© 1997 American Heart Association, Inc.
Articles |
A Partial Estrogen Receptor Agonist With Strong Antiatherogenic Properties Without Noticeable Effect on Reproductive Tissue in Cholesterol-Fed Female and Male Rabbits
From the Novo Nordisk A/S, Novo Allé, 2880 Bagsvaerd (P.H., M.S., N.K., B.G.), Department of Obstetrics and Gynecology, Rigshospitalet, University of Copenhagen (S.O.S.), and the Clinical Institute, Odense University (S.S.), Denmark.
Correspondence to Pernille Holm, MD, Department of Women's Healthcare Biology, Novo Nordisk Park, 2760 Maaloev, Denmark. E-mail PHIm{at}novo.dk
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Abstract Estrogen replacement therapy retards the development of cardiovascular disease and osteoporosis in postmenopausal women. However, long-term unopposed use increases the risk of cancer in endometrium and possibly in breast. The racemic compound ormeloxifene, widely used in India as an antifertility agent, is a partial estrogen receptor agonist with antiosteoporotic properties. The present study was undertaken to investigate the effect of the L-enantiomer (levormeloxifene) and the d-enantiomer (d-ormeloxifene) on the development of atherosclerosis. In a short-term experiment (6 weeks), 4x10 ovariectomized female rabbits were fed a 0.25% cholesterol-enriched diet and the effect on plasma cholesterol levels was studied. In a long-term experiment (13 weeks), 4x15 ovariectomized female and 4x15 sham-operated male rabbits were maintained at a similar plasma cholesterol level of 25 mmol/L and the effect on undamaged and balloon-injured arterial wall was studied. In both experiments, the rabbits were treated with levormeloxifene, d-ormeloxifene, 17ß-estradiol, or placebo, respectively.
In the short-term experiment, levormeloxifene, in contrast to d-ormeloxifene, significantly reduced plasma cholesterol by 30% compared with the placebo group. In the long-term experiment, levormeloxifene, in contrast to d-ormeloxifene, significantly reduced atherosclerosis by 50% in the undamaged arterial wall of both female and male rabbits. Because these rabbits were cholesterol-clamped, the antiatherogenic effect was not mediated via plasma cholesterol lowering. Like estrogen, levormeloxifene did not inhibit atherosclerosis in the endothelium-denuded site of aorta. The antiatherogenic effects of levormeloxifene were thus similar to those of estrogen, but produced in the absence of any noticeable estrogenic effect on uterine or testicular tissue.
Key Words: antiestrogens • atherosclerosis • estrogen • plasma cholesterol • rabbits
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Introduction |
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Atherosclerosis is the major cause of morbidity and mortality in both men and women in the Western world. Several studies suggest that this disease in postmenopausal women can be prevented by the use of estrogen replacement therapy (ERT).1 2 3 In addition to its preventive effect on atherosclerosis, estrogen also prevents postmenopausal bone loss.4 However, to gain the full benefits of estrogen, it must be administered for several years. This raises some concern because long-term unopposed ERT in women is associated with an increased risk of endometrial and perhaps also breast cancer.5 6 The addition of a progestin eliminates the increased risk of endometrial cancer,7 but may not prevent the possible risk of breast cancer.8 Progestins do not appear to attenuate the protective effect of estrogen on atherosclerosis,9 but are associated with either a resumption of regular withdrawal bleeding or spotting, which may limit the number of patients willing to initiate this treatment.
Thus, neither estrogen alone nor estrogen/progestin combinations represent the ideal therapy for postmenopausal women. The ideal therapy would be one with estrogenic effects on the cardiovascular system and bone but without estrogenic effects on endometrium and breast. Such a therapy without influence on reproductive tissue would also be of potential interest for men at high risk of atherosclerosis.
The realization that combined estrogen receptor agonist/antagonists may exhibit some degree of tissue selectivity has intensified the search for the ideal drug for hormone replacement therapy. Tamoxifen has antiosteoporotic and plasma cholesterol-lowering effects in postmenopausal women10 11 and an inhibiting effect on the formation of diet-induced lipid lesions in atherosclerosis-susceptible mice.12 Tamoxifen, however, also stimulates the endometrium strongly.13 14 Raloxifene and droloxifene seem to prevent osteoporosis and reduce plasma cholesterol levels in ovariectomized rats without producing significant effects on the endometrium.15 16
At present, the use of conventional ERT is reserved for postmenopausal women, because this therapy is unacceptable for men due to the development of thromboembolism,17 18 testes atrophy, and impotence. From a medical perspective this is unfortunate, however, since epidemiologic studies indicate that middle-aged men are at higher risk of cardiovascular disease than middle-aged women.
Part of the protective effects of estrogen on atherogenesis rely on favorable actions on plasma concentrations of lipids and lipoproteins.19 20 Epidemiologic studies suggest that estrogen exerts even stronger effects directly on the arterial wall, however, and that these effects constitute more than two thirds of the antiatherogenic action.21 The notion of a direct effect of estrogen on the arterial wall has been supported by studies in monkeys22 23 and rabbits24 25 and for tamoxifen in atherosclerosis-susceptible mice.12
Recently, ormeloxifene (3,4-trans-7-methoxy-2,2-dimethyl-3-phenyl-4-{4-[2-(pyrrolidin-1-yl)ethoxy]phenyl}chroman), a partial estrogen receptor agonist with antiosteoporotic properties in the ovariectomized rat, has been identified.26 Ormeloxifene, which is marketed in India as a birth control pill (tradename centchroman), is a racemic substance. The two stereoisomers have been isolated and named levormeloxifene (L-enantiomer) and d-ormeloxifene (d-enantiomer). The purpose of the present study was to compare the effects of levormeloxifene and d-ormeloxifene with those of estrogen on the development of atherosclerosis in cholesterol-fed female and male rabbits. In the first experiment (plasma lipid experiment) potential effects on plasma cholesterol and lipoprotein distribution were studied. In the second experiment (arterial wall experiment), plasma cholesterol was maintained at the same level in all animals in order to study potential direct effects on the arterial wall. We have previously shown that the antiatherogenic effect of estrogen is abolished by balloon catheter injury in cholesterol-clamped rabbits,27 28 suggesting that an intact endothelium is pivotal for the direct antiatherogenic effect of estrogen. To investigate whether this was also the case for levormeloxifene and/or d-ormeloxifene, all rabbits were balloon injured in the upper thoracic aorta.
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Methods |
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Plasma Lipid Experiment
Surgery
Forty sexually mature, female New Zealand White rabbits (3.0-3.9 kg) from self breeding were used. The rabbits were anesthetized with intravenous pentobarbital, which was given as an initial dose of 25 mg/kg of body weight followed by repeated small doses. A total dosage of about 50 mg/kg of body weight was given to each rabbit. The abdomen was opened through a midline incision and both ovaries were removed. The incision was closed and the animals were allowed to recover before being placed in their individual cages. All experimental procedures of this and the following experiment were conducted in compliance with Danish regulations for experiments on animals.
Treatment
One week after surgery, the rabbits were divided into four
groups having similar baseline values of plasma cholesterol
and weight. The four groups were subcutaneously treated with
levormeloxifene (7.5 mg/kg), d-ormeloxifene (7.5
mg/kg), 17ß-estradiol cypionate (Sigma Chemical Co) (50
µg/kg) or placebo every third day. The dose of estradiol was
chosen because it produces plasma estradiol levels in rabbits
comparable to those found in postmenopausal women.27
Plasma and Dietary Cholesterol
For the first 2 weeks of treatment, the rabbits were fed
100 g of standard rabbit chow daily (Altromin 2113, Lage, Germany)
and plasma cholesterol of each rabbit was determined once a
week. Plasma cholesterol levels were determined by
enzymatic cholesteryl ester hydrolysis and cholesterol
oxidation followed by a color reaction (CHOD-PAP, Boehringer
Mannheim, Mannheim, Germany).
For the next 4 weeks, the rabbits were fed 80 g of a cholesterol-enriched chow, consisting of 72 g of standard rabbit pellets, 8 g of corn oil (NOMECO, Copenhagen, Denmark), and 0.2 g of cholesterol (0.25%) (CHP-UPS, Sigma Chemical Co). The chow was prepared by dissolving cholesterol in heated corn oil and mixing the corn oil/-cholesterol solution thoroughly with the standard rabbit pellets. During the cholesterol-feeding period, the plasma cholesterol level of each rabbit was determined twice a week. The distribution of cholesterol between VLDL (d<1.006 g/mL), IDL (1.006<d<1.019 g/mL), LDL (1.019<d<1.063 g/mL), and HDL (d>1.063 g/mL) was determined after 4 weeks of treatment using ultracentrifugation as described previously.27 All blood samples (2 mL for plasma cholesterol determinations and 10 mL for ultracentrifugation) were drawn from the lateral ear vein into tubes containing Na2EDTA.
Arterial Wall Experiment
Surgery
One hundred and twenty (60 female+60 male) sexually mature New
Zealand White rabbits (2.4-4.0 kg) from Inter Fauna, Huntingdon,
England, were used. Anesthesia was induced as described
above, whereafter the female rabbits were ovariectomized and the male
rabbits sham operated. All rabbits were furthermore subjected to
balloon catheter injury of the upper thoracic aorta according to the
following procedure: the right femoral artery was exposed through a
groin incision and isolated. An arteriotomy was made, and a 4F Fogarty
embolectomy catheter (Baxter Healthcare Corp) inserted and passed until
resistance was met by the aortic arch. The balloon was inflated with
0.6 ml of saline (distension 9.0 mm) and the catheter retracted 3
cm. The balloon was then deflated and the catheter withdrawn. The
artery was ligated and both the groin incision and the midline
abdominal incision closed. Again, the animals were observed during
recovery, whereafter they were placed in their individual
cages.
Treatment
The animals were sorted into eight experimental groups with
similar baseline values of body weight and plasma
cholesterol. Immediately after surgery, all rabbits were
given a single subcutaneous injection of levormeloxifene,
d-ormeloxifene, 17ß-estradiol, or placebo in the same
doses as described above. For the remainder of the experiment, these
compounds were administered orally together with the
cholesterol-enriched chow (levormeloxifene and
d-ormeloxifene, 10 mg/d, and 17ß-estradiol, 4
mg/d). For the addition of hormones, estradiol was dissolved in
ethanol (96%) and poured into the heated corn
oil/cholesterol mixture. Levormeloxifene and
d-ormeloxifene were dissolved in corn oil before being
added. The dose of estradiol was much higher than the subcutaneous one
given in the plasma lipid experiment because the rabbit has a very high
first pass metabolism of estrogen in the liver.
Plasma and Dietary Cholesterol
Cholesterol feeding was initiated 1 week prior to
surgery and hormone treatment. The amount of cholesterol
added to the chow of each rabbit was not constant but continuously
adjusted according to weekly plasma cholesterol
determinations. Each rabbit received 80 g of chow daily,
consisting of 72 g of standard rabbit pellets, 8 g of corn
oil, and 0-0.8 g of cholesterol (0-1%). In this way, all
rabbits were maintained at a similar plasma cholesterol
concentration of 20-25 mmol/L, resulting in all aortas
being exposed to a similar average plasma cholesterol level
(Tables 1
and 2). The distribution of
cholesterol between lipoprotein fractions was determined at
week 8.
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Arterial Blood Pressure
At week 8 the central ear artery of all rabbits was cannulated
for measurement of intraarterial blood pressure. The
animals were allowed to rest quietly for a minimum of 15 minutes before
blood pressure was measured through the arterial cannula
using Baxter pressure transducers connected to HSE pressure couplers
and a PONEMAH data acquisition system.
Plasma Estradiol Concentration
At week 8 the plasma trough concentrations of estradiol of all
animals was measured by specific RIA after extractions and
chromatography.29 The detection limit of
the estradiol assay was 40 pmol/L.
Preparation of Aortic Tissue
At the end of the experiment the rabbits were injected
intravenously with 5 mL of Evans Blue dye (5 mg/mL).
The dye was allowed to circulate for 5 minutes before the rabbits were
killed with an overdose of intravenous pentobarbital
(50-100 mg/mL). Through a needle inserted into the left
ventricle of the heart the systemic circulation was perfused with 500
mL of saline, whereafter the aorta was excised and carefully freed of
adventitia. The balloon-injured site in the upper thoracic aorta was
easily identified by its blue staining. Regeneration was evident as
small islands of white tissue around the branch orifices and as narrow
borders of white tissue lining the proximal and distal edge of the
blue-stained area. The blue-stained site was removed together with
three undamaged sites from the aortic arch, the lower thoracic aorta,
and the upper abdominal aorta, respectively (Fig 1). One specimen of unopened aorta was
taken from each of these four sites for histologic and
immunohistochemical evaluation: After fixation in formalin, the
specimen was embedded in paraffin wax and cut into 3-µm sections. The
remaining tissue was used for determination of cholesterol
content: The aortic sites were opened longitudinally, fixed with pins
on a cork board, and the area of each site outlined on graph paper.
Because atherosclerotic changes should be manifested in the intimal
layer of the artery, the aortic sites were separated into an inner
layer (intima-inner media) and an outer layer (outer media). The outer
layer was discarded, while the inner layer was weighed and stored at
-20°C until chemical analysis.
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Quantification of Atherosclerosis Development
For determination of cholesterol content, aortic
tissue was minced and extracted with at least 20 volumes of
chloroform/methanol (1:1, vol/vol). After the addition of
chloroform, the extract was washed by the Folch
procedure.30 Subsequent to evaporation, the extracted
lipids were redissolved in isopropanol and the cholesterol
content determined by the same enzymatic kit as that used for plasma
cholesterol determinations. The validity of this procedure
has been tested previously.25
For evaluation of intimal thickening, 3-µm sections of the four aortic segments were stained with elastic-van Gieson and elastic-hematoxylin and eosin, respectively. Images of the aortic cross-sections were projected onto the screen of a computer through a video-monitored microscope (Olympus). The image was calibrated and the magnification calculated. On each picture a suitable grid for the chosen magnification was applied. The number of the points over the intima and media was counted and the ratio of intima to total aortic wall (intima+media) was calculated. All measurements were performed by the same independent observer who was unaware of the composition of the treatment groups.
Immunohistochemistry
To identify the types of cells present in the lesions, five
3-µm sections from undamaged (lower thoracic) and balloon-injured
(upper thoracic) aorta were randomly selected from each group and
stained with monoclonal antibodies directed against rabbit
macrophages (RAM11) (DAKO Corp), rabbit smooth muscle 
-actin
(HHF35) (DAKO A/S, Glostrup, Denmark), and rabbit CD43, a T cell
antigen (L11/135) (Serotec, Oxford, England). An avidin-biotin method
was used with these antibodies.
Macrophages and smooth muscle cells were quantitated by use of computerized image analysis (Leica Q500MC, Glostrup, Denmark). The number of stained cells as well as the number of other cells were counted in areas of the intima (magnification x400), and stained cells were expressed as percentage of total number of cells. If possible, cells were counted in four different visual fields with lesions. If lesions were less pronounced, fewer visual fields were counted. In some specimens, it was not possible to find any visual fields with lesions. T-lymphocytes were counted in the whole circumference of the intima and related to the intimal cross-sectional area (number of points). All measurements were performed by the same investigator who was unaware of the composition of the treatment groups.
Statistics
Mann-Whitney's test (two-tailed) was used for comparison
between groups. All values are given as mean±SEM.
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Results |
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Plasma Lipid Experiment
Plasma Cholesterol Levels
During the first 2 weeks of the experiment, when the rabbits were fed a standard chow, plasma cholesterol levels of the four groups remained unchanged between 1 and 2 mmol/L (Fig 2A
). After a few days of
cholesterol feeding, however, the plasma
cholesterol levels started to rise steeply. The plasma
cholesterol level of the levormeloxifene group rose
significantly less than that of the placebo group. The difference
between the plasma cholesterol levels of the two groups
became significant as early as 4 days after initiation of
cholesterol feeding and remained significant for the rest
of the experiment except for the last measurement (P=.08).
The plasma cholesterol levels of the estrogen group were
also significantly lower than those of the placebo group in that
period. There was no significant difference between the plasma
cholesterol-lowering effect of estrogen and
levormeloxifene. d-Ormeloxifene did not influence plasma
cholesterol levels. The percentage distribution of
cholesterol between lipoprotein fractions after 4 weeks of
treatment showed only minor differences between groups (Fig 2B). All
rabbits thrived equally well during the experimental period, and the
final weight did not differ between the four groups (data not
shown).
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Arterial Wall Experiment
Plasma Cholesterol Clamping
We succeeded in maintaining a similar mean concentration of plasma
cholesterol in all rabbits throughout the experimental
period. Calculated as the area under the curve (AUC), the mean plasma
cholesterol concentration was not statistically significant
between the groups (Tables 1
and 2). In the female rabbits, the
levormeloxifene group and the estrogen group had to be given more
dietary cholesterol than the placebo group, although this
was only significant for the levormeloxifene group (P=.03)
(Table 1). The d-ormeloxifene rabbits had a dietary
cholesterol intake similar to that of the placebo rabbits.
In the male rabbits, there was no significant difference in the total
cholesterol intake between the four groups of rabbits
(Table 2). The total food intake was lower for the levormeloxifene
group and the estrogen group compared with the placebo group in both
female and male rabbits, although this difference was not significant
for the male estrogen group (Tables 1 and 2). The total food intake for
the d-ormeloxifene groups was not different from that of the
placebo groups.
The percentage distribution of cholesterol between
lipoprotein fractions measured at week 8 was without differences
between groups in the female and male rabbits except for a minor
decrease in LDL cholesterol in the male estrogen group
(P=.01) (Tables 1 and 2). At week 8 all groups also had the
same mean arterial blood pressure. The plasma trough
concentrations of estradiol measured at week 8 were significantly
higher for the estrogen group than for the three other groups in both
female and male rabbits.
Aortic Atherosclerosis
In the female rabbits, the accumulation of cholesterol
in the levormeloxifene group was less than half that in the placebo
group in the undamaged lower thoracic (P=.002) and upper
abdominal (P=.001) aorta (Fig 3, upper panel). In the upper
abdominal aorta, the inhibition of cholesterol accumulation
was similar to that found in the estrogen group, whereas in the lower
thoracic aorta it was significantly less (P=.03). In the
undamaged aortic arch levormeloxifene had no significant effect. This
was in contrast to the estrogen group, where there was a significant
inhibition of cholesterol accumulation in the aortic arch
also (P=.001). In the balloon-injured site, that is, the
upper thoracic aorta, neither levormeloxifene nor estrogen affected
cholesterol accumulation.
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In the male rabbits, cholesterol accumulation in the
levormeloxifene group was reduced to approximately 50% of that in the
placebo group in the aortic arch (P=.04), the lower thoracic
(P=.005), and upper abdominal site of aorta
(P=.02) (Fig 3, lower panel).
These effects were similar to those of estrogen in all three undamaged
sites. In the balloon-injured site, estrogen and levormeloxifene did
not affect cholesterol accumulation.
d-Ormeloxifene did not affect cholesterol
accumulation in any of the four aortic sites in either female or male
rabbits.
The four aortic sites were also analyzed morphometrically for
intimal thickness in histologic cross-sections. The effects of
levormeloxifene, d-ormeloxifene, and estrogen were similar
to those found for cholesterol accumulation (Fig 4, upper and lower panel).
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Immunohistochemistry
The intimal lesions appeared as circumferential or focal raised
areas composed by closely packed smooth muscle cells and
macrophage-derived foam cells (Fig 5). For the female rabbits, the
cellularity and the number of smooth muscle cells were higher in the
balloon-injured than in the undamaged site, whereas the number of
macrophages was nearly the same (Table 3). This resulted in a lower percentage
of macrophages and a higher percentage of smooth muscle cells
in the balloon-injured upper thoracic aorta than in the undamaged lower
thoracic aorta. In the undamaged site, the cellularity and the number
of smooth muscle cells were higher in the placebo group than in the
three treatment groups, and the number of macrophages was
similar in all four groups. This resulted in a higher percentage of
macrophages in the levormeloxifene and estrogen group than in
the two other groups. These differences, however, did not reach
statistical significance. There was no difference between the groups in
the percentage of smooth muscle cells. T-lymphocytes were only rarely
observed in the intima and with a high coefficient of variance within
each group. Similar trends were observed for male rabbits (data not
shown).
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Reproductive Tissue
The uterine weight of rabbits given levormeloxifene was only
one-fifth of that found in the rabbits given estrogen (3.2±2 g
versus 15.8±1.4 g, P<.0001) (Fig 6, upper panel). Although the uterine
weight of the levormeloxifene group was only 1.2 g higher than
that of the placebo group, this difference was statistically
significant (P<.0001). The uterus weight of the
d-ormeloxifene group was a little less than that of the
placebo group, with no statistical difference between groups.
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The testicular weight of the levormeloxifene rabbits was 4 times higher
than that found in the estrogen rabbits (4.8±0.0 g versus
1.1±0.1 g, P<.0001) (Fig 6, lower panel). The testes
of the levormeloxifene rabbits weighed 1 g less than those of the
placebo rabbits (P<.03). The testes of the
d-ormeloxifene group weighed slightly more than those of the
placebo group, with no statistical difference between the two
groups.
At necropsy no rabbits showed anatomic abnormalities. The final weight
of the male estrogen group was significantly higher than that of the
male placebo group (Table 2). There were no differences in the final
weight between the remaining groups.
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Discussion |
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The main findings of this study are that levormeloxifene, the levorotatory form of a partial estrogen receptor agonist, like estrogen, lowers plasma cholesterol and has a direct antiatherogenic effect on the arterial wall in cholesterol-fed female and male rabbits. d-Ormeloxifene, however, has no antiatherogenic properties, neither on plasma cholesterol levels nor on the arterial wall. Furthermore, and in contrast to estrogen, none of the stereoisomers have any appreciable effect on reproductive tissue.
Effects of Levormeloxifene and d-Ormeloxifene on
Plasma Lipids
Neither levormeloxifene nor estrogen affected plasma
cholesterol levels in the rabbits while they were fed
standard chow. Levormeloxifene significantly reduced the increase in
plasma cholesterol levels induced by
cholesterol-feeding; however, this effect was
indistinguishable from that of estrogen. This
cholesterol-lowering effect most likely reflects a class
effect of partial estrogen receptor agonists, because similar results
have been reported in rats for tamoxifen, raloxifene, and
droloxifene.12 15 16 The plasma
cholesterol-lowering effect of estrogen in
cholesterol-fed rabbits has been attributed to an
upregulation of hepatic LDL receptors, resulting in enhanced uptake of
apolipoprotein (apo) B- and apo E-containing
lipoproteins.31 32 It is likely that the plasma
cholesterol lowering effect of partial estrogen receptor
agonists is due to the same upregulation of the LDL receptor.
Effects of Levormeloxifene and d-Ormeloxifene on
Arterial Wall
In recent years, a number of studies have evaluated the
antiatherogenic effect of estrogens and estrogen/progestin combinations
in the cholesterol-fed rabbit model.24 32 33
However, the identification of a possible direct effect of these
compounds on cholesterol accumulation in the
arterial wall often has been hampered by a varying plasma
cholesterol level between groups due to the
hypocholesterolemic effect of these compounds. This
confounding factor was eliminated in the current study, in which the
plasma cholesterol levels of the rabbits were clamped at
approximately 25 mmol/L throughout the experimental
period.
Despite similar plasma cholesterol levels, lipoprotein distribution, and arterial blood pressure in all groups, levormeloxifene reduced lipid accumulation and intimal thickening in almost all undamaged sites of both the female and male aorta, although this effect was most pronounced in the more distal parts of the aorta. To date, an antiatherogenic effect has been described only for one other partial estrogen receptor agonist, tamoxifen, which has been shown to reduce the formation of diet-induced lipid lesions by 86% in atherosclerosis-susceptible (C57B16) mice.12 However, in that study, tamoxifen significantly reduced the concentration of total plasma cholesterol (11%). Although unlikely to be the major mechanism, this reduction may have contributed to its antiatherogenic effect. In the present experiment, the observed antiatherogenic effect of levormeloxifene could not have been due to effects on plasma cholesterol and may therefore reflect direct actions on the arterial wall.
In the balloon-injured site, however, levormeloxifene and estrogen did not affect atherogenesis in either female or male rabbits. This finding suggests that the direct antiatherogenic effect of levormeloxifene and estrogen on the arterial wall is mediated through a common mechanism. As in the plasma lipid experiment, the estrogen-like direct antiatherogenic effect was confined to the L-enantiomer, since d-ormeloxifene did not modify aortic cholesterol accumulation. It has previously been reported that the ligand binding affinity of d-ormeloxifene to the estrogen receptor is only 13% of that of levormeloxifene.34 These findings suggest that the hypocholesterolemic and the direct antiatherogenic effects of levormeloxifene and estrogen are mediated via estrogen receptors.
The cell types within the aortic lesions were demonstrated by immunohistochemical staining against macrophages, smooth muscle cells, and T-lymphocytes. However, except for the general trend with a lower percentage of macrophages and a higher percentage of smooth muscle cells in tissue with higher cholesterol content, our data do not allow us to be more specific concerning the effect of treatment on the cellularity and composition of the aortic lesions. Since the cholesterol content differs between the various treatment groups we do not know whether the treatment causes the differences in cellularity and composition, which in turn leads to the differences in cholesterol content, or whether it is the other way around.
It has previously been shown that a balloon injury of the same severity as that in the present study removes more than 90% of the endothelium, while the internal elastic membrane and smooth muscle of the media are left intact.35 Consequently, our findings suggest that an intact endothelium is pivotal for the direct antiatherogenic effect of levormeloxifene and estrogen on the arterial wall. Levormeloxifene and estrogen may act on the endothelium through a number of mechanisms. Lately, interest has focused on the ability of estrogen to increase the production of nitric oxide (NO) in the endothelial cells through an induction of NO synthase enzymes.36 37 NO is a potent endogenous vasodilator but also a putative antiatherogenic molecule, being able to inhibit a number of events in the atherosclerotic process.38 Thus one possible mechanism for the direct antiatherogenic effect of estrogen and levormeloxifene on the arterial wall is an increase in the production and release of NO from the endothelium.
As an alternative, it has recently been suggested that the antiatherogenic effect of tamoxifen is not mediated through a sex steroid mimetic mechanism but via an increased level of the cytokine transforming growth factor-ß (TGF-ß) in plasma and the arterial wall.12 TGF-ß inhibits activation of vascular smooth muscle cells and their subsequent uptake of lipoproteins. Whether levormeloxifene has an influence on TGF-ß concentration in the arterial wall is not known at present.
Effects of Levormeloxifene and d-Ormeloxifene on
Reproductive Tissue
Although levormeloxifene, but not d-ormeloxifene,
produced effects very similar to those of estrogen on plasma
cholesterol levels and the arterial wall, there
was a striking difference in the effect of these compounds on
reproductive tissues. In the female levormeloxifene and
d-ormeloxifene groups, the uterine weight was comparable to
that of the placebo group and only about one-fifth of that in the
estrogen group. In the male levormeloxifene and
d-ormeloxifene groups, the testicular weight was four times
higher than that of the estrogen-treated group. Still, levormeloxifene
produced significant increases in uterine weight compared with placebo.
Preliminary histologic studies in rats suggest that this effect is due
to an action on endometrial stroma and myometrium rather than on
glandular epithelial cells, however. In contrast, tamoxifen has a
strong uterotrophic activity both in experimental animal models and in
women.13 14
Taken together, our results show that levormeloxifene, the levorotatory form of a partial estrogen receptor agonist, lowers plasma cholesterol and has a direct antiatherogenic effect on the undamaged arterial wall without producing appreciable effects on uterine tissue in cholesterol-fed female rabbits. This degree of tissue selectivity has so far not been described for any other compound. The lack of effect of d-ormeloxifene on both plasma cholesterol levels and the arterial wall suggests involvement of the estrogen receptor in the mediation of the antiatherogenic effects of levormeloxifene. Because levormeloxifene had a similar beneficial effect on the arterial wall of male rabbits without having noticeable effects on testicular tissue, this highly selective antiatherogenic compound may also be of interest for men.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by grants from the Danish Heart Foundation. The technical help of Hanne Damm and the help of the staff of the animal care unit, Novo Nordisk, Ganløse, are gratefully acknowledged.
Received May 14, 1996; accepted April 1, 1997.
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