Am J Physiol Heart Circ Physiol 292: H2607H2612, 2007.

First published January 19, 2007; doi:10.1152/ajpheart.01107.2006.

Estrogen modulates TNF--induced inflammatory responses in rat aortic

smooth muscle cells through estrogen receptor- activation
Dongqi Xing,* Wenguang Feng,* Andrew P. Miller,1 Nathaniel M. Weathington,2
Yiu-Fai Chen,1 Lea Novak,3 J. Edwin Blalock,2 and Suzanne Oparil1,2
1
Vascular Biology and Hypertension Program, Division of Cardiovascular Disease,
Department of Medicine, 2Department of Physiology and Biophysics, and 3Department
of Anatomic Pathology, University of Alabama at Birmingham, Birmingham, Alabama
Submitted 10 October 2006; accepted in final form 19 January 2007

Xing D, Feng W, Miller AP, Weathington NM, Chen YF, restenotic vessels (18, 22, 28), and is associated with activation

Downloaded from http://ajpheart.physiology.org/ by 10.220.33.2 on September 12, 2017

Novak L, Blalock JE, Oparil S. Estrogen modulates TNF--
induced inflammatory responses in rat aortic smooth muscle cells
through estrogen receptor- activation. Am J Physiol Heart Circ
Physiol 292: H2607H2612, 2007. First published January 19,
2007; doi:10.1152/ajpheart.01107.2006.We have previously
shown that 17-estradiol (E2) attenuates responses to endoluminal
injury of the rat carotid artery, at least in part, by decreasing inflam-
matory mediator expression and neutrophil infiltration into the injured
vessel, with a major effect on the neutrophil-specific chemokine
cytokine-induced neutrophil chemoattractant (CINC)-2. Current
studies tested the hypothesis that activated rat aortic smooth muscle
cells (RASMCs) express these same inflammatory mediators and
induce neutrophil migration in vitro and that E2 inhibits these pro-
cesses by an estrogen receptor (ER)-dependent mechanism. Quiescent
RASMCs treated with E2, the ER-selective agonist propyl pyrazole
triol (PPT), the ER-selective agonist diarylpropiolnitrile (DPN), or
vehicle for 24 h were stimulated with tumor necrosis factor (TNF)-
and processed for real-time RT-PCR, ELISA, or chemotaxis assays
6 h later. TNF- stimulated and E2 attenuated mRNA expression of
inflammatory mediators, including P-selectin, intercellular adhesion
molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1,
monocyte chemoattractant protein (MCP)-1, and CINC-2. DPN dose
dependently attenuated TNF--induced mRNA expression of CINC-
2, whereas PPT had no effect. The anti-inflammatory effects of DPN
and E2 were blocked by the nonselective ER-inhibitor ICI-182,780.
ELISA confirmed the TNF--induced increase and E2-induced inhi-
bition of CINC-2 protein secretion. TNF- treatment of RASMCs
produced a twofold increase in neutrophil chemotactic activity of
conditioned media; E2 and DPN treatment markedly inhibited this
effect. E2 inhibits activated RASMC proinflammatory mediator ex-
pression and neutrophil chemotactic activity through an ER-depen-
dent mechanism.
inflammation; vascular smooth muscle cell; arteries
INFLAMMATION plays an important role in the pathogenesis of
many forms of vascular disease, including atherosclerosis and
the response to acute vascular injury (14, 37). Balloon injury of
arteries has been shown to elicit accumulation of inflammatory
cells, specifically granulocytes (neutrophils) and monocyte/
macrophages, in the adventitia surrounding the injury site
within hours after the insult (17, 28, 40). The appearance of
inflammatory cells is predated by expression of inflammatory
mediators, including adhesion molecules and chemokines, in
acutely injured arteries (25) as well as in atherosclerotic and
of a variety of cell types, including adipocytes and fibroblasts,
in adventitial tissues (20, 28). Since vascular smooth muscle
cells (VSMCs) have been shown to produce adhesion mole-
cules (3) and chemokines (34) when stimulated with cytokines
and because our earlier studies (21) demonstrated that activa-
tion and migration of adventitial fibroblasts can be stimulated
by media conditioned by VSMCs, we hypothesized that
VSMCs are activated early following endoluminal injury, re-
leasing inflammatory mediators that reach the periadventitial
space to recruit leukocytes and are the chief effector cells for
initiation of the early inflammatory response.
Previous studies in our laboratory have characterized an
early estrogen receptor (ER)-dependent anti-inflammatory ef-
fect for 17-estradiol (E2) in this model of vascular injury (2,
25, 40). We have shown that systemic E2 administration
attenuates both expression of inflammatory mediators and
infiltration of leukocytes into balloon-injured carotid arteries of
ovariectomized (OVX) rats at a very early time point postin-
jury (25, 40). In this model, we demonstrated a particularly
robust effect for E2 in modulating neutrophil chemotaxis via
attenuating expression of cytokine-induced neutrophil che-
moattractant (CINC)-2, a member of the cysteine-x-cysteine
(CXC) chemokine family and a potent chemoattractant for
neutrophils in vitro and in vivo (23, 24). Based on our hypoth-
esis that VSMCs are the target cells that initiate the injury
response, the present in vitro study was designed to define
cellular and molecular mechanisms mediating estrogenic anti-
inflammatory effects. We tested the estrogen receptor subtype
(ER or ER) dependence of the anti-inflammatory effect of
E2 by employing two recently developed selective ER and
ER agonists. Based on published observations that 1) tumor
necrosis factor (TNF)- acts as the bodys fire alarm (11)
and induces the rapid recruitment of leukocytes from the circula-
tion in response to many forms of stress, 2) it is elaborated from
diseased bypass grafts and atherosclerotic arteries (8), and 3) our
own finding that TNF- expression is dramatically upregulated in
balloon-injured rat carotid arteries and not affected by E2 admin-
istration (25), we used TNF- as the inflammatory stimulus in the
current study. Since our previous in vivo studies demonstrated
robust estrogenic attenuation of injury-induced CINC-2 expres-
sion and of neutrophil infiltration into injured arteries (25, 40), we
focused on neutrophil activation and chemotaxis in the current in
vitro study.

* D. Xing and W. Feng contributed equally to this study.

Address for reprint requests and other correspondence: D. Xing, 1014 The costs of publication of this article were defrayed in part by the payment
Zeigler Research Bldg., Univ. of Alabama at Birmingham, UAB Station, of page charges. The article must therefore be hereby marked advertisement
Birmingham, AL 35294 (E-mail: dqxing@uab.edu). in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

http://www.ajpheart.org 0363-6135/07 $8.00 Copyright 2007 the American Physiological Society H2607
H2608 ESTROGEN AND INFLAMMATION
MATERIALS AND METHODS
Cell culture. Primary cultures of rat aortic smooth muscle cells
(RASMCs) were derived from 10-wk-old female Sprague-Dawley
rats (Charles River), as previously described (31). Cells were cultured
in complete medium containing phenol red-free DMEM (GIBCO)
supplemented with 10% (vol/vol) FBS, 4 mmol/l L-glutamine, 100
U/ml penicillin, and 100 g/ml streptomycin. Cells were identified as
smooth muscle cells (SMCs) by their characteristic morphology and
positive immunostaining for -smooth muscle actin (-SMA, clone
1A4, DAKO). All experiments were performed using early passage
(5) near-confluent (95%) cultures that were serum starved for 24 h
before treatment.
Initial studies tested whether TNF- could stimulate expression of
relevant inflammatory chemokines and cytokines in RASMCs and
whether pretreatment with E2 could inhibit these inflammatory re-
sponses. In these studies, cells were pretreated with E2 (107 M) or
vehicle (ethanol at a final concentration 0.01%) for 24 h and then
incubated with TNF- (0.12.5 ng/ml) for an additional 6 h.
Subsequent studies assessed the ER subtype dependence of the E2
effect. Cells were pretreated with the selective ER agonist diaryl-
propiolnitrile (DPN) (1010-107 M) (Tocris Cookson, Ellisville,
MO) or the selective ER agonist propyl pyrazole triol (PPT) (1010-
106 M) (OBITER Research, Urbana, IL) for 24 h, and then incubated
with 1 ng/ml TNF- for an additional 6 h. Expression of the neutro-
phil-specific chemokine CINC-2 was assessed in these studies. To
confirm ER dependence of the DPN and E2 effects, subgroups of cells
from the above experiments were exposed to the nonselective ER
antagonist ICI-182,780 (106 M) for 2 h before the E2 (107 M) or
DPN (107 M) pretreatment. For CINC-2 ELISA and chemotaxis
assays, cells were pretreated with E2 (107 M) or vehicle for 24 h,
followed by TNF- (1 ng/ml) treatment for an additional 6 h.
Conditioned media were collected to assess CINC-2 protein concen-
tration and ability to stimulate neutrophil migration.
Real-time quantitative RT-PCR analysis of inflammatory media-
tors. As described previously (25), total RNA was extracted from cells
using TRIzol (Invitrogen, Carlsbad, CA), treated with DNAase I to
remove genomic DNA, and then purified by using an RNA purifica-
tion kit (Invitrogen). The protein- and DNA-free RNA was reverse
transcribed to cDNA using the SYBR Green RT-PCR kit (Applied
Biosystems, Foster City, CA) and specific primers (described in Ref.
25). cDNA was amplified by PCR in the iCycler for 40 cycles, and
relative RNA levels were calculated using the iCycler software and a
standard equation (Applied Biosystems). Unknowns were normalized
using 18S rRNA and then standardized to the mRNA level of
vehicle-treated RASMCs, except for CINC-2, monocyte chemoat-
tractant protein (MCP-1) and P-selectin, which were standardized to
the mRNA level of TNF--treated RASMCs because their mRNA
was undetectable in the vehicle group.
Measurement of CINC-2 protein concentration. The CINC-2
concentration in conditioned media of vehicle control and TNF-
E2-treated cells was assayed with the rat GRO/CINC-2 ELISA kit
(IBL, Japan). Briefly, conditioned media (4 ml) were collected and
concentrated 10-fold using Centricon concentrators (Millipore, Bil-
lerica, MA). Samples were processed according to the manufacturers
instructions. Samples were measured in duplicate, and the CINC-2
concentration was calculated from the standard curve.
Chemotaxis assays. Human myeloid leukemia HL-60 cells (ATCC,
Manassas, VA) were maintained in Iscoves modified medium
(ATCC) supplemented with 10% fetal calf serum, 50 g/ml strepto-
mycin, 2 U/ml penicillin, and 2 mM L-glutamine. For differentiation,
cells (3 105/ml) were incubated in the presence of 1.3% (vol/vol)
DMSO for 46 days (Newburger PE) before assessment of their
chemotactic responsiveness.
Neutrophil chemotactic activity was assayed in a 96-well modified
Boyden chamber (Millipore, Billerica, MA) using differentiated
HL-60 (dHL-60) cells. The bottom wells of the chamber were filled
AJP-Heart Circ Physiol VOL
with 150 l of conditioned medium of vehicle control or TNF-
E2-, TNF- DPN-, or TNF- PPT-treated cells. To test the
CINC-2 dependence of neutrophil chemoattractant activity of condi-
tioned media from TNF--treated cells, anti-CINC-2/ antibody
(R&D System, Minneapolis, MN) at a final concentration of 5 g/ml
or PBS (control) was incubated with conditioned media for 30 min at
room temperature; 150 l of the conditioned media-antibody solution
were added to the bottom wells of the chamber as described. A
polyvinylpyrrolidone-free polycarbonate filter plate with 3-m pores
was placed over the samples, and 100 l of the dHL-60 cell suspen-
sion (2 106 cells/ml) were placed into the upper wells. The
chambers were incubated in humidified air with 5% CO2 at 37C for
90 min. The upper portion was then removed, and four photomicro-
graphs (200) per well were digitally recorded using an Olympus
IX70 microscope and Perkin-Elmer Ultraview image capture equip-
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ment. Cell counts were made from these images. For ease of com-
parison of results between experiments, data were standardized to a
chemotactic index with cell migration to conditioned media of vehi-
cle-treated RASMCs as a baseline (25).
Statistical analysis. Data are expressed as means SE. Statistical
analysis was performed with one-way ANOVA or Students t-test, as
appropriate. Values of P 0.05 were considered significant.
RESULTS
Real-time quantitative RT-PCR analysis showed that TNF-
(0.12.5 ng/ml) dose dependently stimulated expression of
chemokines (CINC-2 and MCP-1) and adhesion molecules
[intercellular adhesion molecule (ICAM)-1, vascular cell ad-
hesion molecule (VCAM)-1, and P-selectin] (results for
CINC-2 shown in Fig. 1). A TNF- concentration of 1 ng/ml
was chosen for further experiments because it was the mini-
mum effective dose for all mediators. All mediators were
expressed at low or undetectable levels in unstimulated vehi-
cle-treated RASMCs (Fig. 2). Pretreatment with E2 (107 M)
significantly inhibited expression of CINC-2, MCP-1,
ICAM-1, VCAM-1, and P-selectin in cells treated with TNF-
(1 ng/ml, Fig. 2). Inhibition of these chemokines and adhesion
molecules expression was abolished by the nonselective ER
Fig. 1. Tumor necrosis factor (TNF)- dose dependently increases mRNA
expression of cytokine-induced neutrophil chemoattractant (CINC)-2 in rat
aortic smooth muscle cells (RASMCs). Cells were grown to subconfluence
(95%) in six-well plates, deprived of serum for 24 h, and then treated with
TNF- (0.52.5 ng/ml) for an additional 6 h. Data, expressed as means SE,
are from real-time quantitative RT-PCR assays and are normalized by 18S
RNA and then standardized to the mean mRNA level of the RASMCs treated
with TNF- at the 0.5 ng/ml dose. UD, undetectable; *P 0.05 vs. vehicle-
treated RASMCs.
292 JUNE 2007 www.ajpheart.org
 ESTROGEN AND INFLAMMATION H2609

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Fig. 2. 17-Estradiol (E2, 107 M) inhibits TNF--induced mRNA expression of chemokines (left) and adhesion molecules (right) in RASMCs, and this effect
is abolished by ICI-182,780. Cells were grown to subconfluence (95%) in six-well plates, deprived of serum for 24 h, pretreated with 107 M E2 or vehicle
for 24 h, and then treated with TNF- (1 ng/ml) for an additional 6 h. ICI-182,780 (106 M) was given to cells at 2 h before E2 treatment in some experiments.
Data, expressed as means SE, are from real-time quantitative RT-PCR assays and are normalized by 18S RNA. Data for P-selectin, CINC-2, and monocyte
chemoattractant protein (MCP)-1 are standardized to the mean mRNA level of the TNF--treated RASMCs. Data for intercellular adhesion molecule (ICAM)-1
and vascular cell adhesion molecule (VCAM)-1 are standardized to the mean mRNA level of the vehicle-treated RASMCs. *P 0.05 vs. respective
vehicle-treated RASMCs; #P 0.05 vs. respective TNF--treated RASMCs.

antagonist ICI-182,780 (106 M), indicating ER dependence
(Fig. 2).
The selective ER- agonist DPN dose dependently inhibited
TNF- (1 ng/ml)-induced expression of CINC-2 over the
concentration range 1010 to 107 M (Fig. 3). This effect was
blocked by treatment with ICI-182,780 (106 M), confirming
its ER dependence (Fig. 3). The selective ER- agonist PPT,
over the dose range of 1010-106 M, had no effect on
CINC-2 expression in TNF- (1 ng/ml)-treated cells (Fig. 4),
suggesting that the anti-inflammatory effects of E2 in
RASMCs are selectively mediated by ER and not ER.
CINC-2 protein concentration was quantified in condi-
tioned media from RASMCs using a double sandwich ELISA
technique (Fig. 5). E2 had no significant effect on CINC-2
protein concentration in conditioned media from vehicle-
treated RASMCs. TNF- increased CINC-2 protein levels
2.7-fold compared with vehicle-treated control cells, and E2
pretreatment completely abolished the TNF--induced in-
crease in CINC-2 expression.
To assess the functional significance of the TNF- and E2-
or DPN-induced alterations in CINC-2 expression, the ability
of conditioned media from cells subjected to these treatments
to stimulate migration of dHL-60 cells was assayed. Chemo-
tactic activity was normalized to conditioned media from
vehicle-treated RASMCs as described in MATERIALS AND METH-
ODS. There was no significant difference in the chemotactic
activity of conditioned media derived from vehicle-treated
versus E2-, DPN-, and PPT-treated cells. TNF- treatment
induced a marked (2.2-fold) increase in dHL-60 cell migration,
and E2 pretreatment of RASMCs significantly attenuated the
TNF--induced effect on dHL-60 cell migration (by 48%, P
0.01) (Fig. 6). DPN, but not PPT, mimicked the inhibitory
effect of E2 on TNF- treatment-induced dHL-60 cell migra-

Fig. 3. The ER-selective agonist diarylpropiolnitrile (DPN) inhibits TNF--

induced CINC-2 mRNA expression in RASMCs, and this effect is abolished
by ICI-182,780. Cells were grown to subconfluence (95%) in six-well plates,
deprived of serum for 24 h, pretreated with DPN (1010-107 M) or vehicle for
24 h, and then treated with TNF- (1 ng/ml) for an additional 6 h. ICI-182,780
(106 M) was given to cells at 2 h before DPN treatment in some experiments.
Data are from real-time RT-PCR assays and normalized by 18S RNA and then
standardized to the mean mRNA level of the TNF--treated RASMCs. Results
are means SE. *P 0.05 vs. vehicle-treated RASMCs; #P 0.05 vs.
TNF--treated RASMCs.
Fig. 4. ER-selective agonist propyl pyrazole triol (PPT) has no effect on
TNF--induced CINC-2 mRNA expression in RASMCs. Cells were grown
to subconfluence (95%) in six-well plates, deprived of serum for 24 h,
pretreated with PPT (1010-106 M) or vehicle for 24 h, and then treated with
TNF- (1 ng/ml) for an additional 6 h. Data are from real-time RT-PCR assays
and normalized by 18S RNA and then standardized to the mean mRNA level
of the TNF--treated RASMCs. Results are means SE. *P 0.05 vs.
vehicle-treated RASMCs.

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H2610 ESTROGEN AND INFLAMMATION
Fig. 5. E2 inhibits TNF--induced CINC-2 protein expression in RASMCs.
Cells were grown to subconfluence (95%) in six-well plates, deprived of
serum for 24 h, pretreated with 107 M E2 or vehicle for 24 h, and then treated
with TNF- (1 ng/ml) for an additional 6 h, and conditioned media were
collected. Data, expressed as means SE, are from a double sandwich ELISA
assay. *P 0.05 vs. vehicle-treated RASMCs; #P 0.05 vs. TNF--treated
RASMCs.
tion (by 69%, P 0.01). To test the CINC-2 dependence of
neutrophil chemoattractant activity of conditioned media from
TNF--treated cells, conditioned media from separate groups
of cells were preincubated with selective anti-CINC-2 anti-
body. Antibody treatment neutralized chemotactic activity of
conditioned media by 63%, verifying its CINC-2 dependence.
DISCUSSION
This in vitro study demonstrates that isolated RASMCs
express high levels of proinflammatory mediators, including
the neutrophil- and monocyte-selective chemokines CINC-2
and MCP-1, when activated by TNF- and that estrogen
inhibits this process via and ER-dependent mechanism. Me-
dia conditioned by activated SMCs directed the migration of
neutrophils, and this effect was partially inhibited by pretreat-
ment with anti-CINC-2 antibody, indicating that the CINC-2
secreted into the conditioned media was functional. Treatment
with an ER-selective agonist dose dependently inhibited
CINC-2 expression and reduced the chemotactic activity of
media conditioned by TNF--treated SMCs. Together, these
data support the concept that arterial SMCs are important
sources of proinflammatory mediators in the setting of acute
vascular injury, triggering a robust inflammatory response that
is amplified by recruitment of neutrophils and monocytes into
the adventitial domain of the injured vessel and by activation
and migration of adventitial fibroblasts into the neointima
(25, 40).
The CXC chemokines are powerful mediators of neutrophil
recruitment. A representative member of the CXC chemokine
family is IL-8, which is a major chemoattractant for neutro-
phils in humans (29, 39). In rats, no homologue of IL-8 has
been identified, and the CXC chemokines that recruit neutro-
phils are termed CINC (1, 26). Four CXC chemokines, includ-
ing CINC (or CINC-1), CINC-2, CINC-2, and MIP-2 (or
CINC-3), have been identified in rats (10, 27, 32). CINC-2 has
two splicing isoforms, CINC-2 and CINC-2 (33), which
differ in the three amino acids at the COOH terminus (27).
Both CINC-2 and CINC-2 are chemotactic for neutrophils,
with an effect at a 10 nM concentration (80 ng/ml) (27). CINCs
are structurally related to one another and share many func-
tions. They have an ability to attract neotrophils and have
AJP-Heart Circ Physiol VOL
effects on other neotrophil functions, including adhesion mol-
ecule expression, intracellular calcium influx, and phagocytosis
(27, 32). Upregulation of CINC-2 has been correlated with
neutrophil infiltration in many disease models, including acute
right ventricular failure following pulmonary embolism (35)
and ischemia-reperfusion injury of the liver (41). The present
study reveals marked estrogenic modulation of CINC mRNA
and protein expression in TNF--stimulated RASMCs, consis-
tent with our previous in vivo reports of attenuated CINC-2
expression and neutrophil infiltration in injured arteries with
E2 treatment. To our knowledge, this report is the first in vitro
study of CINC or CXCR2 ligand regulation by E2 in
RASMCs.
The finding that antibodies to CINC-2 did not completely
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ablate the chemotactic avtivity of supernates from TNF--
treated RASMCs was not unexpected because there are un-
doubtedly other neutrophil chemoattractants in such super-
nates. These might induce leukotriene B4 (9) and the recently
described tripeptide, PGP (38). It is, however, particularly
interesting that about two-thirds of the chemotactic activity of
supernates from TNF--stimulated RASMCs is apparently due
to CINC-2 and that a similar amount of chemotactic activity is
inhibited by E2 (48%) or DPN (69%). This suggests that the
effects of estrogen on neutrophil chemotaxis are likely due to
the ability of estrogen to inhibit CINC-2 production.
In the current study, we employed the selective ER agonist
DPN and the selective ER agonist PPT to define the relevant
ER subtypes involved in the anti-inflammatory effects of E2 on
RASMCs. Because of its selective binding affinity, DPN is 30-
to 70-fold more potent as an agonist of ER than ER (4).
Supporting its anti-inflammatory effects, DPN has been shown
to suppress IL-6 promoter activity in human endothelial cancer
(HEC-1) cells in vitro to the same extent as E2 (15). In a male
rat model of trauma-hemorrhage, DPN, and not PPT, protected
against lung injury, with associated reductions of inducible
nitric oxide synthase and IL-6 expression (42), and restored
cardiac function, with associated upregulation of heat shock
Fig. 6. Neutrophil chemotactic activity of conditioned media from TNF--
stimulated RASMCs is blocked by E2 and DPN. Cells were grown to
subconfluence (95%) in 75-mm2 flasks, deprived of serum for 24 h, and
pretreated with 107 M E2, 107 M DPN, 107 M PPT, or vehicle, respec-
tively, for 24 h followed by TNF- (1 ng/ml) treatment for an additional 6 h.
Conditioned media were collected. Selective anti-CINC-2 antibody (5 g/ml)
partially neutralized neutrophil chemotactic activity of conditioned media from
TNF--treated cells. Results are means SE normalized by vehicle-treated
RASMCs. *P 0.05 vs. vehicle-treated RASMCs; #P 0.05 vs. TNF--
treated RASMCs.
292 JUNE 2007 www.ajpheart.org
 ESTROGEN AND INFLAMMATION
proteins (43). A further study in this model demonstrated that
DPN, in subcutaneous doses of 5 g/kg administered during
resuscitation, inhibited myeloperoxidase activity and ELISA-
measured levels of CINC-1, CINC-3, and ICAM-1 in the lung,
suggesting an important role in regulation of neutrophil infil-
tration after acute injury (44).
A related compound ERB-041 has 200-fold increased bind-
ing activity for ER over ER and is orally active (16).
ERB-041 in oral doses as low as 1 mgkg1 day1 has been
shown to reverse chronic diarrhea and ameliorate colonic
lesions in a transgenic rat model of inflammatory bowel disease
(HLA-B27) and shown to reduce joint inflammation in the
Lewis rat adjuvant-arthritis model (16). In the latter model,
mRNA expression profiling in the spleen, lymph nodes, and
liver, and global analysis of the plasma proteome revealed
disease-related alterations in a large number of genes and
proteins related to immune responses that were completely or
partially reversed by ERB-041 administration (12). When
taken together, these findings support the current study and
suggest the possibility that ER mediates the anti-inflamma-
tory effects of estrogen in some tissues.
PPT is 400 times more potent as an agonist for ER than
for ER (4). PPT in doses of 0.315 mgkg1 day1 admin-
istered subcutaneously has been shown to evoke a number of
physiologically relevant E2-induced tissue responses (uterine
weight gain, prevention of OVX-induced body weight gain and
loss of bone mineral density, reduction in plasma cholesterol
levels, increases in brain progesterone receptor mRNA levels,
and prevention of experimentally induced hot flushes) in rats
(24). In the vasculature, PPT, and not DPN, has been shown to
mediate estrogenic vasodilatory responses at physiological
doses: Acute administration of PPT (1013-107 M) to pre-
contracted aortic rings from intact female rats dose depen-
dently induced an ER-dependent vasodilatory response equal
to that of E2, whereas DPN had no acute effect on vasomo-
tion (4).
Studies with knockout mice support important ER-medi-
ated protective effects on vascular injury (19, 30). Pare et al.
(30) showed that complete ER knockout mice (ERKOST) lost
the protective effects of E2 in a wire injury model. In this
study, the primary outcomes of change in medial area, proteo-
glycan deposition, and VSMC proliferation (by bromode-
oxyuridine staining) were not altered by E2 administration in
the ERKOST animals. In contrast, E2 did protect against wire
injury-induced increases in vascular medial area and prolifer-
ation of VSMCs in ER knockout mice (18). ER, but not
ER, has also been shown to mediate the reendothelialization
effect of E2 after electric carotid injury in mice with targeted
disruptions of ER genes (6). As we have previously demon-
strated, E2 restores endothelial cell function after denudation in
the balloon injury model of the rat carotid artery as well (38),
but this is a late event that occurs 2 4 wk postinjury (6).
In vitro studies support ER subtype and cell type-specific
modulatory effects of E2 on vascular cell function (13). Selec-
tive ER and ER mRNA anti-sense oligomers were used to
examine the ER subtype dependence of E2-induced inhibition
of PDGF-BB-induced p38 and p42/44 mitogen-activated pro-
tein kinase phosphorylation, migration, and proliferation in
porcine SMCs, and endothelial cells. The inhibitory effects of
E2 on porcine SMCs were abrogated by downregulation of
ER protein expression, whereas downregulation of ER had
AJP-Heart Circ Physiol VOL
H2611
no effect. In contrast, downregulation of ER expression in
porcine aortic endothelial cells inhibited E2-induced p38 and
p42/44 mitogen-activated protein kinase activation, whereas
downregulation of ER had no effect. These observations are
relevant to our in vivo model of balloon injury of the rat carotid
artery, because the injured area is denuded of endothelium for
several weeks and the early injury response is driven by
activated SMCs and infiltrating leukocytes. Thus modulatory
effects of ER activation on SMC-initiated inflammatory re-
sponses likely play an important role in inhibiting early inflam-
matory changes in the setting of endoluminal vascular injury.
Limitations of the current study include use of ER agonists
to delineate ER subtype-specific effects in stimulated VSMCs.
These agents, while the best available, are not pure ER or
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ER subtype agonists. While we recognize that perturbation of
ER and ER signaling with pharmacological ER antagonists
and/or siRNA strategies might theoretically provide superior
approaches, no good selective ER and ER antagonists are
available, and small interfering RNA technology has not been
successful in modulating E2 signaling in SMCs in our hands.
We have not tested whether ER activation, as studied here, is
sufficient to attenuate neointima formation in animals sub-
jected to endoluminal injury in vivo. We are aware that other
ER-modulated processes, e.g., reendothelialization, contrib-
ute to the injury response at later time points. Further study is
needed to elucidate the ER subtype-specific effects of E2 in
modulating injury responses in arteries.
Taken together, our in vivo and in vitro results suggest that
medial VSMCs are an important source for the proinflamma-
tory mediators that trigger the response to acute endoluminal
arterial injury. VSMCs of injured arteries are initial targets for
the anti-inflammatory actions of E2. In VSMCs, ER appears
to be the dominated ER subtype that contributes to the anti-
inflammatory effects of E2.
GRANTS
This work was supported, in part, by National Heart, Lung, and Blood
Institute Grants HL-07457, HL-64614, HL-75211, HL-68806; by American
Heart Association Grant AHA-0425455B; and by a Vascular Biology Working
Group Fellows Award.
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