(Circulation. 2000;101:2949.)
© 2000 American Heart Association, Inc.
Basic Science Reports |
Estrogen Attenuates Integrin-ß3–Dependent Adventitial Fibroblast Migration After Inhibition of Osteopontin Production in Vascular Smooth Muscle Cells
From the Departments of Medicine, Vascular Biology and Hypertension Program, and Surgery, Division of Transplantation, University of Alabama at Birmingham, Birmingham.
Correspondence to Guohong Li, MD, PhD, 1024 Zeigler Research Bldg, 703 S 19th St, Birmingham, AL 35294-0007. E-mail ghli{at}uab.edu
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Abstract |
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Background—Previous in vitro studies have suggested that estrogen attenuates the vascular injury response by modulating vascular smooth muscle cell (VSMC) expression of soluble factor(s) directing migration of adventitial fibroblasts. Previous in vivo studies have established a role for osteopontin (OPN) and its integrin receptors after vascular injury. In this study, we examined OPN expression in activated VSMCs, its modulation by estrogen, and its effects on adventitial fibroblast migration. In addition, the relative functional roles of ß1- and ß3-integrin–matrix interactions were examined.
Methods and Results—Primary cultures of VSMCs and adventitial
fibroblasts were derived from female Sprague-Dawley rats.
Serum-activated VSMCs expressed high levels of OPN mRNA and
secreted protein that was effectively inhibited by estrogen treatment
(10-7 mol/L). Compared with VSMCs, fibroblasts expressed
similar levels of integrins 
and ß1 and higher
levels of integrin-ß3. Exogenous OPN (5.0 to 40 µg/mL)
directed fibroblast migration in a dose-dependent fashion.
Anti–ß3-integrin antibody (F11) pretreatment markedly
inhibited adventitial fibroblast migration directed by exogenous OPN or
VSMC-conditioned medium in a dose-dependent manner. In contrast,
anti–ß1-integrin antibody (Ha2/5) did not affect
fibroblast migration. Similarly, pretreatment with either linear or
cyclic RGD peptides (10 to 1000 µmol/L) inhibited fibroblast
migration directed by OPN or VSMC-conditioned medium in a
dose-dependent manner.
Conclusions—These observations suggest that estrogen indirectly attenuates integrin-ß3–dependent adventitial fibroblast migration after inhibition of OPN expression in VSMCs.
Key Words: hormones • osteopontin • cells • muscle, smooth • integrins • vasculature
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Introduction |
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Mounting experimental evidence from a variety of animal models suggests that activation and migration of adventitial cells toward the lumen participate in the response to endoluminal injury of blood vessels.1 2 3 4 5 Recent studies in our laboratory have shown indirectly that estrogen modulates adventitial cell activation and migration into neointima of balloon-injured rat carotid arteries.6 In vitro studies have demonstrated that vascular smooth muscle cells (VSMCs) derived from carotid arteries of female Sprague-Dawley rats release soluble factors that are competent to bind Boyden chamber membranes and promote adventitial fibroblast migration.7 Furthermore, pretreatment of VSMCs with 17ß-estradiol (E2, 10-9 to 10-7 mol/L) resulted in dose-dependent inhibition of adventitial fibroblast migration. The inhibitory effect of estrogen on fibroblast migration was blocked by cotreatment of VSMCs with the estrogen receptor (ER) antagonist ICI 182780 (10-7 mol/L). These findings, coupled with the observation that the ER was detected in early-passage VSMCs, but not in fibroblasts, suggested a novel mechanism of estrogen-mediated vasoprotection: Estrogen modulates VSMC expression and secretion of factor(s) promoting migration of adventitial fibroblasts via an ER-dependent mechanism. Identification of the estrogen-sensitive soluble chemoattractant factor(s) involved in this process is requisite to establish molecular mechanisms regulating estrogen-induced vasoprotection.
Cellular migration involves the collaboration of a number of
variables, which are mediated to a great extent by members of the
integrin family of transmembrane cell adhesion receptors for
extracellular matrix (ECM) molecules.8 9 10 In a variety of
balloon-injured animal models, expression of both osteopontin (OPN) and
one of its specific receptors,
integrin-ß3, has been shown to be
elevated in the blood vessel wall.11 12 13 In addition, both
neutralizing antibodies against OPN and Arg-Gly-Asp (RGD) peptides,
which inhibit cellular integrin-ß3
interactions with OPN, suppressed neointima formation in
these models.13 14 15 16 The present in vitro studies were
designed to determine a potential modulatory role for estrogen relevant
to fibroblast migration and interactions between
integrin-ß3 receptors and OPN. Several
lines of evidence provide a compelling argument that estrogen
indirectly attenuates integrin-ß3–dependent
adventitial fibroblast migration after inhibition of OPN expression in
VSMCs.
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Methods |
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Antibodies, Peptides, and Reagents
Hamster anti-rat ß1 subunit (clone Ha2/5), mouse anti-rat ß3 subunit (clone F11), and mouse IgG1 (
-isotype; negative control) were purchased from
Pharmingen; mouse anti-human subunit (clone VNR139) was
purchased from Gibco/BRL; FITC-conjugated goat anti-mouse IgG and goat
anti-hamster IgG were purchased from KPL, Inc. Cyclic
Gly-Pen-Gly-Arg-Gly-Asp-Ser-Pro-Cys-Ala (GPenGRGDSPCA), linear
Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP), and linear Gly-Arg-Ala-Asp-Ser-Pro
(GRADSP) (negative control) were purchased from GIBCO/BRL.
17ß-Estradiol was purchased from Sigma Chemical Co. Recombinant rat
OPN17 was kindly provided by Dr Pi-Ling Chang (University
of Alabama at Birmingham). Vitronectin, fibronectin, and
type I collagen were purchased from Gibco/BRL.
Cell Culture
Primary cultures of VSMCs (passages 3 to 5) and adventitial
fibroblasts (passages 5 to 10) were established as
described18 19 and maintained in complete medium
containing phenol red–free DMEM, 10% (vol/vol) FBS, 4 mmol/L
L-glutamine, 100 U/mL penicillin, and 100 µg/mL
streptomycin. Individual cell populations were characterized by
morphological appearance, growth behavior, and intrinsic molecular
markers.7
RT-PCR Analysis of OPN mRNA
Subconfluent (
90%) rat VSMCs were treated with
10-7 mol/L E2 or vehicle
(0.01% ethanol) for 24 hours in phenol red–free DMEM plus 5%
(vol/vol) charcoal-treated FBS. Total RNA was extracted from cells and
analyzed by reverse transcriptase–polymerase chain reaction
(RT-PCR) under previously established conditions.20
Synthetic primers specific for rat OPN were sense,
5'-CTCCCGGT-GAAAGTGGCTGA-3' and antisense,
5'-GACCTCAGAAGATG-AACTCT-3'. Synthetic primers for rat GAPDH were
sense, 5'-TGAAGGTCGGTGTGAACGGATTTGGC-3' and antisense,
5'-CATGTAGGCCATGAGGTCCACCAC-3'. Predicted sizes of RT-PCR amplification
products for rat OPN and GAPDH mRNA are 899 and 982 bp,
respectively. OPN mRNA levels were quantified with NIH Image
(version 1.58, NIH) and standardized to levels of GAPDH mRNA from
identical samples.
Western Analysis of OPN Protein
Subconfluent (90%) rat VSMCs were treated (24 hours) with
10-7 mol/L E2 or vehicle
(0.01%, vol/vol, ethanol) for 24 hours in phenol red–free DMEM plus
5% charcoal-treated FBS. VSMC-conditioned medium (1.0 mL) was
incubated (16 hours, 4°C) with 5 µg of rabbit anti-rat polyclonal
anti-OPN antibody (gift of Dr Charles W. Prince, University of Alabama
at Birmingham). Immune complexes were precipitated (1.5 hours, 4°C)
with 50 µL protein A–Sepharose and fractionated by reducing 12.5%
(wt/vol) SDS-PAGE. Proteins were transferred electrophoretically to
PVDF (Immobilon-P, Millipore), blocked (16 hours, 20°C) with 5%
(wt/vol) dry milk in 50 mmol/L Tris-HCl (pH 7.4) containing
150 mmol/L NaCl and 0.05% (vol/vol) Tween 20, and incubated with
monoclonal antibodies against rat OPN (1:1000;
MPIIIB101, Developmental Studies Hybridoma Bank,
University of Iowa). Membranes were probed with horseradish
peroxidase–conjugated goat anti-mouse serum (1:1000, Southern
Biotechnologies). Immunoreactive bands were visualized by treatment
with ECL Western blotting detection reagents (Amersham) according to
the manufacturer’s instructions.
Cell Surface Integrin Analysis by
Fluorescence-Activated Cell Sorter
Integrin profiles on rat VSMC and adventitial fibroblast
surfaces were analyzed by flow cytometry. Subconfluent
(90%) cultures of cells were treated with
10-7 mol/L E2 or vehicle
(0.01%, vol/vol, ethanol) for 24 hours in phenol red–free DMEM
containing 5% (wt/vol) charcoal-treated FBS. Confluent cultures of
cells were trypsinized, washed with PBS containing 0.1% (wt/vol)
NaN3 and 1.0% (vol/vol) FBS, and resuspended at
106 cells/mL. Cells were fixed in 2% (vol/vol)
neutral buffered formalin for 20 minutes and washed. Primary antibodies
against integrin subunits were incubated (4°C, 45 minutes) in the
dark with 105 cells/µg antibody. Cells were
washed, incubated (4°C, 45 minutes) in the dark with FITC-conjugated
goat anti-mouse IgG or goat anti-hamster IgG, washed, and
analyzed for fluorescence by flow cytometer with a
Becton-Dickinson FACScan. Negative controls included the absence of
primary antibody or the use of irrelevant isotype-matched antibody as
the primary reagent.
Migration Assay
Migration assays were performed in Transwell migration
chambers (Costar Corp) essentially as described.7 9 For
the chemotaxis assays, upper-chamber membranes were coated with 0.5 mL
PBS containing 10 mg/mL BSA. OPN protein, resuspended in dilution
buffer (DMEM/0.1%, wt/vol, BSA), was added to the lower chambers at
the indicated concentrations. For the haptotaxis assays, upper-chamber
membranes were coated (4°C; 48 hours) with indicated concentrations
of OPN protein in dilution buffer. The lower chambers contained
dilution buffer only. Haptotaxis assays also included coating of the
upper-chamber membrane with medium conditioned by VSMCs, maintained in
the presence or absence of 10-7 mol/L
E2. In addition, depletion of OPN from medium
conditioned by VSMCs was achieved by immunoadsorption
techniques.21 Briefly, 5 µg of polyclonal anti-OPN or
nonimmune rabbit IgG was incubated (4°C; 16 hours) with 50 µL
protein A–Sepharose (Santa Cruz). After a washing (PBS),
immunocomplexed beads were incubated (4°C; 1 hour) with 1.0 mL
VSMC-conditioned medium. Clarified supernatants were used to coat upper
chambers for fibroblast haptotaxis migration assays. Additional efforts
included reconstitution of immunodepleted supernatants with defined
concentrations of recombinant OPN before upper-chamber membranes were
coated.
Adventitial fibroblasts were grown to confluence, trypsinized briefly (<1 minute), and added to an equal volume of 0.5 mg/mL soybean trypsin inhibitor. Cells were centrifuged, resuspended in dilution buffer, and incubated (30 minutes; 37°C) with or without the indicated concentrations of RGD peptides, integrin-ß1 antibody, integrin-ß3 antibody, or murine IgG1 before migration was initiated. A suspension (200 µL) of fibroblasts (3x104 cells) was added to the upper chamber under defined experimental conditions. Migration was allowed to proceed for 6 hours at 37°C in a humidified incubator, and cells that migrated to the bottom of the filter were fixed with methanol and stained with hematoxylin. Migration was quantified by cell counts of 4 random high-power (x100) fields in each well. Each assay was performed in quadruplicate.
Adhesion Assay
Adhesion studies were performed as described.7
Briefly, either 200 µL of medium conditioned by VSMCs or 100 µL of
PBS containing 10 µg/mL of specific ECM protein was added to
untreated 96-well cluster plates and incubated overnight at 4°C.
Wells were rinsed with adhesion assay buffer (mmol/L: NaCl 140, KCl
5.4, CaCl2 1, MnCl2 0.5,
D-glucose 5.56, and HEPES 10, and 1% wt/vol BSA; pH 7.4),
and nonspecific binding sites were blocked by the addition of 1%
(wt/vol) BSA in PBS for 1 hour at 37°C. Control wells contained
DMEM/0.1% (wt/vol) BSA. Confluent cultures of fibroblasts were
trypsinized minimally (<1 minute), diluted with an equal volume of 0.5
mg/mL soybean trypsin inhibitor, centrifuged,
resuspended in the adhesion assay buffer with or without indicated
concentrations of RGD peptides or anti-ß1,
anti-ß3, or murine IgG1, and incubated for 30
minutes before the adhesion assay. Fibroblasts
(4x104 cells) were plated in wells and incubated
for 45 minutes at 37°C. The assay was terminated by washing (PBS)
adherent cells, which were fixed with 4% (vol/vol)
paraformaldehyde, stained with 1% (wt/vol) crystal
violet, air-dried, and extracted in 10% (vol/vol) acetic acid. Dye
uptake was determined by an ELISA plate reader at 570 nm.
Statistical Analysis
Data are given as mean±SEM. Statistical analyses were
carried out with the CRUNCH statistical package on an IBM-compatible
personal computer. Our primary statistical test was MANOVA. Differences
in mean values due to main effects and their interactions were tested,
with a value of P<0.05 considered statistically
significant.
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Results |
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OPN mRNA expression in rat VSMCs was analyzed by RT-PCR and standardized with GAPDH mRNA from identical samples. Readily detectable steady-state levels of OPN mRNA were expressed in serum-activated VSMCs. E2 treatment (24 hours) of VSMCs induced a dose-dependent decrease of OPN mRNA, such that exposure to 10-7 mol/L E2 resulted in a significant (P<0.01) 52.7% reduction of this transcription product (Figure 1A
). Similarly, high levels of OPN
protein were readily demonstrated by Western analysis of medium
conditioned by serum-activated VSMCs. Low to undetectable
levels of OPN protein were observed in medium conditioned by VSMCs that
had been treated (24 hours) with 10-7 mol/L
E2 (Figure 1B).
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Integrin subunit expression in rat VSMCs and adventitial fibroblasts
was analyzed by analytical
fluorescence-activated cell sorting (Figure 2). VSMCs expressed high levels of
- and ß1-integrin subunits and minimal
levels of ß3-integrin subunit (Figure 2, left). Compared with VSMCs, adventitial fibroblasts expressed
significantly higher levels of ß3-integrin,
similar levels of -integrin, and slightly lower levels of
ß1-integrin (Figure 2, right). The
relative intensity of integrin subunit expression in both VSMCs and
adventitial fibroblasts was
ß1>>ß3.
E2 (10-7 mol/L) treatment
(24 hours) did not alter the expression of -,
ß1-, or ß3-integrin
subunits in either cell type (data not shown).
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Adventitial fibroblast migration was directed in a dose-dependent
manner by OPN, whether added in solution in the lower chamber of the
Transwell apparatus (Figure 3A) or coated on the upper-chamber
membranes (Figure 3B). However, fibroblast migration in response
to membrane-bound OPN (haptotaxis) was more robust than to OPN in
solution (chemotaxis). Pretreatment of fibroblasts with the
anti–ß3-integrin antibody F11 resulted in
dose-dependent reductions in OPN-induced fibroblast haptotaxis, with a
maximal reduction of 73.6% at 40 µg/mL of F11 (Figure 3C). In
contrast, similar treatment with the
anti–ß1-integrin antibody did not affect
OPN-induced fibroblast haptotaxis. Moreover, pretreatment of
fibroblasts with 0.5 mmol/L of either the linear RGD peptide
(GRGDSP) or the cyclic RGD peptide (GPenRGDSPCA) completely blocked
OPN-induced fibroblast haptotaxis (Figure 3C). The inactive
control RGD peptide GRADSP and control murine IgG had no effect on
fibroblast migration directed by OPN.
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Likewise, pretreatment of fibroblasts with the
anti–ß3-integrin antibody F11 reduced
fibroblast migration directed by VSMC-conditioned medium in a
dose-dependent fashion, with a maximal reduction of 40% (compared with
IgG control) at 80 µg/mL of F11 (Figure 4A). In contrast, treatment of
fibroblasts with the anti–ß1-integrin antibody
Ha2/5 did not affect fibroblast migration induced by VSMC-conditioned
medium. Moreover, pretreatment of fibroblasts with either GRGDSP or
GPenRGDSPCA also resulted in a dose-dependent reduction of fibroblast
migration directed by VSMC-conditioned medium, with complete inhibition
observed with 1 mmol/L of either peptide (Figure 4B). In
contrast, equivalent amounts of the inactive control RGD peptide
(GRADSP) had no effect on fibroblast migration directed by
VSMC-conditioned medium. Immunodepletion (polyclonal anti-OPN) of OPN
from medium conditioned by E2-untreated VSMCs
resulted in a significant (P<0.01) 18.8% reduction in
fibroblast haptotaxis (Figure 4C). Pretreatment (24 hours) of
VSMCs with E2 (10-7 mol/L)
resulted in a significant (P<0.01) 37.5% inhibition of
fibroblast migration. However, immunodepletion (anti-OPN) of medium
conditioned by E2-treated VSMCs had no effect on
fibroblast migration. Treatment of medium conditioned by either
E2-untreated or -treated VSMCs with nonspecific,
immobilized IgG had no effect on fibroblast migration. OPN
reconstitution of immunodepleted (anti-OPN) medium, conditioned by
either E2-untreated or -treated VSMCs,
demonstrated a significant (P<0.01), dose-dependent
increase in fibroblast migration.
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The soluble ECM proteins OPN, vitronectin, fibronectin, and
type I collagen induced significant fibroblast adhesion (Figure 5A). Pretreatment of fibroblasts with
either the anti–ß1- or
anti–ß3-integrin antibodies (20 µg/mL) as
well as the control IgG had no effect on fibroblast adhesion to OPN
(Figure 5B). Pretreatment of fibroblasts with 0.25 mmol/L
of either the linear or the cyclic RGD peptide resulted in complete
inhibition of fibroblast adhesion induced by OPN (Figure 5B).
Equivalent amounts of the inactive control peptide had no effect on
fibroblast adhesion to OPN. Similarly, pretreatment of fibroblasts with
either the anti–ß1- or
anti–ß3-integrin antibodies (20 µg/mL) as
well as the control IgG had no effect on fibroblast adhesion to medium
conditioned by VSMCs (Figure 5C). Pretreatment of fibroblasts
with 500 µmol/L of either the linear or the cyclic RGD peptide
resulted in significant, 31% to 38%, inhibition of fibroblast
adhesion to medium conditioned by VSMCs (Figure 5C). Equivalent
amounts of the inactive control peptide had no effect on fibroblast
adhesion to medium conditioned by VSMCs. Medium conditioned by VSMCs in
the presence of 10-7 mol/L
E2 had no effect on fibroblast adhesion, compared
with results obtained with medium conditioned in the absence of
E2 (Figure 5C). In general, the overall
pattern of interventional strategies using antibodies and RGD peptides
had a similar effect on fibroblast adhesion to medium conditioned by
VSMCs in the presence of 10-7 mol/L
E2 (data not shown).
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Discussion |
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The present study delineated a cellular mechanism whereby estrogen may modulate responses to vascular injury. Previously, we provided evidence that estrogen attenuates both adventitial cell migration into the neointima of balloon-injured rat carotid arteries in vivo6 and VSMC expression of factors promoting adventitial fibroblast haptotaxis in vitro.7 Expression of OPN and its
ß3-integrin receptor has been
demonstrated to be markedly elevated after injury to the blood vessel
wall.11 12 13 In addition, neutralizing antibodies against
OPN and a specific RGD peptide (XJ735) antagonist of
ß3 have been shown to suppress
neointima formation in animal models.13 14
Collectively, these observations suggested that OPN plays a regulatory
role in mediating the vascular injury response. Consequently, we
examined the effect of estrogen on these biological relationships in
our experimental, VSMC-dependent adventitial fibroblast migration
model.7 Both this study and previous efforts22 23 24 demonstrated that VSMCs in vitro express OPN mRNA and extracellular protein. The observation that estrogen inhibited VSMC production of OPN mRNA and secreted protein correlated with a significant reduction of adventitial fibroblast haptotaxis in response to membranes treated with medium conditioned by estrogen-treated VSMCs. In addition, immunodepletion of OPN from untreated VSMC-conditioned medium, and not immunodepletion of estrogen-treated VSMC-conditioned medium, attenuated fibroblast migration. The addition of recombinant OPN to either immunodepleted medium from untreated VSMCs or medium from estrogen-treated VSMCs reconstituted the haptotactic fibroblast response in a dose-dependent fashion. In fact, adventitial fibroblast migration in response to membrane-bound recombinant OPN (haptotaxis) was more robust than to OPN in solution (chemotaxis), an observation similar to that reported for VSMC responses to ECM proteins.9 25 Taken together, these findings suggest that OPN is an estrogen-inhibitable factor that may play a pivotal role in estrogen-mediated suppression of neointima formation in animal models of vascular injury.
Additional evidence of this potential was supported by examination of
the role of integrin receptor function. Integrins
ß1, ß3, and
ß5 serve as OPN receptors, in which
adhesion to OPN is regulated by each of these complexes and migration
toward OPN is dependent solely on
ß3.24 In vitro, both VSMCs
and fibroblasts expressed high cell-surface levels of - and
ß1-integrin subunits. However, compared with
VSMCs, adventitial fibroblasts expressed significantly more of the
ß3 subunit, suggesting that the
ß3 complex may be more abundant in these
cells. The observation that the
anti–ß3-integrin antibodies markedly inhibited
fibroblast haptotaxis directly by OPN or VSMC-conditioned medium,
whereas the anti–ß1-integrin antibodies had no
effect, is consistent with
ß3-integrin–mediated adventitial
fibroblast migration toward OPN. However, neither the
anti-ß1 nor anti-ß3
antibodies alone had any effect on fibroblast adhesion directed by OPN
or medium conditioned by VSMCs. This result would be consistent
with the observation that adventitial fibroblasts exhibited significant
adhesion to several ECM proteins, thereby suggesting intrinsic levels
of multiple integrin receptors.
The tripeptide RGD sequence found in several matrix proteins, including
OPN, vitronectin, fibronectin, collagen, and fibrinogen, is
recognized by a variety of integrin receptors, such as
ß1, ß3,
ß5,
5ß1, and
IIbß3.26 27 Synthetic
RGD-containing peptides have been shown to inhibit VSMC migration in
vitro and reduce neointima formation in vivo by preventing
binding of cell-surface integrin receptors (eg,
ß3) to ECM proteins.15 16
However, specific RGD peptides have distinct effects on cell adhesion
and migration to defined substrates. For instance, the linear GRGDSP
and the cyclic GPenGRGDSPCA peptides exerted contrary effects on rat
VSMC adhesion and migration.28 The present study
demonstrated that both cyclic and linear RGD peptides completely
inhibited fibroblast haptotaxis directed by OPN or VSMC-conditioned
medium and fibroblast adhesion to OPN, but only partially (31% to
38%) inhibited fibroblast adhesion to VSMC-conditioned medium. This
observation predicts an RGD peptide effect on cellular migration and
adhesion that is dependent on the heterogeneity of
intrinsic integrin receptors in different cell populations and the
nature of the matrix protein substrate under study. Nevertheless,
adventitial fibroblast haptotaxis to OPN and VSMC-conditioned medium
was mediated in an RGD-dependent manner.
In summary, the present study demonstrated that VSMCs expressed high levels of OPN mRNA and protein that were effectively inhibited by estrogen. Adventitial fibroblast haptotaxis to medium conditioned by estrogen-treated VSMCs was reduced significantly. Compared with VSMCs, adventitial fibroblasts expressed higher levels of ß3-integrin subunits. Pretreatment of fibroblasts with anti-ß3 antibodies or synthetic RGD peptides markedly inhibited fibroblast haptotaxis directed by OPN alone or VSMC-conditioned medium. Immunodepletion of OPN from VSMC-conditioned medium inhibited fibroblast haptotaxis. Reconstitution of either immunodepleted medium or medium conditioned by estrogen-treated VSMCs with recombinant OPN restored fibroblast haptotaxis. Collectively, these results suggest that estrogen indirectly attenuates integrin-ß3–dependent adventitial fibroblast migration after inhibition of OPN expression in VSMCs.
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Acknowledgments |
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This work was supported in part by grants HL-07457, HL-45990, HL-57270, and DK-51629 from the National Institutes of Health and a Grant-in-Aid (9750665N) from the American Heart Association.
Received October 21, 1999; revision received January 13, 2000; accepted January 25, 2000.
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