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Raloxifene and estrogen reduces progression of advanced

atherosclerosis — a study in ovariectomized, cholesterol-fed

rabbits

Nina Hannover Bjarnason

a,

*, Jens Haarbo

a

, Inger Byrjalsen

a

, Peter Alexandersen

a

,

Raymond F. Kauffman

b

, Claus Christiansen

a

aCenter for Clinical and Basic Research,Ballerup By6ej222,2750Ballerup,Denmark bEli Lilly & Company,Lilly Research Laboratories,Indianapolis,IN46285,USA

Received 16 November 1999; received in revised form 6 March 2000; accepted 10 March 2000

Abstract

The present study investigated the effect of raloxifene, a selective estrogen receptor modulator (SERM), on aortic atherosclero-sis in 80 ovariectomized, cholesterol-fed rabbits with pre-induced atheroscleroatherosclero-sis. The animals were fed an atherogenic diet containing 240 mg cholesterol/day for 15 weeks, after this period a baseline control group was sacrificed. Thereafter, oral treatment was initiated with either estradiol 4 mg/day (n=20), raloxifene (210 mg/day) or placebo (n=20). In the treatment period of 39 weeks, the dietary cholesterol content was reduced to 80 mg cholesterol/day. Postmortem evaluation showed a significantly increased uterine weight induced by estradiol treatment (10.391.2 g), whereas raloxifene intervention caused a decreased uterus weight (1.2190.1 g) when compared to placebo (2.4890.47 g). Throughout the study, serum lipids increased in all groups to levels seen in very high risk humans. After 58 weeks the cholesterol content in the aorta was 3.1890.54mmol/cm2 (38% reduction) in the estradiol group, 3.6690.52mmol/cm2(29% reduction) in the raloxifene group and 5.1290.60mmol/cm2 in the placebo group. Analyses of the aortic cholesterol content corrected for time-averaged serum cholesterol revealed that both estradiol and raloxifene therapy significantly reduced the progression of atherosclerosis (PB0.01 for both) as compared to placebo. © 2001 Elsevier Science Ireland Ltd. All rights reserved.

Keywords:Atherosclerosis; Lipids; Cholesterol; Prevention; Estrogen; Raloxifene; Endometrium

www.elsevier.com/locate/atherosclerosis

1. Introduction

The prevalence, morbidity and mortality of cardio-vascular disease in postmenopausal women requires a concerted investigational effort to define efficacious prevention strategies. The increase in cardiovascular events with age in elderly women has been related to the decrease in endogenous estrogen production after menopause [1], and numerous observational data have shown a protective effect of estrogen treatment, partic-ularly in secondary prevention [2 – 4].

The recent null conclusion of the Heart and Estro-gen/progestin Replacement Study (HERS) [5] has

raised doubts about the exact effect of female hormones upon ischemic heart disease and the side effect profile of traditional HRT doses prevents many women from receiving long-term treatment [6]. Raloxifene, a selec-tive estrogen receptor modulator (SERM), is distin-guished from estrogen by its ability to interact with the estrogen receptor either as an estrogen agonist or an-tagonist depending on the target tissue and hormonal milieu [7]. Thus, raloxifene has estrogen-like effects upon bone [8,9] but anti-estrogenic effects on breast [10] and endometrium [8,9]. The influence of raloxifene on cardiovascular risk markers has demonstrated both similarities and differences from that of estrogen [11]. It has previously been shown that both estradiol and raloxifene treatments inhibit atherosclerosis formation in healthy rabbit arteries [12 – 14]. In rabbits with pre-induced formation of atheroma, estradiol inhibited fur-* Corresponding author. Tel.: +45-44-684600; fax: +

45-44-684220.

E-mail address:nb@ccbr.dk (N.H. Bjarnason).

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ther progression of atherosclerosis [15,16]. Thus, it was important also to study the effect of raloxifene in rabbits with pre-induced formation of atheroma, which may mimic mild atherosclerosis as seen in early post-menopausal women.

2. Experimental animals and methods

2.1. Experimental animals

Eighty healthy and sexually mature female rabbits of the Danish Country strain (SSC:CPH), were obtained at Statens Serum Institut, Copenhagen, DK. The rab-bits, which previously had been fed a standard commer-cial rabbit chow, were housed at approved animal facilities (Ledoeje, DK) in standard rabbit cages with a room temperature of 2092°C and a 12-h light cycle. All rabbits had free access to water and were adminis-tered 100 g of chow/day at 08:00 h. The study was approved by the Danish authorities for experimental animals (Department of Justice). Body weight was checked every 4 weeks on the same scale. The chow containing estradiol, raloxifene or placebo was pre-pared, stored and controlled as previously described [13,14].

2.2. Study design

The rabbits (n=80) were allowed to acclimate for 3 weeks, were ovariectomized the following week as de-scribed earlier [13,14] and randomised to either baseline control (n=20), placebo (n=20), estradiol 4 mg/day (n=20) or raloxifene 210 mg/day (n=20). In week 5 the induction of atherosclerosis was initiated by includ-ing 240 mg cholesterol/day in the diet. In week 19 the baseline control group was sacrificed to measure the baseline level of atherosclerosis. To stabilize serum lipids, the amount of cholesterol in the diet was reduced to 80 mg/day from week 20 – 58. In the same time period, the rabbits were treated orally as indicated by randomisation. Blood sampling was performed in weeks 3, 13, 19, 27, 42 and 57, approximately 24 h after the last feed.

2.3. Biochemistry

Serum total cholesterol, high density lipoprotein (HDL)-cholesterol (only at baseline) and triglycerides were determined enzymatically by routine analysis pro-cedures. Serum lipoproteins were fractionated into HDL (density\1.063 g/ml), low density lipoprotein (LDL) (1.019 g/mlBdensityB1.063 g/ml), intermedi-ate density lipoprotein (IDL) (1.006 g/mlBdensityB 1.019 g/ml) and very low density lipoprotein (VLDL) (densityB1.006 g/ml) by ultracentrifugation and

mea-sured in weeks 13, 19, 27, 42 and 57 as previously described [13,14].

2.4. Aortic tissue

The thoracic aorta was freed from the adventitia, opened longitudinally and divided into proximal and distal parts at the level of the first intercostal arteries. After determination of the surface area of the proximal part, the inner layer containing the intima and part of the media was stripped from the underlying outer me-dia. The inner layer, which was used for the assessment, was weighed and stored at −20°C. The total choles-terol and protein content was then determined accord-ing to published procedures [13].

2.5. Uterus and endometrial tissue

The bicorn uterus of the rabbits was cut at the bifurcature, weighed and opened. A sample of endome-trial tissue was excised from the cavity, frozen immedi-ately on solid carbon dioxide and stored at −85°C. For the analyses the tissue was homogenised and a cytosolic fraction and a nuclear extract were prepared as previously described [13]. The estrogen receptor con-tent in the cytosolic fraction and in the nuclear extract was measured by an enzyme immunoassay according to the manufacturer’s instruction (Abbott Laboratories, North Chicago, IL). The cytosolic progesterone binding capacity was measured by a steroid binding assay using dextran-coated charcoal separation [17]. The results of the receptor contents were then normalised to the cyto-solic protein content, which was determined as previ-ously [13] according to Bradford.

2.6. Statistics

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Table 1

Baseline characteristics (mean9S.D.), food consumption and weight gain (mean9S.E.M.)a

S-triglycerid 0.6490.27 0.6790.21 es

Fig. 1. The cholesterol content in the inner proximal layer of the thoracic aorta (mean9S.E.M. ) after correction for time-averaged serum cholesterol is shown for unadjusted aortic cholesterol content (A) and for area-adjusted aortic cholesterol content (B).

The aortic accumulation of cholesterol was signifi-cantly related to time-averaged serum values of total cholesterol, VLDL-cholesterol, IDL-cholesterol and triglycerides, whereas this was not the case for LDL-cholesterol and HDL-LDL-cholesterol. After correction for these values of lipids and lipoproteins, an independent effect of treatment upon aortic atherosclerosis was demonstrated and Fig. 1A and B show the results corrected for serum cholesterol. For area-adjusted aor-tic cholesterol content, the response in the estradiol group was 62% of that of placebo, whereas for ralox-ifene the response was 71% of placebo. The aortic cholesterol content adjusted for protein showed com-parable responses, although only of borderline significance.

The uterine wet weight was significantly increased due to estradiol treatment (10.391.2 g; PB0.001), in contrast to raloxifene therapy, which significantly re-duced the weight of the uterus (1.2190.1 g; PB0.05) as compared to placebo (2.4890.47 g) (Fig. 2A). The endometrial nuclear estrogen receptor content was up-regulated in the raloxifene group (P=0.02), whereas estradiol therapy tended to decrease the number of estrogen receptors in the nucleus (P=0.16) (Fig. 2C). In the cytosol of endometrial cells, both estradiol and raloxifene downregulated estrogen receptors (PB0.001 for both) (Fig. 2D), whereas progesterone receptors were increased in the estradiol group (P=0.02) and 3. Results

At baseline, no differences between the groups in age, body weight, lipids or lipoproteins were seen (Table 1). Three rabbits died prematurely, two in the estradiol group and one in the placebo group, thus 77 rabbits completed the study. The groups were comparable with regard to food intake, weight gain, lipids and lipo-proteins throughout the study (Tables 1 and 2). The cholesterol content for the baseline sacrifice group were 2.8190.44 mmol (unadjusted) and 0.5290.10 mmol/

cm2 (adjusted for aortic surface area), whereas the estradiol group had 24.694.9 mmol and 3.1890.54 mmol/cm2, the raloxifene group had 27.894.6 mmol

Time averaged serum lipids and lipoproteins, total study period (mean9S.E.M.)

Raloxifene Estradiol Placebo P, ANOVA

(N=18) (N=19) (N=20)

17.4191.30 16.8491.79 16.5791.28 NS S-total cholesterol (mmol/l)

11.1091.08 10.1291.33

S-VLDL-cholesterol (mmol/l) 9.2791.05 NS

NS

3.8290.55 4.6190.47

S-LDL-cholesterol (mmol/l) 5.6490.60

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Fig. 2. Uterine weight (A) and endometrial activity as evaluated by the content of progesterone receptors in the cytosol (B), estrogen receptors in the cytosol (C) and by estrogen receptors in the nucleus (D) (mean9S.E.M.).

borderline increased in the raloxifene group (P=0.16) (Fig. 2B).

4. Discussion

The data show a reduction in atherosclerosis progres-sion on atherosclerotic arteries during treatment with raloxifene and estradiol in cholesterol-fed, ovariec-tomized rabbits. This effect is only partially explained by changes in serum lipids and lipoproteins.

The study was designed to evaluate the effect of raloxifene upon pre-existing atherosclerosis, therefore the baseline sacrifice group was added to assess the degree of atherosclerosis at the time of initiation of treatment and included placebo and estradiol groups as negative and positive controls.

A 38% reduction in atherosclerosis formation due to estradiol treatment was comparable to previous sec-ondary prevention studies, which ranged from 24 [16] to 53% [15]. The somewhat milder effect of raloxifene (29% reduction) was also comparable to results from primary prevention experiments in rabbits, where a 30% reduction was found on the same raloxifene dose [14]. This dose was chosen to produce plasma raloxifene levels comparable to those of postmenopausal women during raloxifene therapy [18]. Table 3 shows the accu-mulated results in the rabbit model of estradiol and raloxifene, respectively. These data show a tendency for a less pronounced response when atherosclerosis is present at the initiation of therapy (secondary preven-tion model).

A recent study of conjugated equine estrogens (CEE) and medroxyprogesterone acetate (MPA), involving

2700 women with documented, advanced coronary heart disease, failed to demonstrate preventive effects upon myocardial infarction and cardiac death. In fact, in the first 2 years of the study an increased risk during active treatment was seen whereas the risk decreased during the third and fourth year [5]. Theoretically, this may be due to an initial negative effect upon haemosta-sis, while the effect upon atherosclerotic plaque forma-tion and stability becomes evident thereafter [5]. These results may cast doubt on the value of CEE+MPA upon secondary prevention, but not necessarily on the potential for raloxifene, although both treatment regi-mens have been found to increase the risk of venous thrombosis with a relative risk of 3 [5,10]. Although the current consensus based on observational data is that HRT protects against atherosclerosis, these results are

Table 3

Accumulated results on reduction in aortic cholesterol content in cholesterol-fed ovariectomized rabbits during treatment with HRT or raloxifene compared to placebo

Model

Reference Estradiol (%) Raloxifene (%)

Primary

[12] 66 –

prevention

69 31

Primary [13]

prevention

Primary 45 30

[14]

prevention Secondary

[15] 53 –

prevention

Secondary –

[16] 24

prevention

Secondary 29

Present data 38

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likely to be biased because of baseline differences be-tween women who chose — or chose not — to receive long-term treatment with estrogens. Thus, the effect of HRT in general may be significantly less than originally assumed, but it may also be that only certain popula-tions will benefit or that only particular regimes may have a clinically relevant efficacy.

The effect of raloxifene on CVD is not fully eluci-dated but in postmenopausal women, raloxifene de-creases serum total and LDL-cholesterol, and has a neutral effect on triglycerides and HDL-cholesterol [9,11]. Raloxifene also decreases plasma fibrinogen, but has no effect on markers of fibrinolysis. This was in contrast to conjugated equine estrogens co-adminis-trated with medroxyprogesterone acetate (CEE+

MPA), which had a positive effect on fibrinolysis markers, but no influence on coagulation markers [11]. As suggested by the data, non-lipid mechanisms may be involved in a possible beneficial effect of raloxifene. This is supported by recent data showing that ralox-ifene relaxes rabbit and human coronary arteries acutely by an endothelial-dependent mechanism involv-ing NO [19,20]. Results from a study on levormelox-ifene and estradiol furthermore indicate a role for NO in antiatherogenic effects of estrogen [21]. Taken to-gether, these data may provide a rationale for a hypoth-esis on a non-lipid mechanism of action.

A possible cardioprotective effect of raloxifene in postmenopausal women will be investigated in the RUTH trial, a study in 10 000 women at risk for ischemic heart disease, which is currently in its enrol-ment phase. The study is powered to show a 17.5% reduction in cardiac events due to raloxifene interven-tion and excludes women with increased risk for venous thrombosis and expected to show results in 2005.

The partially estrogenic and non-estrogen effects of raloxifene were confirmed by the uterine data, which elegantly demonstrated that raloxifene exerts both es-trogen agonist and eses-trogen antagonist effects in the same target organ in rabbits. Thus, whereas raloxifene in contrast to estradiol decreases uterine weight and upregulates the nuclear content of estradiol receptors, both therapies increase cytosolic progesterone receptors and decrease cytosolic estrogen receptors. In the MORE trial, a study in 7700 osteoporotic women, raloxifene did not increase the risk of either endome-trial hyperplasia or uterine cancer [10].

In conclusion, in a series of three experiments investi-gating the effect of raloxifene on atherosclerosis in ovariectomized, cholesterol-fed rabbits a 30% reduction has consistently been shown in aortic cholesterol con-tent, which is only partially explained by lipid lowering. While awaiting the results from the RUTH study, possible mechanisms for non-lipid actions of raloxifene should be further studied.

Acknowledgements

The authors thank the staff at the CCBR animal farm for the daily caretaking of the rabbits, Eli Lilly, Indianapolis, US for financial support and supplying raloxifene and Novo Nordisk, Bagsvaerd for supplying 17b estradiol.

References

[1] Colditz GA, Willett WC, Stampfer MJ, Rosner BS, Speizer FE, Hennekens CH. Menopause and the risk of coronary heart disease in women. N Engl J Med 1987;316:1105 – 10.

[2] Sullivan JM, Zwaag RV, Hughes JP, Maddock V, Kroetz FW, Ramanathan KB, Mirvis DM. Estrogen replacement and coro-nary heart disease. Effect on survival in postmenopausal women. Arch Intern Med 1990;150:2557 – 62.

[3] Grodstein F, Stampfer MJ, Manson JE, et al. Postmenopausal estrogen and progestin use and the risk of cardiovascular disease. N Engl J Med 1996;335:453 – 61.

[4] Bush TL, Barrett-Connor E, Cowan LD, et al. Cardiovascular mortality and noncontraceptive use of estrogen in women: re-sults from the lipids research clinics program follow-up study. Circulation 1987;75:1102 – 9.

[5] Hulley S, Grady D, Bush T, Furberg C, Herrington D, Riggs B, Vittinghoff E, for the HERS Research Group. Randomized trial of estrogen plus progestin for secondary prevention of coronary heart disease in postmenopausal women. J Am Med Assoc 1998;280:605 – 613.

[6] Ravnikar VA. Compliance with hormone replacement therapy: are women receiving the full impact of hormome replacement therapy preventive health benefits? Womens Health Issues 1992;2(2):75 – 80 discussion 80 – 82.

[7] Mitlak BH, Cohen FJ. In search of optimal long-term female hormone replacement: the potential of selective estrogen receptor modulators. Horm Res 1997;48(4):155 – 63.

[8] Black LJ, Sato M, Rowley ER, et al. Raloxifene (LY139481HCl) prevents bone loss and reduces serum cholesterol without caus-ing uterine hypertrophy in ovariectomized rats. J Clin Invest 1994;93:63 – 9.

[9] Delmas PD, Bjarnason NH, Mitlak BH, et al. The effects of raloxifene on bone mineral density, serum cholesterol and uter-ine endometrium in postmenopausal women. N Engl J Med 1997;337:1641 – 7.

[10] Cummings SR, Eckert S, Krueger K et al., for the MORE Research Group. The effect of raloxifene on risk of breast cancer in postmenopausal women. J Am Med Assoc 1999;281:2189 – 2197.

[11] Walsh BW, Kuller LH, Paul S, Farmer M, Lawrence JB, Shah AS, Anderson PW. The effect of raloxifene on serum lipids and coagulation factors in healthy, postmenopausal women. J Am Med Assoc 1998;279:1445 – 51.

[12] Haarbo J, Leth-Espensen P, Stender S, Christiansen C. Estrogen monotherapy and combined estrogen-progestogen replacement therapy attenuate aortic accumulation of cholesterol in ovariec-tomized cholesterol-fed rabbits. J Clin Invest 1991;87:1274 – 9. [13] Bjarnason NH, Haarbo J, Byrjalsen I, Kauffman RF,

Chris-tiansen C. Raloxifene inhibits aortic accumulation of cholesterol in ovariectomized, cholesterol-fed rabbits. Circulation 1997;96(6):1964 – 9.

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[15] Haarbo J, Christiansen C. The impact of female sex hormones on secondary prevention of atherosclerosis in ovariectomized, cholesterol-fed rabbits. Atherosclerosis 1996;123:139 – 44. [16] Alexandersen P, Haarbo J, Sandholdt I, Shalmi M, Lawaetz H,

Christiansen C. Norethindrone acetate enhances the antiathero-genic effect of 17b estradiol. Arterioscler Thromb Vasc Biol 1998;18:902 – 7.

[17] Thorpe SM. Steroid receptors in breast cancer: sources of inter-laboratory variation in dextran-charcoal assays. Breast Cancer Res Treat 1987;9:175 – 89.

[18] Geiser J, Ni L, Knadler MP, Henry D, Dere W, Allerheiligen S. The population pharmacokinetics of raloxifene in post-menopausal women. Pharm Sci Supplement 1998;S-518.

[19] Figtree GA, Lu Y-Q, Webb CM, Collins P. Raloxifene acutely relaxes rabbit coronary arteries in vitro by an estrogen receptor and nitric oxide-dependent mechanism. Circulation 1999;100:1095 – 101.

[20] Figtree GA, Lu Y-Q, Webb CM, Collins P. Raloxifene acutely relaxes rabbit and human arteries in vitro by an endothelium-de-pendent mechanism. J Am Coll Cardiol 1999;33:231A – 2A Ab-stract.

[21] Holm P, Korsgaard N, Shalmi M, Andersen HL, Hougaard P, Skouby SO, Stender S. Significant reduction of the antiathero-genic effect of estrogen by long-term inhibition of nitric oxide synthesis in cholesterol-clamped rabbits. J Clin Invest 1997;100:821 – 8.

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