Randomized, Double-Blind, Placebo-Controlled Study on

Effects of Raloxifene and Hormone Replacement Therapy

on Plasma NO Concentrations, Endothelin-1 Levels,
and Endothelium-Dependent Vasodilation in
Postmenopausal Women
Antonino Saitta, Domenica Altavilla, Domenico Cucinotta, Nunziata Morabito, Nicola Frisina,
Francesco Corrado, Rosario D’Anna, Antonino Lasco, Giovanni Squadrito, Agostino Gaudio,
Francesco Cancellieri, Vincenzo Arcoraci, Francesco Squadrito

Abstract—The endothelium is thought to play an important role in the genesis of atherosclerosis, and several lines of evidence
suggest that the effect of an intervention on endothelial function might predict its involvement in coronary disease progression
and in the rate of cardiovascular events. Estrogen has direct effects on the blood vessel wall, indicating that vascular
endothelium may play a key role in the cardiovascular protective effects of hormone replacement therapy (HRT). Raloxifene
relaxes coronary arteries in vitro by an estrogen receptor– dependent and NO-dependent mechanism, thus suggesting that this
selective estrogen receptor modulator could also have beneficial effects on endothelial function. This study compared the
effects of HRT and raloxifene on NO products, endothelin-1 plasma levels, and endothelium-dependent vasodilatation in
postmenopausal women. Healthy postmenopausal women (n⫽90) were enrolled in a double-blind, randomized, placebo-
controlled, 6-month trial. Women were randomly assigned to receive continuous HRT (1 mg 17␤-estradiol combined with 0.5
mg norethisterone acetate), raloxifene (60 mg/d), or placebo for 6 months. Flow-mediated endothelium-dependent
vasodilation of the brachial artery, plasma NO concentrations, and endothelin levels were measured at baseline and after 6
months of therapy. The mean baseline level of NO breakdown products was 26.5⫾10.7 ␮mol/L and increased to 36.3⫾11.4
␮mol/L after 6 months of treatment with raloxifene. The mean baseline plasma endothelin level was 17.3⫾8.9 pg/mL and
decreased to 11.5⫾2.1 pg/mL after 6 months of treatment with the selective estrogen receptor modulator. The mean baseline
Downloaded from http://ahajournals.org by on November 23, 2022

ratio of NO (breakdown products) to endothelin was also significantly increased at the end of treatment with raloxifene.
Postmenopausal women treated with HRT had similar changes in plasma nitrites/nitrates and endothelin levels as well as in
the ratio of NO to endothelin. In contrast, these markers of endothelial function did not change in the placebo-treated women.
Flow-mediated endothelium-dependent vasodilation of the brachial artery was 8.3⫾2.1% at baseline and increased to
12.3⫾2.1% after 6 months of treatment with raloxifene. HRT also caused a significant and similar increase in flow-mediated
endothelium-dependent vasodilation. No change in flow-mediated vasodilation was observed in the participants treated with
placebo. We conclude that raloxifene therapy and HRT influence endothelial function and improve flow-mediated
endothelium-dependent vasodilation to a comparable extent in healthy postmenopausal women at least after a 6-month
treatment period. However, further investigation is warranted to enhance our understanding of the mechanisms of the effect
of raloxifene on vascular function and to determine whether its effect on endothelial function may contribute to the reduction
in cardiovascular-related morbidity and mortality. (Arterioscler Thromb Vasc Biol. 2001;21:1512-1519.)
Key Words: raloxifene 䡲 hormone replacement therapy 䡲 nitric oxide 䡲 endothelin-1 䡲 endothelium

P opulation-based observational studies revealed that the

risk of cardiovascular events in women taking hormone
replacement therapy (HRT) was cut in half, providing support
for estrogen treatment.1–5 Subsequently, a number of clinical
and experimental findings have demonstrated that estrogen
has favorable effects on lipid profiles, hemostatic factors,
endothelial cell function, and vascular reactivity.6 –13
Indeed, the endothelium is thought to play an important
role in the genesis of atherosclerosis, and several lines of
evidence suggest that the effect of an intervention on endo-

Received December 7, 2000; revision accepted April 20, 2001.

From the Department of Internal Medicine (A.S., D.C., N.M., N.F., A.L., G.S., A.G.), the Department of Experimental Medicine and Pharmacology
(D.A., V.A., F.S.), Section of Pharmacology, and the Department of Obstetrics and Gynecology (F. Corrado, R.D., F. Cancellieri), School of Medicine,
University of Messina, Messina, Italy.
Correspondence to Francesco Squadrito, MD, Department of Experimental Medicine and Pharmacology, Section of Pharmacology, School of
Medicine, University of Messina, Azienda Policlinico Universitario, Torre Biologica 5° Piano, Via C. Valeria Gazzi, 98125 Messina, Italy. E-mail
Francesco.Squadrito@unime.it
© 2001 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org

1512
 Saitta et al
thelial function might predict its impact on coronary
disease progression and cardiovascular events. The endo-
thelium has been found to be a complex endocrine and
paracrine organ affecting vasoregulation, smooth muscle
cell proliferation, platelet aggregation, monocyte adhesion,
and thrombosis.14 –19
Endothelial function can be clinically assessed through
measurements of endothelium-dependent vasodilation and
plasma markers, such as NO-derived products, endothelin-1,
von Willebrand factor, thrombomodulin, and monocyte ad-
hesion molecules. In particular, it has been suggested that
postmenopausal women have blunted circulating levels of
NO (nitrite/nitrate), increased plasma levels of endothelin-1,
and impaired endothelial function, measured as brachial
artery flow–mediated vasodilation.20 –22 Improvement in all 3
of these markers of endothelial function has been demon-
strated after estrogen treatment.20 –22
Today, the evidence of the efficacy of estrogen in the
prevention of coronary artery disease has been called into
question because of the negative results of the Heart and
Estrogen/Progestin Interventions trial.23 In this first random-
ized clinical trial performed in women with coronary artery
disease, cardiovascular events were not prevented by combi-
nation therapy of oral estrogen and progesterone, and, sur-
prisingly, early events were actually increased in treated
patients.
Furthermore, unopposed estrogen therapy causes symp-
tomatic and premalignant endometrial hyperplasia, which can
be ameliorated with the addition of progesterone, although at
Downloaded from http://ahajournals.org by on November 23, 2022
a cost of attenuating the lipid-lowering and endothelium-
protecting effects of estrogen.24,25 The modest, yet disturbing,
increased incidence of breast cancer in women taking estro-
gens is another major limitation of this prophylactic thera-
py.26 Angiogenesis, a fundamental process necessary for
tumor genesis as well as atherosclerotic progression, can be
enhanced by estrogen,27 which induces the expression of
cellular adhesion molecules and the organization of endothe-
lial cells into primitive blood vessels, on which tumors
depend. Furthermore, the effect of estrogen on coagulation is
variable. Platelets incubated with estradiol manifest paradox-
ical adherence28 and aggregation,29 although studies in
women with HRT have shown antiplatelet effects.30,31
Given the demonstrated risks of conventional HRT, pre-
clinical and clinical researchers have been in search of
alternatives.
The term selective estrogen receptor modulator has been
applied to compounds that have tissue-specific estrogen
agonist/antagonist properties, such as raloxifene. Raloxifene
(previously called keoxifene) is a benzothiophene derivative
that binds to the estrogen receptor with high affinity.32
Raloxifene has tissue specificity, with estrogen antagonistic
effects on the breast and uterus but with estrogen agonist
effects on bone and plasma cholesterol concentrations.33,34
Raloxifene prevents bone loss without producing uterine
proliferation.35,36 The compound is known to share the
lipid-lowering effects of estrogen,37 and it inhibits aortic
accumulation of cholesterol in ovariectomized cholesterol-
fed rabbits.38 However, there was a lack of effect of choles-
terol on coronary artery atherosclerosis in a cynomolgus
monkey model,39 thus calling into question the use of
raloxifene as prophylactic treatment for coronary artery
Raloxifene and HRT in Postmenopausal Women 1513
disease. However, it has been recently suggested that ralox-
ifene in vitro relaxes coronary arteries by an estrogen recep-
tor– dependent and NO-dependent mechanism,40 thus sug-
gesting that this selective estrogen receptor modulator could
also have beneficial effects on endothelial function. This
result, if confirmed clinically in postmenopausal women,
might predict a positive impact on coronary disease progres-
sion and on the rate of cardiovascular events. The aim of the
present study was to compare the effects of HRT and
raloxifene on NO products, endothelin-1 plasma levels, and
endothelium-dependent vasodilatation in healthy postmeno-
pausal women.
Methods
Participants
After the ethics committee approved the present study, participants
were recruited between those reporting to the Centers for Menopause
of the Department of Internal Medicine and to the Department of
Obstetrics and Gynecology of the University of Messina. All
participants gave informed consent. Participants were healthy ambu-
latory women who were aged 53 to 62 years, had not undergone
surgically induced menopause, had not had a menstrual period in the
preceding year, and had a follicle-stimulating hormone level ⬎50
IU/L and a serum 17␤-estradiol level of ⱕ100 pmol/L. At the
beginning of the study, a complete history, physical examination,
laboratory evaluation (chemistry and hematology panel), and ECG
were performed at the Department of Internal Medicine of the
University of Messina. Exclusion criteria were clinical or laboratory
abnormalities that suggested cardiovascular, hepatic, or renal disor-
ders; coagulopathy; use of oral or transdermal estrogen, progestin,
androgen, or other steroids in the preceding year; a smoking habit of
⬎10 cigarettes per day; or therapy with cholesterol-lowering or
cardiovascular medications. All patients agreed not to alter their diet
and exercise regimens during the study protocol.
Study Protocol
All participants had a baseline cholesterol profile, and plasma NO
and endothelin-1 were measured on samples obtained while the
participants were fasting for at least 16 hours to minimize dietary
effects.
All participants were placed in the supine position in a
temperature-controlled vascular research laboratory. We studied
vascular reactivity in a conduit vessel, the brachial artery. An
imaging study of the brachial artery was performed by using a
high-resolution ultrasound machine that was equipped with a 7.5-
MHz linear-array transducer. Baseline images of the brachial artery
were obtained proximal to the antecubital fossa. Imaging of the
artery was performed longitudinally, allowing clear visualization of
the posterior wall intima-lumen interface and the anterior wall
media-adventitial interface. We assessed endothelium-dependent
vasodilation by measuring the change in the caliber of the brachial
artery during reactive hyperemia, a maneuver that increases flow
through the conduit segment being studied (flow-mediated vasodi-
lation). To create this stimulus, a cuff placed on the upper arm above
the point undergoing measurement was inflated to suprasystolic
pressure for 5 minutes, thereby occluding flow to the forearm. This
results in the dilation of downstream forearm resistance vessels.
After cuff deflation, reactive hyperemia occurs, as brachial artery
blood flow increases to accommodate the dilated resistance vessels.
Imaging of the brachial artery was continually performed for a
5-minute period after cuff deflation until basal conditions were
reestablished. Thereafter, sublingual nitroglycerin (at a dose of 0.4
mg) was administered to assess endothelium-independent vasodila-
tion. The artery was studied for an additional 5 minutes. Blood
pressure and heart rate were monitored continuously throughout the
procedure.
All images were recorded on Super VHS videotape for subsequent
analysis. Image analysis was performed on a personal computer that
was equipped with a video frame grabber. Images recorded on
 1514 Arterioscler Thromb Vasc Biol. September 2001

videotape were analyzed by an investigator blinded to treatment TABLE 1. Clinical Characteristics of 90 Postmenopausal Women
assignment. Previous studies have shown that the peak diameter
change during reactive hyperemia occurs ⬇1 minute after cuff Placebo (N⫽30) Raloxifene (N⫽30) HRT (N⫽30)
deflation and 3 minutes after nitroglycerin administration. We used Age, y 55⫾6 57⫾7 56⫾8
these time points in the present study. Images corresponding to the
Time elapsed after 1.8⫾0.8 1.9⫾0.5 1.4⫾0.7
end of the T wave on a simultaneous ECG were selected and
digitized. Image analysis was then performed by using proprietary menopause, y
analysis software that searched for the shortest distance between the FSH, IU/L 84⫾10 85⫾9 81⫾7
points on the arterial wall, creating 10 to 20 paired measurements Current smokers, n 5 6 7
along a 10-mm length. We measured arterial diameter from the
intima-lumen interface on the posterior wall to the media-adventitial Previous smokers, n 3 3 4
interface on the arterial wall. We calculated brachial artery diameter History of CAD, n 4 8 6
by averaging these paired lumen measurements and reported them in
FSH indicates follicle-stimulating hormone; CAD, coronary artery disease.
millimeters by using calibration factors derived from real-time
Values are mean⫾SD or number of women.
ultrasonography. We used an average of 3 separate measurements for
each condition. To ensure that the blood flow stimulus during
reactive hyperemia was similar during each treatment phase, forearm The mean plasma endothelin-1 level was 17.3⫾8.9 pg/mL
blood flow was measured by venous occlusion strain-gauge pleth-
ysmography with the use of calibrated mercury-in-silastic strain and decreased to 11.5⫾2.1 pg/mL after 6 months of treatment
gauges. Women were randomly assigned to receive continuous HRT with raloxifene (Figure 1). With raloxifene, total and LDL
(1 mg 17␤-estradiol combined with 0.5 mg norethisterone acetate per cholesterol levels significantly decreased, whereas HDL cho-
day), raloxifene (60 mg/d), or placebo for 6 months. The experimen- lesterol levels did not change (Table 2). The ratio of NO
tal protocol was repeated after the end of treatment.
(nitrites/nitrates) to endothelin-1 increased from 2.2⫾1.8 to
Assays 3.4⫾2.1 (Table 2).
Plasma endothelin was measured by using a commercially available In postmenopausal women treated with HRT, the plasma
assay system (Amersham). Blood was drawn, and plasma was levels of NO oxidation products increased, and the levels of
separated. The efficacy of the extraction procedure was 81%. circulating endothelin-1 decreased (Figure 1). The ratio of
Interassay variations were 9%, and intra-assay variations were 5%.
In this assay, cross-reactivities were ⬍5% for endothelin-2, ⬍3% for NO (nitrites/nitrates) to endothelin-1 also increased after
endothelin-3, and ⬍37% for proendothelin. Previously established HRT (Table 2). No significant difference was observed
normal values (⫾SD) for plasma endothelin-1 are 7.1⫾0.1 pg/mL. between raloxifene and HRT regarding these markers of
NO production was assessed by monitoring plasma levels of endothelial function (Figure 1 and Table 2).
nitrites and nitrates, the 2 stable oxidation products of NO metabo-
lism by chemiluminescence detection. Blood samples were centri- HRT significantly reduced total cholesterol and LDL
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fuged at 2500 rpm for 20 minutes at 10°C. The supernatant was cholesterol levels (Table 2), but in contrast to raloxifene, it
removed and stored at ⫺70°C. The assay of NO products was also significantly increased HDL. Placebo administration did
standardized by a calibration curve with the use of known concen- not modify the markers of endothelial function as well as the
trations of nitrate (0.01 to 100 ␮mol/L) obtained from sodium nitrate.
For each measurement, a 4-␮L sample was placed in a reducing lipid profile (Figure 1 and Table 2).
vessel with 5 mL of 0.1 mol/L vanadium II chloride, 1 mol/L
hydrochloric acid, and 100 ␮L antifoaming agent at 90°C. Each
standard was analyzed 3 times, and each plasma sample was
analyzed at least 5 times. The mean value was used for all
subsequent analysis. Total cholesterol, HDL cholesterol, LDL cho-
lesterol, and triglyceride levels were measured in the Azienda
Policlinico Universitario Immunochemistry laboratory.

Statistical Analysis
Data are given as the mean⫾SD. The significance of difference was
assessed by ANOVA, followed by post hoc evaluation. A value of
Pⱕ0.05 was considered statistically significant.

Results
Clinical characteristics
Table 1 shows the clinical data in the 3 randomized groups.
All the groups were of a similar age. No single subject was
⬍1.3 years postmenopausal when treatment was started.
Similar trends were also seen when groups were subdivided
according to smoking status and history of coronary artery
disease. The level of follicle-stimulating hormone was not
significantly different in the 3 groups

Plasma NO Products and Endothelin-1 Levels

The data on NO oxidation products, endothelin-1, and cho-
lesterol are summarized in Figure 1. The mean baseline
nitrite/nitrate level was 26.5⫾10.7 ␮mol/L and increased to
36.3⫾11.4 ␮mol/L after 6 months of treatment with
raloxifene.
Figure 1. Plasma oxidized products of NO and endothelin-1
(ET-1) in postmenopausal women treated for 6 months with pla-
cebo, raloxifene, and HRT. #P⬍0.05 vs placebo; *P⬍0.05 vs
value before the treatment (before).
 Saitta et al Raloxifene and HRT in Postmenopausal Women 1515

TABLE 2. Effect of Placebo, Raloxifene, or HRT on Cholesterol, Estradiol, and

Ratio of NO to ET-1 in Postmenopausal Women
Variable Placebo Raloxifene HRT
Basal
Estradiol level, pmol/L 69.2⫾895 71.3⫾12.5 72.4⫾14.3
Ratio of NO to ET-1 2.4⫾1.1 2.2⫾1.8 2.4⫾1.9
Total cholesterol level, mmol/L 5.8⫾0.2 5.8⫾0.4 5.9⫾0.7
HDL cholesterol level, mmol/L 1.3⫾0.4 1.2⫾0.9 1.1⫾0.9
LDL cholesterol level, mmol/L 3.8⫾0.5 3.9⫾0.7 3.7⫾0.8
Triglyceride level, mmol/L 1.8⫾0.5 1.9⫾0.7 1.7⫾0.5
After therapy
Estradiol level, pmol/L 70.3⫾12.5 83.5⫾15.7 121.2⫾15.7*†
Ratio of NO to ET-1 2.2⫾1.3 3.4⫾2.1*† 3.8⫾2.3*†
Total cholesterol level, mmol/L 5.9⫾0.3 5.4⫾0.2*† 5.6⫾0.3*†
HDL cholesterol level, mmol/L 1.2⫾0.2 1.3⫾0.3 1.5⫾0.2*†
LDL cholesterol level, mmol/L 3.8⫾0.3 3.1⫾0.3*† 3.2⫾0.2*†
Triglyceride level, mmol/L 1.9⫾0.8 1.7⫾0.9 1.9⫾0.6
ET-1 indicates endothelin-1. Values are mean⫾SD for 30 healthy postmenopausal women.
*P⬍0.05 for comparison with placebo; †P⬍0.05 for comparison with corresponding basal value.

Hemodynamic Measurements, Flow-Mediated
Endothelium-Dependent Vasodilation, and
Endothelium-Independent Vasodilation
The effect of placebo, raloxifene, and HRT on blood pressure,
heart rate, forearm blood flow, and forearm vascular resis-
tance is presented in Table 3. Placebo, raloxifene, and HRT
did not affect blood pressure or heart rate.
Downloaded from http://ahajournals.org by on November 23, 2022
last years, several experimental and clinical studies have been
undertaken with the aim of assessing whether raloxifene
therapy is beneficial in the prevention of cardiovascular
disease. The effects of raloxifene on lipid levels were studied
in a group of postmenopausal women randomized to receive
2 doses of raloxifene, oral estrogen, or placebo.41 Like oral
estrogen, raloxifene reduced LDL cholesterol; in the present

Basal forearm blood and basal forearm vascular resistance study, the LDL decreased by 12%. Unlike oral estrogen,
were similar among the several groups and did not change however, raloxifene did not increase HDL. It also did not
after the treatment. The peak forearm blood flow during raise the triglyceride level. Both treatments reduced Lp(a),
reactive hyperemia was similar before and after placebo,
raloxifene, or HRT.
The brachial artery diameter, under basal conditions, was
3.3⫾0.7, 3.6⫾0.8, and 3.8⫾0.5 mm in the placebo, ralox-
ifene, and HRT groups, respectively. The percentage increase
in brachial diameter during reactive hyperemia before the
treatment was 7.5⫾1.8%, 8.3⫾2.1%, and 8.5⫾1.9% in the
placebo, raloxifene, and HRT arms, respectively (Figure 2).
Basal brachial artery diameter did not change significantly
after placebo, raloxifene therapy, or HRT. However, the
changes in brachial artery diameter during reactive hyperemia
were greater after raloxifene treatment or HRT, and no
difference was observed between these 2 active treatments
(Table 3 and Figure 2).
Absolute change in brachial artery diameter was
0.3⫾0.2 mm and 0.7⫾0.4 mm before and after raloxifene
treatment (P⬍0.05), respectively. This suggests an increase
of ⬎100% in brachial artery diameter (Figure 3) after
raloxifene treatment. A similar increase was also observed
after HRT (Figure 3)
Placebo administration did not change endothelium-
dependent vasodilation (Table 3 and Figures 2 and 3).
Furthermore placebo, raloxifene, and HRT did not affect
nitroglycerin and endothelium-independent vasodilation (Ta-
ble 3 and Figure 2).
Figure 2. Flow-mediated endothelium-dependent vasodilation
and nitroglycerin-mediated endothelium-independent vasodila-
Discussion tion in postmenopausal women treated for 6 months with pla-
Raloxifene is a selective estrogen agonist that prevents bone cebo, raloxifene, and HRT. #P⬍0.05 vs placebo; *P⬍0.05 vs
resorption without causing endometrial hyperplasia. In the value before the treatment (before).
 1516 Arterioscler Thromb Vasc Biol. September 2001
Figure 3. Absolute change in brachial artery diameter in post-
menopausal women treated for 6 months with placebo, ralox-
ifene, and HRT. #P⬍0.05 vs placebo; *P⬍0.05 vs value before
the treatment (before).
but the reduction was less in the raloxifene-treated patients;
therefore, this selective estrogen receptor modulator has a less
beneficial effect on lipoprotein profiles than does oral
estrogen.
In a 2-year placebo-controlled study in healthy postmeno-
pausal women, raloxifene and estrogen monotherapy had
similar beneficial effects on LDL cholesterol and fibrinogen
levels, even if they differed in their effects on HDL, choles-
terol, triglycerides, and plasminogen activator inhibitor-1 and
possibly in their effects on prothrombin fragment F1⫹2 and
C-reactive protein.42
Furthermore, in a randomized, double-blind, placebo-
controlled comparison with conjugated equine estrogen, the
effects of a 24-month treatment with orally administered
raloxifene on serum Lp(a), an independent risk factor for
Downloaded from http://ahajournals.org by on November 23, 2022
coronary disease, were investigated.43 Long-term raloxifene
treatment significantly lowered serum Lp(a), thus suggesting
that this therapy might reduce the risk of coronary artery
disease.43 Finally, raloxifene therapy has been shown to
reduce homocysteine levels, another independent risk factor
for the development of cardiovascular disease, in healthy
postmenopausal women in 2 randomized controlled trials.44,45
Several experimental and clinical studies also suggest that
the effect of an intervention on endothelial function might
predict its involvement in coronary disease progression and in
the rate of cardiovascular events. More specifically, the
effects on NO, the predominant relaxing factor, and
endothelin-1, the major constrictor factor, are particularly
important.
NO and endothelin-1 are, in fact, endothelium-derived
vasoactive factors that are important in cardiovascular dis-
ease.46,47 NO causes vasodilation, inhibits platelet aggrega-
tion, suppresses smooth muscle proliferation, and acts as an
antiatherogenic factor.46 – 48 Continuous release of NO from
the endothelium maintains basal vascular tone, and alterations
in NO release allow autoregulation of blood flow.49 Endog-
enous substances and physical forces, such as shear stress,
stimulate the production of NO. Alternatively, endothelin-1
opposes the effect of NO. It causes potent vasoconstriction of
the systemic and coronary vasculature through the endothelin
receptors, increases monocyte adhesion, activates macro-
phages, and promotes vascular smooth muscle cell prolifer-
ation and migration.50
In addition to the opposing effects of NO and endothelin-1
on vascular tone and smooth muscle cell proliferation, the
regulatory mechanisms of these vasoactive factors interact.
NO inhibits the production of endothelin-1 and modulates the
number and the affinity of endothelin-A receptors. NO
synthase is functionally coupled to the endothelin-B recep-
tor.51 In disease states such as hypercholesterolemia, athero-
sclerosis, and congestive heart failure, imbalances between
NO and endothelin-1 (ie, decreased NO breakdown product
concentrations, increased endothelin-1 levels, and reduced
ratio of NO to endothelin-1) are detected and may contribute
to vasomotor abnormalities and vascular remodeling.46 In-
deed, NO and endothelin-1 represent valuable markers for the
assessment of endothelial function, as previously shown.52–54
Estrogen replacement therapy has been shown to signifi-
cantly improve vascular function; indeed, the vascular endo-
thelium has been claimed to play an important role in
mediating the cardiovascular protective effects of
estrogens.10,11,16
Raloxifene possesses in vitro endothelium-dependent ef-
fects, thus raising the possibility that this compound may
have the potential to be a cardioprotective agent, with the
benefit of no increased risk of cancer or other side effects.40
However, the positive endothelium-dependent effects of
raloxifene need to be confirmed and verified clinically in
postmenopausal women. Therefore, we tested the hypothesis
that raloxifene increases the ratio between the plasma total
oxidized products of NO and circulating endothelin-1, in turn,
improving the endothelium-mediated vasodilation. More spe-
cifically, we compared the effects of HRT and raloxifene on
NO breakdown products, endothelin-1 plasma levels, and
endothelium-dependent vasodilatation in healthy postmeno-
pausal women.
The present study shows that in postmenopausal women,
therapy with this selective estrogen receptor modulator or
HRT increases plasma levels of nitrites/nitrates and decreases
plasma endothelin-1, thereby enhancing the ratio of NO to
endothelin-1 and improving endothelium-dependent vasodi-
lation. The endothelium is likely to be responsible for
increased production of NO. As a matter of fact, we also
measured NO production by peripheral blood mononuclear
cells in our subjects either under basal conditions or after in
vitro stimulation with endotoxin. We found no statistical
difference in basal NO release between placebo and either
HRT or raloxifene, thus ruling out the possibility that the
increase in plasma nitrites/nitrates might be due to an
inflammation-mediated activation of the inducible NO syn-
thase (results not shown). Furthermore, after endotoxin stim-
ulation, peripheral blood mononuclear cells harvested from
women treated with HRT or raloxifene had a reduced NO
release compared with women treated with a placebo. Finally,
our patients had no sign of inflammatory or infectious
disease. All these findings, taken together, strongly indicate
that the endothelium is likely to be the source of the increased
plasma levels of nitrites/nitrates after HRT or raloxifene
therapy in our experimental protocol.
The main finding is that these 2 active treatments showed,
at least under these experimental conditions, similar effects.
As far as we know, this is the first report indicating that
raloxifene may improve endothelial function to the same
extent as estrogen.
Several mechanisms may be responsible for this effect. The
therapy with raloxifene may regulate NO by increased pro-
duction of NO synthase, and experimental evidence indicates
 Saitta et al Raloxifene and HRT in Postmenopausal Women 1517

TABLE 3. Effect of Placebo, Raloxifene, and HRT on Hemodynamic Measurements

Variable Placebo Raloxifene HRT
Basal
Blood pressure, mm Hg
Systolic 113⫾9 115⫾11 111⫾14
Diastolic 78⫾8 79⫾8 78⫾12
Mean 94⫾5 92⫾8 96⫾10
Heart rate, bpm 69⫾8 74⫾4 77⫾5
Forearm blood flow, mL 䡠 100 mL tissue⫺1 䡠 min⫺1
Basal 2.7⫾1.3 3.6⫾1.8 2.9⫾1.6
Reactive hyperemia 21⫾4 24⫾6 23⫾3
Forearm vascular resistance, U
Basal 43⫾7 39⫾5 38⫾6
Minimal 5.8⫾1.8 5.1⫾2.1 5.6⫾2.5
Brachial artery diameter, mm
Basal 3.3⫾0.7 3.6⫾0.8 3.8⫾0.5
During reactive hyperemia 3.5⫾1.4* 3.9⫾1.7* 4.1⫾1.9*
After nitroglycerin administration 4.4⫾1.7† 4.1⫾0.5† 4.3⫾0.7†
Brachial artery diameter, % change
During reactive hyperemia 7.5⫾1.8 8.3⫾2.1 8.5⫾1.9
After nitroglycerin administration 12.9⫾3.1 13.5⫾2.8 14.4⫾3.2
After therapy
Blood pressure, mm Hg
Systolic 114⫾11 115⫾13 115⫾14
Diastolic 75⫾9 80⫾12 78⫾12
Mean 92⫾7 95⫾8 96⫾10
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Heart rate, bpm 67⫾4 72⫾5 71⫾6

Forearm blood flow, mL 䡠 100 mL tissue⫺1 䡠 min⫺1
Basal 2.9⫾1.1 3.2⫾2.5 3.3⫾2.9
Reactive hyperemia 23⫾5 26⫾7 25⫾4
Forearm vascular resistance, U
Basal 41⫾6 35⫾7 38⫾6
Minimal 5.2⫾1.1 4.2⫾1.7 4.8⫾2.3
Brachial artery diameter, mm
Basal 3.5⫾0.8 3.4⫾0.6 3.7⫾0.5
During reactive hyperemia 3.8⫾1.1* 4.1⫾1.4† 4.3⫾1.1†
After nitroglycerin administration 4.2⫾1.3† 4.3⫾0.8† 4.6⫾2.3†
Brachial artery diameter, % change
During reactive hyperemia 7.2⫾1.1 12.3⫾2.1‡ 11.5⫾2.8‡
After nitroglycerin administration 13.4⫾2.8 14.4⫾3.3 15.3⫾2.9
Values are mean⫾SD for 30 healthy postmenopausal women.
*P⫽NS vs basal; †P⬍0.05 vs basal; ‡P⬍0.05 vs placebo and pretreatment values.

that raloxifene can act via an estrogen-dependent mechanism
on the expression of endothelial NO synthase to increase
endothelium-dependent relaxation.40 This finding has been
recently confirmed and amplified; indeed, raloxifene acutely
stimulates NO release from human endothelial cells via an
estrogen receptor– dependent acute stimulation of endothelial
NO synthase enzymatic activity.55
Another possible mechanism for the increase in NO seen
with raloxifene is the beneficial effects of estrogen on lipid
metabolism. The therapy lowers total cholesterol and LDL
cholesterol levels; all of these reductions blunt the risk of
cardiovascular events. Because hypercholesterolemia is asso-
ciated with impaired vascular tone (probably secondary to the
decreased bioavailability of NO), decreased cholesterol levels
may increase vascular NO production, and this may be
another mechanism by which raloxifene exerts its effects.
Indeed, the fall in cholesterol levels found in the present study
after HRT and raloxifene therapy is similar to that observed
in previously published studies.56 –58 Thus, raloxifene may
increase systemic NO levels through multiple mechanisms,
thus improving endothelial function.
However, the improved endothelial function may also be
the result of the increased ratio between NO and
endothelin-1; as a matter of fact, endothelin-1 concentration
 1518 Arterioscler Thromb Vasc Biol. September 2001
decreased in response to raloxifene therapy. The mechanism
by which the selective estrogen receptor modulator may
affect endothelin-1 concentrations remains, at the moment, a
matter of speculation. We can only hypothesize that ralox-
ifene increases NO bioavailability and, in turn, reduces
endothelin-1 levels. In other words, the reduction in the
plasma levels of this potent vasoconstrictor may be an
NO-mediated phenomenon that is due to the ability of the
latter to modulate the production of endothelin-1.
The raloxifene-induced changes in endothelial function
shown in the present study (ie, increase in plasma NO total
oxidized products, decrease in endothelin-1 levels, and im-
provement in endothelial flow–mediated vasodilation) are
similar to previously reported data and demonstrate an ability
to modify cardiovascular mortality and morbidity over the
long term.59 – 61 This evidence further stresses the importance
of the present findings.
In conclusion, the present study shows that raloxifene
therapy increased the ratio between NO and endothelin-1 and
improved flow-mediated endothelium-dependent vasomotion
to the same extent as estrogen in postmenopausal women.
Further investigation is warranted to enhance our understand-
ing of the mechanisms of the effect of raloxifene on vascular
function and to determine whether its effect on endothelial
function may contribute to the reduction in cardiovascular-
related morbidity and mortality.
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