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(Circulation. 1996;94:175-181.)
© 1996 American Heart Association, Inc.

Articles

Different Effects of Estrogen and Progesterone on Experimental Atherosclerosis inFemale Versus Male Rabbits

Quantification of Cellular Proliferation by Bromodeoxyuridine

Hartmut Hanke, MD; Sybille Hanke, MD; Gerald Finking, MD; Alexandra Muhic-Lohrer, MD; Alfred O. Muck, PhD, MD; Friedrich W. Schmahl, MD; Rainer Haasis, MD; Vinzenz Hombach, MD

the College of Medicine, Department of Internal Medicine, Division of Cardiology (H.H., S.H., V.H.), University of Ulm, and the College of Medicine, Department of Social Medicine--Atherosclerosis Research (G.F., F.W.S.), the Department of Obstetrics and Gynecology (A.O.M.), and the Department of Internal Medicine, Division of Cardiology (A.M.-L., R.H.), University of Tubingen, Germany.

Correspondence to Hartmut Hanke, MD, Department of Internal Medicine, Division of Cardiology, Robert-Koch-Str 8, 89081 Ulm, Germany.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background The aim of the present study was to compare the effect of estrogen and progesterone on the development of experimental atherosclerosis in female versus male rabbits to assess possible sex-specific differences.

Methods and Results A total of 32 female and 32 male New Zealand White rabbits were ovariectomized or castrated. In addition to a 0.5% cholesterol diet, the rabbits received estradiol alone (1 mg/kg body wt [BW] per week), progesterone alone (25 mg/kg BW per week), or combined estradiol-progesterone in these dosages during 12 weeks. Ovariectomized female and castrated male rabbits served as control groups without hormone treatment. Before excision of the vessels, bromodeoxyuridine labeling was performed to determine the extent of cellular proliferation in the atherosclerotic lesions. The aortic arch was analyzed immunohistologically and morphometrically. An inhibitory effect of estrogen on intimal plaque size was found in female rabbits compared with the ovariectomized control group (0.7±0.5 versus 3.7±2.5 mm², P<.002; proliferating cells, 3.1±1.8% versus 8.5±2.6%, P<.002). In combination with progesterone, however, estrogen was not able to reduce intimal plaque size or cellular proliferation. In contrast, estradiol in castrated male rabbits was not associated with an inhibitory effect on cellular proliferation or intimal thickening compared with controls (estrogen treatment, 7.6±2.1% proliferating cells and 2.8±1.0 mm² neointima; control group, 7.2±2.1% cellular proliferation and 2.9±1.2 mm² intimal thickening).

Conclusions Our data suggest that the atheroprotective effect of estrogen is probably due to a mechanism that is present in female rabbits only.

Key Words: atherosclerosis • hormones • bromodeoxyuridine • muscle, smooth • hypercholesterolemia

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Premenopausal women have a lower incidence of CHD than men of the same age, but after menopause the incidence in women approaches that in men.^{1 2 3}

A reduction of cholesterol content in the aorta by estrogen was first described in 1942 by Ludden et al⁴ in an experimental study, and it is now widely believed that ovarian estrogen is responsible for the sex-related protection in CHD.^{5 6 7 8}

Little is known, however, about the exact mechanism(s) of estrogen action in the arterial vessel wall. Premenopausal women have lower total cholesterol serum levels, higher HDL levels, and lower LDL levels, and the vasoprotection compared with men was first generally attributed to these marked differences in lipid serum levels. In recent years, however, a variety of direct and indirect estrogen effects of the vascular vessel wall on metabolism have been demonstrated, ie, suppression of collagen synthesis, inhibition of LDL oxidation, attenuation of coronary and aortic LDL accumulation, and calcium antagonist properties.^{9 10 11 12 13 14 15 16}

Intimal SMC proliferation is a key event in the process of atherosclerosis, but little is known about the direct effect of female sex hormones on SMC proliferation in vivo. Thus, the purpose of the present study was to determine the proliferative activity of SMCs and the development of atherosclerotic lesions in the arterial vessel wall, modulated by estrogen, progesterone, and a combined estrogen-progesterone therapy in an animal model. As described previously,¹⁷ the quantification of cellular proliferation was performed by the use of BrdU labeling.

Recently published data suggest a possible involvement of arterial estrogen receptors in the beneficial action of estrogen.^{18 19} Our hypothesis was that there are probably sex-specific differences in the action of estrogen and progesterone and that these could be shown experimentally in vivo with female and male rabbits.

	Methods

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Animals and Study Protocol
A total of 40 female and 40 male New Zealand White rabbits (Charles River, Kißlegg, Germany), 12 weeks of age (see the Table for weights) were used for the present study. The rabbits were housed individually with 12-hour light periods.

View this table: [in this window] [in a new window]   Table 1. Weight and Plasma Cholesterol and Plasma Hormone Levels of Rabbits

Under general anesthesia (ketamine-HCl and xylazine-HCl), either bilateral ovariectomy in 32 female rabbits or total castration in 32 male rabbits was performed. After ovariectomy or castration, the rabbits were randomized into 8 groups of 8 rabbits each. All rabbits subsequently received a 0.5% cholesterol diet (Altromin Inc) for 12 weeks to induce atherosclerotic lesions.

Two groups, one made up of female rabbits and one of male rabbits, were not ovariectomized or castrated. Both groups of intact rabbits also received a 0.5% cholesterol diet but no hormone treatment.

Estrogen and Progesterone Treatment
Estradiol valerate (Progynon Depot 10, Schering AG; dosage, 1 mg/kg BW per week), progesterone capronate (Proluton, Schering AG; 25 mg/kg BW per week), or combined estrogen/progesterone (in above dosages) was administered intramuscularly once a week to groups of eight rabbits each. This protocol was carried out in corresponding groups of ovariectomized female and castrated male rabbits. Eight female ovariectomized and eight male castrated rabbits served as two control groups receiving no hormone therapy.

Determination of Plasma Parameters
Blood samples from each rabbit were collected into EDTA-containing tubes before ovariectomy or castration and at the end of the experiment after beginning the cholesterol diet. Blood was drawn from the intact rabbits at the same time points.

After centrifugation of the samples at 3000 rpm at 4°C for 15 minutes, the total plasma cholesterol concentration was measured by a standard enzymatic method (Boehringer Inc).

Estradiol and progesterone plasma levels were measured by RIA with standard commercially available kits (Biermann Inc and Diagnostic Products Corp).

BrdU Labeling
To determine the extent of cells undergoing DNA synthesis, BrdU, a thymidine analogue, was used. As described earlier in detail,¹⁷ 100 mg BrdU per 1 kg BW and 75 mg deoxycytidine per 1 kg BW (both from Sigma GmbH) were given as subcutaneous neck depots 18 hours before the rabbits were euthanatized. In addition to this neck depot, 30 mg BrdU per 1 kg BW and 25 mg deoxycytidine per 1 kg BW were injected intramuscularly 18 and 12 hours before excision of the vessels.

Fixation and Histological Preparation
After the final blood collection, the rabbits were euthanatized. Subsequently, the proximal aortic arch and a small intestinal mucosa segment were excised from each rabbit and immersion-fixed in 0.1 mol/L cacodylate-buffered 2% paraformaldehyde solution for at least 24 hours. The excised samples were then embedded in paraffin and prepared for histological examination.

Each aorta was serially sectioned (4-µm slices) until the maximal thickness of each lesion was reached. At plaque maximum, four sections per lesion were then analyzed histologically and immunohistologically.

The incorporation of BrdU during the 18 hours of the labeling period in the DNA of replicating cells allowed determination and quantification of cellular proliferation in the arterial segments. A monoclonal antibody against BrdU (Bio Cell Consulting) was used to identify these proliferated cells. Immunohistological detection of BrdU-labeled cells was performed in cross sections with the biotin avidin method²⁰ and combined staining with hemalaune.

For quantification of the number of cells undergoing DNA synthesis, BrdU staining was performed in one cross section at the lesion maximum of the aortic arch.

The histological sections were examined (quantification of cellular proliferation and morphometric analysis) by an independent investigator blinded to the type of treatment protocol. The percentage of proliferating cells was calculated by counting all BrdU-labeled cells and the total intimal cell number in four diametrically arranged segments by use of an occular grid. The total number of counted cell nuclei per section ranged from 635 to 7072 cells.

Estimation of the small intestinal mucosa labeling index was used in each rabbit to check the incorporation of BrdU in replicating cells. Evidence of DNA synthesis usually is found in 30% of all cells in the small intestinal mucosa. Because of insufficient BrdU labeling, two rabbits were excluded from further quantification of cellular proliferation in the arterial vessel wall.

To identify neointimal cells as SMCs, immunohistological staining (biotin avidin method) in one additional serial cross section per lesion was performed with a monoclonal antibody against -actin (Fa Renner). -Actin is known to be a highly specific marker for smooth muscle cells.^{21 22}

In addition to the immunochemical staining of BrdU and -actin, hematoxylin and eosin and elastica–van Gieson staining was performed in the two remaining serial cross sections.

In cross sections after elastica–van Gieson staining, the extent of the intimal cross-sectional area at the region of maximal plaque size was measured morphometrically (in millimeters squared) by use of a digital image analyzer with a software package from Bilaney Consulting Inc.

Statistical Evaluation
Results are expressed as mean±SD. The Wilcoxon rank-sum test was used to determine the significance of differences comparing the different study groups. After Bonferroni adjustment, differences were considered significant at a value of P.0083.

	Results

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Morphological Results
Based on histomorphological criteria, the basic structure and composition of the plaques were essentially the same in female and male rabbits. Staining of -actin in all vessels in female and male rabbits showed similar concentric proliferation of plaques, with most SMCs toward the lumen and foam cells located centrally (Fig 1).

View larger version (157K): [in this window] [in a new window]   Figure 1. Histological cross section of a rabbit aorta from the ovariectomized control group. Staining of -actin identifies vascular SMCs predominantly in the luminal part, whereas foam cells are located in the central part of the plaque (magnification x31).

In ovariectomized female control rabbits receiving no hormones, the mean cross-sectional intimal area at the region of maximal plaque size was 3.7±2.5 mm² (Fig 2). Under estrogen therapy, the intimal area was significantly reduced (0.7±0.5 mm², P<.002) compared with the ovariectomized control group (Fig 2).

View larger version (116K): [in this window] [in a new window]   Figure 2. A and B, Cross sections of a rabbit aortic arch after ovariectomy and feeding of a 0.5% cholesterol diet for 12 weeks. Note the extent of the atherosclerotic plaque, separated into a luminal layer with a large number of SMCs, and a layer below consisting primarily of foam cells. C and D, Histological cross section of the aortic arch of a rabbit treated with 17ß-estradiol and a 0.5% cholesterol diet for 12 weeks. Compared with ovariectomized controls (A and B), the intimal plaque size is reduced. However, foam cells also are located predominantly in the central part of the plaque (A and C, elastica–van Gieson stain; B and D, hematoxylin and eosin stain). I indicates intima; l.e.i., lamina elastica interna; and M, media (bars=200 µm).

Both progesterone alone and combined estrogen-progesterone application had no effect on intimal plaque size (4.0±2.3 and 3.4±2.4 mm²; Fig 3). Intact female rabbits without hormone treatment displayed an intimal plaque of 3.6±1.1 mm², which was similar to that in the ovariectomized control group.

View larger version (11K): [in this window] [in a new window]   Figure 3. Bar graph showing the extent of neointimal plaque size (in mm2) in the female groups studied (values are expressed as mean±SD). E indicates estrogen; P, progesterone.

In castrated rabbits, the maximal intimal plaque size was 2.9±1.2 mm²; in intact male rabbits, 3.2±1.4 mm². Application of estradiol resulted in an intimal plaque size of 2.8±1.0 mm², which was similar to that in the castrated control group.

Interestingly, progesterone therapy was associated with considerably increased intimal thickening compared with castrated control rabbits (5.5±3.5 mm²) although the increase was not statistically significant.

After combined estrogen-progesterone application, a mean intimal plaque size of 3.2±1.5 mm² was measured, which was not statistically significantly different from that in the castrated control group or the male group treated with progesterone alone (Fig 4).

View larger version (13K): [in this window] [in a new window]   Figure 4. Bar graph showing the extent of neointimal plaque size (in mm2) in the male groups studied (values are expressed as mean±SD). Abbreviations as in Fig 3.

Quantification of Cells Undergoing DNA Synthesis
In ovariectomized rabbits, quantification of cellular proliferation in the intima revealed that 8.5±2.6% of cells were undergoing DNA synthesis. Under estrogen therapy, immunohistological quantification displayed a significant reduction in proliferating cells (3.1±1.8%, P<.002) compared with the ovariectomized control group. After progesterone alone and combined estrogen-progesterone administration, there was no significant change in the number of proliferating cells compared with ovariectomized controls. In addition, cellular proliferation in intact rabbits was comparable to the ovariectomized control group (Fig 5).

View larger version (13K): [in this window] [in a new window]   Figure 5. Bar graph showing the number of proliferating SMCs undergoing DNA synthesis (in %) in the female groups studied (values are expressed as mean±SD). Abbreviations as in Fig 3.

In male rabbits receiving estrogen, progesterone, or combined estrogen-progesterone therapy and in intact rabbits, no significant changes in cellular proliferation were observed compared with controls. In castrated controls and males receiving progesterone or combined estrogen-progesterone, proliferating cell numbers were similar to those in corresponding female groups (Figs 5 and 6).

View larger version (14K): [in this window] [in a new window]   Figure 6. Bar graph showing the number of proliferating SMCs undergoing DNA synthesis (in %) in the male groups studied (values are expressed as mean±SD). Abbreviations as in Fig 3.

Staining of -actin in serial cross sections of BrdU-stained sections in female and male rabbits revealed homogeneously arranged actin filaments predominantly in the luminal part of the intima, where most of the proliferating cells were located (Fig 7), confirming that most of the BrdU-positive cells were SMCs.

View larger version (139K): [in this window] [in a new window]   Figure 7. Serial cross section of the luminal part of the same vessel as shown in Fig 1. Staining of BrdU-positive cells shows proliferating cells in the luminal part of the plaque, confirming that most of the proliferating cells are SMCs (magnification x125).

Plasma Cholesterol, Estrogen, andProgesterone Levels
The initial and final values of weight, plasma cholesterol level, and plasma hormone level from the experiment are presented in the Table.

Within 12 weeks, total cholesterol levels rose from 54±11 to 1836±385 mg/dL in ovariectomized controls, whereas in castrated male controls, total cholesterol rose from 55±24 to 2012±330 mg/dL, which is not significantly different from that in females. In all corresponding hormone-treated female and male groups, no significant differences in the total cholesterol levels were observed.

Under estrogen treatment, 17ß-estradiol plasma levels were similar after either estrogen alone or combined estrogen-progesterone treatment in both female and male groups. Compared with the intact nonovariectomized, untreated female rabbits, the estrogen levels achieved in our study were approximately 10-fold higher under estradiol treatment (the Table).

Progesterone application also was associated with similar 17-OH-progesterone plasma levels in female and male study groups that received either progesterone alone or the combined estrogen-progesterone regimen.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
This study was designed to investigate the effects of estrogen, progesterone, and continuous combined estrogen/progesterone therapy on atherosclerosis and cellular proliferation in female versus male rabbits.

Our hypothesis, that a sex-specific estrogen action in the process of atherosclerosis might exist, was assessed by a direct comparison of female with male rabbits in regard to different effects of female sex hormones on the development of experimental atherosclerosis.

An atheroprotective effect of estrogen has been found in a variety of animal models, including chickens, primates, and rabbits.^{4 11 23 24 25} It is difficult to find an appropriate model because atherosclerosis does not develop naturally in animals as it does in humans.

For the present study, the hypercholesterolemic rabbit was chosen. It has become one of the most useful experimental models for atherosclerosis research since it was first described by Anitschkow²⁶ in 1914. The hypercholesterolemic rabbit model has the distinct advantage of rapid and reliable induction of atherosclerotic lesions within an 8- to 12-week period.^{11 27 28}

Our study design allowed direct comparison of ovariectomized female rabbits with castrated male rabbits, which served as the basic model for the present study.

In agreement with other recent studies,^{9 10} we also found an antiatherosclerotic effect of estrogen in female rabbits, independent of a significant influence on plasma cholesterol concentrations.

In combination with progesterone, the extent of intimal plaque formation in female rabbits was not reduced by estrogen in the current study, suggesting that progesterone can exert an inhibitory influence on the effect of estrogen directly.

However, this progesterone effect on estrogen action might vary strongly with the application modus of progesterone. Interestingly, Adams et al²³ found that the inhibitory effect of estrogen on experimental atherosclerosis in monkeys was not affected by cyclic-administered progesterone, whereas in our study a continuous combined application of estrogen and progesterone was used. In addition, the progesterone effect on estrogen action might also be explained by the higher progesterone plasma levels in our study. Adams et al²³ found progesterone levels of 9 ng/mL using a subcutaneous Silastic implant in monkeys, whereas in our experimental setting, progesterone levels during the week after intramuscular injection ranged from 27 ng/mL (peak value by hour 12) to 2 to 3 ng/mL, the lowest level immediately before the next scheduled progesterone application (unpublished data).

Progesterone alone in the female group had no effect on plaque development or SMC proliferation. Application of progesterone in male rabbits, however, was associated with a considerable increase in intimal plaque size compared with castrated controls and rabbits treated with estrogen and progesterone combined. From this observation, it might be speculated that progesterone exerts an atheroprogressive effect in male rabbits that is not apparent under combined estrogen application. However, these interesting findings did not reach a statistically significant probability value, possibly because of the relatively high SD in the male group treated with progesterone alone.

Cell proliferation is present during all stages of the atherosclerotic process, as documented in studies with ³H-thymidine incorporation into cells in atherosclerotic lesions in hypercholesterolemic rabbits.^{29 30 31} Similar to the detection of proliferating cells by autoradiography in these studies, the technique of BrdU labeling used in our study also allows the identification and quantification of cells undergoing DNA synthesis but by immunohistochemical methods with paraffin-embedded sections. In contrast to simple morphometric measurements of atherosclerotic plaque, the visualization of cells actually proliferating in the plaques provides additional information about growth dynamics in the lesions.

The present study is the first report based on a single unified experimental setting to show a sex-specific difference of estrogen action and its effects on cellular proliferation and development of atherosclerotic lesions.

Interestingly, the cross-sectional intimal area and the extent of cellular proliferation clearly were greater in ovariectomized female than in castrated male rabbits as a trend, although this difference was not quite statistically significant. In addition, proliferation rate and intimal plaque size of intact female and male rabbits with a normal sex steroid hormone status also revealed no statistically significant differences.

Estrogen could not reduce intimal plaque size in atherosclerotic male rabbits, whereas the identical dosage of estrogen in female rabbits resulted in a significant reduction of intimal thickening.

According to these histomorphological results, the cellular turnover, assessed by the quantification of the number of cells undergoing DNA synthesis, was significantly reduced in female rabbits under estrogen therapy. Using immunohistological staining of -actin in serial cross sections, we identified most of the proliferating cells in the luminal part of the plaque as SMCs. Our data suggest an inhibitory effect of estrogen on vascular SMCs, supporting a recently published study by Vargas et al,³² who used cultured SMCs.

Interestingly, no inhibitory effect of estrogen on cellular proliferation and plaque development was observed in male rabbits. Our present experiments do not provide an explanation for these marked differences in female and male rabbits. However, a sex-different metabolism of the administered sex hormones, which might result in different sex hormone plasma levels, can be excluded because female and male rabbits had comparable estrogen and progesterone plasma levels in the corresponding groups.

One possible explanation for the estrogen effect on atherosclerosis might be related to an estrogen-specific pathway, such as a specific interaction with sex hormone at the receptor level.

Estrogen and progesterone receptors have been identified in arterial endothelial cells and SMCs in the baboon³³ and in human coronary arteries.^{19 34} In ovariectomized baboons, 17ß-estradiol was apparently able to affect the intracellular distribution of cardiovascular estrogen receptors and increase the cytoplasmic concentration of progesterone receptors, suggesting that these estrogen receptors in the baboon are physiologically functional.³³

Interestingly, Vargas et al³² found in vitro a complete block of the inhibitory effect of 17ß-estradiol on SMC proliferation by phenol red, a weak estrogen agonist that is believed to occupy the estrogen receptor.^{35 36}

Additionally, in a recent necropsy study, a relationship between the presence of estrogen receptors and the absence of atherosclerosis in premenopausal women was demonstrated, suggesting that these receptors may play a functional role in coronary atheroprotection.¹⁹

Considering these data and suggesting a predominantly receptor-mediated action of estrogen in the process of atherosclerosis, we can draw the following conclusions from our study: (1) progesterone could influence and inhibit the beneficial effect of estrogen, probably by affecting arterial sex hormone receptors; (2) estrogen has failed to inhibit atherosclerosis in male rabbits because a sex-specific pathway, which is present in female rabbits only; (3) to develop an effective secondary preventive hormone replacement regimen for women with CHD, our model might be useful for testing different progestogens in regard to their atherogenic potency and their effect on estradiol action during combined administration in female rabbits; and (4) further experimental studies are needed to explain the exact mechanism(s) and pathway(s) of estrogen action in the arterial vessel wall during the process of atherosclerosis.

	Acknowledgments

 
The RIAs of 17ß-estradiol and 17-OH-progesterone were kindly done by Dr H. Seeger from the Department of Obstetrics and Gynecology (Division of Clinical Pharmacology, Prof Dr T.F. Lippert), University of Tubingen, Germany. We also thank Prof Dr K. Dietz from the Institute of Biometry and Statistics, University of Tubingen, for his advice and help with the statistical analysis. We gratefully acknowledge the excellent technical assistance of Hannelore Bacher, Mechthilde Heilig, Mechthild Holz, and Gisela Kaletta in histological preparations, immunohistological methods, and lipid analysis.

	Selected Abbreviations and Acronyms

 

BrdU = bromodeoxyuridine BW = body weight CHD = coronary heart disease RIA = radioimmunoassay SMC = smooth muscle cell

	Footnotes

 
Presented in part at the 67th Scientific Sessions of the American Heart Association, Dallas, Tex, November 14-17, 1994, and published previously in abstract form (Circulation. 1994;90[pt 2]:I-291).

Received October 23, 1995; revision received January 3, 1996; accepted January 4, 1996.

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