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(Circulation. 2000;102:III-281.)
© 2000 American Heart Association, Inc.
Myocardial Protection and Vascular Biology |
Selective Estrogen Receptor Modulator Idoxifene Inhibits Smooth Muscle Cell Proliferation, Enhances Reendothelialization, and Inhibits Neointimal Formation In Vivo After Vascular Injury
From the Departments of Cardiovascular Pharmacology (T.-L.Y., L.V.-C., J.-L.G., X.L., J.C., B.S., R.W., E.H.O.) and Experimental Toxicology (C.S.L., P.K.N., K.A.G.), SmithKline Beecham Pharmaceuticals, King of Prussia, Pa, and Division of Emergency Medicine, Thomas Jefferson University (X.L.M.), Philadelphia, Pa.
Correspondence to Tian-Li Yue, PhD, Cardiovascular Pharmacology, SB, 709 Swedeland Rd, UW-2510, King of Prussia, PA 19406. E-mail Tian-Li_Yue{at}sbphrd.com
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Abstract |
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Background—Idoxifene (ID) is a tissue-selective estrogen receptor modulator (SERM). The pharmacological profile of ID in animal studies suggests that it behaves like an estrogen receptor (ER) agonist in bone and lipid metabolism while having negligible ER activity on the reproductive system. It is unknown whether ID retains the vascular protective effects of estrogen.
Methods and Results—In cultured vascular smooth muscle cells
(VSMCs), ID inhibited platelet-derived growth factor–induced DNA
synthesis and mitogenesis with IC50 values of 20.4 and 27.5
nmol/L, respectively. Treatment with ID resulted in S-phase cell cycle
arrest in serum-stimulated VSMCs. ID 1 to 100 nmol/L significantly
protected endothelial cells from tumor necrosis
factor-
(TNF-)–induced apoptosis in vitro. Virgin
Sprague-Dawley rats ovariectomized 1 week before the study were treated
with ID (1 mg · kg-1 ·
d-1) or vehicle by gavage for 3 days before
balloon denudation in carotid artery. The SMC proliferation in injured
vessels was determined by immunostaining for
proliferating cell nuclear antigen (PCNA). The number of PCNA-positive
SMCs was reduced by 69%, 82%, and 86% in the media at days 1, 3 and
7, respectively, and by 78% in the neointima at day 7
after injury in ID- versus vehicle-treated group
(P<0.01). ID significantly enhanced
reendothelialization in the injured carotid arteries as
determined by Evans blue stain and immunohistochemical analysis
for von Willebrand factor. In the former assay, the
reendothelialized area in injured vessels was 43% in
ID-treated group versus 24% in the vehicle group
(P<0.05); in the latter assay, the numbers of von
Willebrand factor–positive cells per cross section increased
from 24.8 (vehicle) to 60.5 (ID) (P<0.01) at day 14
after injury. In addition, the production of nitric oxide from
excised carotid arteries was significantly higher in ID-treated than
the vehicle group (8.5 versus 2.7 nmol/g, P<0.01).
Finally, ID treatment reduced neointimal area and the ratio
of intima to media by 45% and 40%, respectively
(P<0.01), at day 14 after balloon angioplasty.
Conclusions—The results indicate that ID beneficially modulates the balloon denudation–induced vascular injury response. Inhibition of VSMC proliferation and acceleration of endothelial recovery likely mediate this protective effect of ID.
Key Words: idoxifene • vasculature • endothelium • cells • restenosis
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Introduction |
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The vascular protective effect of estrogen was first shown in population studies in humans in which estrogen replacement therapy demonstrated a protective effect on atherosclerotic vascular diseases in postmenopausal women,1 2 as later confirmed in ovariectomized (Ovx) monkeys. The vascular protective effect has since been documented in more detail in animal models and in vitro. The recent demonstration of estrogen receptor (ER) expression by vascular smooth muscle cells (VSMCs)3 and endothelial cells4 has further suggested that estrogen may act directly on vascular tissue in addition to its cholesterol-lowering effect. Estrogen has been found to promote vasodilation both in humans and in experimental animals, accompanied by improved endothelial function.5 Estrogen also exerts a direct inhibitory effect on smooth muscle by inhibiting calcium influx and reduces SMC proliferation that may attenuate the progression of atherosclerotic lesions.5
Idoxifene (pyrrolidino-4-iodofamaxifene) is a novel selective estrogen
receptor modulator (SERM) that is defined as a compound that has
estrogen agonism on the desired target tissues such as bone and has
antagonism or minimal agonism in reproductive tissue such as the
breast or uterus. Idoxifene has a 2.4- to 5-fold-greater affinity for
ER than tamoxifen while being significantly less
uterotrophic.6 Idoxifene also binds to ERß (unpublished
data). A recent study7 has demonstrated that idoxifene is
an agonist through the estrogen response element and exhibits
postreceptor effects similar to estrogen in bone-forming osteoblasts.
Idoxifene can both prevent and arrest the loss of bone mineral density
that occurs in the axial and appendicular skeleton in the Ovx rat.
Idoxifene protected against Ovx-induced loss of bone mineral density in
both the lumbar spine and the proximal tibia by suppressing the
increase in bone turnover that occurs on estrogen withdrawal. Moreover,
idoxifene showed an effect similar to estrogen by stimulating
osteoclast apoptosis. These pleiotropic effects of idoxifene
could contribute to the maintenance of bone homeostasis.
However, idoxifene differs from estrogen in a tissue-specific manner.
In human endometrial cells, in which estrogen is a potent agonist
through the estrogen response element, idoxifene has negligible agonist
activity and was able to block estrogen-induced gene expression in
endometrial cells, which is in agreement with observations in the
rat.7 The pharmacological profile of idoxifene in animal
studies suggests that it behaves like an ER agonist in bone and lipid
metabolism while having negligible or no ER agonist
activity on the uterus at doses that prevent bone loss. Therefore,
idoxifene may provide a better profile for the treatment of
postmenopausal diseases. However, it is unknown whether this SERM still
retains the vascular protective effect of estrogen, an important
therapeutic benefit for postmenopausal women who are known to be at
increased risk for atherosclerotic cardiovascular
diseases.
The present study was undertaken to explore the effect of idoxifene on balloon denudation–induced vascular injury response in Ovx rats in which idoxifene has been demonstrated to prevent bone loss while having negligible effect on the reproductive system. A well-established model of carotid artery balloon injury8 was used because the cellular processes controlling the vascular response to injury are well characterized in this model and some of them may be also involved in atherosclerotic vascular diseases. The effects of idoxifene on injury-induced SMC proliferation, reendothelialization, and neointimal formation were studied at a dose that is effective for prevention of bone loss in the Ovx rat.7 In addition, the effects of idoxifene on VSMC proliferation and endothelial cell apoptosis in vitro were also examined.
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Methods |
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Cell Culture
Female human coronary SMCs (FHCSMCs) from an adult (age, 39 years) were purchased from Clonetics (lot 16810), and female rat aortic SMCs (FRASMCs) were isolated from medial explants from the thoracic aorta of female Sprague-Dawley rats as described previously.9 The cells under passage 6 were used in this study. Bovine pulmonary arterial endothelial cells (BPAECs) (passages 17 through 20) were obtained from the American Type Culture Collection.
DNA Synthesis
DNA synthesis in FHCSMCs was assessed by measurement of the
incorporation of [3H]thymidine (New England
Nuclear) as reported previously.9 FHCSMCs were grown in
24-well plates in DMEM phenol-free media containing 10%
charcoal-stripped FBS (Hyclone) and subsequently rendered quiescent
with DMEM media without FBS for 48 hours. Idoxifene (SmithKline
Beecham) 0.001 to 1 µmol/L or vehicle was added 15 minutes
before the FCSMCs were challenged with platelet-derived growth
factor (PDGF; 1 nmol/L; Boehringer Mannheim) for 24 hours.
[3H]thymidine (1 µCi per well) was added for
a further 4-hour incubation. DNA synthesis was assessed by measuring
the radioactivity incorporated into the trichloracetic acid–insoluble
fraction of the cells. The results were from 5 separate experiments
performed in triplicate.
Cell Proliferation Study
FHCSMCs were prepared as above and treated with vehicle or
idoxifene (0.01 to 1 µmol/L) for 15 minutes before the addition
of PDGF (1 nmol/L), and incubation was continued for 5 days. The basal
level of cell proliferation was measured from the cells treated with
vehicle only. At the end of incubation, cells were harvested and
counted.9 The results were from 5 separate experiments
performed in triplicate.
Cell Cycle Study by Fluorescence-Activated Cell Sorting
Analysis
FRASMCs were cultured in 6-well plates and made quiescent for 72
hours in phenol-free DMEM without FBS. Idoxifene (100 nmol/L) or
vehicle was added to the cells 30 minutes before the addition of FBS
(10% final) and incubated for 48 hours. At the end, cells were
suspended in methanol (1x106 cells/mL) and
stained with propidium iodide. The DNA content of the cells was
analyzed by a Becton-Dickinson FACSCalibur flow
cytometer. Fluorescent measurements collected from the cells
were analyzed by the Cellquest (BDIS) data
analysis software, followed by cell cycle analysis of
DNA histograms with ModFitLT.10
Morphological Assessment and Quantification of Apoptosis in
Endothelial Cells
BPAECs were cultured in phenol-free DMEM containing 2%
estrogen-free serum for 24 hours and treated with vehicle or idoxifene
(100 nmol/L) for 30 minutes before the addition of tumor necrosis
factor- (TNF-; 5 ng/mL) and incubation continued for the
indicated times (time dependence), or the cells were treated with
vehicle or idoxifene at the concentrations indicated in Figure 3B
for 30 minutes before the addition of TNF- (5 ng/mL) and
incubation continued for 24 hours (concentration dependence). To
quantify endothelial cells undergoing
apoptosis, cell monolayers were fixed and stained with Hoechst
33324 (molecular probe). The morphological features of
apoptosis (cell shrinkage, chromatin condensation, blebbing,
and fragmentation) were monitored by fluorescence microscopy.
At least 500 cells from 13 randomly selected fields per dish were
counted as described previously.11
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Rat Carotid Artery Balloon Angioplasty
All procedures were reviewed and approved by the Animal Care and
Use Committee at SmithKline Beecham Pharmaceuticals. Virgin female
Sprague-Dawley rats (Charles River) at 7 to 9 months of age (weight,
350 to 400 g) were bilaterally ovariectomized 1 week before
study7 and then treated orally with either idoxifene (1
mg/kg, once a day) or vehicle for 3 days before balloon angioplasty.
Idoxifene was suspended in 1% aqueous solution of carboxymethyl
cellulose as described previously.7 Left common carotid
artery balloon angioplasty was performed under aseptic
conditions,8 and daily oral administration of idoxifene or
vehicle was continued after surgery until the animal was killed. The
animals were killed on days 1, 3, 7, and 14 after surgery for
proliferating cell nuclear antigen (PCNA) study or at day 14 for
reendothelialization or neointimal
formation studies (see below).
Neointimal Formation
The extent of neointimal formation was quantified
histologically in left common carotid arteries 14 days
after balloon angioplasty as described previously.8 After
the carotid arteries were removed from each rat, 
0.5-cm segments
from both ends were removed before embedding in paraffin wax. This
approach avoided removal of the areas close to edges where the damage
could be low and give false-positive results. Four equidistant sections
per artery were measured for entire circumference, lumen, media, and
neointima with a Bioscan Optimas cell imaging
system.8
Evaluation of SMC Proliferative Activity in Injured Carotid Artery
(PCNA Assay)
The injured arterial segments were harvested, and
the SMC proliferative activity was evaluated by immunohistochemical
analysis for PCNA as described previously.12 13
The extent of proliferative SMCs was determined by counting
PCNA-positive SMCs in intima and media. Four sections from each artery
and 10 fields per section were reviewed and scored by a board-certified
pathologist. The percentage of PCNA-positive cells versus total counted
cells was calculated.
Evaluation of Reendothelialization by Evans
Blue Staining
Two weeks after angioplasty, the Ovx rats treated with idoxifene
(1 mg · kg-1
· d-1) or vehicle were
anesthetized and injected with sterile Evans blue dye via the
femoral vein. A half-hour later, the animal was perfused with saline in
situ at a pressure of 90 mm Hg until the effluent ran clear,
followed by 5 minutes of fixation with 100% methanol. The carotid
arteries were harvested as described above, incised longitudinally, and
then pinned to a tray containing wax for photography with a dissecting
microscope. Planimetric analysis with an Optimas 6.2 Image
analysis program was performed. The
reendothelialized area was defined macroscopically as
the area not stained with Evans blue dye.
Reendothelialization was expressed as
reendothelialized area versus the total denuded area as
described previously.13
Detection of Reendothelialization by von
Willebrand Factor Immunohistochemistry
The carotid arteries were harvested and processed as described
above. Tissue (5-µm) tissue slides were deparaffinized, rehydrated,
and placed in PBS with 0.05% Tween. The primary antibody used was a
rabbit anti–von Willebrand factor (DAKO) followed by an
anti-rabbit IgG biotinylated secondary (Vector Laboratories). Tissues
were incubated in a dilution of streptavidin-hrp (DAKO) followed by
3'3'-diaminobenzidine (DAB). The slides were counterstained with
hematoxylin, dehydrated, and placed in coverslips. Six fields per
section and 4 sections per artery were counted (6 rats per group).
Measurement of Nitric Oxide From Vessel Segments
To determine whether functional recovery of the
endothelium was accelerated by idoxifene, the
production of nitric oxide by excised carotid
arterial segments was measured by modification of a method
reported previously.14 The balloon-injured carotid
arteries were collected from the Ovx rats at day 14 after injury.
Vascular segments were rinsed with PBS, cleaned of connective tissue,
and homogenized in PBS (1:100, wt:vol). The nitric oxide
concentrations in the supernatant were quantified by a
chemiluminescence detector (Sievers 270B NO Analyzer).
Data Analysis
Statistical analysis was performed by Dunnett’s test
with a program provided by MicroComputer Specialists. A value of
P<0.05 was considered significant.
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Results |
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Inhibition of PDGF-Stimulated DNA Synthesis and Proliferation in FCSMCs
In FCSMCs, PDGF stimulated a significant increase in [3H]thymidine incorporation into the cells that was similar to that reported previously.9 The count-per-minute values in vehicle and PDGF-treated FCSMCs were 719±78 and 1616±162 (P<0.01, n=5), respectively. Idoxifene exerted a concentration-dependent inhibition of PDGF-stimulated [3H]thymidine incorporation with an IC50 value of 20.4 nmol/L (Figure 1A
). As shown in Figure 1B, idoxifene inhibited PDGF-induced FCSMC proliferation
(IC50=27.5 nmol/L).
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Inhibition of Serum-Induced Cell Cycle Progression in
FRASMCs
Serum-stimulated cell cycle progression in quiescent FRASMCs and
the effect of idoxifene are shown in the Table
(n=3). In the presence of 100 nmol/L idoxifene, the entering of
SMCs into G2/M from S phase was completely
abrogated with a concomitant increase in the percentage of cells in S
phase, indicating that idoxifene arrested the cells in S phase. Figure 2 is a representative
measurement of DNA distribution in serum-stimulated FRASMCs in the
absence or presence of 100 nmol/L idoxifene.
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Idoxifene Protects Endothelial Cells From
TNF-–Induced Apoptosis In Vitro
Endothelial cells exposed to TNF- (5 ng/mL;
Genzyme) and stained with Hoechst 33324 displayed morphological
features of apoptotic cell death: condensed chromatin,
fragmented nuclei, and blebbing of the plasma membrane as reported
previously11 (data not shown). Figure 3A
demonstrates a time dependence of
TNF-–induced apoptosis in endothelial cells
and the protective effect of idoxifene at 100 nmol/L. Figure 3B
shows a concentration-dependent protection by idoxifene of
TNF-–induced endothelial cell apoptosis.
The number of apoptotic cells over the basal level was reduced
by 66.3% in the presence of 100 nmol/L idoxifene.
Inhibition of Transluminal Balloon Injury–Induced Carotid Artery
SMC Proliferation
Proliferative SMCs in the injured carotid arterial
segments harvested on days 1, 3, 7, and 14 after injury were evaluated
by histochemical analysis for PCNA. In vehicle-treated animals,
the number of PCNA-positive SMCs in the media was significantly
increased on days 1, 3 (peak), and 7 after injury and then reduced to
an undetectable level (Figure 4A). In the
idoxifene-treated group, an increased number of PCNA-positive SMCs was
observed on day 3 after injury but to a much smaller degree compared
with idoxifene-treated rats (Figure 4A). A remarkable increase
in the number of PCNA-positive SMCs was also demonstrated in the intima
7 days after injury in vehicle-treated rats, which was much higher than
the number of PCNA-positive SMCs in idoxifene-treated group at the same
time (Figure 4B). Figure 4C is a
representative photomicrograph demonstrating PCNA
immunostaining in the intima and media on days 3 and 7
after balloon injury in idoxifene- (A and B) and vehicle- (C and D)
treated rats.
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Idoxifene Enhances Reendothelialization Detected by
Evans Blue Stain and Von Willebrand Factor Immunohistochemical
Analysis
Figure 5 (top) illustrates the
examples of left carotid arteries harvested from sham-operated or
balloon angioplasty rats treated with vehicle or idoxifene. Evans blue
staining identifies segments of each injured carotid artery that has
not been reendothelialized. No staining was found in
sham-operated vessel (A), but almost the entire area of the artery
harvested immediately after denudation was stained by Evans blue (B).
At 2 weeks after denudation, the area of
reendothelialization in vehicle-treated artery (C) was
significantly smaller than that in idoxifene-treated artery (D). The
bottom panel of Figure 5 shows the quantitative results. The
percentage of area in injured carotid arteries covered by
endothelium was nearly 2-fold higher in the
idoxifene-treated group than that observed in the vehicle-treated group
(P<0.05, n=12).
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Figure 6 shows the results of
immunohistochemical study for von Willebrand factor (factor VIII).
Sections of uninjured left carotid arteries showed a linear
immunofluorescence staining for factor VIII in the
endothelium but not in the SMCs or fibroblasts of the
media, as shown in the top panel (A). The linear staining for factor
VIII was not observed in the cross section of the left carotid artery 1
day after denudation (B). The cross section of the injured carotid
artery from the idoxifene-treated rats (D) but not from the
vehicle-treated rat (C), showed a linear positive staining for factor
VIII on the surface of the intima. The bottom panel is the quantitative
measurement of the number (per section) of endothelial
cells (factor VIII–positive cells) in the left carotid arteries from
idoxifene- and vehicle-treated rats (6 fields per section, 4 sections
per rat). The number of endothelial cells per section
was increased from 24.8±2.2 (vehicle) to 60.5±2.1 (idoxifene)
(P<0.01, n=6).
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Nitric Oxide Production Is Increased in
Idoxifene-Treated Arteries
Nitric oxide production by the injured left carotid
arteries was significantly increased from 2.71±0.67 (vehicle) to
8.52± 1.31 (idoxifene) nmol/g tissue (P<0.01, n=12 to 13)
at 14 days after balloon angioplasty as shown in Figure 7. There was no difference in the plasma
levels of nitric oxide between the 2 groups.
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Idoxifene Reduces Neointimal Formation After Carotid
Denudation in the Ovx Rat
The total neointimal area of the left carotid arteries
measured 2 weeks after balloon denudation was 0.13±0.01
mm2 in vehicle-treated animals compared with
0.07±0.01 mm2 in the idoxifene-treated
group (45% reduction, P<0.01, n=15). The calculated
intima/media ratios also displayed a significant decrease in the
idoxifene-treated animals compared with the vehicle group
(P<0.01) (Figure 8, top). The
bottom panel of Figure 8 shows representative
cross sections of left carotid arteries from a sham-operated rat (A), a
vehicle-treated rat (B), and an idoxifene-treated rat (C)14 days after
angioplasty.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The concept of SERM has been demonstrated for a number of compounds, including idoxifene. However, the molecular mechanisms for the tissue-specific effect of SERM are not well understood. Two types of ER (ER
and ERß) have been identified, and their tissue
distribution and functions have been the focus of intense
studies.15 16 Adding to the complexity, recent studies
have indicated that transcriptional regulation by ER depends on a
variety of coactivators and corepressors, and more novel
proteins may be involved.17 18 The effect of SERM on these
cofactors is not known. Because of this complexity, the profile of
tissue-specific effects of each SERM is still unpredictable and was
based mainly on animal and clinical studies. The objective of the
present study is to find whether idoxifene, a new SERM, retains
vascular protective effects of estradiol.
VSMC proliferation is one of the major vascular remodeling processes
after vascular injury that may participate in the vaso-occlusive
disorders associated with multiple vascular diseases.19 In
the present study, the antiproliferative effects of idoxifene on
VSMCs were demonstrated in vitro and in vivo. Medial SMC proliferation
appeared immediately after balloon injury and peaked at day 3 (Figure 4), which is consistent with the observation reported
previously.20 Proliferative VSMCs also appeared in the
intima 5 to 7 days after injury when the intimal formation was clearly
demonstrated. The selective ER modulator idoxifene significantly
reduced the number of proliferative VSMCs in both the media and intima.
In vitro studies further demonstrated a direct inhibitory
effects of idoxifene on PDGF-stimulated VSMC proliferation, similar to
that reported in 17ß-estradiol–treated VSMCs.21
We were interested in exploring further the hypothesis that ER may
exert an effect on cell cycle progression in VSMCs. The data
presented in the Table and Figure 2 indicate
clearly that cells treated with idoxifene encountered an S-phase arrest
accompanied by a gradual decrease in SMCs from the
G2/M phase. The data agree with the results
obtained from in vitro [3H]thymidine
incorporation and proliferation studies. The cell cycle progression of
SMCs from S to G2/M phase was hindered by
idoxifene leading to an overall reduction in the total number of cells
undergoing cell division. As a result,
[3H]thymidine incorporation was decreased
significantly in idoxifene-treated SMCs.
Endothelial cells affect the homeostasis of the vessel wall in terms of vasomotor tone, platelet and monocyte adhesion, growth of SMCs, and extracellular matrix production and thereby provide an antithrombotic and anti-inflammatory barrier for the normal vessel wall. Disruption of the anatomic and functional integrity of the endothelium has been postulated as a mechanism for the initiation of atherosclerosis.19 Recent data have suggested that after arterial injury, acceleration of reendothelialization by 17ß-estradiol is associated with attenuation of intimal hyperplasia.22 23 Accordingly, we performed a series of experiments to determine whether idoxifene accelerates reendothelialization after arterial injury. Two weeks after balloon injury, the idoxifene-treated animals showed more rapid recovery of the endothelium and its barrier function as demonstrated by exclusion of Evans blue dye and immunohistochemical study for factor VIII in the vessel. Furthermore, recovery of endothelial function, manifested as enhanced nitric oxide production, was also shown to be accelerated by idoxifene treatment. The data are consistent with recent studies that demonstrated that 17ß-estradiol accelerated reendothelialization after arterial injury.22 23
It has been suggested that increased endothelial cell
turnover mediated through accelerated apoptosis plays a role in
endothelial disruption.24 To further
explore the mechanisms for the enhancement of
reendothelialization by idoxifene, we studied the
effect of idoxifene on TNF-–induced apoptosis in
endothelial cells. TNF- is known to be secreted by
macrophages, activated T cells,25 and SMCs
after vascular injury.26 TNF-, as a
proapoptotic factor for endothelial cells, has
been demonstrated in vitro in human and nonhuman
species.24 27 The reported upregulation of TNF-
expression in animal models of arterial injury by balloon
angioplasty26 and in human coronary artery
restenotic lesions28 provides further evidence
that regulation of the expression of this cytokine may be
functionally important in vivo.29 The data
presented in Figure 3 demonstrate the protection by
idoxifene of endothelial cell apoptosis induced
by TNF-. The protective effect was even observed at 1 nmol/L
idoxifene. However, the maximal protection by idoxifene was 66%, and
increasing the concentration of idoxifene did not further enhance the
protective effect, indicating that not all proapoptotic signals
implicated in TNF-–induced apoptosis were affected by
idoxifene. Our data are in accordance with a recent study that
demonstrated the protective effect of 17ß-estradiol against
TNF-–induced apoptosis in cultured
endothelial cells.29 It has been suggested
that estradiol, by protection of endothelial cell from
apoptosis and enhancement of endothelial cell
adhesion, may help migrating cells to colonize in injured vessel and
aid in reendothelialization.24 This could
also be a mechanism for idoxifene in enhancing
reendothelialization.
The enhanced regeneration of endothelial cells by idoxifene appeared to contribute to suppression of intimal hyperplasia. Animals in the idoxifene-treated group showed less intimal thickening than those in the vehicle-treated group, consistent with the reported data of 17ß-estradiol in the same model.30 31 The notion that this observed differential in neointimal thickening truly represents a differential degree of intimal hyperplasia is supported by the results of PCNA immunostaining. The number of PCNA-positive SMCs in both media and neointima was significantly higher in the vehicle-treated than idoxifene-treated group. The inverse relationship between reendothelialization and neointimal SMC proliferation indicates that neointimal thickening thus developed in association with delayed reendothelialization, and the vascular protection provided by idoxifene is most likely through inhibition of VSMC proliferation and acceleration of reendothelialization.
In summary, the present study has demonstrated for the first time the beneficial vascular remodeling effect of idoxifene in a balloon denudation rat model. Both inhibition of SMC proliferation and acceleration of endothelial regeneration likely mediate this vascular protection. The favorable effect of idoxifene on vessel wall injury, together with its cholesterol-lowering action,7 would be expected to have implications for clinical benefit. The data from the present study also suggest that SERM is able to retain the favorable vascular protective effect while avoiding unwanted effects of estrogen on the reproductive system. This could be another therapeutic advantage to the use of SERM in postmenopausal women.
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