estrogen/female sex on vascular tissue.
Conclusions
This study confirms a previous finding that female rabbits develop
less-extensive diet-induced atherosclerosis than
males.16 More importantly, it suggests that this
relative protection of female rabbits is independent of sex differences
in plasma cholesterol and lipoproteins but vitally
dependent on the state of the arterial
endothelium, because it is present in aortas with
intact endothelium, absent in aortas with regenerated
endothelium, and even reversed in aortas denuded of
endothelium. An endothelium-dependent
mechanism for the gender gap in the development of
atherosclerosis may involve
mononuclear-endothelial cell binding, which is
significantly greater in aortic segments from female than from male
rabbits even after just 2 weeks of cholesterol feeding and
which shows a regional variation similar to that seen for
atherosclerosis.
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Acknowledgments |
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This study was supported by grants from the Danish Heart Foundation and from Jenny Vissing's Fund.
Received March 27, 1998; revision received August 7, 1998; accepted August 19, 1998.
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