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Vascular Pharmacology 40 (2004) 261–268

Chronic effects of toremifene on the vasculature

of menopause-induced rats

Jorge Gonzalez-Perez, Marıa J. Crespo*

Department of Physiology, University of Puerto Rico-School of Medicine, GPO Box 365067 San Juan, PR 00936-5067, Puerto Rico

Received 25 October 2003; accepted 20 January 2004

Abstract

During menopause, women have a higher propensity for developing cardiovascular diseases. Recent studies have shown that treatment

with selective estrogen receptor modulators (SERMs) improves cardiovascular status in menopausal women. The mechanisms involved,

however, have not been elucidated. The present study evaluates the effect of toremifene (10 mg/kg/day), a new member of SERM family, on

the vasculature of ovariectomized (Ovx) Sprague–Dawley rats that have been treated with the drug for a 4-week period. Age-matched sham,

Ovx-untreated, and Ovx 17h-estradiol-treated rats were used as controls. Aortic rings from treated and untreated animals were used to

determine vascular responses to norepinephrine, acetylcholine, and sodium nitroprusside. Systolic blood pressure (SBP), plasmatic nitric

oxide (NO) concentration, estrogen levels, aortic wall thickness, and cholesterol profiles were also determined. Toremifene displaces the

concentration–response curve (CRC) for the acetylcholine-induced relaxation to the left and increases the Emax by 34% (from 59.2F 4.2% in

Ovx-untreated to 90.2F 3.1% in Ovx-treated rats, n = 9, P< .05). Toremifene increases the Emax (by 22%) without modifying the EC50 for

the NE-induced contraction. In addition, toremifene amplifies the relaxing responses to sodium nitroprusside compared to Ovx-untreated

group (P< .05). SBP was significantly reduced in the Ovx toremifene-treated group when compared to the Ovx-untreated group (124F 3.5

mm Hg for Ovx toremifene-treated vs. 161F 4.3 mm Hg for Ovx-untreated, n = 10, P < .05). Rats treated chronically with toremifene also

exhibited a significantly higher plasmatic NO levels, and a decrease in basal resting tension and aortic wall thickness. The drug, however, did

not affect the plasmatic high-density lipoprotein (HDL)/total cholesterol ratio. These results suggest that chronic administration of toremifene

improves cardiovascular performance in menopause-induced rats by reversing endothelial dysfunction and decreasing vascular resting tone.

Thus, use of toremifene may help to diminish total peripheral resistance and improve cardiovascular status in Ovx rats.

D 2004 Elsevier Inc. All rights reserved.

Keywords: Toremifene; Endothelial dysfunction; Vascular reactivity; Ovariectomized rats

1. Introduction

Epidemiological studies have demonstrated that the in-

cidence of cardiovascular diseases increases with the onset

of menopause (de Kleijn et al., 2002; Matthews et al.,

2001). The increase appears to be related to the diminish-

ing concentration of estrogen that characterizes this stage

(Mercuro et al., 2001; Scuteri and Ferrucci, 2003). Estro-

gen has been demonstrated to exert a protective effect on

the vascular wall (Rubanyi et al., 2002; Prorock et al.,

2003). Hormone replacement therapy (HRT) has been

reported to improve vascular function and to decrease

the risk factors of developing cardiovascular complications

1537-1891/$ – see front matter D 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.vph.2004.01.005

* Corresponding author. Fax: +1-787-753-0120.

E-mail address: mcrespo@rcm.upr.edu (M.J. Crespo).

in menopausal women and animal models (Higashi et al.,

2001). A recent report, however, indicates that this therapy

increases cardiovascular complications in healthy meno-

pausal women (Rossouw et al., 2002). Interestingly, the

use of selective estrogen receptor modulators (SERMs),

synthetic compounds that exhibit estrogenic-associated

properties, has not been associated with the cardiovascular

side effects observed with HRT. In fact, some authors

report that the incidence of cardiovascular events during

menopause decreases in patients that are treated with

SERMs (Blum and Cannon, 2001; Saitta et al., 2001).

Alternative therapies are clearly needed to treat meno-

pause-associated cardiovascular complications. Thus,

SERMs needs to be further evaluated as one promising

therapy.

SERMs form a family of drugs that was originally

designed to treat breast cancer (Mitlak and Cohen, 1999;

J. Gonzalez-Perez, M.J. Crespo / Vascular Pharmacology 40 (2004) 261–268262

Simoncini and Genazzani, 2000). SERMs are now used

also to treat postmenopausal osteoporosis (Nuttall et al.,

1998). These modulators bind with high affinity to the

estrogen receptors and have tissue-specific effects (Katze-

nellenbogen et al., 2001). Toremifene, a chlorinated anti-

estrogen compound that is structurally identical to

tamoxifen, was developed in 1979 and received FDA

approval for the treatment of breast cancer in 1997. The

cardiovascular profile of toremifene has not been fully

investigated, although acute administration of toremifene is

known to interfere with the vascular wall of both intact

and menopause-induced rats, promoting a state of vascular

relaxation (Gonzalez-Perez and Crespo, 2003). The effect

of chronic treatment with toremifene on the cardiovascular

system, however, has not been investigated. The present

study was designed to characterize the effects of chronic

administration of toremifene (10 mg/kg/day) on the vas-

culature of menopause-induced rats. We present evidence

indicating that both the vascular and hemodynamic status

of these animals improve after 4 weeks of treatment with

toremifene.

2. Materials and methods

2.1. Animal model

Adult, 12-week-old female Sprague–Dawley rats (Hill-

top Lab Animals, Scottdale, PA) were used in the present

study. The rats were divided into four groups: age-matched

sham, ovariectomized (Ovx) untreated, Ovx-treated for 4

weeks with 17h-estradiol (1.0 mg/kg/day), and Ovx-treated

for 4 weeks with toremifene (10 mg/kg/day). Animals

were housed in groups of two to three per cage. Water

and food (Harlan Rodent Diet, 18% protein) were provided

ad libitum. The rats were maintained on a normal light–

dark cycle (12L:12D), with lights off at 5 p.m. and in a

temperature-controlled room. All experiments were ap-

proved by the Institutional Animal Care and Use Commit-

tee, and adhered to the guidelines established by the

National Institutes of Health (USA) and the American

Veterinary Medical Association.

2.2. Bilateral ovariectomy and implant preparation

Surgeries were performed when the rats were approxi-

mately 3 months of age, by which time all animals had

reached sexual maturity. Prior to any surgical procedure, the

animals received a combined solution of ketamine (70 mg/

ml) and xylazine (10 mg/ml) intraperitoneally. After com-

plete anesthesia was achieved, one patch of skin on the

dorsum was shaved and disinfected. The ovaries were

removed via a small incision. On the same day, silastic

implants were placed subcutaneously in the dorsal region of

the neck. Following recovery, the rats were returned to their

home cages and monitored for infections and surgical

complications. After 4 weeks, the ovariectomized rats were

sacrificed and the thoracic aorta was removed following the

protocol described below.

The implants were prepared using medical-grade silastic

tubing (Dow Corning, Midland, MI, USA) 0.078 in. internal

diameter, 0.125 in. outer diameter and 0.78 in. length for

toremifene (Iino et al., 1993) and 0.058 in. internal diameter,

0.077 in. outer diameter and 0.20 in. length for 17-hestradiol (Kelner et al., 1977; Meisel et al., 1987). The

implants were adjusted to release approximately 1.0 mg/kg/

day of 17-h estradiol and 10 mg/kg/day of toremifene. The

concentration of toremifene in the plasma produced by this

rate of release was determined previously (Bridges, 1984;

Iino et al., 1993; Johnston et al., 1997). Estrogen implants

produced comparable plasma levels to those found during

the proestrus phase (35.0–52.0 pg/ml, Dupon and Kim,

1973). Empty implants were used in sham and ovariecto-

mized control groups.

2.3. Isometric tension studies

The same day of the experiment, animals were weighed

and anaesthetized. The descending thoracic aorta was

removed and placed in Krebs’ bicarbonate solution (com-

position in mmol/l: 118 sodium chloride, 2.5 calcium

chloride, 5 potassium chloride, 1.1 magnesium sulfate,

25 sodium bicarbonate, 1.2 potassium monobasic phos-

phate and 10 glucose, pH = 7.4). The connective tissue

adjacent to the adventitia of the aorta was carefully

removed, avoiding damage to the smooth muscle and the

endothelium. Aortic rings, approximately 5 mm in length,

were obtained from the proximal segment of each aorta.

The rings were suspended horizontally between two stain-

less-steel wires and mounted in a two-hook 50-ml organ

chamber (Radnoti, Monrovia, CA). The wires were

connected to a force-displacement transducer (Grass, mod-

el FT03C) which was attached to a DC preamplifier (Grass

model 7P1F). The signal was analyzed with a data

acquisition card (National Instruments, PC-LPM-16/PnP)

and recorded utilizing a LabView program. The rings were

subjected to a resting tension of 2.0 g. Our laboratory

determined previously that this tension is optimal for these

experiments (Crespo et al., 1996). Once the optimal

tension was reached, the aortic rings were subjected to a

1-h equilibration period.

Concentration–response curves (CRCs) for acetylcho-

line and sodium nitroprusside were constructed to determine

the chronic effect of toremifene on the endothelium-depen-

dent and endothelium-independent relaxation, respectively.

Aortic rings were precontracted with 1.0 Amol/l norepineph-

rine. When the maximal contractile plateau was reached,

CRCs were generated for acetylcholine (from 0.1 nmol/l to

10 Amol/l), and the relaxation developed after each dose

was recorded. Following the completion of each CRC, the

aorta was fully relaxed with 1.0 Amol/l sodium nitroprus-

side, washed, and stabilized for 20 min. A similar

J. Gonzalez-Perez, M.J. Crespo / Vascular

procedure was followed for the sodium nitroprusside

(from 0.1 nmol/l to 10 Amol/l) experiments. For each

experiment, the relaxation was expressed as a percentage

of the relaxation achieved for each individual concentra-

tion relative to the maximal contraction induced by 1.0

Amol/l norepinephrine.

To determine the effect of toremifene on vascular con-

tractility, cumulative CRC for the norepinephrine-induced

contraction (from 0.1 nmol/l to 10 Amol/l) were conducted

in rings from treated and untreated animals. The tension

developed by the rings was recorded after each individual

dose, and the contractility was expressed as the tension (g)

divided by the dry mass (mg) of the rings. In separate

experiments, basal resting tension was recorded after the

aortic rings were equilibrated in the absence of any drug.

Basal resting tension (g) was defined as the difference

between the tension registered in the aorta before and after

a 15-min period. A similar set of experiments was con-

ducted in the presence of 1 mmol/l of the nitric oxide (NO)

synthase inhibitor NG-nitro-L-arginine (L-NAME). The pur-

pose of these latter experiments was to evaluate the possible

involvement of NO in basal tension after chronic treatment

with toremifene.

2.4. Determination of systolic blood pressure

Systolic blood pressure (SBP) was determined in 10

animals from each experimental group. Briefly, blood

pressure was recorded by placing a pressure cuff on the

tail and inflating the cuff to a pressure of 250 mm Hg. A

piezoelectric sensor, located in the distal side of the cuff,

was connected to a microcomputer system (RTBP-2000,

Kent Scientific, Litchfield, CT) that processed the signals.

The data were recorded and analyzed using the program

LabView. With this setup, consecutive readings of SBP

were obtained from the same animal. In order to obtain

an accurate SBP value for each animal, we used the

mean value of 5 measurements, separated by 3-min

intervals.

2.5. Determination of plasmatic NO

The production of NO by the endothelium was assessed

indirectly by measuring the nitrite/nitrate concentration in

plasma using the Griess reagent (1% sulfanilamide in 5%

H3O4 and 0.1% napthylethlenediamine dihydrochloride, in a

ratio of 1:1). Blood was obtained and centrifuged at 3000

rpm for 5 min. Plasma samples were stored overnight in a

freezer. On the day of the experiment, an aliquot of 750 Al ofplasma was mixed with 750 Al of the Griess reagent,

protected from light, and maintained at room temperature

for 15 min. The concentration of nitrite/nitrate in the

samples was determined spectrophotometrically at 540

nm. For every NO assay, a standard curve was performed,

using sodium nitrite (NaNO2) as an NO source (Kauser et

al., 1998).

2.6. Determination of lipid and hormonal profiles

To determine the lipid profiles, blood samples were

obtained from rats in each experimental group. Blood was

centrifuged at 5000 rpm for 5 min, and the plasma was

collected and stored at � 70 jC. Plasmatic high-density

lipoprotein (HDL) levels were determined using a com-

mercial kit (Isospin, Sigma Diagnostic) that precipitates

vLDL and LDL. An aliquot of 500 Al of plasma was

mixed with 100 Al of phosphotungstate and magnesium

chloride at room temperature for 5 min, and then centri-

fuged at 3000 rpm for 5 min. A 50-Al aliquot from the

supernatant was mixed with 1.0 ml enzymatic cholesterol

esterase/cholesterol oxidase commercial kit (Sigma Diag-

nostic) at 37 jC for 15 min in a temperature-controlled

bath. The resulting colored product was quantified spec-

trophotometrically at 500 nm. An HDL standard curve was

prepared using a calibrator provided by Sigma. To deter-

mine total cholesterol, an aliquot of 10 Al of plasma was

mixed with 1.0 ml of reagent (Sigma Diagnostic) which

contained a mixture of cholesterol esterase and cholesterol

oxidase, and incubated in a water bath at 37 jC for 15

min. The mixture was quantified spectrophotometrically at

500 nm. Total cholesterol concentration was determined

using standard calibrators provided by Sigma. Triglyceride

levels were calculated using a method described by Frie-

dewald et al. (1972).

Plasmatic17h-estradiol levels were determined using

I125-RIA kits (ICN Pharmaceuticals; double antibody).

The serum aliquot required was 50 Al. Briefly, after 1.5

h of incubation with the primary antibody at 37 jC,separation of bound and free 17h-estradiol was achieved

with a secondary antibody. For each sample, the hormon-

al level was determined by interpolation from a standard

curve prepared in triplicate using calibrators (Gonzalez et

al., 2001).

2.7. Histological techniques

Thoracic aortas from toremifene-treated, Ovx-untreated,

and sham rats were removed and stored at � 70 jC. Aorticrings, approximately 2 mm in length, were obtained from

the proximal segment of each aorta. The rings were sec-

tioned at 20 Am with a cryostat, collected on RNase-free

subbed slides, and stained with Masson’s Trichrome. Thick-

ness of the ring wall was measured at 10 different locations

using the MCID M5+ Image Analysis software (Imaging

Research, Ontario, Canada) and a mean thickness value was

obtained.

2.8. Statistical analysis

Results are presented as the meanF S.E.M. EC50 values

were determined by graphic analysis (GraphPAD, Califor-

nia, USA). Statistical comparisons between groups were

performed with Student’s t test when only two variables

Pharmacology 40 (2004) 261–268 263

Table 1

General characteristics of experimental animals

Groups Weight (g) Estradiol

(pg/ml)

SBP

(mm Hg)

NO (AM)

Sham 255.0F 2.3 21.5F 3.7 118.0F 2.5 12.0F 0.7

Ovx 309.0F 6.9 * 9.2F 0.4 ** 161.0F 4.3 * 8.8F 0.6 *

Estrogen 235.0F 8.7 50.8F 4.1 120.0F 11.1 10.0F 1.4

Toremifene 239.0F 4.7 8.8F 1.2 124.0F 3.5 14.1F 3.2

The values shown are the meansF S.E.M. of 10 experiments per group.

* P < .05 when compared to all groups.

** P < .05 when compared to the sham group.

J. Gonzalez-Perez, M.J. Crespo / Vascular Pharmacology 40 (2004) 261–268264

were compared, and with the analysis of variance when

more than two variables were compared. Values were

considered statistically significant at P < .05.

Fig. 1. Concentration– response curves for the acetylcholine-induced

relaxations of aortic rings from sham, Ovx-untreated, Ovx 17h-estradiol-treated, and Ovx toremifene-treated rats. The values shown are the

3. Results

General characteristics of toremifene-treated, 17h-estra-diol-treated, Ovx-untreated, and sham rats are summarized

in Tables 1 and 2. During the study period, body weight of

Ovx rats treated with toremifene was lower than in the

Ovx-untreated group (P < .05), but similar to the other

groups. Estrogen implants (1 mg/kg/day) produced a plas-

matic hormone concentration similar to the concentration

reported during the proestrus phase (Dupon and Kim,

1973). This concentration, however, was higher than the

average concentration during all phases of the estrous cycle

that was reported from sham animals, whose value ranged

from 35.0 to 52.0 pg/ml (Dupon and Kim, 1973). SBP was

found to be significantly higher in Ovx untreated rats

(161F 4 mm Hg) than in sham (118F 2 mm Hg) or

17h-estradiol-treated rats (120F 11 mm Hg). Toremifene

treatment lowered the blood pressure of Ovx rats to

124F 3 mm Hg. In addition, plasmatic NO concentration

was higher in the toremifene group (14.1F 3.2 AM) than

in the sham group (12.0F 0.7 AM). Total cholesterol and

triglyceride values decreased significantly in Ovx rats

after chronic treatment with the drug (Table 2). The

effect of toremifene on the ratio of HDL to total

cholesterol did not change when compared to the ratio

of the sham group or the Ovx-untreated group. In

contrast, this ratio was significantly increased in the

17h-estradiol-treated group (n = 7, P < .05).

Table 2

Lipid profile of experimental animals

Groups Cholesterol

(mg/dl)

Triglycerides

(mg/dl)

% HDL/total

Sham 119.0F 25.0 153.0F 69.0 34.0F 4.0

Ovx 141.0F 13.0 * 284.0F 53.0 * 33.0F 3.0

Estrogen 109.0F 14.0 148.0F 62.0 44.0F 6.0 *

Toremifene 91.0F 7.0 154.0F 46.0 32.0F 5.0

The values shown are the meansF SEM of 7 experiments per group.

*P< .05 when compared to all groups.

3.1. Effect of toremifene on endothelial-dependent

relaxation

As depicted in Fig. 1, the CRC for acetylcholine was

displaced to the left in rings from toremifene-treated rats.

Chronic treatment with this drug increased the maximal

relaxation achieved with 10 AM acetylcholine by approx-

imately 34% (from 59.2F 4.2% to 90.2F 3.1%, n = 9,

P < .05). Moreover, the EC50 value from the CRC was

decreased compared with the values from the Ovx-untreat-

ed group (from 580F 188 nM for Ovx-untreated to

211F 93 nM for toremifene-treated group, n = 9, P < .05).

No differences were observed in EC50 values between

toremifene-treated, sham, and 17h-estradiol-treated groups.

In addition, toremifene treatment significantly decreased

meansF S.E.M. of nine experiments per group.

Fig. 2. Basal resting tension of aortic rings from sham, Ovx-untreated, Ovx

17h-estradiol-treated, and Ovx toremifene-treated rats. The values shown

are the meansF S.E.M. of 10 experiments per experimental group. Note

that the basal relaxation induced by toremifene was fully blocked by L-

NAME. *P < .05 when compared to the sham group, **P < .05 when

compared to all groups.

Fig. 3. Concentration–response curves for sodium nitroprusside-induced

relaxation in aortic rings from sham, Ovx-untreated, Ovx 17h-estradiol-treated, and Ovx toremifene-treated rats. The values shown are the

meansF S.E.M. of 10 experiments per group.

Fig. 5. Wall thickness of aortic rings from sham, Ovx-untreated, and Ovx

toremifene-treated rats. The values shown are the meansF S.E.M. of four

experiments per group. *P < .05 when compared to the control and

toremifene groups.

J. Gonzalez-Perez, M.J. Crespo / Vascular Pharmacology 40 (2004) 261–268 265

basal resting tension by approximately 84% compared

to the Ovx-untreated group (� 0.044F 0.011 g vs.

� 0.0069F 0.005 g, Ovx; n = 10, P < .05; Fig. 2). Basal

relaxation was fully abolished in all groups by incubation

with 1 mM L-NAME.

3.2. Effect of toremifene on endothelial-independent

relaxation

Chronic administration of toremifene to Ovx rats dis-

placed to the left the entire CRC for sodium nitroprusside

when compared to results from Ovx-untreated rats (Fig. 3).

The EC50 value from the toremifene-treated group (21F 6.5

nM) decreased compared to the value from Ovx-untreated

rats (66F 11 nM, n = 10, P < .05). No significant differences

in this parameter were observed between toremifene-treated,

sham, and 17h-estradiol-treated groups.

Fig. 4. Concentration– response curves for norepinephrine-induced con-

tractions in aortic rings from sham, Ovx-untreated, Ovx 17h-estradiol-treated, and Ovx toremifene-treated rats. The values shown are the

meansF S.E.M. of 10 experiments per group.

3.3. Effect of toremifene on norepinephrine-induced vascu-

lar contractility

Daily administration of toremifene to Ovx rats over a 4-

week period increased the maximal contraction (Emax)

achieved with norepinephrine by 22% when compared to

the Ovx-untreated group (n = 10, P < .05; Fig. 4). Neverthe-

less, the CRC for toremifene-treated, sham, and 17h-estra-diol-treated groups were superimposable. The EC50 value,

however, was not modified by the treatment.

3.4. Effect of toremifene on aortic wall thickness

Histological studies performed on 20 Am sections of the

descending thoracic aorta of Ovx rats showed that the

thickness of the media was significantly reduced after

toremifene treatment (Fig. 5). The thickness of the media

was 0.073F 0.0008 mm in toremifene-treated rats vs.

0.083F 0.0017 mm in Ovx-untreated animals (n = 4,

P < .05). The thickness of the media after toremifene treat-

ment was similar to values obtained for age-matched sham

animals (0.067F 0.0009 mm).

4. Discussion

Chronic treatment with toremifene improves the car-

diovascular status of Ovx rats by improving endothelial

function, decreasing blood pressure, preventing vascular

hypertrophy, and modifying the serum lipid profile. This

study provides evidence that, in addition to its well-

documented value in cancer treatment, toremifene may

J. Gonzalez-Perez, M.J. Crespo / Vascular Pharmacology 40 (2004) 261–268266

be beneficial to the cardiovascular system following

menopause.

The potentiation on the acetylcholine-induced relaxation

observed in Ovx rats after chronic treatment with toremifene

is similar to the potentiation described previously in Ovx

rats after acute treatment with this drug (Gonzalez-Perez and

Crespo, 2003). Other members of the SERM family that are

structurally related to toremifene share this characteristic. In

Ovx rats, idoxifene potentiates the relaxation induced by

acetylcholine in the mesenteric arteries (Ma et al., 2000),

and arzoxifene increases acetylcholine-induced relaxation in

the aorta (Rahimian et al., 1997). Toremifene decreases the

EC50 value for acetylcholine, suggesting a direct effect of

the drug on muscarinic receptors. In this context, tamoxifen,

another member of the SERM family, is reported to interact

with these receptors in membrane fractions from human

urinary bladder and rabbit myometrium (Batra, 1990).

Additional studies are needed, however, to determine if

toremifene also interacts with muscarinic receptors.

Chronic toremifene administration significantly de-

creases basal resting tone in vessels. This effect is com-

parable to that observed in rings from menopause-induced

rats after acute incubation with the drug (Gonzalez-Perez

and Crespo, 2003). L-NAME incubation fully abolished

basal relaxation observed following both chronic and acute

treatment with toremifene. This finding suggests that

toremifene interacts with the NO production system at

the endothelial level. Moreover, the high plasmatic NO

concentration found in treated rats supports the hypothesis

that toremifene increases endothelial NO production. Sim-

ilarly, idoxifene increases plasmatic NO levels in Ovx

Sprague–Dawley rats (Ma et al., 2000). Several mecha-

nisms may be involved in the increased production and/or

actions of NO observed following toremifene treatment.

The drug may improve endothelial function by increasing

the constitutive endothelial NO synthase (eNOS) expres-

sion or activity. Interactions with eNOS have been de-

scribed for other members of the SERM family. Acute

administration of raloxifene increases eNOS activity in

human umbilical endothelial cells (Simoncini and Genaz-

zani, 2000). In addition, chronic administration of either

acolbifene or raloxifene increases eNOS activity and

expression in aortic rings from Ovx rats (Simoncini et

al., 2002; Rahimian et al., 2002). Toremifene may also

improve endothelial dysfunction by acting as an antioxi-

dant agent. Most of the members of the SERM family are

known antioxidants. Raloxifene decreases superoxide pro-

duction (Wassmann et al., 2002), and tamoxifen blocks

lipid peroxidation (Dubey et al., 1999) in smooth muscle

cells from rat aortic rings. Droloxifene inhibits lipid

peroxidation in rat liver microsomes (Wiseman et al.,

1992) and toremifene decreases the level of lipid perox-

idation in rats (Ahotupa et al., 1997). Toremifene, by

acting as an antioxidant, may increase NO bioavailability,

thereby increasing endothelial function. Further experi-

ments, however, are needed to corroborate this idea.

The contractile responses of aortic rings from Ovx rats to

norepinephrine decrease compared to the responses after

norepinephrine treatment in sham and 17h-estradiol-treatedgroups. This finding agrees with previous reports, which

show that bilateral ovariectomy decreases vascular reactivity

to norepinephrine in uterine arteries (Wight et al., 2000) and

aortic rings (Zamorano et al., 1995; Cheng and Gruetter,

1992). In contrast, toremifene amplifies norepinephrine-

induced contraction in the vasculature of Ovx rats. The

adrenergic responses under the conditions of chronic tor-

emifene treatment are similar to the responses observed after

17-h estradiol treatment (Cheng and Gruetter, 1992; Acs et

al., 2001; Varbıro et al., 2000, 2002). Unlike chronic

replacement, however, acute treatment with toremifene

decreases the responses of rat aortic tissue to norepinephrine

stimulation (Gonzalez-Perez and Crespo, 2003). The mech-

anisms for this discrepancy are not currently evident.

Nevertheless, the combined observations presented above

suggest that toremifene has a time-dependent effect on the

vasculature that requires further analysis.

Absence of estrogen decreases the sensitivity of smooth

muscle to sodium nitroprusside in the mesenteric arteries of

rats (Minoves et al., 2002). In contrast, toremifene supple-

mentation amplifies this response in Ovx rats. The later

finding indicates that vascular smooth muscle is an addi-

tional target for SERM actions that needs to be considered.

Nevertheless, treatment for 4 days with idoxifene does not

modify the vascular response of mesenteric arteries to

sodium nitroprusside (Ma et al., 2000). The discrepancy

between toremifene and idoxifene in their effects on vascu-

lar smooth muscle may be related to differences in the

treatment periods or the chemical structures of the drugs.

Toremifene decreases both total cholesterol and trigly-

cerides levels, but the HDL/total cholesterol ratio is not

improved. This drug has a similar effect on cholesterol and

triglyceride levels in women receiving treatment for breast

cancer (Joensuu et al., 2000; Saarto et al., 1996; Gylling et

al., 1995). Droloxifene also decreases cholesterol levels in

healthy postmenopausal women (Herrington et al., 2000),

suggesting that lowering cholesterol and triglyceride levels

is a common property of the SERM family. Numerous

reports link a decrease in sexual hormone concentration

with a significant increase in blood pressure in animal

models (Meyer et al., 1997; Hernandez et al., 2000),

menopausal women (Scuteri and Ferrucci, 2003), and wom-

en devoid of sex steroids (Stoney et al., 1997). The

increased blood pressure is normalized, however, if estrogen

replacement therapy is offered (De Meersman et al., 1998;

Sasaki et al., 2000). In our study, we demonstrate that

toremifene normalizes the increased blood pressure ob-

served in rats after bilateral ovariectomy. This beneficial

effect could be the combined result of the actions of the drug

on endothelial function, basal resting tension, smooth mus-

cle sensitivity to NO, and the lipid profile. In addition, the

regression of vascular hypertrophy observed after toremi-

fene treatment may be also correlated with the decrease in

J. Gonzalez-Perez, M.J. Crespo / Vascular Pharmacology 40 (2004) 261–268 267

blood pressure. Indeed, vascular wall thickness has been

correlated with smooth muscle cell hypertrophy, hyperpla-

sia, accumulation of connective tissue, and hypertension

(Adams et al., 1995). Thus, treatment with toremifene, in

addition to restoring smooth muscle function also may

influence structural changes in the vascular wall.

This is the first study to assess the effects of chronic

toremifene administration on the cardiovascular system of

Ovx rats. We demonstrate that a marked improvement

occurs in the cardiovascular status of menopause-induced

rats, possibly due to the beneficial effects of the drug on

vascular function and lipid profile.

Acknowledgements

This work was supported by grants from the National

Institutes of Health (RR-03051, 2 SO6 GM08224 MBRS-

SCORE and RISE Program), and a Porter Fellowship from

the APS.

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