Estrogen Inhibits Vascular Smooth Muscle Cell–Dependent Adventitial Fibroblast Migration In Vitro
Background—Mounting experimental evidence suggests that estrogen treatment protects against neointima formation in response to vascular injury in vivo. Previous studies have suggested that this process includes the activation and migration of adventitial fibroblasts. The present in vitro study was designed to establish a mechanism whereby estrogen attenuates migration of adventitial fibroblasts.
Methods and Results—Primary cultures of vascular smooth muscle cells (VSMCs) and adventitial fibroblasts were derived from female Sprague-Dawley rats. Reverse transcriptase–polymerase chain reaction and Western blotting were used to determine that expression of the estrogen receptor (ER) was restricted to early-passage VSMCs. Migration of transduced (retrovirally mediated) fibroblasts was determined by counting the number of blue lacZ-expressing cells attached to Boyden-type chambers preconditioned under defined experimental conditions. Compared with growth medium alone, chambers treated with medium conditioned by VSMCs demonstrated a 2-fold increase in fibroblast migration, suggesting that VSMCs release soluble factor(s) competent to bind the Transwell membrane and promote fibroblast migration. In contrast, treatment of VSMCs with 17β-estradiol (10−9 to 10−7 mol/L) before preconditioning of the chamber induced a dose-dependent inhibition of fibroblast migration. Cotreatment of VSMCs with 17β-estradiol and the ER antagonist ICI-182780 (10−7 mol/L) blocked the inhibitory effect of estrogen on fibroblast migration.
Conclusions—These observations suggest a novel mechanism of hormonal vasoprotection by which estrogen directly modulates VSMC expression of factor(s) controlling migration of adventitial fibroblasts via an ER-dependent mechanism.
Participation of the adventitia in the response to vascular injury has been suggested by pathological findings of adventitial activation (inflammation and fibrosis) in coronary arteries of victims of fatal coronary artery disease.1 2 In some individuals who died suddenly at an early age, adventitial inflammation appeared to antedate intimal disease.1 Furthermore, neointima formation and/or atherosclerotic lesions have been observed in response to adventitial injury in various animal models,3 4 raising the possibility of alternative pathways of the vascular injury response. More recently, endoluminal injury of a porcine coronary artery was shown to result in significant remodeling of the adventitia.5 Activated adventitial cells were suggested to migrate into the neointima and transform into a myofibroblast phenotype.6 These findings predict that adventitial fibroblasts may play an important role after vascular injury.
A variety of animal models have demonstrated the inhibitory effects of estrogen on the vascular injury response. The balloon-injured rat carotid artery model has been used extensively in our own laboratory to study this issue.7 8 Early preliminary studies with this model have suggested that estrogen treatment attenuates both adventitial activation and the potential translocation of adventitial fibroblasts into medial and neointimal compartments. The present in vitro study was designed to provide insight into potential cellular and molecular mechanisms of adventitial cell migration that could be induced by endoluminal vascular injury and inhibited by estrogen. Primary cultures of adventitial fibroblasts from female rat carotid arteries were derived, characterized, and evaluated for their migratory behavior in response to conditioned medium from vascular smooth muscle cells (VSMCs) pretreated in the presence and absence of estrogen. Several lines of evidence suggest that estrogen indirectly attenuates fibroblast migration after estrogen receptor (ER)-dependent modulation of VSMC release of a soluble chemoattractant factor(s).
Cell Culture and Transduction
Primary VSMCs were prepared from the thoracic aorta of intact 10-week-old female Sprague-Dawley rats.9 The adventitial layer of aorta was dissected away with a scalpel blade, and the endothelium was removed with a cotton swab. The tissue explants were incubated in DMEM (Life Technologies) supplemented with 10% (vol/vol) heat-inactivated FBS (Hyclone), 4 mmol/L l-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin (complete medium). This medium did not contain phenol red because of the recognized estrogenic effects of this compound. VSMCs were allowed to grow out from the tissue, which was removed after 8 to 10 days. Population doublings were monitored, and unless otherwise indicated, only early, stabilized passages (from 3 to 6) were used for these studies.
Primary adventitial fibroblasts were recovered from the carotid arteries of intact female Sprague-Dawley rats by use of a solid support consisting of nonwoven, multifilament, angel-hair fibers of expanded polytetrafluoroethylene (PTFE) prepared as described.10 11 These fibers were surgically implanted into the neck cavity of the rat, adjacent to the carotid adventitia. Two to 3 weeks after implantation, the visible PTFE sponge was surgically removed and digested with collagenase to recover resident cells.10 11 Explanted cells were seeded and expanded in culture with complete medium.
Subconfluent (70% to 80%) primary adventitial fibroblasts (passage 3 to 4) were treated with retroviral particles encoding β-galactosidase (lacZ) as described.12 Transduced adventitial fibroblasts were isolated by fluorescence-activated cell sorter (FACS), plated, and expanded in complete medium for subsequent analyses. Expression of lacZ was monitored with the chromogenic substrate Bluo-gal.
In Situ Immunohistochemical Analysis
VSMCs and adventitial fibroblasts were seeded (1×104 cells) onto glass coverslips and allowed to attach (16 hours) in complete medium. Attached cells were washed in PBS and maintained (48 hours) under normal culture conditions. Cells were fixed (15 minutes) in 4% (vol/vol) phosphate-buffered formalin and incubated (15 minutes) with 1% (vol/vol) H2O2 in methanol. Fixed cells were washed with 50 mmol/L Tris-HCl (pH 7.5) containing 0.1 mol/L NaCl and 0.05% (vol/vol) Tween 20 (TTBS) and incubated (37°C, 45 minutes) in a humidified chamber with specific monoclonal and polyclonal antibodies (Table 1⇓) diluted in TTBS according to the manufacturers’ recommendations (Boehringer Mannheim, Serotec, Dako, Novacastra Laboratories, Sigma, Calbiochem, ICN). Antibody binding was detected with a Quick Staining Kit (Dako) using either swine anti-rabbit or swine anti-mouse horseradish peroxidase (HRP)–conjugated secondary antibodies. Peroxidase-stained samples were developed with 0.5 mg/mL 3,3′-diaminobenzidine and counterstained with Mayer’s hematoxylin (Sigma). Chromogenic development of stained samples permitted analysis of positive brown staining by light microscopy. Four coverslips per antibody staining of individual cell populations were analyzed, and ≥5 fields (≈250 cells) per coverslip were counted.
Measurement of NADPH and NADH Oxidase Activity
Confluent VSMCs and adventitial fibroblasts were washed in PBS and pretreated (4 hours) with serum-free DMEM in the absence or presence of 10−7 mol/L angiotensin II. Treated cells were washed (PBS), scrape-harvested in 15 mL ice-cold PBS, and centrifuged (10 minutes, 4°C) at 1500 rpm. Pelleted cells were resuspended and prepared by Dounce homogenization in 1.0 mL lysis buffer (4°C) containing protease inhibitors (20 mmol/L potassium phosphate [pH 7.0], 1 mmol/L EGTA, 10 μg/mL aprotinin, 0.5 μg/mL leupeptin, 0.7 μg/mL pepstatin, and 0.5 mmol/L phenylmethylsulfonyl fluoride). Protein concentration was determined by the Bradford assay (Pierce). NADPH and NADH oxidase activities were measured by a modified luminescence assay13 in 50 mmol/L phosphate buffer (pH 7.0) containing 1 mmol/L EGTA, 150 mmol/L sucrose, 100 μmol/L of either NADPH or NADH, and cellular homogenate (20 to 40 μg total protein). The reaction was initiated by the addition of 150 μL of lucigenin (final concentration 500 μmol/L). Photon emission from reduced lucigenin was measured every 15 seconds with a 5-second delay in a luminometer (model Optocomp I). NADPH and NADH oxidase activities were expressed by relative light units and normalized to protein content of each sample run in quadruplicate.
Nucleic Acid Extraction and Analysis
Total RNA was analyzed by reverse transcriptase–polymerase chain reaction (RT-PCR) techniques under previously established conditions.12 Synthetic DNA amplimers specific for rat ER-α were sense, AATTCTGACAATCGACGCCAG and antisense, GTGCTTCAACATTCTCCCTCCTC. Synthetic DNA amplimers specific for rat GAPDH were sense, TGAAGGTCGGTGTGAACGGATTTGGC and antisense, CATGTAGGCCATGAGGTCCACCAC. RT-PCR products were analyzed by 2% (wt/vol) agarose gel electrophoresis. After electrophoresis, amplification products were stained with ethidium bromide, visualized with UV light, and standardized to levels of amplified GAPDH mRNA.12
Protein Extraction and Western Analysis
Whole-cell extracts of rat VSMCs, adventitial fibroblasts, and human breast cancer cells (MCF-7) were prepared by releasing cells from monolayer cultures with 2× SDS gel sample buffer (4% wt/vol SDS, 12% wt/vol sucrose, 10% vol/vol β-mercaptoethanol, 0.125 mol/L Tris [pH 6.8], and 0.05% wt/vol bromophenol blue). All samples were sonicated for 1 second and heated at 90°C for 5 minutes before electrophoresis. Proteins were fractionated under reducing conditions by routine 10% (wt/vol) SDS-PAGE. Resolved proteins were transferred electrophoretically to nitrocellulose membranes (Amersham), which were probed (1 hour) with purified ER-21 (0.5 μg/mL) in the presence or absence of a 50-fold molar excess of a synthetic HER1–21 peptide14 and followed by HRP–conjugated goat anti-rabbit IgG (1:2000 dilution, Sigma). Immunoreactive bands were visualized by treatment with ECL Western blotting detection reagents (Amersham) according to the manufacturer’s instructions.
VSMCs (8×104 cells/well) were seeded in 24-well plates (Costar) in complete medium and grown to 70% to 80% confluence (Figure 1A⇓). Subconfluent VSMCs were washed in PBS, fed DMEM containing 5% (vol/vol) FBS, and maintained for 24 hours (Figure 1B⇓). Where experimentally indicated, the medium contained 17β-estradiol (10−9 to 10−7 mol/L) or vehicle (0.01% ethanol). To determine an ER-dependent effect, VSMCs were pretreated (4 hours) with the estrogen antagonist ICI-182780 (10−7 to 10−5 mol/L) or vehicle (0.01% ethanol) before 17β-estradiol exposure. VSMC-conditioned medium was added to a new well and incubated (16 hours) with a Transwell insert (8-μm pores) that had been precoated (16 hours) with 0.1% (wt/vol) gelatin (Figure 1C⇓). The conditioned Transwell chamber was moved to a separate well containing 0.7 mL DMEM supplemented with 0.1% (wt/vol) BSA (Figure 1D⇓).
Transduced adventitial fibroblasts were grown to 70% to 80% confluence in complete medium, washed (PBS) twice, detached with 0.1% (wt/vol) trypsin, and resuspended (1.5×105 cells/mL) in DMEM supplemented with 0.1% (wt/vol) BSA. A suspension (0.2 mL) of transduced fibroblasts was pipetted into the conditioned Transwell chamber and incubated (4 hours) at 37°C (Figure 1D⇑). After incubation (Figure 1E⇑), nonmigratory fibroblasts were removed carefully from the upper face of the Transwell chambers with a cotton swab. Transwell membranes were fixed in 0.2% (vol/vol) glutaraldehyde and stained with the chromogenic substrate Bluo-gal. LacZ-expressing (blue) fibroblasts that had migrated and adhered to the Transwell membrane were microscopically counted. In all cases, 4 separate high-power fields were counted per membrane, and all experiments were run in duplicate.
VSMCs and adventitial fibroblasts were seeded in 24-well plates in complete medium and grown to 70% to 80% confluence. Subconfluent cells were washed (PBS), fed DMEM containing either 5% (vol/vol) or 0.5% (vol/vol) FBS in the presence or absence of 10−8 mol/L 17β-estradiol, and maintained (24 hours) at 37°C. Media conditioned by individual cell populations under defined experimental conditions (n=5/condition) were collected separately. Aliquots (100 μL/well) of conditioned medium were added to individual wells of 96-well cluster plates and incubated (16 hours) at 4°C. Each treated well was washed gently with adhesion assay buffer (140 mmol/L NaCl, 5.4 mmol/L KCl, 1 mmol/L CaCl2, 1 mmol/L MgCl2, 0.5 mmol/L MnCl2, 5.56 mmol/L d-glucose, 10 mmol/L HEPES [pH 7.4], and 1% wt/vol BSA), and nonspecific binding sites were blocked (1 hour, 37°C) with 1% (wt/vol) BSA.
VSMCs and adventitial fibroblasts were grown to 70% to 80% confluence in complete medium, washed, detached with 0.1% (wt/vol) trypsin, and resuspended (4×105 cells/mL) in adhesion assay buffer. A suspension (0.1 mL) of cells was pipetted into individual wells previously treated with conditioned medium and incubated (45 minutes; 37°C). Nonadherent cells were removed by washing (PBS) and swabbing of the sides of the well. Adherent cells were fixed with 4% (vol/vol) paraformaldehyde and stained with 1% (wt/vol) crystal violet, followed by air-drying and extraction with 10% (vol/vol) acetic acid. Dye uptake was quantified by an ELISA plate reader, and results were determined as the mean total absorbance at 570 nm.
Within each in vitro experiment, cells were matched for lineage, population doublings, and days before or after confluence to avoid differences related to cell culture variables. Statistical analyses were carried out with the Crunch statistical package on an IBM-compatible personal computer. Our primary statistical test was multivariate ANOVA. Differences in mean values due to main effects and their interactions were tested, with a value of P<0.05 considered statistically significant.15
The fiber implant presumably signals the proximal cells of the tunica adventitia, which migrate into the implant site and produce molecular products competent to orchestrate relevant biological cascades reflective of typical wound healing.10 11 Histological examination of the fiber implant itself suggests that this response is composed of cells recruited from the host in which by 14 to 21 days after implantation, the entire fiber bundle is permeated with invading fibroblasts, which dominate the implant stroma. Consequently, it was reasoned that surgical removal of the fiber implants at this time would provide an explant enriched in adventitial cells.
Microscopic examination was used to compare morphological appearances of expanded populations of VSMCs and adventitial fibroblasts derived from their respective explants. Primary VSMCs displayed a typical monolayer phenotype exhibiting a characteristic hill-and-valley growth pattern. In contrast, stromal fibroblasts displayed a spindle-like, bipolar and tripolar morphology exhibiting small processes at each pole and a smooth cell border. Compared with primary VSMCs (doubling time=27.7 hours), primary adventitial fibroblasts (doubling time=19.4 hours) exhibited a growth advantage under normal culture conditions. In situ immunohistochemical analysis (Table 1⇑) of both primary cell populations failed to demonstrate the appearance of cells with either endothelial (vWF, QBEnd/40, UEA-1), lymphatic (MAC-1, ED-1, CD5), epithelial (NCL-EMA, cytokeratin), or mesothelial (HBME-1) markers. Instead, expanded populations of both VSMCs and adventitial cells demonstrated positive staining for markers (vimentin, α-SM actin, actin, HHF35, myosin) typically found in smooth muscle cells but often shared with fibroblasts. After expansion in culture, activated adventitial fibroblasts failed to demonstrate expression of desmin, an observation consistent with previous efforts.6
Both VSMCs and adventitial fibroblasts exhibited NADH and NADPH oxidase activities, each of which was stimulated (>1.4-fold) after treatment with angiotensin II (Table 2⇓). Compared with NADPH oxidase activity, both VSMCs (>7-fold) and adventitial fibroblasts (>9-fold) demonstrated significantly higher levels of NADH oxidase activity. However, compared with VSMCs, adventitial fibroblasts consistently demonstrated significantly higher levels of both NADPH (>2-fold) and NADH (>2.5-fold) oxidase activity. Collectively, these results are consistent with previous studies.13 16 17
To rigorously track the migratory behavior of adventitial fibroblasts, primary cells were transduced, selected by 2 rounds of FACS, and expanded for biochemical analyses. This approach routinely resulted in an adventitial fibroblast preparation in which >95% of the cells expressed readily detectable levels of lacZ. Compared with primary adventitial fibroblasts, transduced cells exhibited a similar phenotype, growth behavior, expression of characteristic markers (Table 2⇑), and high angiotensin II–inducible levels of NADPH/NADH oxidase. At least 4 separate explant preparations of primary and transduced fibroblast populations were examined, and each provided consistent results.
A characteristic RT-PCR product (344 bp) was identified for the ER-α gene in early passages (from 3 to 6) of VSMCs (Figure 2⇓). Late passages (from 13 to 18) of VSMCs lost their ability to express ER-α mRNA. Early-passage nontransduced and transduced adventitial fibroblasts both failed to demonstrate detectable expression of ER-α mRNA (Figure 2⇓). Early passages of VSMCs expressed readily detectable cellular levels of immunoreactive ER-α protein with an apparent molecular mass of ≈65 kDa (Figure 3⇓). In contrast, transduced adventitial fibroblasts failed to express ER-α protein. Western analysis of late-passage VSMCs and nontransduced adventitial fibroblasts failed to demonstrate the appearance of immunoreactive ER-α protein (data not shown). Collectively, these results are consistent with RT-PCR analyses and confirm that expression of ER-α mRNA and protein is restricted to early passages of primary rat VSMCs.
Transduced, lacZ-expressing fibroblasts demonstrated minimal migration to Transwell membranes preconditioned with serum-free medium (Figure 4A⇓). Preconditioning of the membrane with complete medium increased the number of migrating transduced fibroblasts. Treatment of the Transwell membrane with medium conditioned by VSMCs induced a statistically significant (P<0.01), nearly 2-fold increase in the number of migrating fibroblasts. Treatment of VSMCs with 17β-estradiol inhibited adventitial fibroblast migration in a dose-dependent manner over the range of 10−9 to 10−7 mol/L. Addition of 17β-estradiol (10−7 mol/L) to medium conditioned by late-passage VSMCs had no effect on adventitial fibroblast migration. Furthermore, there was no evidence of lacZ-expressing cells either in the culture medium of the lower chamber or attached to the bottom of the lower chamber.
The inclusion of the selective ER antagonist ICI-182780 (10−6 mol/L) alone in medium conditioned by VSMCs had no effect on fibroblast migration (Figure 4B⇑). However, pretreatment of VSMCs with the ER antagonist produced a dose-dependent inhibition of the estradiol effect, such that a 100-fold molar excess completely abrogated the ability of 10−8 mol/L 17β-estradiol to attenuate VSMC-induced migration of adventitial fibroblasts. 17β-Estradiol (10−7 to 10−9 mol/L) pretreatment of ER-negative adventitial fibroblasts had no effect on their migration (Figure 5⇓). In addition, medium conditioned by primary adventitial fibroblasts, treated in the presence or absence of 10−8 mol/L 17β-estradiol, had no effect on fibroblast migration (Figure 5B⇓).
The migration assay alone does not determine whether the ER-dependent estrogen effect is on chemotaxis or attachment or both. Consequently, we investigated primary cell adhesion to medium conditioned by either VSMCs or adventitial fibroblasts in the absence or presence of 10−8 mol/L 17β-estradiol (Figure 6⇓). Compared with preconditioning with serum-free medium, treatment of plates with medium conditioned by VSMCs induced a significant increase in the number of adhering fibroblasts (Figure 6A⇓) or VSMCs (Figure 6B⇓). Likewise, treatment of plates with medium conditioned by adventitial fibroblasts induced a significant increase in the number of adhering fibroblasts or VSMCs. Inclusion of 10−8 mol/L 17β-estradiol during medium preconditioning had no significant effect on either fibroblast or VSMC adhesion regardless of the cellular source of attachment factors.
This study provides a first indication that adventitial fibroblasts in vitro have the capability to migrate in response to a soluble factor(s) secreted by activated VSMCs and that estrogen indirectly inhibits this process in a dose- and ER-dependent manner. These findings suggest a cellular mechanism for the previous observations that adventitial cells are activated and migrate into neointima after in vivo endoluminal injury. Whether the signaling pathway responsible involves diffusion of chemoattractant/mitogenic factors from damaged VSMCs through the medium and external elastic lamina to the adventitia and/or delivery of circulating mediators to the adventitia via the vasa vasorum is uncertain.
Evidence that fibroblasts can serve as targets for chemoattractant/growth factors comes mainly from studies of wound healing.18 19 20 21 The wound-healing process is associated with early activation of fibroblasts, which subsequently proliferate, migrate, and differentiate into myofibroblasts. Similar cellular responses have been described in a variety of other pathological conditions associated with fibrogenesis and organ remodeling.21 22 23 24 Accordingly, it has been hypothesized that this fibroblast response is a general feature of tissue repair and therefore may also occur during vascular injury.21 The observation that “nonmuscle” cells are found in the media of large arteries and veins of the dog and have a higher proliferative capacity than VSMCs25 has questioned the traditional belief that undifferentiated VSMCs are entirely responsible for neointima formation in injured blood vessels.26
Systemic estrogen influences the development of intraperitoneal scarring by suppressing connective tissue formation in a murine model of peritoneal adhesion formation.27 In contrast, postmenopausal hormone replacement therapy increases the dermal content of collagen and limits age-related increases in skin extensibility.28 In vitro studies have shown that estrogen influences processes critical to wound repair, such as cellular proliferation and cytokine production.29 30 Furthermore, there is evidence that the estrogen effect on wound healing may be mediated by the action of estrogen-stimulated cytokine release.30 These studies suggested that estrogen provided an indirect effect on dermal fibroblasts by stimulating responses in other cell types invading the wound site. In studies reported here, a similar indirect effect of estrogen is suggested by which ER-dependent modulation of VSMC release of a soluble factor(s) attenuates adventitial fibroblast migration. Additional in vitro and in vivo efforts will be necessary to identify the soluble factor(s) involved in this hormone-mediated vasoprotective process.
This work was supported in part by grants HL-07457, HL-45990, and HL-52070 from the National Heart, Lung, and Blood Institute and a Grant-in-Aid (9750665N) from the American Heart Association. The authors thank Marla Kolarik for her assistance in the preparation of the manuscript and Danielle Barstad for her technical assistance in the Western blotting experiments.
- Received January 27, 1999.
- Revision received May 27, 1999.
- Accepted June 2, 1999.
- Copyright © 1999 by American Heart Association
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