Effects of estradiol, cyproterone acetate, tibolone and raloxifene on uterus and aorta atherosclerosis in oophorectomized cholesterol-fed rabbits

Maturitas 45 (2003) 59 /66 www.elsevier.com/locate/maturitas

Effects of estradiol, cyproterone acetate, tibolone and raloxifene on uterus and aorta atherosclerosis in oophorectomized cholesterol-fed rabbits A. Sanjua´n a, C. Castelo-Branco a,*, M. Colodro´n a, C. Ascaso d, J.J. Vicente a, J. Ordi c, E. Casals b, I. Mercade´ b, G. Escaramı´s d, J.A. Vanrell a a

Section of Gynecologic Oncology, Department of Gynecology and Obstetrics, Menopause Clinic, Hospital Clı´nic i Provincial, University of Barcelona, c/Villarroel 170, 08036 Barcelona, Spain b Department of Biochemistry, Hospital Clı´nic i Provincial, University of Barcelona, Barcelona, Spain c Department of Pathology, Hospital Clı´nic i Provincial, University of Barcelona, Barcelona, Spain d Department of Public Health, University of Barcelona, Barcelona, Spain Received 13 May 2002; received in revised form 7 January 2003; accepted 27 January 2003

Abstract Background: Different hormonal replacement regimens are used for treating climacteric complaints; however, not all of them have the same clinical profile. Cardiovascular disease (CVD) is a major health problem and tibolone, raloxifene, estradiol (alone or with cyproterone acetate) have been added to cholesterol-fed rabbits to study atherosclerosis. Methods: A total of 48 cholesterol-fed New Zealand white rabbits were studied for 4 months. Forty rabbits underwent bilateral ovariectomy and the other eight were sham operated (group S). The ovariectomized rabbits were allocated to five groups of eight animals each receiving tibolone (Group T, 6 mg/day), raloxifene (R, 35 mg/day), estradiol valerate (E, 3 mg/day), estradiol valerate plus cyproterone acetate (EC, 3
/0.5 mg/day, respectively), and no treatment for the control group (C). The sham group received no treatment too. Results: After 4 months the percentage of the extent of atherosclerosis in the aorta was 30.4% in C group, 24.5% in S group, 10.2% in T group, 30.3% in R group, 17.9% in E group and 28.1% in EC group (P B/0.05 T vs. C, R, EC). The aortic cholesterol content compared with aortic weight was 8.55 mg/mg in C group, 11.97 mg/mg in S group, 1.86 mg/mg in T group, 3.82 mg/mg in R group, 2.86 mg/mg in E group and 5.24 mg/mg in EC group (P B/0.05 T vs. EC, C, S; R vs. C, S; E vs. C, S). Uterine weights in grams were: 1.89 (C group), 2.24 (S), 7.38 (T), 1.94 (R), 9.92 (E), and 5.94 (EC); P B/0.05 (C, S, R, vs. T, E, EC; T vs. E; EC vs. T, E). Conclusion: Our study showed a decrease in the extent of aortic atherosclerosis in oophorectomized cholesterol-fed rabbits treated with tibolone or estradiol, and a decrease in aortic cholesterol content in rabbits treated with tibolone, raloxifene and estradiol. However, rabbits treated with tibolone showed an increased uterine weight, which is contrary to that observed in humans. # 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cyproterone acetate; Estradiol; Experimental atherosclerosis; Raloxifene; Tibolone

* Corresponding author. Tel.:
/34-93-227-5534; fax:
/34-93-227-9325. E-mail addresses: [email protected], [email protected] (C. Castelo-Branco). 0378-5122/03/$ - see front matter # 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0378-5122(03)00086-0

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1. Introduction Cardiovascular disease (CVD) is the leading cause of death in postmenopausal women in developed countries. Epidemiological and clinical data [1] suggest that hormone replacement therapy (HRT) has a protective effect against CVD. In patients with intact uterus, HRT usually combines estrogens with progestogens in order to avoid endometrial hyperplasia [2] or uses drugs that do not stimulate the endometrium, such as tibolone [3] or raloxifene [4]. Work done on experimental animals [5] has shown that tibolone has a potential role in the prevention of CVD despite its effect on endometrial proliferation [6]. Contradictory data has been reported on the effects of raloxifene on atherosclerosis [7 /9] without affecting endometrial tissue. Estradiol has cardiovascular actions that may be important for controlling the development of atherosclerosis [10], such as antioxidative properties, effects on endothelial cells, and direct actions on the atheroma plaque [11]. Finally, cyproterone acetate is a 17-OH-progesterone derivative introduced in HRT because of its antiandrogenic effect on lipid metabolism and its absence of effects on weight and arterial pressure [12]. However, its real effect on atherosclerosis is unknown. On the other hand, doubts have appeared on the real effect of HRT on the cardiovascular risk, since recent randomized clinical trials [13 /16] did not yield positive data. The present study was designed to investigate the effect of these hormonal compounds on the extent of atherosclerosis and the endometrial tissue, and to compare tibolone versus raloxifene for the first time.

2. Methods 2.1. Experimental design Forty-eight healthy, sexually mature, female New Zealand White rabbits, weighing between 2.5 and 3.5 kg, were included in this study. The rabbits were housed at approved animal facilities in standard rabbit cages at a room temperature of

209/2 8C and a 12-h light:12-h dark cycle. After the 2-week acclimatization period and under general anesthesia (Ketamine-HCl 30 /40 mg/kg i.m. and Xylazine-HCl 5 /8 mg/kg i.m.), 40 rabbits underwent bilateral oophorectomy through the abdominal route. Prior to surgery and anesthesia, all animals were sedated with chlorpromazine (2 mg/kg i.m.) and premedicated with atropine (0.05 mg/kg s.c.). The rabbits were randomly divided into six groups (T, R, E, EC, S, and C). After oophorectomy, the rabbits from group T received 6 mg/day of tibolone, those from group R received 35 mg/day of raloxifene, those from group E received 3 mg/day of estradiol valerate, those from group EC received the same dose of estradiol plus 0.5 mg/day of cyproterone acetate, and those from group C received no hormone therapy and served as controls. Finally, the rabbits from group S were sham operated and received no therapy. The treatments were administrated during 12 weeks mixed with the rabbit chow. All the hormonal compounds were obtained directly from the pharmaceutical laboratories. The hormonal doses were chose according to previous studies for estradiol, tibolone, and raloxifene and for cyproterone acetate an equivalent dose to estradiol was choused.

2.2. Rabbit chow All rabbits were fed 100 g chow/day from the first week throughout the study. During the acclimatization period, the rabbits were given a standard commercial rabbit chow (Pan Lab SA, Barcelona, Spain) for 2 weeks. After surgery, the rabbits were given the same standard chow for 2 more weeks and subsequently received either a hypercholesterolemic diet (groups S and C) or a hypercholesterolemic diet plus hormone treatment (groups T, R, E and EC) mixed with the chow (Pan Lab SA). All the animals followed this last diet during the next 12 weeks. In the hypercholesterolemic diet, cholesterol was added to the normal diet at a percentage of 0.5 g per 100 g of food. The rabbits were weighed every week throughout the study and food consumption was recorded daily by weighing leftover chow.

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2.3. Laboratory analyses Blood samples were drawn from the ear marginal vein of all rabbits in the morning after an overnight fast. Laboratory analyses were performed during the last week of the acclimatization period (i.e. just before surgery), during week 5 (before beginning the hypercholesterolemic diet), and subsequently every 3 weeks until week 16, when the rabbits were euthanized. Total cholesterol (CHOL), VLDL-cholesterol and triglycerides (TG) were measured. VLDL-cholesterol was determined by ultracentrifugation. CHOL and TG were measured by enzymatic methods (CHODPAP and TGO-PAP methods; Technicon Instr Corp., New York, USA). Data were expressed with area under the curve from the six blood samples done. 2.4. Necropsy The rabbits were euthanized with an intravenous injection of 10% pentobarbital solution (100 mg/kg body weight) and the uterus and aortas were dissected free and cleaned for further studies.

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tion to all other pixels similar to the previous ones, thus making it possible to identify all the atheroma plaques. By using the ‘‘Histogram’’ of the ‘‘Image’’ menu, we obtained the total number of pixels corresponding to damaged areas, after which the whole aorta area was marked and the total number of pixels quantified. The percentage of the atherosclerotic area was estimated by dividing the number of pixels in the damaged area by the number of pixels in the aorta. 2.6. Aorta cholesterol quantification The inner layer of the aorta containing the intima and part of the media was stripped from the underlying outer media. The inner layer was dried and weighed. The whole aortic tissue was minced and homogenized using a Politron (Kinematica AG, Littau-Luzern, Switzerland). Cholesterol was isolated using the Folch procedure [17]. Total cholesterol was determined using an enzymatic procedure (CHOD-PAP; Bayer Diagnostics, Tarrytown, NY) and the value measured was adjusted for the aortic weight. 2.7. Statistical analysis

2.5. Aortic atherosclerosis The aorta samples were cut longitudinally, stretched onto a piece of cardboard and then fixed in 4% formaldehyde for 24 h. Then, the aorta samples were washed in 70% alcohol and then stained for 20 min in Herxheimer solution. Herxheimer solution was prepared beforehand with 500 ml of 70% ethanol, 500 ml acetone and 5 g of Sudan IV. The aorta samples were then washed in 80% ethanol for 20 min and distilled water for 1 h to eliminate the excess of staining. This method provided a clear view of the plaques, which were of a strong red color. In order to measure the fatty streaks, the aortas were dissected free and divided into two parts: thoracic (including aortic arch) and abdominal aorta. Color photographs were made of each segment of the aorta. These images were processed with an image analysis program (Olympus Microimage 3.0). Several small (10 /12 pixels) areas of red atherosclerotic tissue were selected and the computer was asked to extend the selec-

The results were expressed as mean9/S.D. Logarithmic transformations were done when variables were not symmetric. ANOVAS and ANCOVAS studied statistical differences between the groups. If variances were not homogeneous after logarithmic transformation, non-parametric tests were used. For multiple comparisons, the Student /Newman /Keuls correction was used. Significance was established at P B/0.05. The results were analyzed using a statistical package software (SPSS version 9.0).

3. Results All the animals passed the 2-week acclimatization period without incidences. Therefore, the animals were randomized into six groups of eight. Out of the 48 rabbits included, one rabbit from group R was sacrificed due to a vertebral fracture and two rabbits from group EC did not finish the

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study because of pneumonia by Pasteurella multocida and fatal diarrhea. All the other rabbits ate all the food that was offered to them. Body weight increased during the study from an initial mean value of 2900 g to a final value of 3500 g. At the end of the study, the rabbits from group T showed a body weight increase that was lower than the increase measured in other groups (Table 1). The extension of the atheroma plaque was lower in the T group than in groups C, R, and EC. The animals from group E showed a markedly lower plaque extension, although the differences were not significant. There were no differences between all the other groups (Table 2). No differences were observed between the weight values of all the aorta samples obtained from the six groups (data not shown). The rabbits from groups T, R and E showed a significantly lower aortic cholesterol content than rabbits from groups C and S, whereas rabbits from group EC showed a lower content that was not significantly different (Table 2). The plasma lipid levels were higher in all six groups after introduction of the cholesterol-rich diet. When the lipid pattern was expressed as an area under the curve (AUC), a lower cholesterol level was found in group T compared with the

Table 2 Percentage of whole aortic surface covered by atherosclerosis and determination of aortic cholesterol in function of the aortic weight (%) of total aorta surmg aortic cholesface with atherosclerosis terol/mg aorta Control (n/8) Sham (n /8) Tibolone (n /8) Raloxifene (n / 7) Estradiol (n/7) Estradiol-cyproterone (n /6)

30.479/12.2 24.519/16.1 10.219/6.8* 30.319/18.0

8.559/4.63 11.979/11.33 1.869/0.72a 3.829/2.14b

17.919/10.1 28.169/7.97

2.869/1.1c 5.249/0.91

There is a significative correlation between both variables (correlation of Pearson: r/0.68 (P B/0.0001). *P/0.02 (T vs. C, R, EC). a P B/0.000 (T vs. EC, C and S). b P B/0.000 (R vs. C and S). c P B/0.000 (E vs. C and S).

other groups (except for group EC) and a lower VLDL-CHOL level compared with group C. Besides, a higher triglyceride level was found in group R compared with groups E, EC and S. There were no differences between groups C and S (Table 3). The analysis of the relationship between the aortic cholesterol content and the area under the Table 3 Area under the curve for total cholesterol, cholesterol-VLDL and triglycerides

Table 1 Body and uterine weight in the different groups Group

Basal body weight

Final body weight

Uterine weight

C (n /8) S (n/8) T (n/8) R (n/7) E (n/8) EC (n/ 6)

2956.19/226.7 2917.09/178.2 2914.09/242.2 3040.69/166.6 2918.69/244.8 3065.69/109.5

3566.59/230.3 3505.29/229.1 3206.39/292.3b 3455.79/167.2 3624.29/156.5 3655.59/123.1

1.899/0.6a 2.239/1.2a 7.379/1.3* 1.949/0.3a 9.99/1.8 5.99/0.9c

Control Sham Tibolone Raloxifene Estradiol Values are means9/S.D. expressed in grams. One rabbit from the group S was excluded from the analysis of uterine weight because its value was out of range. C: control, S: sham, T: tibolone, R: raloxifene, E: estradiol and EC: estradiol
/ cyproterone acetate. *P B/0.000 (T vs. E). a P B/0.000 (C, S, R vs. T, E and EC). b P /0.003 (T vs. C, S, R, E, and EC). c P B/0.000 (EC vs. T and E).

Estradiol-cyproterone

Cholesterol

Chol-VLDL Triglycerides

14 226.19/ 2210.0 13 180.19/ 3034.8 8910.09/ 4335.2* 14 501.09/ 3048.1 14 640.59/ 2338.4 11 587.79/ 1977.9

7972.19/ 1691.7 7524.89/ 2351.6 4456.39/ 3586.0a 7221.29/ 1385.7 7593.89/ 1854.6 5822.09/ 1192.9

3378.09/ 1104.3 2311.69/599.8 2974.19/ 2986.6 4634.59/ 852.8b 1877.59/574.1 2271.19/898.6

Results are mean9/S.D. expressed in mg/dl. *P B/0.003 (T vs. C, S, R and E). a P /0.027 (T vs. C). b P/0.013 (R vs. S, E and EC).

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curve for total cholesterol using regression lines suggested that the aortic cholesterol content was related to plasma cholesterol levels in groups C and S but not in the other groups. Therefore, cholesterol content was unrelated to plasma lipid levels in animals from treated groups (Fig. 1). The weight of the uterus was higher in rabbits from estradiol therapy group than in rabbits from the other groups. The rabbits from group EC had a lower uterus weight than rabbits from groups E and T. Uterus weight values were lower in animals from groups C, S, and R. There were no differences between groups S and C (Table 1). The histological examination of the uterus showed a marked increase of endometrial glands in groups E, T, and EC.

4. Discussion In order to evaluate more accurately the effect of HRT on the progression of experimental

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atherosclerosis we used two different methods. We studied the extension of the atheroma plaque and also we studied the composition of the plaque through quantification of the aortic cholesterol content. Tibolone decreased the aortic atheromatosis extension and cholesterol content, thus allowing the stabilization of the atheromatic plaque. These results agree with previous reports by Zandberg et al., which suggested an important role of tibolone in experimental primary cardiovascular prevention [5,18]. The rabbits treated with estradiol showed a marked tendency to a lower extension of the atheroma plaque. However, the small number of cases provide the study with a low statistical power. Our results agree with Zandberg et al., who reported a decrease in plaque extension with subcutaneous estradiol but only non-significant differences when estradiol was administered orally [5,18]. The research conducted with transdermal estradiol have yielded contradictory data [19,20].

Fig. 1. Relationship between aortic cholesterol (mg chol/mg aorta) and the area under the curve for total plasmatic cholesterol. These data suggest that aortic cholesterol was related to plasma cholesterol for groups C and S but not for the other groups.

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Besides, the rabbits treated with estradiol showed a decrease in aortic cholesterol content. This positive effect of estradiol is also in accordance with previous reports [7,21,22] and suggests a benefit of estradiol in primary cardiovascular prevention. However, the addition of progestins to the estrogen supplementation therapy for endometrial protection may counteract the beneficial effects of estradiol, and our results cast doubts on the hypothetical benefits on atheromatosis derived from adding cyproterone acetate to estradiol. Previous reports studied the cardiovascular effects of different progestogenic compounds with discordant results [21,23 /26]. The combined treatment with cyproterone acetate gave results below those derived from administering estradiol alone, and therefore, from the point of view of CVD prevention cyproterone acetate does not seem a good progestogen for combination with estradiol. According to our results, raloxifene does not reduce the size of the atheroma plaques but decreases the aortic cholesterol content. These results conciliate two conflicting previous reports: the decrease in cholesterol content with raloxifene determined by Bjarnason et al. [7] and the absence of differences in plaque extension reported for raloxifene by Clarkson et al. [9]. In a recent study by Bjarnason et al., the highest dose of raloxifene (210 mg/day) did not increase the effect of a dose of 70 mg/day, although the effect was greater with 70 mg/day than with 35 mg/day [8]. Clarkson [9] used a dosage that made it possible to reach raloxifene plasma levels similar to those in humans, and they did not find any favorable effect of raloxifene on plaque extension in monkey aortas. This seems to suggest that the discordance of results is more due to a problem in the quantification method than to a problem in the dosage used in the study. According to previous results on humans and rabbits [27,28], the reduction in cholesterol content may stabilize the atheroma plaque, and although raloxifene does not decrease the size of the atheroma plaque it may render it much more stable. This may be crucial to prevent acute myocardial infarction, unstable angina, peripheral vascular disease, stroke, and aortic aneurysm

formation [29], and is in agreement with the neutral effect of raloxifene on plasmatic CRP as a marker of inflammation [30,31], and with a positive direct effect of raloxifene on vascular reactivity [32]. Lipid levels were responsible for the accumulation of cholesterol in the aorta in rabbits from groups S and C; however, in the other groups the quantity of aortic cholesterol was not related to cholesterol plasma levels, thus suggesting that hormonal treatments may reduce the cholesterol content of atheroma plaques through a mechanism unrelated to the lipid levels (Fig. 1). Our study confirms previous data, as in the rabbit model ovariectomy does not lead to differences in the results between sham and control groups [21,33]. All these events are influenced by the low basal estrogen levels in rabbits [7] and thus the sham group is not present in recently published studies [18,34]. The effects of tibolone on uterine weight and endometrial tissue, which are the opposite of those found in humans, are a major source of concern. The dose of tibolone used in these experiments was chosen in order to obtain drug plasma levels similar to those in humans [5]. This different endometrial effect could be due to the rabbit uterus converting tibolone preferably to its estrogenic compound than to its gestagenic compound, contrary to what happens in humans [6]. Alternatively, the high tibolone dose administered to the animals could promote conversion of the drug to its estrogenic compound. A recent study by Zandberg et al. found less endometrial proliferation with a dose of 0.15 mg but also a lower effect on atheroma plaque [18]. According with these results we found two studies with monkeys and tibolone where a tibolone dose similar to a human dose had a minimal effect on endometrium and no effect on coronary atherosclerosis [35,36]. All these results give us the idea that in fact the dose we used of tibolone (6 mg) was so high. The uterine effects of raloxifene, estradiol and cyproterone acetate agree with the results obtained in humans [4]. In conclusion, this is the first study that compares tibolone and raloxifene. Tibolone and raloxifene are hormones used to prevent postmenopausal osteoporosis [37,38] without affecting

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endometrial tissue [3,39]. Although the doses of raloxifene used in our study could be debatable, it seems clear that raloxifene does not decrease plaque extension. The reduction of aortic cholesterol content will probably be greater at higher raloxifene doses [8]. However, it seems likely that it will not decrease it as much as tibolone. The effects of tibolone on the endometrial tissue of rabbits is the only drawback that this drug has when compared with raloxifene, and when we try to extrapolate the results to humans. It seems that there is a very important dose dependent effect. Our results with estradiol confirm previous positive reports, and we intend to continue our search for a good progestogen that does not interfere with estradiol [16].

Acknowledgements We would like to thank Montse Tortosa for her collaboration in the study of pathology samples and Carme Cleries for her help with the management of animals. We also thank Martin CullellYoung for the English revision of the manuscript. This study was supported in part by a grant from the Hospital Clinic i Provincial, awarded to Dr Alex Sanjua´n.

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