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(Circulation. 1999;100:1095-1101.)
© 1999 American Heart Association, Inc.

Basic Science Reports

Raloxifene Acutely Relaxes Rabbit Coronary Arteries In Vitro by an Estrogen Receptor–Dependent and Nitric Oxide–Dependent Mechanism

Gemma A. Figtree, BSc; Ying-qing Lu, PhD; Carolyn M. Webb, PhD; Peter Collins, MD, FRCP

From Cardiac Medicine, National Heart and Lung Institute, Imperial College School of Medicine, London, UK, and Department of Physiology, University of Sydney, Australia (G.A.F.).

Correspondence to Dr Peter Collins, Cardiac Medicine, National Heart and Lung Institute, Imperial College School of Medicine, Dovehouse St, London SW3 6LY, UK. E-mail peter.collins{at}ic.ac.uk

	Abstract

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Background—Selective estrogen receptor modulators (SERMs) have been defined as compounds that display tissue specificity with regard to estrogenic effects. They appear to share the beneficial effects of estrogen on bone and lipids but are not associated with an increased risk of breast or uterine carcinoma. Estrogen relaxes coronary arteries and has long-term protective effects on the vascular system. The effect of SERMs on the coronary vasculature is unknown. We therefore investigated the effects of the SERM raloxifene on isolated rabbit coronary arteries.

Methods and Results—–Rings of coronary artery from adult male and nonpregnant female New Zealand White rabbits were suspended in organ baths containing Krebs solution; isometric tension was then measured. Raloxifene induced coronary arterial relaxation in male and female coronary arteries by an endothelium-dependent and estrogen receptor–dependent mechanism involving nitric oxide. Raloxifene also had a direct calcium antagonistic effect on the coronary myocyte. Estrogen, however, induced only endothelium-independent coronary arterial relaxation. The endothelium-dependent component of relaxation induced by raloxifene 10^-6 mol/L resulted in almost 100% (79±7% versus 43±3%, P<0.001) more relaxation than that induced by estrogen 10^-6 mol/L.

Conclusions—These data demonstrate that raloxifene has vascular relaxing properties. The surprising finding is that the receptor-dependent effects via the endothelium are observed in coronary arteries from both male and female animals.

Key Words: raloxifene • arteries • endothelium • nitric oxide

	Introduction

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The risk of coronary heart disease and osteoporosis in postmenopausal women is reduced if they are current users of hormone replacement therapy.^{1 2} Estrogen has direct relaxing properties on blood vessels, including coronary arteries from animals and humans in vivo.^{3 4 5} Some of the mechanisms discovered may contribute to a protection against atheroma development.⁶

In rabbit coronary artery rings in our laboratory, acute estrogen-induced relaxation has been shown to be endothelium-independent.^{3 7} Rings without endothelium relaxed to a degree similar to rings with endothelium, and inhibition of nitric oxide synthase (NOS) did not alter the relaxation response.³ Calcium antagonism at the level of the coronary myocyte was shown to be a mechanism of relaxation by estrogen.⁸ This effect of estrogen was not dependent on the classic estrogen receptor.⁴ Studies have also shown an enhancement of endothelium-dependent responses in a variety of vessels.^{9 10 11 12 13}

Chronic exposure to estrogen can result in an increase in the release of NO from the vascular endothelium.^{11 14 15 16} This effect is estrogen receptor–dependent,¹⁶ supported by the identification of estrogen receptor in human coronary endothelial cells.¹⁷ Estrogen therefore has both receptor-dependent and non–receptor-dependent effects on blood vessels that contribute to its vasorelaxing properties and possibly its long-term cardioprotective effects.

Despite the epidemiological evidence that postmenopausal hormone therapy is cardioprotective,^{1 18} it is estimated that <10% of women who might benefit from a reduction of cardiovascular disease are actually taking it.¹⁹ The major reasons for this are fear of estrogen-induced breast and uterine cancer, as well as resumption of menses, mastodynia, and weight gain.²⁰ Raloxifene hydrochloride (LY139481), a selective estrogen receptor modulator, prevents bone loss without producing uterine proliferation.^{21 22 23 24 25} Raloxifene is known to share the lipid-lowering effects of estrogen,^{22 26 27} and it inhibits aortic accumulation of cholesterol in ovariectomized cholesterol-fed rabbits.²⁸ There was, however, a lack of effect of raloxifene on coronary artery atherosclerosis in a cynamolgus monkey model.^28a The effects of raloxifene on coronary artery vasoreactivity have not been investigated. The mixed estrogen agonist/antagonist profile that it exhibits in different tissues may also result in its action on different sites on the vessel wall, which may contribute to vasodilation. The purpose of this study was to investigate the possible relaxing effects of raloxifene on the coronary artery in vitro and the role of endothelial modulation and calcium antagonism in isolated rabbit coronary arterial preparations.

	Methods

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Animals and Tissues
Adult male or nonpregnant female New Zealand White rabbits (2.5 to 3.0 kg) were killed by an overdose of pentobarbitone (60 mg/kg) and heparin (150 U/kg). The heart was removed, and epicardial coronary arteries were dissected free of connective tissue. Arterial rings were prepared, and in alternate rings, the endothelium was removed by gentle rubbing with a wooden probe. Each ring (3 to 4 mm long) was suspended horizontally between 2 parallel stainless steel hooks for the measurement of isometric tension in individual organ baths containing 10 mL modified Krebs solution at 37°C, bubbled with 95% O₂ and 5% CO₂. The composition of the modified Krebs solution was as follows (mmol/L): NaCl 118.3, KCl 4.7, CaCl₂ 2.5, MgSO₄ 1.2, K₂PO₄ 1.2, and glucose 11.1. Coronary arterial rings were allowed to stabilize for 90 minutes under a resting tension of 1 g before being contracted. Preparations were exposed to maximally effective concentrations of a contractile agonist (K⁺, 30x10^-3 mol/L) to ensure stabilization of the vascular smooth muscle. The agonist was then removed and the ring reequilibrated. The presence or absence of endothelium was always verified by observation the relaxation response to acetylcholine.

Effect of Raloxifene and 17ß-Estradiol on Precontracted Rabbit Coronary Arteries
Coronary arterial rings with or without endothelium from male and female rabbits were contracted with potassium (K⁺, 30x10^-3 mol/L) or prostaglandin F₂ (PGF₂, 3x10^-6 mol/L). Raloxifene or the equivalent volume of DMSO solvent was added 7 minutes after the addition of constrictor agents or when the contraction was stable. Raloxifene was added in increasing concentrations at half-log increments, and dose-response curves were performed. Segments not exposed to raloxifene but exposed to the DMSO solvent acted as time-matched controls. In different rabbit coronary arterial rings, 17ß-estradiol was added at log increments from 10^-7 to 10^-5 mol/L.

Effect of N-Nitro-L-Arginine Methyl Ester on Relaxation Induced by Raloxifene
N-Nitro-L-arginine methyl ester (L-NAME) is an inhibitor of synthesis of endothelium-derived relaxing factor from L-arginine in vascular endothelial cells.²⁹ Rings with endothelium were incubated with L-NAME 10^-4 mol/L for 20 minutes before precontraction with PGF₂ or K⁺. A concentration-response curve to increasing concentrations of raloxifene was repeated.

Role of Classic Intracellular Estrogen Receptor in Raloxifene-Induced Relaxation
To examine the possible role of the classic estrogen receptor in mediating raloxifene-induced relaxation, rings with and without endothelium were incubated in ICI 182,780, a specific estrogen receptor antagonist, at 10^-5 mol/L for 20 minutes before precontraction in PGF₂. Measurements of the responses to increasing concentrations of raloxifene were then repeated.

Role of Potassium Channels in Raloxifene-Induced Relaxation
To examine the possible role of potassium conductance on raloxifene-induced coronary relaxation, barium chloride, a nonspecific inhibitor of potassium channels,³⁰ at 3x10^-3 mol/L was added to coronary arterial rings with and without endothelium 20 minutes before being contracted with PGF₂. The response to increasing concentrations of raloxifene was then measured.

Effect of Raloxifene on Calcium Channels
Rabbit coronary arterial rings without endothelium were incubated in calcium-free solution containing 0.5 mmol/L EGTA for 10 minutes. Calcium concentration–dependent contraction curves were then performed in high-K⁺ (80x10^-3 mol/L) depolarization medium. Rings were readjusted in modified Krebs solution for 20 minutes before being incubated with raloxifene for 30 minutes. The calcium concentration–dependent contraction curves were then repeated. Both the control and raloxifene calcium-dependent contraction curves were also repeated after incubation in barium chloride for 20 minutes

Drugs
The following drugs were used: raloxifene hydrochloride (LY139481; dissolved in DMSO, gift from Eli Lilly Research Laboratories, Indianapolis, Ind), L-NAME (Sigma), PGF₂ (Sigma), pentobarbitone (Sigma), ICI 182,780 (ICI). All drugs were analytical grade.

Statistical Analysis
All results are expressed as mean±SEM. Relaxation is expressed as percentage relaxation of contraction induced by PGF₂ 3x10^-6 mol/L or K⁺ 30x10^-3 mol/L. The results were analyzed by ANOVA. Each group was compared with the time-matched DMSO solvent control, with the Student-Newman-Keuls test for multiple comparisons. A value of P0.05 was considered statistically significant.

	Results

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Effect of Raloxifene on Precontracted Coronary Arteries and the Effect of Endothelium Removal
Raloxifene induced significant dose-dependent relaxation of coronary arterial rings with endothelium compared with time-matched DMSO solvent controls precontracted by PGF₂ (Figure 1) or K⁺. Relaxation occurred in rings without endothelium; however, relaxation in rings without endothelium was significantly less than in rings with endothelium (Figure 2, top). There was no significant difference in relaxation between rings precontracted by PGF₂ and K⁺ (n=8 to 13; P>0.05) (n indicates number of animals).

View larger version (32K): [in this window] [in a new window]   Figure 1. Relaxing effect of raloxifene (-8 to -5.5 log mol/L) on rabbit coronary artery rings with endothelium (With endo) precontracted with PGF2 3x10-6 mol/L. Data are expressed as percentage relaxation of contraction induced by PGF2 (mean±SEM). Control indicates time-matched DMSO solvent controls. n=11 to 13. Significant differences vs control: *P<0.05, ***P<0.001.

View larger version (27K): [in this window] [in a new window]   Figure 2. Top, Raloxifene-induced relaxation (-7, -6.5, -6, and -5.5 log mol/L) in endothelium-intact (Control) vs endothelium-denuded (No endo) rabbit coronary arteries (n=11 to 13). Significant differences vs control: *P<0.05, **P<0.01, ***P<0.001. Bottom, 17ß-Estradiol–induced relaxation (-7, -6 and -5 log mol/L) in endothelium-intact (Control) vs endothelium-denuded (No endo) rabbit coronary arteries (n=7 to 11). NS indicates no significant difference vs without endothelium.

17ß-Estradiol induced significant dose-dependent relaxation of coronary arterial rings with and without endothelium. There was no difference in the relaxation in rings with or without endothelium (Figure 2, bottom).

K⁺ 30x10^-3 mol/L and PGF₂ 3x10^-6 mol/L induced comparable contractile responses in rings with endothelium (0.93±0.1 and 0.98±0.1 g, respectively; P>0.05) and without endothelium (0.78±0.1 and 0.86±0.1 g, respectively; P>0.05). The difference between contraction in rings with or without endothelium was not significant for rings contracted by either K⁺ or PGF₂ (P>0.05).

The time course of the relaxation response was longer for raloxifene than for 17ß-estradiol. Relaxation curves were performed over 90 minutes, whereas similar relaxation occurred over 10 to 15 minutes with estrogen (Figure 3).

View larger version (14K): [in this window] [in a new window]   Figure 3. Original tracings to demonstrate time course of relaxation in K+-contracted coronary arterial rings to 17ß-estradiol 10-7 to 10-5 mol/L (top tracing) and raloxifene 10-7 to 10-5 mol/L (bottom tracing) in rings with endothelium.

There were no differences between relaxation in arteries from male or female rabbits in rings with endothelium contracted in PGF₂ (P>0.05) (Figure 4). Rings from male and female rabbits showed similar significant reduction of relaxation in rings denuded of endothelium.

View larger version (21K): [in this window] [in a new window]   Figure 4. Comparison of relaxant effect of raloxifene in rabbit coronary artery rings from male rabbits (n=5) with (Control) and without (No endo) endothelium to rings from female rabbits (n=7) with (Control) and without (No endo) endothelium. No significant difference existed between male and female either with or without endothelium (P>0.05). Significant differences in comparison with rings with endothelium: *P<0.05, ***P<0.001.

Effect of L-NAME on Raloxifene-Induced Relaxation
Incubation with L-NAME 30x10^-6 mol/L for 20 minutes inhibited raloxifene-induced relaxation in rabbit coronary arterial rings with endothelium precontracted with PGF₂ (Figure 5, top). There was no significant difference between rings with endothelium incubated in L-NAME and endothelium-denuded rings (P>0.05) (Figure 5, bottom).

View larger version (28K): [in this window] [in a new window]   Figure 5. Top, Effect of NOS inhibitor L-NAME 10-4 mol/L on raloxifene-induced relaxation in rings with endothelium (n=9 to 11). Control is raloxifene-induced relaxation in rings with endothelium (n=11 to 13). Significant differences in comparison with control rings: *P< 0.05, **P<0.01. Bottom, Effect of NOS inhibitor L-NAME 10-4 mol/L on raloxifene-induced relaxation in rings with endothelium incubated in L-NAME vs control rings with endothelium (Control) and control rings without endothelium (No endo). No significant difference existed between control rings with no endothelium and rings with endothelium incubated in L-NAME (n=11 to 13; P>0.05).

Effect of ICI 182,780 on Raloxifene-Induced Relaxation
Incubation with the specific estrogen receptor antagonist ICI 182,780 at 10^-5 mol/L for 20 minutes inhibited raloxifene-induced relaxation in rings with endothelium but not in rings without endothelium (Figure 6).

View larger version (31K): [in this window] [in a new window]   Figure 6. Top, Effect of incubation with estrogen receptor antagonist ICI 182,780 (n=6) on raloxifene-induced relaxation in rabbit coronary artery rings with endothelium vs control rings (With endo; n=11 to 13). Significant differences vs corresponding controls: *P<0.05, ***P<0.001. Bottom, Effect of incubation in ICI 182,780 on raloxifene-induced relaxation in rabbit coronary artery rings with (n=6) and without (n=6) endothelium vs control rings (With endo and No endo, n=11 to 13). Significant differences vs corresponding controls: *P<0.05, ***P<0.001.

Effect of Barium Chloride on Raloxifene-Induced Relaxation
Incubation with the nonspecific potassium channel inhibitor barium chloride 3x10^-6 mol/L for 20 minutes inhibited raloxifene-induced relaxation in rings with an intact endothelium precontracted with PGF₂ but not in rings without endothelium (Figure 7).

View larger version (27K): [in this window] [in a new window]   Figure 7. Top, Effect of barium chloride (BaCl) on relaxation induced by raloxifene in rabbit coronary arteries with endothelium (n=3) vs control (n=11 to 13). Significant differences vs corresponding controls: **P<0.01. Bottom, Effect of BaCl on relaxation induced by raloxifene in rabbit coronary arteries with (n=3) and without (n=6) endothelium. Significant differences vs corresponding controls (n=11 to 13): **P<0.01.

Effect of Raloxifene on Calcium Concentration–Dependent Contractile Responses
In rings without endothelium, incubation in raloxifene (-6.5 and -6 log mol/L) shifted the calcium concentration–dependent contraction curves in high-K⁺ (80 mmol/L) depolarization medium to the right compared with control. Maximal contraction was also reduced (Figure 8). This shift was not reversed by incubation in the nonspecific potassium channel inhibitor barium chloride 3x10^-6 mol/L for 20 minutes (n=4; P>0.05).

View larger version (21K): [in this window] [in a new window]   Figure 8. Effect of raloxifene -6.5 (n=7) and -6 (n=9) log mol/L on calcium concentration–dependent contraction curves in rabbit coronary arteries without endothelium vs control (n=11). Data are expressed as percentage of maximal contraction induced by calcium in controls (mean±SEM). Significant differences vs corresponding controls: *P<0.05, ***P<0.001.

	Discussion

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We have demonstrated that raloxifene induces relaxation in precontracted male and female rabbit epicardial coronary arteries, with and without endothelium. This relaxation was significantly greater in rings with endothelium than in rings without endothelium, which is a distinct difference from relaxation induced by estrogen in this preparation (Figure 2). At a concentration of 10^-6 mol/L, raloxifene caused 100% more relaxation in rings with endothelium than in rings without endothelium (Figure 2). Inhibition of raloxifene-induced relaxation in endothelium-intact rings was achieved by incubation with L-NAME, ICI 182,780, and barium chloride. Raloxifene shifted the calcium concentration–dependent contraction curve to the right in endothelium-denuded rings. The endothelium-dependent contribution to relaxation was greatest at lower concentrations of raloxifene (-6.5 and -6 log mol/L). However, as the concentration of raloxifene was increased, relaxation occurred by a combined effect on the endothelium and smooth muscle. Endothelium-denuded arterial rings relaxed to 100% at greater concentrations by a direct effect of raloxifene on the vascular smooth muscle myocyte.

Studies have also shown an enhancement of estrogen-induced endothelium-dependent responses in a variety of vessels.^{9 10 11 12 13} It is important to stress, however, that in all these studies, the animals were pretreated for a number of days before the demonstration of an enhancement of endothelium-dependent relaxation of vessels in vitro in organ bath experiments. This is in distinct contrast to these data on raloxifene, which resulted in acute responses after exposure to raloxifene in the organ bath. There was no requirement for pretreatment of the animals to produce an endothelium-dependent vascular relaxing effect. NO is synthesized in the endothelium from L-arginine by NO synthase³¹ and can diffuse rapidly to smooth muscle, causing relaxation via stimulation of soluble guanylate cyclase and a subsequent increase in cGMP.³² By blocking NO synthesis with L-NAME, we were able to completely abolish the difference in relaxation to raloxifene between rings with and without endothelium, implying that the endothelial contribution to raloxifene-induced relaxation is dependent on NO.

The specific estrogen receptor antagonist ICI 182,780 also inhibited raloxifene-induced relaxation in rings with endothelium, again to a level identical to relaxation in rings without endothelium. ICI 182,780 had no effect on raloxifene-induced relaxation in coronary artery rings denuded of endothelium, suggesting that the classic estrogen receptor mediates endothelium-dependent but not endothelium-independent relaxation. Raloxifene appears to interact with the estrogen receptor and may increase the expression of NOS, thereby increasing endothelium-derived NO production. We used an equivalent maximal concentration of ICI 182,780 (10^-5 mol/L), which in single-cell studies has specific anti–estrogen receptor antagonism.³³ Support for a short-term (5-minute) activation of eNOS, mediated by estrogen receptor- functioning in a nongenomic manner via mitogen-activated protein, has recently been published.^33a The mechanism of action of raloxifene in our experiments could easily involve this mechanism. We were unable to distinguish between an effect on the classic estrogen receptor- and the newly discovered estrogen receptor-ß.³⁴

Potential-sensitive calcium channels are activated by depolarization of the plasma membrane when the extracellular potassium concentration is increased. Raloxifene relaxed coronary arterial rings contracted by high extracellular potassium both with and without endothelium. This suggests that it may have an inhibitory effect on potential-sensitive calcium channel activation in the smooth muscle cell. Raloxifene also induced relaxation of coronary arteries precontracted with PGF₂ (an agonist of receptor-operated calcium channels), indicating that raloxifene has relaxing effects on contraction induced by activation of both receptor-operated and potential-operated calcium channels. The shift of the calcium concentration–dependent contraction curve to the right by raloxifene suggests that raloxifene may be a calcium antagonist in these preparations and may be a mechanism involved in the direct smooth muscle relaxation induced by raloxifene. Calcium antagonism may have been a result of decreased probability of voltage-gated calcium channels being open secondary to a change in other ion conductances and consequent hyperpolarization. To investigate this, rings were incubated in the nonspecific potassium channel blocker barium chloride. The shift in calcium concentration–dependent contraction curve to the right was not reversed by incubation in barium chloride, suggesting that modulation of potassium channel conductance does not play a role in the direct calcium antagonistic effect of raloxifene.

Another possible mechanism of relaxation by raloxifene may involve modulation of ion channel conductance in the smooth muscle in response to NO release from the endothelium. This could cause hyperpolarization of the cell, decreasing calcium influx through voltage-gated calcium channels and resulting in relaxation. In this study, we demonstrated that raloxifene-induced, endothelium-dependent relaxation is sensitive to inhibition by the nonspecific potassium channel inhibitor barium chloride, but endothelium-independent relaxation is not. This occurred only at a raloxifene concentration of 10^-6 mol/L, a dose that induces maximal NO-dependent relaxation. Investigations with estrogen have shown that its ability to open potassium channels in vascular smooth muscle cells may be partly dependent on the endothelium and increased production of NO.¹⁶ NO activates guanylate cyclase, elevates intracellular cGMP, and stimulates G kinase in the coronary myocyte.^{35 36} G kinase is able to activate large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels.^{35 37} The inhibition of NO-induced coronary artery dilation by the BK_Ca blocker iberiotoxin^{16 16 38} further supports the idea that endothelium-dependent estrogen-induced relaxation may depend on NO activation of BK_Ca channels. A similar mechanism may explain the sensitivity of endothelium-dependent relaxation by raloxifene to the nonspecific potassium channel blocker barium chloride. An increase in conductance of the BK_Ca channels by NO is a means by which raloxifene could indirectly decrease calcium influx. Hyperpolarization would then result in a decreased probability of voltage-operated calcium channels being open and relaxation.

Raloxifene took longer to induce relaxation than comparable studies using estrogen.^{3 39} Relaxation curves were performed over 90 minutes versus 10 to 15 minutes for similar studies with estrogen (Figure 3). The relaxant effect of raloxifene was not reversed over periods of up to 5 hours, compared with estrogen, for which initial precontraction levels were achieved after 30 minutes. These observations are consistent with the finding of estrogen-receptor involvement in the endothelium-dependent relaxation by raloxifene. This is an effect that is not observed in vitro for estrogen in this model³ and is not involved in the calcium antagonistic effect. Further studies are needed to investigate this phenomenon, because it may represent an advantage in long-term treatment. Steroid-induced events, which are mediated via classic intracellular receptors, occur over periods of minutes to hours. Thus, in our investigation, initial relaxation may be due to early direct actions of raloxifene, followed by the prolonged, estrogen receptor–mediated relaxation. It is also well recognized that tissues such as breast tumor can actively accumulate similar moieties, such as tamoxifen, to concentrations much greater than those found in the plasma.^{40 41} No such data exist in vascular tissue; however, this could partially explain the extended time course of action compared with estrogen in this coronary arterial model, which may involve stimulation of the receptor and transcription of mRNA.

Limitations
In vitro and in vivo concentrations do differ with regard to potency. In the present study, we have demonstrated a relaxant effect of raloxifene on coronary arterial ring preparations at concentrations 50 times greater than the plasma concentrations achieved by administration of raloxifene to patients. However, this model does not include effects of other circulating hormones, blood volume, or coronary circulation. In the case of 17ß-estradiol, the lowest concentrations shown to be effective in relaxing rabbit coronary arterial rings in vitro are less than for raloxifene 10^-6 mol/L.^{3 42} Plasma levels of estrogen similar to those with administered raloxifene (10^-9 mol/L) are achieved during hormone replacement therapy, and long-term beneficial effects in the cardiovascular system have been reported at these concentrations.^{43 44} The actions demonstrated are short-term effects of raloxifene; long-term effects in vivo may differ.

Conclusions
Coronary heart disease is one of the most important causes of morbidity and mortality in developed countries, in terms of both its prevalence and its human and economic cost. We have shown that raloxifene is able to act both via the endothelium and directly on the vascular smooth muscle to induce relaxation of epicardial coronary arterial rings from both male and female animals. The former mechanism involves interaction with the classic estrogen receptor at the level of the endothelium and stimulation of NO. Raloxifene also has the ability to directly antagonize calcium influx in the smooth muscle cell, achieving smooth muscle cell relaxation independent of the endothelium. These in vitro findings with raloxifene in epicardial coronary arterial preparations would support further investigation of this compound in vivo. The mechanisms discovered in the present study raise the possibility that raloxifene may have the potential to be a cardioprotective agent, with the benefit of no increased risk of cancer and other side effects. Further work in humans may identify a therapeutic role for raloxifene in reducing cardiovascular disease in postmenopausal women and the significant mortality and morbidity associated with it.

Received December 15, 1998; revision received April 20, 1999; accepted April 28, 1999.

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