This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Elhage, R. Articles by Bayard, F. Search for Related Content PubMed PubMed Citation Articles by Elhage, R. Articles by Bayard, F.

(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:2679-2684.)
© 1997 American Heart Association, Inc.

Articles

17ß-Estradiol Prevents Fatty Streak Formation in Apolipoprotein E–Deficient Mice

R. Elhage; J.-F. Arnal; M.-T. Pieraggi; N. Duverger; C. Fiévet; J.-C. Faye; ; F. Bayard

From INSERM U397 (R.E., J.-F.A., J.-C.F., F.B.) and INSERM U466 (M.-T.P.), Institut L. Bugnard, Toulouse Cédex, France; RPR-Gencell, Atherosclerosis Department (N.D.), Vitry sur Seine Cédex, France; and INSERM U325 et Serlia (C.F.), Institut Pasteur, Lille Cédex, France.

Correspondence to Dr F. Bayard, INSERM U397, Institut L. Bugnard, 1 avenue Jean Poulhès, 31054 Toulouse Cédex, France. E-mail bayard{at}rangueil.inserm.fr

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Abstract The reality of the atheroprotective effect of estrogens is still a matter of debate, and its unknown mechanisms could involve favorable changes in blood lipids and lipoproteins and/or direct action at the level of the arterial wall. We used the recently developed animal model of atherosclerosis constituted by apolipoprotein E–deficient mice in an attempt to clarify these issues. Male and female animals, fed a low-fat chow diet, were treated with increasing doses of 17ß-estradiol (E₂) after castration and compared with testosterone treated and uncastrated (intact) animals. Total serum cholesterol, LDL-cholesterol, and HDL-cholesterol concentrations decreased under E₂ treatment in each sex and were weakly correlated with lesion area. However, a highly significant correlation between lesion area and serum E₂ levels also suggested a direct action of E₂ on cells of the vascular wall. A dose-response curve analysis revealed that these activities were sex-dependent, with females being nearly twice as sensitive to E₂ as males. It also revealed that the atheroprotective activity was recruited at higher E₂ concentrations than those needed by other E₂ target tissues such as uterus or functions such as apoA-1 and LDL production and/or clearance rates.

Key Words: fatty streak formation • apolipoproteins

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Although most retrospective epidemiological studies suggest that estrogen replacement therapy is effective in preventing arterial disease in postmenopausal women,¹ the significance of these observations is still a matter of debate since a selection bias toward healthier women for hormone treatment may have influenced the reported beneficial effect.² Some authors would recommend a widespread use³ but others feel such universal preventive hormonal therapy for postmenopausal women unwarranted at present, preferring to wait for the completion of randomized clinical trials.^{2 4} In the meantime, women, especially those who already have dyslipidemia or heart disease, will need to make decisions based on what is known. In this context, experimental studies strongly suggest the reality of the atheroprotective effect in monkey, rabbit, and swine^{5 6 7 8 9 10} but cannot yet explain the mechanisms involved, whether these be favorable changes in blood lipids and lipoproteins³ and/or direct action at the level of the arterial wall.¹¹

Apolipoprotein E–deficient (apoE KO) mice have recently been generated by gene targeting.^{12 13 14} These animals develop pronounced hypercholesterolemia on a normal chow diet, with chylomicron and very low lipoprotein remnant accumulation in plasma probably resulting from the abnormal receptor-mediated lipoprotein removal,^{15 16} an increase in atherogenic lipoprotein retention by matrix components, as well as slowing down of the reverse cholesterol transport from the arterial wall.^{17 18} Under these conditions, the mice develop lesions similar to those seen in humans, with foam cell–rich fatty and fibrous lesions including both proliferating smooth muscle cells and calcium deposits. Bourassa et al¹⁹ recently reported the atheroprotective effect of 17ß-estradiol (E₂) in this mouse model. However, these authors used a higher E₂ dose than the substitutive one, defined as the lowest dose maintaining a normal uterine weight. Moreover, they did not consider that estrogen treatment in mice, in contrast to humans, decreases plasma HDL-cholesterol and apolipoprotein A-1 (apoA-1) concentrations,^{20 21} which may contribute to the development of an atherogenic lipoprotein profile.^{22 23} Indeed, previous reports have shown that C57BL/6 female mice on an atherogenic diet developed more extensive fatty streak formation than their male littermates,^{24 25} which was tentatively attributed to a higher level of HDL under the influence of testosterone.

The aim of the present study was therefore to investigate the influence of increasing doses of E₂ on aortic lesion formation in apoE KO mice. Since it was expected that E₂ would be unable to induce favorable changes in the blood lipoprotein profile, it was hypothesized that any measurable atheroprotective effect of E₂ would be mediated at the level of the vascular wall. Testosterone treatment was also included to compare the effects on the lipoprotein profile.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Animals
Seventy apoE KO mice of either sex were obtained by breeding 3 couples bought from the Jackson Laboratory (6th generation of backcross from 129/B6 F1 heterozygotes to C57BL/6). They were housed in stainless-steel cages in groups of 5, kept in a temperature-controlled facility on a 12-hour light-dark cycle, and fed normal laboratory mouse chow containing 4.3% fat and 0.02% cholesterol. All experimental protocols were performed in accordance with the recommendations of the French Accreditation of Laboratory Animal Care. At 4 weeks of age, mice were anesthetized and randomized to either a sham operation (10 females and 10 males designated as intact animals) or bilateral gonadectomy (60 mice of each sex). The castrated animals, 10 animals in each group, were administered either 17ß-estradiol, testosterone, or placebo-containing 60-day time-release pellets (Innovative Research of America). The 17ß-estradiol pellets contained 0.01, 0.05, 0.1, or 0.5 mg, releasing 0.17, 0.83, 1.66, or 8.3 µg/d, respectively. The 7.5-mg testosterone pellets released 125 µg/d. They were implanted subcutaneously into the back of the animals using a sterile trochar and forceps. The body weight was determined 8 weeks later, after a 16-hour fast. Blood was collected by retroorbital bleeding and the serum separated by centrifugation for 10 minutes at 12 000g at 4°C. Mice were given an overdose of ketalar. The heart, together with the ascending aorta, and the uterus in females, was removed.

Serum Hormone Concentrations
Radioimmunoassay kits for 17ß-estradiol and testosterone were used following manufacturer instructions (Sorin Biomedica). Hormone levels were assayed for each individual mouse in a same series of assays. The intra-assay coefficient of variability was 4.5% for 17ß-estradiol and 7.8% for testosterone. The assay sensitivity, defined as 15% displacement of labeled tracer, was 0.5 pg E₂ and 12.5 pg testosterone.

Lipid Analyses
Serum total cholesterol concentrations were measured using Boehringer-Mannheim Biochemicals enzymatic assay kits. Serum lipoprotein separation was achieved by ultracentrifugation micromethod as described by Brousseau et al.²⁶ HDL-cholesterol content was also determined after selective precipitation of apoB-containing lipoproteins with phosphotungstic acid/magnesium chloride (Boehringer-Mannheim) according to Melhum et al.²⁷ Comparative analysis of the two methods showed a highly significant linear correlation coefficient (n=60, r=.836, P<.001). Serum apoA-1 concentrations were measured in castrated and 0.01- to 0.1-mg E₂-treated animals by immunonephelometry using mouse specific antibodies.²⁷

Evaluation of Fatty Streak Formation
The lesions were estimated according to Paigen et al.²⁸ Briefly, the heart and ascending aorta were washed in phosphate-buffered saline and fixed with phosphate-buffered paraformaldehyde (4%, pH 7.4) for 24 hours. Each heart was frozen on a cryostat mount with OCT compound (Tissue-Tek), and stored at -70°C. One hundred sections of 10-µm thickness were prepared from the top of the left ventricle, where the aortic valves were first visible, up to a position in the aorta where the valve cusps were just disappearing from the field. After drying for 2 hours, the sections were stained with oil red O and counterstained with Mayer's hematoxylin. Ten sections out of the 100, each separated by 90 µm, were used for specific morphometric evaluation of intimal lesions using a computerized Biocom morphometry system. The first and most proximal section to the heart was taken 90 µm distal to the point where the aorta first becomes rounded. Lipid droplets <500 µm² as well as those located in the media were excluded from the measurements. The mean lesion size (expressed in µm²) in these 10 sections was used to evaluate the lesion size of each animal. The coded slides were examined blind in two separate analyses by the same examiner and gave consistent results (r=.94). Cellular composition was also appreciated on successive sections analyzed either by oil red O staining or by immunohistochemistry using the rat monoclonal anti-mouse macrophages/monocytes (No. MCA519, Serotec) and IgG rabbit anti-rat IgG FITC conjugate (Biosys).

Statistics
The results are presented as mean±SE. Because of a large scatter in the individual values of lesion area, a logarithmic transformation was performed and used for statistics and graphic presentations. The significance of differences between means was tested by using ANOVA comparison of the castrated mice implanted with placebo or 0.01-, 0.05-, 0.1-, or 0.5-mg estradiol pellets (ie, five groups for each sex). Bonferroni's post hoc test was used to determine significant differences between two groups. Differences between the castrated mice implanted with placebo and 7.5 mg testosterone, or intact mice, and between intact male and female groups were also tested by using ANOVA. Correlations between different parameters were analyzed using simple linear regressions. The respective role of 17ß-estradiol treatment and of sex on the lesions was analyzed by comparing the correlation coefficients from the regression curves of lesion area in relation to serum E₂ (by the least squares method) and by a two-factor ANOVA on the groups responsible for the dose-effect. A value of P<.05 was considered as significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Effect of E₂ and Testosterone on Body Weight and Lipids
As shown in Tables 1 and 2, using a 50-µL aliquot, serum E₂ concentrations could not be detected in intact animals or in animals receiving the lowest E₂ dose pellets; they were measurable under higher E₂ treatment and were not different in either sex. In females, uterine weight was completely restored by the lowest E₂ dose pellets. Similarly, large variations in testosterone concentrations were observed in intact males, with many of the values lower than the detection limit of the assay (Table 2), whereas testosterone levels were constantly detectable in treated animals with no difference between male and female castrated-supplemented animals.

View this table: [in this window] [in a new window]   Table 1. Effects of 17ß-Estradiol and Testosterone Treatment on Body Weight, Serum Hormone, Total Cholesterol, VLDL-, LDL-, and HDL-Cholesterol Levels in Female Mice

View this table: [in this window] [in a new window]   Table 2. Effects of 17ß-Estradiol and Testosterone Treatment on Body Weight, Serum Hormone, Total Cholesterol, VLDL-, LDL-, and HDL-Cholesterol Levels in Male Mice

The mean body weight decreased significantly under the highest dose of E₂ treatment in both males and females when compared with castrated animals. Weights were not different with the other doses of E₂ and testosterone treatment.

As shown in Table 1 and 2, total serum cholesterol was higher in intact males than in intact females; it decreased under the 0.05- to 0.5-mg doses of E₂ and testosterone treatment in males and under the 0.5-mg E₂ dose and testosterone in females. LDL- and VLDL-cholesterol followed a similar pattern although variations were not significantly different for VLDL-cholesterol. In the placebo and E₂-treated groups, total cholesterol (r=-0.38, P<.001 for the whole groups; r=-0.35, P<.01 for females and r=-0.40, P<.001 for males, respectively) and LDL-cholesterol were negatively correlated with serum E₂ concentrations (r=-0.41, P<.0001 for the whole groups; r=-0.33, P<.03 for females and r=-0.48, P<.001 for males, respectively); a negative correlation was also observed between VLDL-cholesterol and serum E₂ concentrations in the whole groups (r=-0.25, P<.02) and in males (r=-0.28, P<.05) but not in females (P=.14).

HDL-cholesterol increased significantly after ovariectomy but not after orchidectomy; it was unaffected by testosterone treatment and decreased significantly under E₂ (0.05 to 0.5 mg) treatment. In the placebo and E₂-treated groups, a negative correlation was observed between HDL-cholesterol and serum E₂ concentrations in the whole groups and in females (r=-0.31, P<.001 and r=-0.33, P<.05, respectively) but was of borderline significance in males (P=.054). As shown in Fig 1, serum apoA-1 concentrations also decreased maximally under the 0.05 mg E₂ dose in females but only under the 0.1 mg dose in males. Indeed, serum HDL-cholesterol and apoA-1 concentrations in the 0.01- to 0.1-mg E₂-treated mice of either sex were highly correlated (r=.80, P<.0001).

View larger version (15K): [in this window] [in a new window]   Figure 1. Serum apolipoprotein A-1 (Apo A1) concentrations (g/L, mean±SE) under increasing doses of 17ß-estradiol treatment; 0.01, 0.05, and 0.1 mg indicates amount of E2 released over a 60-day period (*P<.05 vs castrated mice).

Effect of 17ß-Estradiol and Testosterone on Fatty Streak Formation
As shown in Fig 2, fatty streak areas were slightly higher in intact and castrated females compared with intact and castrated males, but the difference was not statistically significant. The lesions decreased in a dose-dependent manner in each sex (P<.0001). In E₂-treated females, significantly lower lesions were noted with the 0.05-mg dose, and the maximal effect was reached at the dosage of 0.1 mg and then plateaued at 0.5 mg. In males, a higher dose (0.1 mg) was required to significantly reduce the fatty streak formation, which continued to decrease under 0.5 mg.

View larger version (14K): [in this window] [in a new window]   Figure 2. Mean lesion area in castrated, E2-treated, testosterone-treated, and intact mice. Each point represents the average area (±SE) of intimal lipid staining material in 10 aortic sections from individual animals. Mean serum 17ß-estradiol concentrations and corresponding molarities resulting from the E2 pellet content are also shown for female mice. nd indicates not detectable. (*P<.05 vs castrated mice).

In the placebo and E₂-treated groups, lesion area was positively correlated with total serum cholesterol (r=.43, P<.0001 for the whole groups; r=.50, P<.001 and r=.37, P<.01 for females and males, respectively) and with LDL-cholesterol in the whole groups (r=.33, P<.005) and males (r=.41, P<.02) but not in females (P=.08). Lesion area was also positively correlated with VLDL-cholesterol in the whole groups (r=.24, P<.03) and with HDL-cholesterol in the whole groups (r=0.22, P<.05) and in females (r=0.35, P<.03) but not in males (P=.63). Taking into account that a maximal effect was reached at the dose of 0.1 mg E₂ in females and assigning a null value when the E₂ concentration was undetectable, a significant negative high correlation was observed between lesion area and serum E₂ concentrations in the placebo and 0.01- to 0.1-mg–treated animals (r=- 0.70, P<.0001 for the whole groups; r=-0.78, P<.0001 for females and r=-0.65, P<.0001 for males). Furthermore, a significant difference in the slopes (t=2.289, df=78, P=.025) suggested an interaction of sex and E₂ on lesion depth (Fig 3). This suggestion verified when a two-factor ANOVA was performed on the groups given 0.01- to 0.1-mg pellets, which induced a dose-dependent decrease in lesions. The dose-effect relationship differed between males and females (P=.03), with no interaction between sex and dose (P=.8).

View larger version (16K): [in this window] [in a new window]   Figure 3. Fatty streak area as a function of E2 in castrated mice receiving placebo or 0.01- to 0.1-mg E2-releasing capsules. A significant negative high correlation was observed between lesions and E2 concentration in females (r=-0.78, P<.0001) and in males (r=-0.65, P<.0001) as well as a significant difference in the slopes (P=.025).

The lesions also decreased under testosterone treatment, although to a lesser extent than under E₂ treatment.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
In first considering the systemic effects of E₂ treatment, we observed that 0.5-mg pellets (corresponding to 8.33 µg/d) induced significant decreases in body weight and total cholesterol concentration and could have modified body composition and/or food intake.²⁹ Treatments with 0.05- and 0.1-mg pellets (corresponding to 0.83 to 1.66 µg/d) did not alter food intake (data not shown) but were still able to decrease serum cholesterol in male mice. Similar results had been obtained by Tang et al²⁰ and Srivastava et al²¹ in the C3H/HeJ strain but not in C57BL/6. These observations were therefore unexpected, as C57BL/6 had been used to generate the apoE KO mice; they may reflect a genetic heterogeneity introduced by the use of the 129 strain³⁰ and concur with the report by Bourassa et al.¹⁹ This group also concluded that the effects on plasma cholesterol could only partially explain the beneficial effect of estrogen.¹⁹ This conclusion is further supported by the present data if one examines the evolution of serum HDL-cholesterol and apoA-1 concentrations, which were not considered by these authors but probably contributed to the majoration of an atherogenic profile.^{22 23 24} These effects on serum HDL-cholesterol and apoA-1 concentrations agree with those reported by Tang et al and Srivastava et al in normal male C57BL/6J mice but differ from a previous study by Paigen et al,²⁴ in which testosterone was administered to ungonadectomized females and concluded that the increasing level of HDL-cholesterol was directly induced by testosterone.

In contrast to the relatively weak correlation with total cholesterol and the different lipoprotein subfractions, a highly significant correlation was observed between lesion area and serum E₂ levels, strongly suggesting a direct action of E₂ on cells of the vascular wall. Endothelial cells could be involved through regulation of permeability to lipoproteins, leukocyte adhesion and chemotaxis, cytokine production, and/or superoxide anion production, but the monocyte/macrophage lineage itself could also be the site of this action. In fact, all vascular aortic wall cells, including cells from the inflammatory/immune system,³¹ are estrogen target cells, which express estrogen receptors as well as aromatase, estradiol-17ß hydroxysteroid dehydrogenase, and 17-ketoreductase enzyme activities.^{32 33 34} In a recent communication, Rubanyi et al reported that disruption of the estrogen receptor gene in mice significantly modified vascular function, reducing basal release of endothelium-derived nitric oxide and increasing smooth muscle reactivity to KCl (10th International Congress of Endocrinology. 1996. ORG18-1. Abstract). These data favor the involvement of estrogen receptors in the mechanism of E₂ action at the level of the vascular wall. The dose-response curves obtained in the present study, show an "inhibitory plasma concentration 50," inducing half a decrease in lesion area, in the range of 0.05 nmol/L, which is consistent with this hypothesis. This analysis also clearly shows that the estrogen-dependent activities that mediate the atheroprotective effect are recruited at higher hormonal concentrations than are needed by other E₂ target tissues such as uterus³⁵ or functions such as apoA-1 (compare Figs 1 and 2), LDL (Table 2), or VLDL production and/or clearance rates.¹⁹ Although E₂ serum concentrations in this range have been reported by some authors during the estrous cycle,^{36 37} such concentrations are probably only reached during pregnancy.³⁵ Moreover, this sensitivity differs between female and male animals, as shown in Figs 2 and 3. In their recent report, Bourassa et al¹⁹ also described the atheroprotective effect of estrogens in this mouse model. Although both sets of data are in agreement in general, the E₂ doses used (6 to 28 µg/d, generating plasma estradiol concentrations from 100 to 900 pg/mL) did not allow these authors to characterize the peculiar tissue and sex specificity of the effect. The molecular basis for such tissue and sex-specific responses to E₂ is not yet understood and may involve not only differences in chromatin structure as well as estrogen receptor dynamics and characteristics^{38 39} but also synergy between progesterone and estrogen.⁴⁰ With regard to humans, the incidence of E₂ on serum HDL-cholesterol and apoA-1 concentrations as well as absence of apoE probably make this mouse model irrelevant for studies of lipoprotein metabolism. However, this model is useful for definition of the target in the vascular wall, and the data presented should be considered in defining future clinical trials of postmenopausal replacement therapy and possible pharmacological studies in males.

The lower but also significant atheroprotective effect of testosterone is more difficult to interpret. When administered in a continuous fashion, testosterone decreased total serum and LDL-cholesterol, suggesting an effect on lipoprotein production or metabolism. A direct effect at the level of the vascular wall, as a result of testosterone aromatization, should also be considered. Further work, using variable concentrations and non aromatizable androgens, should clarify these issues.

In conclusion, we confirm that 17ß-estradiol is effective in preventing the development of fatty streaks in female and male apoE KO mice. Moreover, this animal model allowed us to approach the respective roles of blood lipids and lipoproteins and the direct action on cells of the arterial wall and to define a peculiar tissue and sex specificity. This model is well adapted to characterization of the molecular mechanisms that mediate sex steroid hormone effects on the atherosclerosis process.

	Acknowledgments

 
This work was supported in part by INSERM, the Ministère de la Recherche et de la Technologie, the Fondation de France, and the Conseil Régional Midi-Pyrénées. We thank Drs M. Plantavid and B. Perret for steroid radioimmunoassays, J.P. Charlet for statistical analysis, S. Estaque and J.-C. Thiers for histologic lesion analysis, and G. Tatinian, R. Baritaud, and M. Larribe for technical and secretarial assistance.

Received December 31, 1996; accepted May 13, 1997.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Stampfer MJ, Colditz GA. Estrogen replacement therapy and coronary disease: a quantitative assessment of the epidemiological evidence. Prev Med. 1991;20:47-63.[Medline] [Order article via Infotrieve]
2. Posthuma WF, Westendorp RG, Vandenbroucke JP. Cardioprotective effect of hormone replacement therapy in postmenopausal women: is the evidence biased? Br Med J. 1994;308:1268-1269.[Abstract/Free Full Text]
3. Lobo RA, Speroff L. International consensus conference on postmenopausal therapy and the cardiovascular system. Fertil Steril. 1994;61:592-595.[Medline] [Order article via Infotrieve]
4. Barrett-Connor E. Postmenopausal estrogen and heart disease. Atherosclerosis. 1995;118:S7-S10.
5. Hough IL, Zilversmit DB. Effect of 17ß-estradiol on aortic cholesterol content and metabolism in cholesterol-fed rabbits. Arteriosclerosis. 1986;6:57.[Abstract/Free Full Text]
6. Adams MR, Kaplan JR, Manuck SB, Koritnik DR, Parks JS, Wolfe MS, Clarkson TB. Inhibition of coronary artery atherosclerosis by 17-ß estradiol in ovariectomized monkeys: lack of an effect of added progesterone. Arteriosclerosis. 1990;10:1051-1057.[Abstract/Free Full Text]
7. 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.
8. Wagner JD, Clarkson TB, St Clair RW, Schwenke DC, Shively CA, Adams MR. Estrogen and progesterone replacement therapy reduces LDL accumulation in the coronary arteries of surgically postmenopausal cynomolgus monkeys. J Clin Invest. 1991;88:1995-2002.
9. Hayashi T, Fukuto JM, Ignarro LJ, Chaudhuri G. Basal release of nitric oxide from aortic rings is greater in female rabbits than in male rabbits: implications for atherosclerosis. Proc Natl Acad Sci U S A. 1992;89:11259-11263.[Abstract/Free Full Text]
10. Keaney JF, Shwaery GT, Xu AM, Nicolosi RJ, Loscalzo J, Foxall TL, Vita JA. 17ß-Estradiol preserves endothelial vasodilator function and limits low-density lipoprotein oxidation in hypercholesterolemic swine. Circulation. 1994;89:2251-2259.[Abstract/Free Full Text]
11. Bush TL, Barrett-Connor E, Cowan LD, Criqui MH, Wallace RB, Suchindran CM, Tyroler HA, Rifkind BM. Cardiovascular mortality and noncontraceptive estrogen use in women: results from the Lipid Research Clinic's Program Follow-up Study. Circulation. 1987;75:1102-1109.[Abstract/Free Full Text]
12. Plump AS, Smith JD, Hayek T, Aalto-Setälä K, Walsh A, Verstuyft JG, Rubin EM, Breslow JL. Severe hypercholesterolemia and atherosclerosis in apolipoprotein E-deficient mice created by homologous recombination in ES cells. Cell. 1992;71:343-353.[Medline] [Order article via Infotrieve]
13. Zhang SH, Reddick RL, Piedrahita JA, Maeda N. Spontaneous hypercholesterolemia and arterial lesions in mice lacking apolipoprotein E. Science. 1992;258:468-471.[Abstract/Free Full Text]
14. van Ree JH, van den Broek WJAA, Dahlmans VEH, Groot PHE, Vidgeon-Hart M, Frants RR, Wieringa B, Havekes LM. Hofker MH. Diet-induced hypercholesterolemia and atherosclerosis in heterozygous apolipoprotein E-deficient mice. Atherosclerosis. 1994;111:25-37.[Medline] [Order article via Infotrieve]
15. Ishibashi S, Herz J, Maeda N, Goldstein JL, Brown MS. The two-receptor model of lipoprotein clearance: tests of the hypothesis in `knockout' mice lacking the low density lipoprotein receptor, apolipoprotein E, or both proteins. Proc Natl Acad Sci U S A. 1994;91:4431-4435.[Abstract/Free Full Text]
16. Mortimer BC, Beveridge DJ, Martins IJ, Redgrave TG. Intracellular localization and metabolism of chylomicron remnants in the livers of low density lipoprotein receptor-deficient mice and apoE-deficient mice. J Biol Chem. 1995;270:28767-28776.[Abstract/Free Full Text]
17. Saxena U, Ferguson E, Bisgaier CL. Apolipoprotein E modulates low density lipoprotein retention by lipoprotein lipase anchored to the subendothelial matrix. J Biol Chem. 1993;268:14812-14819.[Abstract/Free Full Text]
18. Hayek T, Oiknine J, Brook JG, Aviram M. Role of HDL apolipoprotein E in cellular cholesterol efflux: studies in apo E knockout transgenic mice. Biochem Biophys Res Comm. 1994;205:1072-1078.[Medline] [Order article via Infotrieve]
19. Bourassa PAK, 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.[Abstract/Free Full Text]
20. Tang J, Srivastava RAK, Krul ES, Baumann D, Pfleger BA, Kitchens RT, Schonfeld G. In vivo regulation of apolipoprotein A-1 gene expression by estradiol and testosterone occurs by different mechanisms in inbred strains of mice. J Lipid Res. 1991;32:1571-1585.[Abstract]
21. Srivastava RAK, Baumann D, Schonfeld G. In vivo regulation of low-density lipoprotein receptors by estrogen differs at the post-transcriptional level in rat and mouse. Eur J Biochem. 1993;216:527-538.[Medline] [Order article via Infotrieve]
22. Pàszty C, Maeda N, Verstuyft J, Rubin EM. Apolipoprotein AI transgene corrects apolipoprotein E deficiency-induced atherosclerosis in mice. J Clin Invest. 1994;94:899-903.
23. Plump AS, Scott CJ, Breslow JL. Human apolipoprotein A-1 gene expression increases high density lipoprotein and suppresses atherosclerosis in the apolipoprotein E-deficient mouse. Proc Natl Acad Sci U S A. 1994;91:9607-9611.[Abstract/Free Full Text]
24. Paigen B, Holmes PA, Mitchell D, Albee D. Comparison of atherosclerosis lesions and HDL-lipid levels in male, female, and testosterone-treated female mice from strains C57BL/6, BALB/c, and C3H. Atherosclerosis. 1987;64:215-221.[Medline] [Order article via Infotrieve]
25. Warden CH, Hedrick CC, Qiao J-H, Castellani LW, Lusis AJ. Atherosclerosis in transgenic mice overexpressing apolipoprotein A-II. Science. 1993;261:469-472.[Abstract/Free Full Text]
26. Brousseau T, Clavey V, Bard JM, Fruchart JC. Ultracentrifugation micromethod for separation of serum lipoproteins and assays of lipids, apolipoproteins and lipoprotein particles. Clin Chem. 1993;39:960-964.[Abstract/Free Full Text]
27. Melhum A, Staels B, Duverger N, Tailleux A, Castro G, Fievet C, Luc G, Fruchart JC, Olivecrona G, Skretting G, Auwerx J, Prydz H. Tissue-specific expression of the human gene for lecithin:cholesterol acyltransferase in transgenic mice alters blood lipids, lipoproteins and lipases towards a less atherogenic profile. Eur J Biochem. 1995;230:567-575.[Medline] [Order article via Infotrieve]
28. Paigen B, Morrow A, Holmes PA, Mitchell D, Williams RA. Quantitative assessment of atherosclerotic lesions in mice. Atherosclerosis. 1987;68:231-240.[Medline] [Order article via Infotrieve]
29. Wade GN, Gray JM. Gonadal effects on food intake and adiposity: a metabolic hypothesis. Physiol Behav. 1979;22:583-593.[Medline] [Order article via Infotrieve]
30. Smithies O, Meada N. Gene targeting approaches to complex genetic diseases: atherosclerosis and essential hypertension. Proc Natl Acad Sci U S A. 1995;92:5266-5272.[Abstract/Free Full Text]
31. Libby P, Hansson GK. Involvement of the immune system in human atherogenesis: current knowledge and unanswered questions. Lab Invest. 1991;64:5-15.[Medline] [Order article via Infotrieve]
32. Gulshan S, McCruden AB, Stimson WH. Estrogen receptors in macrophages. Scand J Immunol. 1990;31:691-697.[Medline] [Order article via Infotrieve]
33. Bayard F, Clamens S, Meggetto F, Blaes N, Delsol G, Faye JC. Estrogen synthesis, estrogen metabolism and functional estrogen receptors in rat arterial smooth muscle cells in culture. Endocrinology. 1995;136:1523-1529.[Abstract]
34. Bayard F, Clamens S, Delsol G, Blaes N, Maret A, Faye JC. Oestrogen synthesis, oestrogen metabolism and functional oestrogen receptors in bovine aortic endothelial cells. In: Non-reproductive Actions of Sex Steroids. Symposium. Chichester: Wiley; 1995:122-138.
35. Bergman MD, Schachter BS, Karelus K, Combatsiaris EP, Garcia T, Nelson JF. Up-regulation of the uterine estrogen receptor and its messenger ribonucleic acid during the mouse estrous cycle: the role of estradiol. Endocrinology. 1992;130:1923-1930.[Abstract]
36. Grainger DJ, Witchell CM, Metcalfe JC. Tamoxifen elevates transforming growth factor-beta and suppresses diet-induced formation of lipid lesions in mouse aorta. Nat Med. 1995;1:1067-1073.[Medline] [Order article via Infotrieve]
37. Sullivan TR, Karas RH, Aronovitz M, Faller GT, Ziar JP, Smith JJ, Odonnell TF, Mendelsohn ME. Estrogen inhibits the response-to-injury in a mouse carotid artery model. J Clin Invest. 1995;96:2482-2488.
38. Bergman MD, Karelus K, Felicio LS, Nelson JF. Tissue differences in estrogen receptor dynamics: nuclear retention, rate of replenishment, and transient receptor loss vary in hypothalamus, pituitary, and uterus of C57BL/6J mice. Endocrinology. 1987;121:2065-2074.[Abstract]
39. Kuiper GGJM, Enmark E, Pelto-Huikko M, Nilsson S, Gustafsson JA. Cloning of a novel estrogen receptor expressed in rat prostate and ovary. Proc Natl Acad Sci U S A. 1996;93:5925-5930.[Abstract/Free Full Text]
40. Medina KL, Kincade PW. Pregnancy-related steroids are potential negative regulators of B lymphopoiesis. Proc Natl Acad Sci U S A. 1994;91:5382-5386.[Abstract/Free Full Text]

This article has been cited by other articles:

				X. Game, J. Allard, G. Escourrou, P. Gourdy, I. Tack, P. Rischmann, J.-F. Arnal, and B. Malavaud Estradiol increases urethral tone through the local inhibition of neuronal nitric oxide synthase expression Am J Physiol Regulatory Integrative Comp Physiol, March 1, 2008; 294(3): R851 - R857. [Abstract] [Full Text] [PDF]

				P. Gourdy, A. Schambourg, C. Filipe, V. Douin-Echinard, B. Garmy-Susini, B. Calippe, F. Terce, F. Bayard, and J.-F. Arnal Transforming Growth Factor Activity Is a Key Determinant for the Effect of Estradiol on Fatty Streak Deposit in Hypercholesterolemic Mice Arterioscler. Thromb. Vasc. Biol., October 1, 2007; 27(10): 2214 - 2221. [Abstract] [Full Text] [PDF]

				J. Bourghardt, G. Bergstrom, A. Krettek, S. Sjoberg, J. Boren, and A. Tivesten The Endogenous Estradiol Metabolite 2-Methoxyestradiol Reduces Atherosclerotic Lesion Formation in Female Apolipoprotein E-Deficient Mice Endocrinology, September 1, 2007; 148(9): 4128 - 4132. [Abstract] [Full Text] [PDF]

				J. Kitayama, F. M. Faraci, S. R. Lentz, and D. D. Heistad Cerebral Vascular Dysfunction During Hypercholesterolemia Stroke, July 1, 2007; 38(7): 2136 - 2141. [Abstract] [Full Text] [PDF]

				R. Elhage, P. Gourdy, J. Jawien, L. Brouchet, C. Castano, C. Fievet, G. K. Hansson, J.-F. Arnal, and F. Bayard The Atheroprotective Effect of 17{beta}-Estradiol Depends on Complex Interactions in Adaptive Immunity Am. J. Pathol., July 1, 2005; 167(1): 267 - 274. [Abstract] [Full Text] [PDF]

				R. K. Dubey, B. Imthurn, M. Barton, and E. K. Jackson Vascular consequences of menopause and hormone therapy: Importance of timing of treatment and type of estrogen Cardiovasc Res, May 1, 2005; 66(2): 295 - 306. [Abstract] [Full Text] [PDF]

				R. Elhage, P. Gourdy, L. Brouchet, J. Jawien, M.-J. Fouque, C. Fievet, X. Huc, Y. Barreira, J. C. Couloumiers, J.-F. Arnal, et al. Deleting TCR{alpha}{beta}+ or CD4+ T Lymphocytes Leads to Opposite Effects on Site-Specific Atherosclerosis in Female Apolipoprotein E-Deficient Mice Am. J. Pathol., December 1, 2004; 165(6): 2013 - 2018. [Abstract] [Full Text] [PDF]

				L. Garidou, S. Laffont, V. Douin-Echinard, C. Coureau, A. Krust, P. Chambon, and J.-C. Guery Estrogen Receptor {alpha} Signaling in Inflammatory Leukocytes Is Dispensable for 17{beta}-Estradiol-Mediated Inhibition of Experimental Autoimmune Encephalomyelitis J. Immunol., August 15, 2004; 173(4): 2435 - 2442. [Abstract] [Full Text] [PDF]

				T. M. Doherty, L. A. Fitzpatrick, D. Inoue, J.-H. Qiao, M. C. Fishbein, R. C. Detrano, P. K. Shah, and T. B. Rajavashisth Molecular, Endocrine, and Genetic Mechanisms of Arterial Calcification Endocr. Rev., August 1, 2004; 25(4): 629 - 672. [Abstract] [Full Text] [PDF]

				T. A. Henriques, J. Huang, S. S. D'Souza, A. Daugherty, and L. A. Cassis Orchidectomy, But Not Ovariectomy, Regulates Angiotensin II-Induced Vascular Diseases in Apolipoprotein E-Deficient Mice Endocrinology, August 1, 2004; 145(8): 3866 - 3872. [Abstract] [Full Text] [PDF]

				A. Villablanca, D. Lubahn, L. Shelby, K. Lloyd, and S. Barthold Susceptibility to Early Atherosclerosis in Male Mice Is Mediated by Estrogen Receptor {alpha} Arterioscler. Thromb. Vasc. Biol., June 1, 2004; 24(6): 1055 - 1061. [Abstract] [Full Text] [PDF]

				A. K. Death, K. C. Y. McGrath, M. A. Sader, S. Nakhla, W. Jessup, D. J. Handelsman, and D. S. Celermajer Dihydrotestosterone Promotes Vascular Cell Adhesion Molecule-1 Expression in Male Human Endothelial Cells via a Nuclear Factor-{kappa}B-Dependent Pathway Endocrinology, April 1, 2004; 145(4): 1889 - 1897. [Abstract] [Full Text] [PDF]

				P. Y. Liu, A. K. Death, and D. J. Handelsman Androgens and Cardiovascular Disease Endocr. Rev., June 1, 2003; 24(3): 313 - 340. [Abstract] [Full Text] [PDF]

				F. C. W. Wu and A. von Eckardstein Androgens and Coronary Artery Disease Endocr. Rev., April 1, 2003; 24(2): 183 - 217. [Abstract] [Full Text] [PDF]

				J. B. Hodgin and N. Maeda Minireview: Estrogen and Mouse Models of Atherosclerosis Endocrinology, December 1, 2002; 143(12): 4495 - 4501. [Abstract] [Full Text] [PDF]

				C. Pendaries, B. Darblade, P. Rochaix, A. Krust, P. Chambon, K. S. Korach, F. Bayard, and J.-F. Arnal The AF-1 activation-function of ERalpha may be dispensable to mediate the effect of estradiol on endothelial NO production in mice PNAS, February 19, 2002; 99(4): 2205 - 2210. [Abstract] [Full Text] [PDF]

				A. Mortensen, V. Breinholt, T. Dalsgaard, H. Frandsen, S. T. Lauridsen, J. Laigaard, B. Ottesen, and J.-J. Larsen 17{beta}-Estradiol but not the phytoestrogen naringenin attenuates aortic cholesterol accumulation in WHHL rabbits J. Lipid Res., May 1, 2001; 42(5): 834 - 843. [Abstract] [Full Text]

				J. C. Escola-Gil, O. Jorba, J. Julve-Gil, F. Gonzalez-Sastre, J. Ordonez-Llanos, and F. Blanco-Vaca Pitfalls of Direct HDL-Cholesterol Measurements in Mouse Models of Hyperlipidemia and Atherosclerosis Clin. Chem., September 1, 1999; 45(9): 1567 - 1569. [Full Text] [PDF]

				U. Brehme, B. Bruck, N. Gugel, M. Wehrmann, S. Hanke, G. Finking, F. W. Schmahl, and H. Hanke Aortic Plaque Size and Endometrial Response in Cholesterol-Fed Rabbits Treated With Estrogen Plus Continuous or Sequential Progestin Arterioscler. Thromb. Vasc. Biol., August 1, 1999; 19(8): 1930 - 1937. [Abstract] [Full Text] [PDF]

				A. Maret, S. Clamens, I. Delrieu, R. Elhage, J.-F. Arnal, and F. Bayard Expression of the Interleukin-6 Gene Is Constitutive and Not Regulated by Estrogen in Rat Vascular Smooth Muscle Cells in Culture Endocrinology, June 1, 1999; 140(6): 2876 - 2882. [Abstract] [Full Text]

				M. M. Marsh, V. R. Walker, L. K . Curtiss, and C. L. Banka Protection against atherosclerosis by estrogen is independent of plasma cholesterol levels in LDL receptor-deficient mice J. Lipid Res., May 1, 1999; 40(5): 893 - 900. [Abstract] [Full Text]

This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Elhage, R. Articles by Bayard, F.