Activation of estrogen receptor-alpha reduces aortic smooth muscle differentiation.
ABSTRACT Women are at high risk of dying from unrecognized cardiovascular disease. Many differences in cardiovascular disease between men and women appear to be mediated by vascular smooth muscle cells (SMC). Because estrogen reduces the proliferation of SMC, we hypothesized that activation of estrogen receptor-alpha (ERalpha) by agonists or by growth factors altered SMC function. To determine the effect of growth factors, estrogen, and ERalpha expression on SMC differentiation, human aortic SMC were cultured in serum-free conditions for 10 days. SMC from men had lower spontaneous expression of ERalpha and higher levels of the differentiation markers calponin and smooth muscle alpha-actin than SMC from women. When SMC containing low expression of ERalpha were transduced with a lentivirus containing ERalpha, activation of the receptor by ligands or growth factors reduced differentiation markers. Conversely, inhibiting ERalpha expression by small interfering RNA (siRNA) in cells expressing high levels of ERalpha enhanced the expression of differentiation markers. ERalpha expression and activation reduced the phosphorylation of Smad2, a signaling molecule important in differentiation of SMC and initiated cell death through cleavage of caspase-3. We conclude that ERalpha activation switched SMC to a dedifferentiated phenotype and may contribute to plaque instability.
- [Show abstract] [Hide abstract]
ABSTRACT: Arterial elastic properties and wave reflections constitute left ventricular pulsatile afterload and are directly related to cardiovascular morbidity and mortality. During the aging process, elastic arteries stiffen and increase pulse wave velocity, causing the reflected pressure wave to arrive at the heart during systole, and augment systolic and pulse blood pressure, resulting in left ventricular hypertrophy. The incidence of cardiovascular disease is much greater in men aged 30-50 years compared with women of a similar age. Among women, the incidence of cardiovascular disease increases dramatically after menopause, which occurs at an average age of 52 years. This observation has lead to the belief that sex hormones produced premenopausally impede or delay the progression of cardiovascular disease. Therefore, it appears logical that the administration of female sex hormones, hormone treatment (HT), estrogen alone or with progesterone should provide some degree of cardiovascular protection. This idea was supported by experiments in animals and isolated arterial segments demonstrating that HT increases plasma nitric oxide, decreases endothelin-1 levels and causes smooth muscle relaxation. Also, 17β-estradiol administration decreases collagen and increases elastin in the aortic wall of rats. These experimental studies suggest an improvement in both elastic and muscular artery properties and favorabe modifications of arterial wave reflection characteristics. Furthermore, HT improves lipoprotein metabolism and reduces coronary artery plaque formation in animal models. Unfortunately, observational and interventional studies in postmenopausal women that have evaluated the impact of HT on cardiovascular changes have produced inconsistent and inconclusive results. Most studies agree that arterial stiffness increases after menopause, partly due to advancing age and reduced estrogen production. Results from most studies that were designed to investigate the effects of HT on arterial properties have shown a selective decrease in elastic artery stiffness with little effect on muscular arteries. This beneficial effect was observed only if estrogen alone was administered. The main objective of this review is to discuss the ill effects of arterial stiffness in general and attempt to translate information from previous experimental studies to those in postmenopausal women and explain the beneficial effects of HT on arterial stiffness and improvement in left ventricular afterload.Expert Review of Endocrinology & Metabolism 08/2007; 2(5):653-665.
- [Show abstract] [Hide abstract]
ABSTRACT: Sex steroid hormones differently control the major physiological processes in male and female organisms. In particular, their effects on vascular smooth muscle cells (VSMCs) migration are at the root of sex/gender-related differences reported in the cardiovascular system. Several exogenous substances, defined endocrine disruptor chemicals (EDCs), could interfere with these androgen and estrogen effects; however, the sex/gender-related susceptibility of VSMC motility to EDCs is completely unknown. Here, the effect of naturally occurring (naringenin, Nar) and synthetic (bisphenol A, BPA) EDCs on male and female VSMC motility has been evaluated. 17β-estradiol (E2, 0.1 nM to 1 µM) induced a dose-dependent inhibition of motility in female-derived VSMC. In contrast, neither dihydrotestosterone (DHT, 0.01 to 100 nM) nor the common precursor of sex steroid hormones, testosterone (Tes, 0.01 to 100 nM) modified male-derived VSMC motility. Estrogen receptor (ER) β subtype-dependent activation of p38 was necessary for the E2 effect on cell motility. High BPA concentration prevented E2 effects in female-derived cells being without any effect in male-derived cells. Nar mimicked E2 effects on female-derived cells even in the presence of E2 or BPA. Intriguingly, Nar also inhibited the male-derived VSMC mobility. This latter effect was prevented by ERβ inhibitor, but not by the androgen receptor (AR) inhibitor. As a whole, ERβ-dependent signals in VSMC results more susceptible to the impact of EDCs than AR signals suggesting a possible high and overall susceptibility of female to EDCs. However, several male-derived cells, including VSMC, express ERβ which could also serve as target of EDC disruption in male organisms. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.Journal of Cellular Physiology 12/2013; · 3.87 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: The anti-inflammatory and neuro-protective effects of estrogen on multiple sclerosis (MS) have been reported in previous studies. Evidence has been found that estrogen can inhibit axonal loss in the MOG-induced experimental autoimmune encephalomyelitis (EAE) model of MS. Rho-kinase (ROCK) mediates axonal growth-inhibitory signals via the Rho/ Rho-kinase pathway. The inhibition of ROCK decreases axonal loss in EAE. However, there is no study reporting the association between estrogen and ROCK in MS and EAE. We examined the anti-inflammatory and axonal protective effects of estrogen and explored the probable mechanism whereby estrogen inhibits ROCK through ERα in EAE rats. Results show that 17β-estradiol (E2) can significantly avoid the loss of neurological function in EAE rats. E2 also decreased the inﬁltration of inﬂammatory cells and cytokines including IL-1β, TNF-α and IL-17, but increased the expression of IL-4. E2 inhibited the expression of ROCK and NF-200 in EAE rats. The inhibitory effect on ROCK was abolished when non-selective estrogen receptor(ER) antagonist ICI 182780 was added to E2. Furthermore, the expression of ROCK was inhibited by ERα-selective ligand agonist propyl pyrazole triol (PPT). ERβ-selective ligand WAY-202041 has no effect on the expression of ROCK. These observations suggest that estrogen inhibits the expression of ROCK in EAE rats, and that the inhibitory effects are mediated by ERα rather than ERβ.Synapse 02/2013; · 2.43 Impact Factor
Activation of Estrogen Receptor-? Reduces Aortic Smooth
Christine R. Montague, Melissa G. Hunter, Mikhail A. Gavrilin, Gary S. Phillips,
Pascal J. Goldschmidt-Clermont, Clay B. Marsh
Abstract—Women are at high risk of dying from unrecognized cardiovascular disease. Many differences in cardiovascular
disease between men and women appear to be mediated by vascular smooth muscle cells (SMC). Because estrogen
reduces the proliferation of SMC, we hypothesized that activation of estrogen receptor-? (ER?) by agonists or by
growth factors altered SMC function. To determine the effect of growth factors, estrogen, and ER? expression on SMC
differentiation, human aortic SMC were cultured in serum-free conditions for 10 days. SMC from men had lower
spontaneous expression of ER? and higher levels of the differentiation markers calponin and smooth muscle ?-actin
than SMC from women. When SMC containing low expression of ER? were transduced with a lentivirus containing
ER?, activation of the receptor by ligands or growth factors reduced differentiation markers. Conversely, inhibiting ER?
expression by small interfering RNA (siRNA) in cells expressing high levels of ER? enhanced the expression of
differentiation markers. ER? expression and activation reduced the phosphorylation of Smad2, a signaling molecule
important in differentiation of SMC and initiated cell death through cleavage of caspase-3. We conclude that ER?
activation switched SMC to a dedifferentiated phenotype and may contribute to plaque instability. (Circ Res.
Key Words: apoptosis ? cardiovascular disease ? gene expression ? nuclear receptors
?smooth muscle differentiation
nosed disease is lower among women.1Women have higher
rates of stable angina, high blood pressure, congestive heart
failure, and stroke but have less angiographic evidence of
atherosclerotic plaques and have fewer myocardial infarc-
tions than men.1The inhibition of collagen production,
smooth muscle proliferation, and endothelial dysfunction by
estrogen may delay the formation of plaques in women until
after menopause.2Hormone replacement therapy started more
than 10 years after menopause increases a woman’s risk for
myocardial infarction,3,4although therapy initiated near
menopause may be more effective in preventing coronary
heart disease.5Additionally, because of gender differences in
symptoms during acute coronary events and in response to
interventional strategies, it is difficult to correctly diagnose
and treat women.6,7
A few studies comparing vascular wall properties and
disease presentation of men and women with symptoms of
coronary artery disease provide insight into the complicated
effects of female hormones and their receptors in vascular
cells. Although women with acute coronary syndromes are
often free of angiographically visible stenoses, testing of
ince 1984, more women than men have died from
cardiovascular diseases, although the prevalence of diag-
coronary flow reserve demonstrates endothelial and smooth
muscle dysfunction.8,9Younger women who die from coro-
nary artery thrombosis are more likely than men or postmeno-
pausal women to have plaque erosion, rather than rupture of
a lipid-rich plaque.10,11Plaque erosions are characterized by
loss of endothelial cells covering a nonocclusive, smooth
muscle cell (SMC)- and hyaluronan-rich plaque with few
inflammatory cells or type I collagen.12It is speculated that
migration of dedifferentiated SMC and expression of hyalu-
ronan weakens endothelial cell adhesion and predisposes the
coronary arteries for thrombotic events.12
After menopause, women experience a dramatic rise in
aortic stiffness, which may cause hypertension.13In those
who develop coronary artery disease, the plaques become
more numerous with larger lipid cores and thinner fibrous
caps marked by calcification.11It is uncertain how matrix
deposition and plaque stability are affected by the lack of
estrogen or by growth factors that activate the estrogen
Because SMC are responsible for many of the differences
in coronary disease noted between men and women, such as
microvessel dysfunction, plaque erosion, and matrix deposi-
tion, we sought to understand the role of ER? in smooth
Original received June 2, 2006; revision received July 3, 2006; accepted July 17, 2006.
From the Department of Medicine (C.R.M., M.G.H., M.A.G., C.B.M.), Ohio State University College of Medicine, Columbus; Center for Biostatistics
(G.S.P.), Ohio State University, Columbus; and School of Medicine (P.J.G.-C.), University of Miami, Fla.
Correspondence to Clay B. Marsh, The Ohio State University Heart and Lung Research Institute, Division of Pulmonary and Critical Care Medicine,
473 W 12th Ave, Columbus, OH 43210. E-mail Clay.Marsh@osumc.edu
© 2006 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.orgDOI: 10.1161/01.RES.0000238376.72592.a2
muscle differentiation in estrogen- or growth factor–rich
environments to mimic gender or menopausal effects. In this
study, we obtained aortic SMC from male and female donors
and determined the effects of ER? expression, estrogen, and
growth factors on differentiation, survival, and adherence of
Materials and Methods
Explantation and SM ?-Actin Detection
Following informed consent, sections of aorta were obtained from
heart transplant donors and recipients at The Ohio State University
Medical Center, as approved by the institutional review board. Aortic
slices were stripped of endothelium and adventitia, rinsed, and cut
into small bits. The average age (?SEM) for females and males
examined in this study was 32.6?6.39 and 47.4?8.62, respectively
(n?5 each). No statistical difference in age was observed between
the donors and recipients or between genders. The SMC were
expanded in growth media with amphotericin and gentamicin (Clo-
netics/Cambrex, Walkersville, Md, and Cascade Biologics, Portland,
Ore), then tested for smooth muscle (SM) ?-actin expression using
FACS Calibur flow cytometer (BD Biosciences, San Jose, Calif).
Cell populations containing at least 85% positive staining for SM
?-actin were used for subsequent studies.
Real-Time PCR for ER?
ER? mRNA was analyzed by real-time PCR in SMC from 5 male
donors and 5 female donors that were starved for 5 days to allow
ER? upregulation. TaqMan primers and probe designed by Primer 3
were synthesized by Applied Biosystems (Foster City, Calif). The
following primers were used to detect ER?: forward, 5?-
agctcctcctcatcctctcc-3?; reverse, 5?-tctccagcagcaggtcatag-3?; and
probe 5?-6FAM-tcaggcacatgagtaacaaaggca-TAMRA-3?. RNA was
isolated using NucleoSpin RNA II (BD Clontech, Mountain View,
Calif), and cDNA generated using random hexamers (Invitrogen,
Carlsbad, Calif). A 111-bp product from ER? was amplified over 40
cycles with 18S RNA as internal control using the ABI PRISM 7700
Sequence Detection System (Applied Biosystems).
Cloning of ER? into EGFP-pLenti6/V5 Plasmid
The pLenti6/V5-D-TOPO vector (Invitrogen) was engineered to
contain an enhanced green fluorescent protein (EGFP) surrounded by
additional restriction sites and designated pLenti-EGFP (generously
provided by Mark Wewers, Ohio State University). cDNA for ER?
was amplified by PCR from a pBK-CMV/ER? plasmid kindly
provided by Robert Brueggemeier (Ohio State University), introduc-
ing EcoRI and EcoRV restriction sites. EGFP was removed from
pLenti-EGFP by digestion with EcoRI and EcoRV and replaced with
ER? to generate pLenti-ER?. pLenti vector lacking EGFP was used
as a control. Purified pLenti-ER? or empty vector control (3 ?g)
were transfected with 2 ?g of pMD.G and 10 ?g of pCMV?R8.2
helper plasmids (kindly provided by Dr K. Boris-Lawrie, Ohio State
University) into HEK293FT cells according to the directions for the
ViraPower Lentiviral Expression System. Virus secreted into the
media was concentrated (Vivaspin 100 000 MWCO; Vivascience,
Germany) and titered in SMC cultures, with blasticidin (2 ?g/mL)
for selection. SMC were then transduced with the virus for each
experiment at approximately 5 multiplicity of infection and incu-
bated overnight in growth media containing 6 ?g/mL polybrene.
Transfection of Small Interfering RNA Plasmids
SMC (1?106) were transfected with 10 ?g of control or ER? small
interfering RNA (siRNA) plasmid (Panomics, Redwood City, Calif)
using nucleofection (Amaxa, Gaithersburg, Md). Transfection effi-
ciency was monitored using 2 ?g of pmaxGFP plasmid (Amaxa).
SMC Differentiation and Activation
Differentiation experiments were performed on SMC in the follow-
ing groups, seeded in an 8-well plate as noted: native cells expressing
endogenous ER? (7?104cells per well), cells with low expression of
ER? to be transduced with ER? lentivirus (8?104), and cells with
high ER? levels transfected with ER? siRNA (1.8?105). After
recovery, the cells were starved overnight in phenol red and
serum-free basal media (EBM-PRF) (Clonetics/Cambrex) and ex-
posed for 10 days to vehicle control (either 4 ?mol/L HCL or
1:400 000 dilution ethanol), 17?-estradiol (10 nmol/L; Sigma, St
Louis, Mo), the ER? agonist propyl pyrazole triol (PPT) (10 nmol/L;
Tocris Cookson, Ellisville, Mo), epidermal growth factor (EGF) (10
ng/mL, R&D Systems, Minneapolis, Minn), platelet-derived growth
factor-BB (PDGF-BB) (10 ng/mL; R&D Systems), or transforming
growth factor-?1 (TGF?1) (5 ng/mL; R&D Systems) in EBM-PRF.
Agonists or vehicle controls were added each day and then cells were
lysed in cell lysis buffer (Cell Signaling Technology, Danvers,
Mass). Samples of the culture media at the end of the experiment
were quantitated for active TGF?1 by ELISA (Quantikine, R&D
Systems). Activation studies were performed on SMC stably trans-
duced with pLenti control or ER? (7?104cells per well or 2?105
cells per 25-cm2flask), incubated for the times indicated using
agonists as listed above, then lysed with cell lysis buffer or CelLytic
NuCLEAR Extraction Kit (Sigma). Equal protein amounts (20 to 50
?g) were subjected to Western blot analysis and detected with
SuperSignal West Femto Maximum Sensitivity Substrate (Pierce,
Rockford, Ill) and the Fluor S-Max system (Bio-Rad, Hercules,
Calif). Smooth muscle (SM) ?-actin, ?-actin, and calponin antibod-
ies were obtained from Sigma. Antibodies to phospho-ER? and
cleaved caspase-3 were from Cell Signaling Technology. Cyclin D1
(clone DCS-6), Erk2 and ER? (HC20) antibodies were obtained
from Santa Cruz Biotechnology (Santa Cruz, Calif).
ER? Transcriptional Activation
Stably transduced SMC were transfected with an estrogen response
element (ERE) reporter construct producing secreted alkaline phos-
phatase (SEAP) (Clontech/BD Biosciences) using Effectene (Qia-
gen, Valencia, Calif). The SEAP signal was obtained over 3 days and
normalized as a percentage of the maximum signal achieved.
Immunofluorescence for ER?
Virally transduced SMC were fixed in 70% ETOH, permeabilized
and blocked with 0.05% triton/1% goat serum. Cells were incubated
overnight with ER? antibody (Ab-16, Lab Vision-Neomarkers,
Fremont, Calif) in 1% goat serum. ER? was detected with Alexa
Fluor 568 anti-rabbit secondary antibody (Molecular Probes, Invitro-
gen) and a DP-11 digital camera connected to an IX-50 inverted
microscope with 10? objective (Olympus, Melville, NY).
Phase contrast images were taken using identical settings at day 10
of the differentiation experiments using the DP-11 digital camera and
IX-50 inverted microscope with 4? objective (Olympus). Quantity
One colony counting software (Bio-Rad) was used to detect live cells
(gray) but exclude apoptotic cells (white). Numbers were normalized
to vehicle control samples for each cell population or control
Real-time PCR results for ER? expression were analyzed using
longitudinal regression over 10 experiments to test the difference in
? cycle times, which are normally distributed. Western blot densi-
tometry ratios for contractile proteins in starved or PDGF-stimulated
SMC from 6 people were compared using a mixed model regression
to account for correlation within cell lines. Densitometry values from
the remaining immunoblots were normalized to loading controls and
by the vehicle control sample for the control group, compared by
2-factor ANOVA (Stata version 9; StataCorp, College Station, Tex),
and pairwise comparisons were adjusted using the Holm’s method.15
478 Circulation Research
September 1, 2006
Low-Level Expression of ER? in Human Aortic
Estrogen receptors are present in healthy aortic SMC and
regulate growth.2Because genes that affect cell growth often
change cell differentiation, and ER? enhances proliferation in
transformed cells, we hypothesized that the expression and
activation of SMC ER? modulated cell differentiation. The
ER? mRNA level, stated as a fold induction above the SMC
population containing the lowest level of ER?, was ?4.3
times higher on average for female donors than for male
donors (P?0.001, Figure 1A). By comparison, serving as a
positive control, the ER? level for the breast cancer line
MCF7 was ?1000 times higher than SMC containing the
lowest ER? levels, whereas as a negative control, the colon
cancer cell line HT29 had little to no ER? detected by PCR.
For subsequent studies, we used SMC from either the female
donor with the greatest amount of ER? or the male donor
with the lowest ER? expression. We confirmed proportional
ER? protein expression in these 2 cell populations (Figure 1B).
SMC Containing ER? Failed to Differentiate
Because growth factors activate ER? and TGF?1 causes
differentiation of SMC,16,17SMC expressing the highest ER?
levels and the lowest ER? levels were treated with these
growth factors as well as ER? ligands (PPT and 17?-
estradiol). As shown, cells with high ER? levels had little
expression of the differentiation markers SM ?-actin or
calponin except in the presence of TGF?1 (Figure 2A). In
Figure 1. Aortic SMC from female donors have more ER? than
cells from male donors. A, Aortic SMC from 5 male and 5
female tissue donors were lysed after 5 days of starvation to
analyze ER? mRNA levels by real-time PCR. ER? mRNA levels
for SMC, the colon cancer cell line HT29 (negative control), and
the ER?-positive breast cancer cell line MCF7 (positive control)
were expressed as fold induction above the cell population
which consistently had the lowest levels of ER? expression. B,
SMC expressing the lowest and highest levels of mRNA for ER?
were starved in basal media for 10 days and Western blotted for
ER? expression. ?-Actin was detected as a loading control. M
indicates male; F, female.
Figure 2. Differentiation of aortic SMC varies inversely to ER?
expression. SMC with the highest (HI) or the lowest (LO) ER?
expression were exposed for 10 days in EBM-PRF to vehicle
(VEH) control, 17?-estradiol (ESTR) (10 nmol/L), PPT (10 nmol/
L), EGF (10 ng/mL), PDGF-BB (10 ng/mL), or TGF?1 (5 ng/mL).
A, Western blotting was performed on protein lysates to deter-
mine expression of contractile proteins calponin and SM
?-actin, with Erk2 as a loading control. A representative blot is
shown. B, Protein expression was quantitated by densitometry,
normalized by the Erk2 band, and stated as a percentage of the
low-ER? vehicle control condition (?, low-ER? SMC; f, high-
ER? SMC; mean?SEM of 4 replicates). C, SMC from 3 male
donors and 3 female donors were grown for 10 days in EBM-
PRF with either PDGF-BB or vehicle control. Western blotting
was performed to detect calponin, and differentiation was calcu-
lated as a ratio of the intensity for the vehicle-treated to the
PDGF-treated samples (multiple replicates: ? n?6 female, n?7
male, ? mean?SEM).
Montague et alEstrogen Receptor Dedifferentiates Smooth Muscle
contrast, cells with lower levels of ER? retained both SM
?-actin and calponin in all conditions except when incubated
with EGF or PDGF (Figure 2A). We observed that low ER?
cells had significantlygreater
(P?0.0001 overall) and greater amounts of SM ?-actin
(P?0.0001 overall) compared with high ER?-expressing
cells (Figure 2B). Individual comparisons are as shown in
Because SMC from the low ER? (male) donor had more
differentiation markers than the high ER? (female) donor, we
further characterized basal differentiation of SMC from other
male or female donors (n?3 each). As shown in Figure 2C,
vehicle control–stimulated SMC from male donors had high
levels of calponin but lost much of this marker on PDGF
stimulation, similar to cells in Figure 2A. In contrast, female
donor SMC expressed only low levels of calponin in either
condition. Consequently, the average calponin ratio was
significantly higher for SMC from men than for SMC from
Transduction of ER? Inhibited
Because ER? expression correlated with SMC dedifferentia-
tion, we examined whether induced expression of ER? in low
ER?-containing cells directly inhibited SMC differentiation.
Transduction efficiency was determined by ER? immunoflu-
orescent staining (Figure 3A).Transduction of ER? lowered
calponin (P?0.0001) and SM ?-actin (P?0.0001) expression
overall. ER?-expression reduced SM ?-actin in response to
17?-estradiol, PPT, and TGF?1 treatment and reduced cal-
ponin in response to vehicle, 17?-estradiol, or PPT (Figure
3B and 3C, probability values as shown). In contrast to these
cell markers, cyclin D increased on transduction of ER?
Interruption of ER? by siRNA Augmented
Because high native levels of ER? correlated with low levels
of SMC differentiation markers, we next reduced ER?
expression through siRNA to enhance their differentiation
program. High transfection efficiency was obtained using
pmaxGFP plasmid DNA in cotransfections (Figure 4A). A
reduction in ER? protein was confirmed in the siRNA-
transfected cells compared with the empty vector control
We found that reduced ER? expression led to higher levels
of differentiation markers. ER? siRNA upregulated calponin
expression overall (P?0.0038), with significant pairwise
difference occurring in TGF?1-treated cells (Figure 4B and
C). Although ER? siRNA slightly raised SM ?-actin levels in
cells incubated with TGF?1, the increase was not significant
Ligand Activation of ER? Inhibited
We next investigated ligand-dependent ER? activation by
17?-estradiol and PPT, and the ligand-independent activation
by EGF and PDGF. A low level of ER? phosphorylation was
observed in the ER?-transduced cells in the vehicle-treated
condition, whereas 17?-estradiol and PPT preferentially
phosphorylated ER?S118, and EGF and PDGF phosphorylated
ER?S167(Figure 5A). TGF?1 caused no activation above
vehicle control of either serine residue.
In contrast to the activation by phosphorylation seen with
EGF and PDGF, only 17?-estradiol and PPT activated
transcription of an ERE reporter construct (Figure 5B,
P?0.0013 for 17?-estradiol and P?0.0008 for PPT com-
pared with vehicle). Activation for up to ten days with EGF,
Figure 3. SMC dedifferentiate following transduction of ER?.
SMC transduced with either an empty vector (pLenti) or ER?
cDNA were exposed for 10 days in EBM-PRF with various stim-
uli as indicated in Materials and Methods and Figure 2. A,
Immunofluorescent staining of ER? illustrates the efficiency of
transduction. B, Western blots demonstrate expression of ER?,
loss of calponin and SM ?-actin contractile proteins, and
upregulation of cyclin D1 in the ER?-transduced cells. C, Calpo-
nin and SM ?-actin densitometry were expressed as a percent-
age of the pLenti vehicle control (VEH) (?pLenti or fER?,
mean?SEM of calponin [n?4] or SM ?-actin [n?5]). ESTR indi-
September 1, 2006
PDGF-BB or TGF?1 caused no detectable signal above
vehicle control samples (data not shown). As a partial
explanation for the transcriptional inactivity of ER? phos-
phorylated by growth factors, we found that stimulating the
cells with 17?-estradiol, but not EGF, PDGF-BB or TGF?1,
for 20 to 60 minutes caused nuclear translocation of ER?
(Figure 5B and data not shown).
Because our differentiation analysis suggested that TGF?1
elevated SMC differentiation markers in the presence of ER?
but full expression of these markers required lower levels of
ER?, we examined whether ER? inhibited TGF? signaling
by interfering with Smad activation, as previously de-
scribed.18Because 17?-estradiol or PPT potently reduced
SMC differentiation, we determined whether 17?-estradiol
inhibited TGF?1 signaling through Smads. TGF?1 induced
the phosphorylation of Smad2 in pLenti or ER?-transduced
SMC for up to 60 minutes (Figure 5C). However, Smad2
phosphorylation was reduced if the ER?-transduced cells
were preincubated with 17?-estradiol 30 minutes before
activation. The relevance of TGF?1 to the differentiation of
SMC was examined by measuring whether the cells sponta-
neously produced TGF?1 and whether this production corre-
lated to cellular differentiation. Active TGF?1 was detected
in the supernatant of pLenti-transduced SMC at 49.0?27.9
pg/mL in the vehicle control condition and 67.2?45.4 and
110.7?42.1 after 17?-estradiol or PPT incubation, respec-
tively (no significant differences, n?2, mean?SEM). Trans-
duction of ER? in the cells did not alter TGF?1 production
suggesting ER? expression altered the response to TGF?1
(45.6?23.1, 49.8?33.6 and 112.0?39.7 for vehicle, 17?-
estradiol, and PPT exposed cells, respectively, n?2).
Ligand Activation of ER? Initiated Apoptosis
Because estrogen inhibits the growth of SMC and causes
apoptosis,2,19we examined the initiation of apoptosis in the
presence of ER? agonists. Indeed, SMC transduced to ex-
press ER? had evidence of caspase-3 activity when stimu-
lated with 17?-estradiol or PPT (Figure 6A, P?0.0231 and
P?0.0646, respectively; n?2).
The effects of ER? expression on cell growth were
apparent by cell detachment when ER?-transduced SMC
were treated with 17?-estradiol (Figure 6B, lower right) or
PPT (not shown), indicating that cells were undergoing
apoptosis. Consequently, fewer ER?-transduced cells were
counted after 17?-estradiol or PPT treatment compared with
pLenti-transduced cells (P?0.0006 overall; Figure 6D, i).
Consistent with this observation, cells natively expressing
high levels of ER? had significantly lower cell densities than
the low-ER? cells when treated with 17?-estradiol or PPT
(P?0.0003 overall; Figure 6D, ii). Finally, a small increase in
cell density was found overall (P?0.0274) when the high-
ER? SMC were transfected with ER? siRNA, although no
individual paired comparisons were significant (Figure 6C
and 6D, iii).
The present study extends the role of ER? in vascular SMC
beyond its ability to inhibit growth. To understand differences
in SMC status between men and women, we characterized
aortic SMC differentiation and ER? expression in these 2
groups. We detected significantly higher levels of ER? in
SMC from our female donors compared with SMC from male
donors. The inverse was true for differentiation markers,
however, as cells from men expressed greater levels of SM
?-actin and calponin protein under starved conditions, pro-
Figure 4. Reduction of ER? expression by siRNA induces differ-
entiation. SMC expressing high levels of ER? were transfected
with either empty vector or plasmid producing ER? siRNA. A,
Transfection of pmaxGFP into high-ER? SMC illustrated the
high-transfection efficiency. Reduction in ER? protein levels by
siRNA in cells starved for 10 days was demonstrated by West-
ern blotting. ?-Actin was used as a loading control. B, Trans-
fected cells were exposed for 10 days in EBM-PRF with stimuli
as indicated in Materials and Methods. Western blotting was
performed to detect the differentiation markers calponin and SM
?-actin. C, Calponin and SM?-actin densitometry as a percent-
age of the empty vector vehicle (VEH) control condition was cal-
culated (mean?SEM of 4 replicates, transfected with either
empty vector [?] or ER? [f] siRNA). ESTR indicates
Montague et alEstrogen Receptor Dedifferentiates Smooth Muscle
viding a connection between ER? expression and
We analyzed the effect of 2 ER? ligands and 3 growth
factors on cell populations containing the lowest and highest
levels of ER?. SMC differentiation markers remained high
for cells natively expressing low amounts of ER? whether
incubated with vehicle, ER? agonists, or TGF?1. However,
EGF and PDGF decreased SM ?-actin and calponin levels in
these cells, similar to published accounts.20In contrast, cells
expressing high native ER? had a low level of SM ?-actin
and calponin under most conditions except when treated with
TGF?1. Similar findings were observed in cells virally
transduced with ER?, which resulted in their dedifferentia-
tion. Only TGF?1 could partially overcome the inhibitory
effect of ER?. These data indicated that ER? may play a role
in causing the low contractile protein levels detected in SMC
from women. The ability of ER? to inhibit differentiation was
unexpected, because ER? is known to inhibit growth and
would be expected to induce differentiation. In contrast, ER?
caused an increase in cyclin D1 expression, indicating that
growth inhibition did not align with quiescence. To confirm
this biological role for ER?, we found that reduction in ER?
resulted in greater contractile protein expression, especially in
the presence of TGF?1.
Several possible pathways could be involved in the re-
duced SMC differentiation caused by ER?. Inhibition of cell
cycle regulators and activation of proliferation genes such as
cyclin D are known to occur in ER?-positive breast cancer
cells exposed to 17?-estradiol.21Similar changes in SMC
could induce a phenotypic switch from differentiated to
proliferating or migratory SMC. Alternatively, ER? may
inhibit transcription by shunting coactivator proteins such as
p300/CBP away from other transcription factors, some of
which are necessary for smooth muscle gene expression.22–24
ER? activates transcription at estrogen response elements on
DNA, but is known to suppress the TGF?1/Smad pathway by
binding to and repressing Smad2 and -3, positive regulators
of contractile protein transcription in SMC.18,25In agreement,
Smad2 phosphorylation was inhibited by estrogen in ER?-
transduced SMC in the current study. Because the SMC
released detectable levels of active TGF?1, the ability of ER?
to inhibit Smad-regulated differentiation is a likely mecha-
nism of action.
Cytoplasmic signaling pathways activated by ER? includ-
ing phosphatidylinositol 3-kinase and Akt, growth factor
receptor autophosphorylation, mitogen-activated protein ki-
nases (MAPKs), and src kinases can contribute to SMC
dedifferentiation.19,26–28A positive-feedback loop also exists
in which S118 of ER? is phosphorylated by 17?-estradiol
and MAPK, whereas S167 of ER? is phosphorylated through
the Akt pathway.16,29,30Depending on the stimulus, we saw
preferential phosphorylation of ER? epitopes in SMC, indi-
Figure 5. Ligand activation of ER?
causes nuclear translocation and inhibi-
tion of Smad2 phosphorylation. SMC
transduced with pLenti or ER? virus
were stably selected with blasticidin and
activated with the various stimuli as indi-
cated previously. A, Western blots from
cells activated for 20 minutes were
sequentially immunoblotted with anti-
bodies to phospho-ER? (S167),
phospho-ER? (S118), ER?, and Erk2,
with stripping of the membrane between
antibodies. Shown is a representative
image from 2 experiments. B, Stably
transduced cells were transfected with
an estrogen response element reporter
construct producing secreted alkaline
phosphatase (SEAP) and were treated as
described for up to 3 days. Aliquots of
the media were analyzed for SEAP each
day, and luminescent signal was normal-
ized as a percentage of the maximum
signal achieved on day 3 (mean?SEM of
4 experiments). Western blotting of
nuclear and cytosolic lysates from SMC
transduced with ER? and activated for
20 minutes as indicated demonstrated
nuclear translocation of ER? when acti-
vated by 17?-estradiol (ESTR). C, SMC
stably transduced with pLenti or ER?
were preincubated with 17?-estradiol (?)
or ETOH (-) for 30 minutes, then acti-
vated with TGF?1 for the time indicated.
Phosphorylated Smad2 and ?-actin as a
loading control were detected by West-
ern blotting (representative of 2
482 Circulation Research
September 1, 2006
cating that upstream and downstream signaling events likely
differed in these cells. Only ER? ligands caused nuclear
translocation and transcriptional activity at an ERE.
Many studies show that 17?-estradiol induces apoptosis
through ER? in SMC.2,19We found that 17?-estradiol and
PPT significantly reduced cell density of native and trans-
duced cells expressing high levels of ER?, whereas inhibition
of ER? by siRNA increased cell density.
Our results may explain some differences in coronary
events in women and men. ER? activation in an affected
coronary artery may cause the dedifferentiation and migration
of SMC into the intima, causing microvessel dysfunction.
Our observation of apoptosis of SMC after estrogen exposure
could partly explain why postmenopausal hormone replace-
ment therapy causes higher rates of myocardial infarction
through thinning of collagen and rupture of plaques.
We acknowledge the advice of Arthur R. Strauch and Tim D.
Eubank, both of Ohio State University.
Sources of Funding
This work was supported by NIH grants HL63800-05, HL67176-04,
HL70294-03 and HL066108-04 (to C.B.M.); and NIH Individual
National Research Service Award 5F32 HL09550 and American
Heart Association Ohio Affiliate Postdoctoral Fellowship Award
9920597V (to C.R.M.).
1. American Heart Association. Heart Disease and Stroke Statistics—2005
Update. Dallas, Tex: American Heart Association; 2005. Available at:
2. Mendelsohn ME, Karas RH. Molecular and cellular basis of cardiovas-
cular gender differences. Science. 2005;308:1583–1587.
3. Rossouw JE, Anderson GL, Prentice RL, LaCroix AZ, Kooperberg C,
Stefanick ML, Jackson RD, Beresford SA, Howard BV, Johnson KC,
Kotchen JM, Ockene J. Risks and benefits of estrogen plus progestin in
healthy postmenopausal women: principal results from the Women’s
Health Initiative randomized controlled trial. JAMA. 2002;288:321–333.
4. Hulley S, Grady D, Bush T, Furberg C, Herrington D, Riggs B,
Vittinghoff E. Randomized trial of estrogen plus progestin for secondary
prevention of coronary heart disease in postmenopausal women. Heart
and Estrogen/progestin Replacement Study (HERS) Research Group.
5. Grodstein F, Manson JE, Stampfer MJ. Hormone therapy and coronary
heart disease: the role of time since menopause and age at hormone
initiation. J Womens Health (Larchmt). 2006;15:35–44.
6. Patel H, Rosengren A, Ekman I. Symptoms in acute coronary syndromes:
does sex make a difference? Am Heart J. 2004;148:27–33.
7. Clayton TC, Pocock SJ, Henderson RA, Poole-Wilson PA, Shaw TR,
Knight R, Fox KA. Do men benefit more than women from an interven-
tional strategy in patients with unstable angina or non-ST-elevation myo-
Figure 6. ER? ligands cause apoptosis
of aortic SMC. A, SMC virally trans-
duced with either pLenti or pLenti-ER?
were exposed for 10 days in EBM-PRF
with various stimuli as indicated in Mate-
rials and Methods. The cleaved or active
form of caspase-3 was detected at 17
and 19 kDa by SDS-PAGE Western
blots. B, Representative phase-contrast
photographs were taken of SMC con-
taining low ER? levels transduced with
either pLenti or ER? and treated with
vehicle (VEH) or 17?-estradiol (ESTR) for
10 days. C, Representative photographs
are shown of cells with high endogenous
levels of ER? transfected with either
control or ER? siRNA plasmid and
treated with vehicle or 17?-estradiol for
10 days. D, Phase-contrast photographs
were quantitated for pixel density of live
(gray) cells in the following groups:
pLenti-transduced (?) or ER?-
transduced (f) cells activated by ER?
agonists (n?5) (i); SMC natively express-
ing low (?) or high (f) ER? levels (n?4)
(ii); or SMC expressing high ER? levels
transfected with empty (?) or siRNA (f)
plasmid (n?4) (iii).
Montague et alEstrogen Receptor Dedifferentiates Smooth Muscle
cardial infarction? The impact of gender in the RITA 3 trial. Eur Heart J.
8. Bugiardini R, Bairey Merz CN. Angina with “normal” coronary arteries:
a changing philosophy. JAMA. 2005;293:477–484.
9. Hochman JS, Tamis JE, Thompson TD, Weaver WD, White HD, Van de
WF, Aylward P, Topol EJ, Califf RM. Sex, clinical presentation, and
outcome in patients with acute coronary syndromes. Global Use of
Strategies to Open Occluded Coronary Arteries in Acute Coronary Syn-
dromes IIb Investigators. N Engl J Med. 1999;341:226–232.
10. Farb A, Burke AP, Tang AL, Liang TY, Mannan P, Smialek J, Virmani
R. Coronary plaque erosion without rupture into a lipid core. A frequent
cause of coronary thrombosis in sudden coronary death. Circulation.
11. Burke AP, Farb A, Malcom G, Virmani R. Effect of menopause on plaque
morphologic characteristics in coronary atherosclerosis. Am Heart J.
12. Kolodgie FD, Burke AP, Farb A, Weber DK, Kutys R, Wight TN,
Virmani R. Differential accumulation of proteoglycans and hyaluronan in
culprit lesions: insights into plaque erosion. Arterioscler Thromb Vasc
13. Waddell TK, Dart AM, Gatzka CD, Cameron JD, Kingwell BA. Women
exhibit a greater age-related increase in proximal aortic stiffness than
men. J Hypertens. 2001;19:2205–2212.
14. Rozen S, Skaletsky HJ. Primer3 on the WWW for general users and for
biologist programmers. In: Bioinformatics Methods and Protocols:
Methods in Molecular Biology. Krawetz S, Misener S, eds. Totowa, NJ:
Humana Press; 2000;132:365–386.
15. Sankoh AJ, Huque MF, Dubey SD. Some comments on frequently used
multiple endpoint adjustment methods in clinical trials. Stat Med. 1997;
16. Kato S, Endoh H, Masuhiro Y, Kitamoto T, Uchiyama S, Sasaki H,
Masushige S, Gotoh Y, Nishida E, Kawashima H. Activation of the
estrogen receptor through phosphorylation by mitogen-activated protein
kinase. Science. 1995;270:1491–1494.
17. Hautmann MB, Madsen CS, Owens GK. A transforming growth factor
beta (TGFbeta) control element drives TGFbeta-induced stimulation of
smooth muscle alpha-actin gene expression in concert with two CArG
elements. J Biol Chem. 1997;272:10948–10956.
18. Matsuda T, Yamamoto T, Muraguchi A, Saatcioglu F. Cross-talk between
transforming growth factor-beta and estrogen receptor signaling through
Smad3. J Biol Chem. 2001;276:42908–42914.
19. Mori-Abe A, Tsutsumi S, Takahashi K, Toya M, Yoshida M, Du B,
Kawagoe J, Nakahara K, Takahashi T, Ohmichi M, Kurachi H. Estrogen
and raloxifene induce apoptosis by activating p38 mitogen-activated
protein kinase cascade in synthetic vascular smooth muscle cells. J
20. Dandre F, Owens GK. Platelet-derived growth factor-BB and Ets-1 tran-
scription factor negatively regulate transcription of multiple smooth
muscle cell differentiation marker genes. Am J Physiol Heart Circ
21. Frasor J, Danes JM, Komm B, Chang KC, Lyttle CR, Katzenellenbogen
BS. Profiling of estrogen up- and down-regulated gene expression in
human breast cancer cells: insights into gene networks and pathways
underlying estrogenic control of proliferation and cell phenotype. Endo-
22. Speir E, Yu ZX, Takeda K, Ferrans VJ, Cannon RO III. Competition for
p300 regulates transcription by estrogen receptors and nuclear factor-
kappaB in human coronary smooth muscle cells. Circ Res. 2000;87:
23. Cao D, Wang Z, Zhang CL, Oh J, Xing W, Li S, Richardson JA, Wang
DZ, Olson EN. Modulation of smooth muscle gene expression by asso-
ciation of histone acetyltransferases and deacetylases with myocardin.
Mol Cell Biol. 2005;25:364–376.
24. Nishihara A, Hanai JI, Okamoto N, Yanagisawa J, Kato S, Miyazono K,
Kawabata M. Role of p300, a transcriptional coactivator, in signalling of
TGF-beta. Genes Cells. 1998;3:613–623.
25. Hu B, Wu Z, Phan SH. Smad3 mediates transforming growth factor-beta-
induced alpha-smooth muscle actin expression. Am J Respir Cell Mol
26. Haynes MP, Li L, Sinha D, Russell KS, Hisamoto K, Baron R, Collinge
M, Sessa WC, Bender JR. Src kinase mediates phosphatidylinositol
3-kinase/Akt-dependent rapid endothelial nitric-oxide synthase activation
by estrogen. J Biol Chem. 2003;278:2118–2123.
27. Kahlert S, Nuedling S, van Eickels M, Vetter H, Meyer R, Grohe C.
Estrogen receptor alpha rapidly activates the IGF-1 receptor pathway.
J Biol Chem. 2000;275:18447–18453.
28. Kawai-Kowase K, Sato H, Oyama Y, Kanai H, Sato M, Doi H,
Kurabayashi M. Basic fibroblast growth factor antagonizes transforming
growth factor-beta1-induced smooth muscle gene expression through
extracellular signal-regulated kinase 1/2 signaling pathway activation.
Arterioscler Thromb Vasc Biol. 2004;24:1384–1390.
29. Chen D, Washbrook E, Sarwar N, Bates GJ, Pace PE, Thirunuvakkarasu
V, Taylor J, Epstein RJ, Fuller-Pace FV, Egly JM, Coombes RC, Ali S.
Phosphorylation of human estrogen receptor alpha at serine 118 by two
distinct signal transduction pathways revealed by phosphorylation-
specific antisera. Oncogene. 2002;21:4921–4931.
30. Campbell RA, Bhat-Nakshatri P, Patel NM, Constantinidou D, Ali S,
Nakshatri H. Phosphatidylinositol 3-kinase/AKT-mediated activation of
estrogen receptor alpha: a new model for anti-estrogen resistance. J Biol
September 1, 2006