© 1997 American Heart Association, Inc.
Articles |
Gender Differences in Intima-Media Permeability to Low-Density Lipoprotein at Atherosclerosis-Prone Aortic Sites in Rabbits
Lack of Effect of 17ß-Estradiol
From the Department of Pathology, Bowman Gray School of Medicine of Wake Forest University, Winston-Salem, N.C. 27157-1072
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Abstract |
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Abstract Premenopausal women are protected from coronary heart disease, and premenopausal nonhuman primates are protected from atherosclerosis, the underlying cause of coronary heart disease. Estrogen is thought to account for this protection in females, and part of this protection is independent of the effects on risk factors, including lipoprotein levels. This study considered the hypothesis that reduced intima-media permeability to low-density lipoproteins (LDL) may account for the protection from atherosclerosis and coronary heart disease in premenopausal females and that this effect might be mediated by estrogen. Intima-media permeability to LDL was determined in male and female rabbits made hypercholesterolemic by feeding them 0.5% cholesterol for 8 days. The diet of half of the female rabbits was supplemented with 17ß-estradiol (4 mg/d) during cholesterol feeding and the preceding 4 weeks. Estrogen treatment in the female rabbits did not influence the intima-media permeability to LDL. However, intima-media permeability to LDL for branch sites of the abdominal aorta and aortic arch (regions highly susceptible to atherosclerosis) was 43% and 38% lower, respectively, in male rabbits than in female rabbits: (2.93±0.39 µL/h/g, (n=8), vs 6.28±0.86 µL/h/g, (n=16), P<.001, and 4.69±0.28 µL/h/g, (n=8) vs 7.57±0.75 µL/h/g, (n=16), P <.02). In contrast, intima-media permeability to LDL in 7 of 8 aortic sites relatively resistant to atherosclerosis did not differ between male and female rabbits. These data suggest that the protection from atherosclerosis associated with female sex and estrogen is mediated by mechanism(s) other than reduction in intima-media permeability to LDL.
Key Words: estrogen • artery • lipoprotein • arterial permeability • intima-media permeability
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Introduction |
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Premenopausal women and women who receive estrogen replacement therapy are relatively protected from coronary heart disease and other clinical consequences of atherosclerosis.1 2 3 Studies in experimental animals, including the rabbit,4 5 6 7 have demonstrated that estrogens inhibit atherosclerosis. The fact that the risk of cardiovascular disease is similar in men and postmenopausal women8 suggests that a protective effect of estrogen plays a role rather than a harmful effect of testosterone. In support of this concept, a carefully conducted study in rabbits9 showed that testosterone had no effect on the development of atherosclerosis. Importantly, the protection from coronary heart disease3 and atherosclerosis4 5 6 10 11 12 that both endogenous and exogenous estrogen appears to confer is determined in part by mechanism(s) that are independent of effects on lipids, lipoproteins, and all other known risk factors for atherosclerosis. This observation suggests the possibility that estrogens may inhibit atherosclerosis or clinically significant coronary heart disease by direct effect(s) on the arterial wall.
Most, if not all, of the cholesterol found in atherosclerotic lesions derives from plasma lipoproteins.13 14 15 16 This suggests that processes which modulate interaction of lipoproteins with the arterial wall may influence the development of atherosclerosis. Previous studies in rabbits have shown that the rates of LDL metabolism and accumulation of undegraded LDL are increased in the aortic arch and branch sites of the descending thoracic aorta and the abdominal aorta,17 18 19 the aortic sites that are most susceptible to atherosclerosis.20 21 22 23 Metabolism of lipoproteins by arterial cells may promote atherosclerosis, and previous studies have shown that estrogen treatment reduces arterial metabolism of both the protein24 and lipid4 components of lipoproteins, including LDL.24 However, those studies were not designed to investigate the mechanism(s) by which estrogens inhibit arterial lipoprotein metabolism.
In order for arterial cells to degrade LDL and other lipoproteins, these lipoproteins must be transported into the artery. Earlier investigations suggested that the increased rate of LDL degradation at branch sites of the abdominal aorta may be partly explained by increased IM permeability of that arterial site to LDL.18 19 A study in normal female rabbits25 demonstrated that exogenous treatment with 17ß-estradiol had little or no effect on the IM permeability to LDL in the aortic arch and thoracic aorta, but this study did not investigate other arterial sites.
The purpose of the present study was to investigate in rabbits the influence of gender and estrogen on IM permeability to LDL at aortic sites susceptible to atherosclerosis and resistant to atherosclerosis. The pulmonary artery was also studied, since it is highly permeable to plasma proteins26 and LDL27 28 and is variably reported to be either similar to the aortic arch in susceptibility to atherosclerosis or less susceptible to atherosclerosis than the entire aorta (reviewed in References 28 and 2928 29 ). Because atherosclerosis only develops to a significant degree in the presence of hypercholesterolemia, rabbits were studied after an 8-day period of cholesterol feeding to induce hypercholesterolemia. Three groups of rabbits were studied: male rabbits, female rabbits, and female rabbits treated with 17ß-estradiol (4 mg/d).
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Methods |
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Experimental Groups
Young, sexually mature male and female New Zealand White rabbits were obtained from Robinson Services, Inc. Female rabbits were randomly assigned to no treatment or treatment with 17 ß-estradiol, a natural estrogen10 and a potent inhibitor of experimental atherosclerosis.5 6 Estrogen treatment was included to facilitate detection of an effect of estrogen.
Rabbits were fed rabbit chow (Prolab, Agway) for about 1 week. The diet was then supplemented with either 2.5% corn oil vehicle (untreated female rabbits and male rabbits) or 0.0040% 17ß-estradiol in the same amount of corn oil (estrogen-treated female rabbits). After 4 weeks, the diet of all rabbits was supplemented with 0.5% cholesterol. Estrogen treatment was continued for those receiving the hormone before addition of cholesterol to the diet. At all times, rabbits received 100 g of their respective diets each day. This provided the estrogen-treated rabbits with 4 mg 17ß-estradiol/day.5 6 Blood samples were collected after an overnight fast at intervals during the study.30 Intima-media permeability to LDL was studied after feeding cholesterol eight days, when rabbits were hypercholesterolemic but when arterial cholesterol concentrations were expected to be unchanged from values in normal rabbits.30
Isolation of LDL for Reinjection Studies
LDL (1.020<d<1.060 g/mL) for labeling was isolated at
4°C by centrifuging the d<1.080 fraction31 isolated
from fresh plasma through a KBr density gradient [modified from
Terpstra et al32 ]. Blood from female New Zealand white
rabbits fed rabbit chow was collected in 1 mg/mL disodium EDTA,
1 µmol/L D-phenylalanyl-L-prolyl-arginine chloromethyl
ketone, and 25 Kallikrein inhibitory units/mL aprotinin;
0.5 mmol/L phenylmethylsulfonyl fluoride and
20 µmol/L BHT were added to plasma. LDL was washed by
recentrifugation through the same density gradient.
Isolated LDL was dialyzed against 0.15 mol/L NaCl, 20
mmol/L sodium phosphate, pH 7.4 containing 2 mmol/L
disodium EDTA18 19 supplemented with 500
µmol/L ascorbic acid and 20 µmol/L
BHT33 (Buffer A) in the dark. Protein was determined as
described by Peterson.34
Labeling and Characterization of LDL
Three LDL preparations were used. One aliquot (4.9 to 13.8 mg
protein) of each LDL preparation was labeled with 125I-TC
(4.6 to 6.3 nmol TC and 1.4 to 3.0 mCi/mg protein) with cyanuric
chloride.35 36 The second aliquot of LDL (10.8 to 26.6 mg
protein) was labeled with 131I-TC (5.1 to 5.6 nmol TC and
0.75 to 1.4 mCi/mg protein). Labeled LDLs were dialyzed against Buffer
A. Labeled LDLs were sterilized by filtration17 and
injected 2 to 3 days after labeling and 5 to 6 days after isolation.
Specific activities were 1633 to 2572 cpm/ng for
125I-TC-labeled LDL and 122 to 445 cpm/ng for
131I-TC-labeled LDL. Labeled LDLs were characterized as
previously described.17 18 19 37 The procedures for
radioiodination and use and disposal of labeled LDL were approved by
the Office of Health Protection of Bowman Gray School of Medicine of
Wake Forest University.
IM Permeability to LDL
IM permeability to LDL was studied during two intervals in most
rabbits. Rabbits were first injected with 125I-TC-labeled
LDL (5.15±0.72x108 cpm/kg, mean±SEM, n=12) or
131I-TC-labeled LDL (1.54±0.23x108 cpm/kg,
n=12), respectively, followed 0.50±0.01 hour later by injection of the
same LDL preparation labeled with the alternate isotopes
(2.41±0.59x108 cpm/kg 131I-TC-labeled LDL,
n=10, or 6.13±1.39x108 cpm/kg
125I-TC-labeled LDL, n=12, respectively). Similar numbers
of male rabbits, untreated female rabbits, and estrogen-treated female
rabbits were studied with each preparation of labeled
LDL.19 Serial plasma samples were collected until the
study was terminated at 1.04±0.01 hours (n=23) after the first or the
only injection of labeled LDL.19 Rabbits were sacrificed
and immediately perfused with buffer, followed by half strength
Karnovsky's fixative as previously described.19 Uteri
were removed from some of the female rabbits. These procedures were
approved by the Animal Care and Use Committee of Bowman Gray School of
Medicine of Wake Forest University.
Arterial Sampling
The aorta30 and pulmonary artery trunk were
removed and fixed in half strength Karnovsky's fixative for an
additional 24 hours.19 35 The aortic arch and the
descending thoracic and abdominal aortas were separated.30
Adventitial tissue was removed, and arterial segments were
opened longitudinally and pinned flat.17 18 19 Branch sites
were collected from the thoracic and abdominal aortas.30
The remaining descending thoracic and abdominal aortas were each
divided into four segments of equal length. The aortic samples were
photographed before and after removal of the branch
sites.17 18 19
The surface areas of selected arterial sites were measured.17 18 19 Arterial thickness was estimated by dividing the arterial surface area by the product of the arterial sample weight and the density of fixed arterial tissue.38
Radioassay
Total and TCA-soluble 125I and 131I
radioactivity in plasma, lipoprotein fractions, and
arterial samples was determined17 18 19 in a
well-type gamma counter with a 3-inch crystal (Cobra II autogamma,
Packard).
Lipids and Lipoproteins
Lipoproteins were isolated from plasma in blood samples
collected immediately before sacrifice.19 Lipoproteins in
plasma were also separated by agarose gel
electrophoresis.39 Plasma cholesterol and
triglyceride concentrations in plasma and lipoprotein
fractions were determined as previously
described.30 40
IM Permeability to LDL
IM permeability to LDL was determined as described by a method
that provides a measure of IM permeability that is independent of the
plasma LDL concentration.19
Statistical Methods
Data for the three groups of rabbits were compared by
ANOVA.41 The two degrees of freedom for comparisons among
groups were divided into comparison of male versus all female rabbits
and estrogen-treated female rabbits versus female rabbits not given
estrogen. When the effect of estrogen treatment was not significant,
estrogen-treated and untreated female rabbits combined were compared
with the male rabbits by independent samples t
tests41 or multiple measures ANOVA (multiple
arterial sites).42 Plasma lipid concentrations
after the three dietary periods were compared with those of the same
rabbits before treatment with paired t tests41
and the Bonferroni criteria to account for multiple
comparisons.43 P<.05 was considered
significant.
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Results |
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Body Weight, Uterine Weight, and Plasma Lipids
Body weights of rabbits did not differ among the groups before treatment or at the end of the study (Table 1
). However, uterine weights of rabbits
treated with 17ß-estradiol were greater than those of untreated
female rabbits, indicating an effective dose of this estrogen on the
uterus. Plasma cholesterol and triglyceride
concentrations did not differ significantly among the groups at any
time during the study (Table 2). However,
plasma cholesterol concentrations were greatly elevated by
cholesterol feeding.
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Labeled LDL
Polyacrylamide gel electrophoresis of delipidated
LDL indicated that only 3.6±0.3% and 2.4±0.2% of
125I-TC and 131I-TC labels, respectively, were
associated with apolipoprotein E, similar to values noted in earlier
studies.17 18 19 For the three LDL preparations, 97.2±0.5%
of 125I-TC and 96.8±0.5% of 131I-TC,
respectively, co-migrated with unlabeled LDL on agarose gel
electrophoresis. Radioactivity soluble in 10% TCA accounted for
1.3±0.2% and 2.4±0.5% of 125I-TC and
131I-TC labels, respectively, while 10.2±1.0% and
9.6±0.7% of these labels could be extracted with
chloroform/methanol.44
At the end of the study, only 2.2±0.2% (n=20) of the protein-bound label remaining in plasma after circulating 0.5 hour could be isolated in the d<1.020 g/mL fraction, while only 2.8±0.4% (n=21) of this label showed alpha mobility on agarose electrophoresis. For the label that circulated 1 hour, the corresponding values were 2.6±0.3% (n=22) and 3.9±0.6% (n=23), respectively, suggesting that 93% to 95% of the label injected on LDL remained in the LDL fraction at the end of the experiment. Therefore, the arterial radioactivity was considered adequate for the calculation of IM permeability to LDL. Comparison of IM permeability to LDL in the same rabbit indicated that values measured during 0.5 hour were slightly greater than those measured during 1 hour, but the values did not differ significantly (by paired t test) in 33 of 36 instances (3 experimental groups x 12 arterial sites). Thus, values of IM permeability obtained from the 0.5 and 1 hour experiments in each animal were averaged to determine a single value of IM permeability for each arterial site.
Effect of Estrogen Treatment on IM Permeability to LDL
Table 3 summarizes data on IM
permeability to LDL for all 12 arterial sites studied in
untreated female rabbits and those treated with 17ß-estradiol.
Estrogen did not influence IM permeability to LDL in any
arterial site studied. Thus, in the comparison below, data
for both untreated and 17ß-estradiol treated–female rabbits were
combined for comparison with male rabbits.
Atherosclerosis-susceptible branch sites were compared
with the adjacent uniform
atherosclerosis-resistant aorta (T2 to T4
combined for thoracic branch sites, A1 and A2 combined for abdominal
branch sites).
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IM Permeability to LDL in
Atherosclerosis- Resistant Aortic Sites in
Male and Female Rabbits
Figure 1 shows data on IM
permeability to LDL for 8 aortic segments, representing the
entire length of the descending thoracic and abdominal aortas but
excluding the atherosclerosis-susceptible branch sites
that had been removed. IM permeability to LDL did not differ between
male and female rabbits for 7 of the 8 aortic sites.
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Differences Between IM Permeability to LDL at Arterial
Sites Susceptible and Resistant to Atherosclerosis
Compared with IM permeability to LDL in the adjacent
atherosclerosis-resistant descending thoracic
aorta, IM permeability to LDL in thoracic branch sites was increased in
female rabbits but not different in male rabbits (Fig 2A). Compared with IM permeability to
LDL in the adjacent atherosclerosis-resistant
aortic sites, IM permeability to LDL in abdominal branch sites and the
aortic arch was increased in both female and male rabbits (Figs 2B and 3A). In each of these instances, the difference between IM permeability
of adjacent atherosclerosis-susceptible and
atherosclerosis-resistant arterial
sites was greater in female rabbits than in male rabbits.
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Gender Differences in IM Permeability to LDL for
Atherosclerosis-Susceptible Arterial Sites
IM permeability to LDL in both branch sites of the abdominal aorta
(Fig 2B
) and in the aortic arch (Fig 3A) was lower in male rabbits than in
female rabbits. There was a smaller but not significant trend in the
same direction for branch sites of the descending thoracic aorta.
The Contribution of Arterial Thickness to Differences
in IM Permeability to LDL
Arterial thickness did not differ between male and
female rabbits and was not influenced by treatment with 17ß-estradiol
(Table 4). Per unit surface area, IM
permeability to LDL was greater for the aortic arch than for the
descending thoracic aorta in both female and male rabbits (Fig 3B). IM permeability was greater for
the aortic arch than for the pulmonary artery in female rabbits
only (Fig 3B). IM permeability of the
atherosclerosis-susceptible aortic arch was lower in
male rabbits compared with female rabbits, while IM permeability of the
pulmonary artery and the relatively
atherosclerosis-resistant descending thoracic
aorta did not differ between genders.
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Discussion |
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The purpose of this study was to investigate the influence of gender on IM permeability to LDL in arterial sites susceptible to and resistant to atherosclerosis and to consider whether differences in IM permeability might be mediated by estrogen. Therefore, not only male and female rabbits were studied but also female rabbits treated with exogenous 17ß-estradiol at a level that reduces experimental atherosclerosis.5 6
Measurement of IM permeability to LDL is complicated, and this study, as all such studies, has some limitations. The possibility cannot be excluded that some of the radiolabeled LDL found in the IM entered from the adventitial surface, as suggested by other investigators.45 However, other studies indicate that the intima and inner media17 35 degrade LDL at much higher rates than do the outer media and adventitia, suggesting that the earlier studies with directly iodinated LDL45 overestimated the contribution of LDL entry from adventitia to medial LDL radioactivity. In this study, IM permeability was measured with LDL labeled with TC, a label that remains trapped in lysosomes after cellular degradation.36 Thus, measurements of IM permeability in this study were not reduced by cellular degradation.
A previous study showed that IM permeability of rabbits was similar when it was measured during 0.5 hour and 1 hour with TC-labeled LDL,46 suggesting that the amount of LDL leaving the arterial wall was minimal during 1 hour and that contamination by adherent plasma was minimal. Under such circumstances, data can be appropriately used to calculate IM permeability with the caveat that measurements would overestimate IM permeability to the extent that labeled LDL enters the media from the adventitia during the experimental period. In this study, IM permeability during 0.5 hour was found to be slightly greater than that measured during 1 hour in the same animal. Since differences were not significant for almost all (33 of 36) arterial sites, IM permeability measurements during the two periods were averaged. However, conclusions regarding the effects of gender and estrogen were similar regardless of which average values of IM permeability or values measured during 0.5 or 1 hour were used.
In this study, 17ß-estradiol was found to have no effect on IM permeability to LDL in any of the 12 arterial sites studied. These results agree with the results of other studies which showed that 17ß-estradiol did not influence IM permeability to LDL in the thoracic aorta of rabbits fed a chow diet25 and that 17ß-estradiol did not influence IM permeability to lipoprotein cholesterol ester in atheromatous thoracic aorta of rabbits independent of a reduction in atherosclerosis.4 The findings of the present study extend those observations to rabbits made hypercholesterolemic by feeding cholesterol for 8 days and to both atherosclerosis-susceptible and atherosclerosis-resistant aortic sites. After feeding cholesterol for 8 days, atherosclerosis-susceptible aortic sites in rabbits have been found to contain few, if any, foam cells18 and to have cholesterol concentrations indistinguishable from those of normal rabbits.30
Despite no effect of exogenous 17ß-estradiol on IM permeability to LDL in female rabbits, IM permeability differed between male and female rabbits. Differences were noted for the aortic arch and the branch sites of the abdominal aorta, two aortic sites very susceptible to early atherosclerosis.20 21 22 23 There was little, if any, difference in IM permeability to LDL for aortic sites resistant to atherosclerosis. Interestingly, IM permeability of the susceptible aortic sites in male rabbits was about half that of the corresponding sites in female rabbits. As far as it is known, gender differences in IM permeability to LDL have not been previously investigated.
The observation that IM permeability to LDL at
atherosclerosis-susceptible aortic sites was lower in
male rabbits than female rabbits was surprising. This finding suggests
that the difference in IM permeability of
atherosclerosis-susceptible aortic sites of male and
female rabbits is either (1) independent of estrogen or (2) completely
expressed in the presence of the low circulating levels of estrogen in
unstimulated female rabbits.7 Confirmation of reduced IM
permeability in atherosclerosis-susceptible aortic
sites in male rabbits compared with female rabbits and explanation(s)
for this difference require further study. However, this difference was
not due to differences in arterial thickness between male
and female rabbits (Table 4).
The finding of this study raises several questions. First, are male rabbits more susceptible to atherosclerosis than female rabbits, as is the case with male humans8 and male nonhuman primates?47 Second, do aortic sites most susceptible to atherosclerosis differ between male and female rabbits? Numerous investigators have studied atherosclerosis separately in male rabbits and female rabbits. However as far as I am aware, only two studies provide quantitative data for both male and female rabbits.48 49 After comparable intervals of treatment with the same concentration of dietary cholesterol, one study reported reduced atherosclerosis in the thoracic aorta of female rabbits compared with male rabbits48 while the other study observed no difference between genders.49 Also, another study demonstrated that treatment with exogenous testosterone did not exacerbate atherosclerosis in castrated male rabbits with matched plasma cholesterol concentrations.9 Furthermore, studies in humans2 3 and nonhuman primates47 have indicated that most, if not all, of the female protection from atherosclerosis47 and cardiovascular disease2 3 can be accounted for by the effects of estrogens. Because unstimulated female rabbits have estrogen levels similar to those of postmenopausal women,7 50 it seems reasonable that female rabbits may be less protected from atherosclerosis than females of other species, such as premenopausal women or nonhuman primates. However, there is no evidence of increased susceptibility to atherosclerosis in female rabbits compared with male rabbits. Most of the studies that have characterized the distribution of atherosclerotic lesions in hypercholesterolemic rabbits20 21 22 23 have been performed in male rabbits.20 21 22 Those studies showed the same distribution of atherosclerotic lesions in male rabbits as that observed in female rabbits in another study.18 Thus, it is most probable that susceptibility to atherosclerosis is similar in male and female rabbits and that arterial sites with increased susceptibility to atherosclerosis are the same in both genders.
There is some concordance between the regional variation in IM permeability to LDL and the regional variation in susceptibility to atherosclerosis in both male and female rabbits. Most, if not all, of the cholesterol found in atherosclerotic lesions derives from plasma lipoproteins,13 14 15 16 which must be transported into the artery to contribute to arterial cholesterol accumulation. However, as shown in this study, IM permeability to LDL in atherosclerosis-susceptible aortic sites in female rabbits (with or without estrogen treatment) was greater than that of the corresponding aortic sites in male rabbits. Because estrogen inhibits atherosclerosis in rabbits,4 5 6 7 this finding suggests that female gender or estrogen influences the relationship between lipoprotein transport into the artery and arterial cholesterol accumulation. In support of this possibility, a previous study in rabbits showed that treatment with 17ß-estradiol reduced metabolism of lipoprotein cholesterol ester by atheromatous thoracic aorta, independent of effects on IM permeability.4 In addition, it is possible that removal mechanisms, such as efflux of cholesterol from arterial cells, may be less efficient in arteries of male rabbits compared with female rabbits.
The findings of this study confirmed the previously reported gradient
from proximal to distal aortic sites for IM permeability to
LDL27 51 and the high IM permeability of the
pulmonary artery to LDL.27 28 Because the
pulmonary artery was one of the arterial sites most
permeable to LDL (Table 3, Fig 3), and was
similarly permeable to LDL in male and female rabbits, the
susceptibility of this arterial site to
atherosclerosis is relevant for consideration. In
normotensive humans, the pulmonary artery is resistant
to atherosclerosis.52 In contrast, in
normotensive rabbits, the susceptibility of the pulmonary artery to
atherosclerosis has been variably reported to be either similar to that
of the aortic arch or less than that of the entire aorta (reviewed in
references 28 and 29). Recent studies provided evidence suggesting that
the pulmonary artery of rabbits is indeed susceptible to
atherosclerosis but that relative susceptibility of aortic arch and
pulmonary artery to atherosclerosis differs at different stages of
atherogenesis.29 Thus, the lower IM permeability to LDL
for atherosclerosis-susceptible aortic sites of male
rabbits compared with female rabbits does not appear to extend to the
pulmonary artery.
In summary, regional differences in IM permeability to LDL were found in macroscopically normal aortic sites of male and female rabbits. Differences in IM permeability to LDL between atherosclerosis-susceptible and atherosclerosis-resistant aortic sites, which have been previously described in female rabbits and were confirmed in the present study, were attenuated in male rabbits. Treatment with exogenous 17ß-estradiol had no influence on IM permeability to LDL in female rabbits. However, atherosclerosis-susceptible aortic sites in female rabbits were more permeable to LDL than the corresponding aortic sites in male rabbits. These results suggest that the protection from atherosclerosis provided by female sex and estrogen is mediated by mechanism(s) other than effects on IM permeability to LDL.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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The author is grateful to Christina Talbert and Elizabeth Ann Jordan for their skillful technical assistance. The author is an Established Investigator of the American Heart Association. This study was supported by National Institutes of Health grant No. HL45027.
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Footnotes |
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Ccorrespondence and reprints to Dawn C. Schwenke, Ph.D., Department of Pathology, Bowman Gray School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157-1072.
Received August 15, 1996; accepted November 26, 1996.
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