Estrogen Attenuates Integrin-β3–Dependent Adventitial Fibroblast Migration After Inhibition of Osteopontin Production in Vascular Smooth Muscle Cells
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 β1- and β3-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 αν and β1 and higher levels of integrin-β3. Exogenous OPN (5.0 to 40 μg/mL) directed fibroblast migration in a dose-dependent fashion. Anti–β3-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–β1-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-β3–dependent adventitial fibroblast migration after inhibition of OPN expression in VSMCs.
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 17β-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-ανβ3, 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-ανβ3 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-ανβ3 receptors and OPN. Several lines of evidence provide a compelling argument that estrogen indirectly attenuates integrin-β3–dependent adventitial fibroblast migration after inhibition of OPN expression in VSMCs.
Antibodies, Peptides, and Reagents
Hamster anti-rat β1 subunit (clone Ha2/5), mouse anti-rat β3 subunit (clone F11), and mouse IgG1 (κ-isotype; negative control) were purchased from Pharmingen; mouse anti-human αν 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. 17β-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.
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 κ 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 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-β1 antibody, integrin-β3 antibody, or murine IgG1 before migration was initiated. A suspension (200 μL) of fibroblasts (3×104 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 (×100) fields in each well. Each assay was performed in quadruplicate.
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-β1, anti-β3, or murine IgG1, and incubated for 30 minutes before the adhesion assay. Fibroblasts (4×104 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.
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.
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⇓).
Integrin subunit expression in rat VSMCs and adventitial fibroblasts was analyzed by analytical fluorescence-activated cell sorting (Figure 2⇓). VSMCs expressed high levels of αν- and β1-integrin subunits and minimal levels of β3-integrin subunit (Figure 2⇓, left). Compared with VSMCs, adventitial fibroblasts expressed significantly higher levels of β3-integrin, similar levels of αν-integrin, and slightly lower levels of β1-integrin (Figure 2⇓, right). The relative intensity of integrin subunit expression in both VSMCs and adventitial fibroblasts was β1>αν>β3. E2 (10−7 mol/L) treatment (24 hours) did not alter the expression of αν-, β1-, or β3-integrin subunits in either cell type (data not shown).
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–β3-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–β1-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.
Likewise, pretreatment of fibroblasts with the anti–β3-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–β1-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.
The soluble ECM proteins OPN, vitronectin, fibronectin, and type I collagen induced significant fibroblast adhesion (Figure 5A⇓). Pretreatment of fibroblasts with either the anti–β1- or anti–β3-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–β1- or anti–β3-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).
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 ανβ3-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 ανβ3 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 ανβ1, ανβ3, and ανβ5 serve as OPN receptors, in which adhesion to OPN is regulated by each of these complexes and migration toward OPN is dependent solely on ανβ3.24 In vitro, both VSMCs and fibroblasts expressed high cell-surface levels of αν- and β1-integrin subunits. However, compared with VSMCs, adventitial fibroblasts expressed significantly more of the β3 subunit, suggesting that the ανβ3 complex may be more abundant in these cells. The observation that the anti–β3-integrin antibodies markedly inhibited fibroblast haptotaxis directly by OPN or VSMC-conditioned medium, whereas the anti–β1-integrin antibodies had no effect, is consistent with ανβ3-integrin–mediated adventitial fibroblast migration toward OPN. However, neither the anti-β1 nor anti-β3 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 ανβ1, ανβ3, ανβ5, α5β1, and αIIbβ3.26 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, ανβ3) 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 β3-integrin subunits. Pretreatment of fibroblasts with anti-β3 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-β3–dependent adventitial fibroblast migration after inhibition of OPN expression in VSMCs.
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.
- Copyright © 2000 by American Heart Association
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