Cardiovascular Research

Cardiovascular Research 1999 43(3):788-797; doi:10.1016/S0008-6363(99)00159-5
© 1999 by European Society of Cardiology

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Copyright © 1999, European Society of Cardiology

Estrogen receptor- gene transfer into bovine aortic endothelial cells induces eNOS gene expression and inhibits cell migration

Enqing Tan¹, Milind V Gurjar, Ram V Sharma and Ramesh C Bhalla^*

Department of Anatomy and Cell Biology, and The Cardiovascular Center, The University of Iowa College of Medicine, Iowa City, IA 52242, USA

^* Corresponding author. Tel.: +1-319-335-7718; fax: +1-319-335-71980 ramesh-bhalla{at}uiowa.edu

Received 17 August 1998; accepted 23 April 1999

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Objectives: It has been suggested that estrogen may improve endothelial cell function to delay the onset of atherosclerosis in pre-menopausal females, though its mechanism of action is not fully understood. We examined the hypothesis that human estrogen receptor- (ER) gene transfection improves the endothelial cell function. Methods: A replication deficient adenoviral vector was used to transfect the ER gene into bovine aortic endothelial cells (BAEC) and a GFP gene containing vector was used as control. Expression of the eNOS gene was determined by Northern blot analysis and enzyme activity assay; cell migration was assayed using a Transwell apparatus; and tyrosine phosphorylation of FAK was estimated by Western blot analysis. Results: ER gene transfection of endothelial cells produced a 2–3-fold increase in eNOS mRNA and protein levels as well as a significant increase (P<0.05) in NOS activity as measured by citrulline assay and nitrite accumulation in the media in response to bradykinin stimulation. Treatment of cells with estrogen blocking agent ICI 182780 inhibited eNOS induction in response to ER transfection. ER gene transfection significantly inhibited (P<0.05) bFGF-induced chemotactic migration of endothelial cells but increased cell attachment to fibronectin, laminin, and type I and IV collagens. ER gene transfer also inhibited bFGF-stimulated tyrosine phosphorylation of FAK. Conclusion: Our results suggest that the atheroprotective effects of estrogen may in part be mediated by ER-induced upregulation of eNOS gene expression and maintenance of endothelial cell function and integrity.

KEYWORDS Ad5/RSV, Replication deficient adenovirus sero type 5 construct containing Rous Sarcoma Virus promoter sequence; BAEC, bovine aortic endothelial cells; eNOS, endothelial nitric oxide synthase; FAK, focal adhesion kinase; GFP, green fluorescent protein; ER, human estrogen receptor ; LNA, nitro L-arginine; MEM, minimal essential medium; NO, nitric oxide; PBS, phosphate buffered saline

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
The atheroprotective effects of estrogen on the coronary vasculature are supported by numerous epidemiological as well as estrogen replacement studies [1,2]. In addition, extensive experimental evidence suggests that estrogen significantly decreases myointimal hyperplasia and vascular smooth muscle cell proliferation [2,3]. However, the mechanisms of estrogen action in the blood vessel wall are not fully understood. Recent studies suggest that nitric oxide (NO) plays an important role in estrogen-mediated vascular protection [4]. However, efforts aimed at demonstrating estrogen-mediated eNOS gene induction in cultured endothelial cells have been inconclusive [5–11]. It is possible that this failure could be due to the lack of or low levels of estrogen receptor expression in cultured cells [7,10]. It has been recently demonstrated that expression of estrogen receptor decreases in endothelial cells with passage and culture conditions [5]. This may explain the inconsistent results of estrogen-mediated eNOS expression in cultured endothelial cells [5].

The effects of estrogen on the angiogenic behavior of endothelial cells have been recently reported in several studies. Estrogen appears to inhibit tumor necrosis factor alpha (TNF)-induced programmed cell death of endothelial cells [12]. Similarly, estrogen enhances endothelial cell attachment, migration, proliferation, and tube-like structure formation [13,14]. In contrast, estrogen inhibits bFGF-stimulated endothelial cell proliferation [15]. Several studies have suggested that altered interactions between vascular endothelial cells lining the lumenal surface of blood vessels and their underlying matrix will likely damage the integrity of the vascular endothelium and its functions [16,17]. Therefore, it is possible that estrogen acts as a survival factor for endothelial cells by increasing interactions with matrix proteins.

Observations made in our laboratory have shown that eNOS gene transfection inhibits PDGF-BB-induced vascular smooth muscle cell migration and tyrosine phosphorylation of focal adhesion kinase (FAK) [18]. Recent evidence suggests that FAK plays a key role in mediating cell adhesion, spreading, migration and proliferation [19]. Tyrosine phosphorylation of FAK in endothelial cells is stimulated by various stimuli, including bFGF [20], suggesting that FAK tyrosine phosphorylation may be involved in the signal transduction pathway of these growth factors in endothelial cells. However, whether the tyrosine phosphorylation of FAK in endothelial cells in response to bFGF stimulation is regulated by estrogen receptors has not been investigated.

The effects of estrogen are mediated via binding to estrogen receptors (ER) that act as ligand-activated transcription factors [21]. Two types of estrogen receptors have been described (ER and ERβ), and they are present in most tissues including arterial cells [2,13,22–25]. Although, estrogen-dependent ER activation has been shown to repress [26], as well as to induce [1,27] a number of target genes in endothelial and vascular smooth muscle cells, the relative importance of ER and ERβ in regulating endothelial and vascular smooth muscle cell function is not known. Therefore, to better understand ER-mediated endothelial cell function and to explore the underlying mechanisms, we have overexpressed the human ER gene in bovine aortic endothelial cells (BAEC). Our results demonstrate that overexpression of the ER gene in BAEC markedly upregulates eNOS gene expression, inhibits bFGF-induced cell migration and tyrosine phosphorylation of FAK, and increases cell attachment to various extracellular matrix (ECM) proteins.

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
2.1 Materials
Chemicals and materials were obtained from the following sources: RNA STAT-60 RNA/mRNA isolation reagent (TEL-TEST ‘B’, INC., Freindswood, TX); L-[2,3,4,5-³H]-arginine (63 Ci/mmol) and [-³²P]-dCTP (3000 Ci/mmol), Amersham; Dowex AG50W-X8 (200-mesh), Bio-Rad; random primed DNA labeling kit, Boehringer Mannheim; ICI 182780 was a generous gift from Zeneca pharmaceuticals, Macclesfield, UK. Water-soluble 17β-estradiol, nitro-L-arginine (LNA), as well as other chemicals not listed were the highest grade available from Sigma. Trypsin 0.25%/EDTA 0.1%, Medium 199, and other cell culture additives were purchased from the Cell Culture Center of the University of Iowa. Bovine endothelial nitric oxide synthase cDNA was kindly provided by Dr. Thomas M. Michel, Brigham and Women’s Hospital, Boston, MA [28] and full-length human ER cDNA was a generous gift from Dr. Geoffrey L. Greene, University of Chicago [29].

2.2 Adenoviral vectors
We have used an adenoviral vector, Ad5/RSVER, carrying the ER gene to overexpress ER in endothelial cells. Adenoviral vectors carrying reporter gene for green fluorescent protein (Ad5/RSVGFP) was used as control vector. Adenoviral vectors containing ER and GFP genes were prepared by the University of Iowa Vector Core as described elsewhere [18,30]. The DNA constructs of this virus comprise almost a full-length copy of the adenovirus genome in which the ER or GFP expression cassette is incorporated at the site of E1 region deletions. In this cassette, a RSV (Rous sarcoma virus) promoter to drive their transcription precedes transgenes. A polyadenylation sequence of SV40 is cloned downstream of transgenes. For each vector, high titer adenovirus stocks were prepared and triple plaque purified to ascertain that recombinant viral vectors were free from wild type virus contamination. Virus titer was determined by plaque assay on HEK 293 cells and virus was suspended in phosphate buffered saline (PBS) containing 3% sucrose and stored at –70°C.

2.3 Cell culture
Bovine aortic endothelial cells were cloned and cultured in the tissue culture facility at the University of Iowa Cardiovascular Center as described elsewhere [31] and were used between 4 and 9 passages. Endothelial cells were cultured in Medium 199 containing 10% fetal calf serum (FCS) and regular supplements (MEM vitamins, MEM amino acids, L-glutamine, sodium bicarbonate, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2.5 µg/ml fungizone). Hormone-free medium was prepared with phenol red-free M199 and charcoal-treated FCS as well as other regular supplements. FCS was treated with dextran-coated charcoal (Sigma) to remove the steroid hormones. Cells were kept in phenol red-free medium after ER gene transfer for the duration of experiments to decrease the estrogenic activity of phenol red confounding the experimental results.

2.4 Adenovirus-mediated gene transfer into BAEC
Adenovirus-mediated gene transfer into BAEC was performed as described elsewhere [18,30,32]. Briefly, confluent cells, grown in 100 mm cell culture dishes, were incubated with 50 MOI (multiplicity of infection) of Ad5/RSVER or Ad5/RSVGFP in 1.5 ml serum-free M199 containing 0.15% BSA for 2 h at 37°C. One MOI is defined as one pfu (plaque forming unit) of virus per cell. Then, 6.5 ml of M199 containing 1% charcoal-treated FCS was added and cells were re-incubated at 37°C for 48 h in the absence or presence of estradiol (3.6 nmol/l). For green fluorescent protein expression and immunocytochemical localization of eNOS protein, cells were grown on 18-mm glass coverslips and transfected in a similar manner.

2.5 Northern blot analysis
Total RNA was extracted using RNA STAT60 RNA isolation reagent and Northern blot analysis was performed [33]. RNA was quantified spectrophotometrically. Denatured RNA (10 µg) was separated by electrophoresis and transferred onto a nylon membrane. The eNOS cDNA and estrogen receptor cDNA probes were radiolabeled by the random primed DNA labeling technique as described by the manufacturer (Boehringer Mannheim) and the blots were prehybridized and hybridized with radiolabeled probes. Signals were visualized through autoradiography. The relative intensities of the RNA bands were quantified by scanning densitometry using Adobe Photoshop Version 3.0 and a UMAX Gemini D-16 scanner. The densitometric data are presented as mRNA/18s RNA ratios.

2.6 Western blot analysis
Cells were washed three times in cold PBS and lysed for 10 min at 4°C using RIPA buffer containing 1x PBS pH 7.4, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 10 mmol/l sodium orthovanadate, 10 mmol/l PMSF, 1 µmol/l aprotinin [18,32]. The cells were then scraped, pipetted and the lysates were collected. After centrifugation at high speed for 10 min, the supernatant was aliquoted and stored at –20°C. The protein content of all samples was determined using the Bradford assay (Bio-Rad). A 10-µg amount of protein per lane was separated on 7.5% SDS–PAGE gels and transferred to PVDF membrane (Amersham). The membranes were blocked in 5% (w/v) non-fat dry milk in TBST containing 20 mmol/l Tris–HCl pH 7.6, 137 mmol/l NaCl, and 0.05% (v/v) Tween-20 and incubated at room temperature for 1 h with mouse monoclonal anti-estrogen receptor antibody (1:400 dilution, Santa Cruz), mouse monoclonal anti-FAK antibody (1:1000; Transduction Laboratories, Lexington, KY), or polyclonal rabbit anti-eNOS antibody (1:400; Santa Cruz) [18,32]. Signals were visualized by incubation with anti-mouse or anti-rabbit IgG horseradish peroxidase conjugate (1:10 000 dilution, Sigma) for 1 h followed by ECL chemiluminescence detection [18,32]. For blotting phosphotyrosine-containing proteins monoclonal antiphosphotyrosine antibody RC20 horseradish peroxidase conjugate was used and 1% (w/v) bovine serum albumin was used for blocking the non-specific binding as suggested by the manufacturer (Transduction Laboratories).

2.7 Citrulline assay
The eNOS enzyme activity was measured using total cell homogenates by the conversion of L-[³H]-arginine to L-[³H]-citrulline according to the published procedure [32,34]. The enzyme activity assays were carried out at 37°C for 15 min and the amount of generated L-[³H]-citrulline was measured by liquid scintillation counting. Specific calcium dependent NOS activity was determined by estimating the difference between L-[³H]-citrulline produced in tubes containing calcium–calmodulin and those containing both calcium–calmodulin and 200 µmol/l LNA and expressed as pmol/min per 10⁷ cells.

2.8 Cell migration assay
Cell migration was measured using a Transwell migration apparatus (Costar Inc., Cambridge, MA) according to the published procedure [14]. The PVP-free filters with 8-µm pores were coated with gelatin on both top and bottom surfaces (5 µg each side). Cells were lifted with trypsin/EDTA, washed once in phenol red-free M199/0.15% BSA and resuspended at a concentration of one million cells per ml. To start the assay 100 000 cells in 100 µl were loaded to the upper chamber of the Transwell and 300 µl M199/0.15% BSA containing 10 ng/ml bFGF (R&D, Minneapolis, MN) was loaded into the lower chamber of the Transwell. The Transwell were incubated for 10–12 h at 37°C. Estradiol (3.6 nmol/l) was added in both upper and lower chambers for estradiol treatment. At the end of the incubation, the cells on the top surface of the filters were wiped off and the migrated cells on the bottom surface of the filters were fixed and stained with hematoxylin, and counted in eight random 400x microscope fields for each filter. The results are expressed as the mean±SEM of the number of cells per field.

2.9 Cell adhesion assay
Cell adhesion assay was performed as previously described [14]. GFP- and ER-transfected cells were allowed to express the transfected genes for 48 h. The cells trypsinized, pelleted, and resuspended in serum-free, steroid-free, and phenol red-free medium. Various extracellular matrix proteins (ECM) including laminin, fibronectin, type I or type IV collagen were prepared, diluted with sterile ddH₂O to 6 µg/100 µl, respectively, and coated onto 48-well plates (Costar, MA) (100 µl/well). The plates were dried at room temperature for 4–6 h. Cells were plated onto the coated plates (10 000 cells/well) and allowed to attach for 4 h at 37°C. At the end of incubation, the cells were rinsed twice with PBS, trypsinized, and counted using a Coulter cell counter. Results were expressed as the mean±SEM of the cell number per well.

2.10 Statistical analysis
Statistical analysis was carried out using Student’s t-test or ANOVA and differences between the treatment groups were considered significant at P<0.05.

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
3.1 Efficiency of adenovirus-mediated ER gene transfer in BAEC
To overexpress ER in cultured endothelial cells, adenoviral vectors were employed due to their high efficiency in transducing recombinant genes into both dividing and non-dividing cells and their unique property of lack of integration into host cell DNA. The high efficiency of adenovirus-mediated gene transfer was confirmed by transfecting confluent BAEC with 50 MOI viral vectors containing GFP gene. Fluorescent microscopy observations demonstrated 70–80% of cells expressing high level of GFP protein (data not shown). Thus, in all other experiments we have used 50 MOI of either control vectors (Ad5/RSVGFP or Ad5/RSVlacZ) or ER vector (Ad5/RSVER) to transfect BAEC.

The expression of ER in transfected cells was verified at both mRNA and protein levels. Transfection of BAEC with 50 MOI of viral construct containing ER gene produced a marked increase in ER protein level measured by Western blot analysis (Fig. 1(A)), while the cells transfected with GFP-expressing control vectors showed undetectable levels of ER protein. Northern blot analysis also showed that the ER mRNA level was markedly increased in ER-transfected cells compared with GFP-transfected cells (Fig. 1(B)). These results demonstrate that adenoviral vectors are highly efficient in overexpressing ER gene in cultured endothelial cells.

View larger version (72K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Expression of ER gene in BAEC. The cells grown were transfected with adenoviral vectors carrying ER gene or control gene (GFP) as described in the Methods. Panel (A) shows a representative autoradiogram demonstrating high level of estrogen receptor protein in ER-transfected cells by Western blot analysis. Panel (B) shows a representative autoradiogram demonstrating high levels of estrogen receptor mRNA in ER-transfected cells by Northern blot analysis. Ethidium-bromide stained 18s RNA was used to compare RNA loading in different lanes.

 
3.2 Effects of ER gene transfer on eNOS gene expression and enzyme activity in BAEC
To evaluate the functional aspect of the overexpressed ER and to test their effect on eNOS gene expression, eNOS protein and mRNA levels were estimated by Western and Northern blot analyses. Estrogen did not increase the eNOS protein level in GFP-transfected cells as shown by Western blotting (Fig. 2(A)). However, ER gene transfer increased the eNOS protein level by 2–3-fold compared to GFP-transfected cells and this increase was not affected by the presence of estradiol (Fig. 2(A) and (B)). These results, together with the observation that the inhibitory effect of ER gene transfer on BAEC migration was not affected by the presence of estradiol, suggest that the ER transferred in BAEC is functional in the absence of its ligand estradiol. Therefore, in most of the subsequent experiments, estradiol was omitted. The eNOS mRNA level evaluated by Northern blot analysis was increased by 2–3-fold in ER-transfected cells compared to GFP-transfected cells (Fig. 2(C) and (D)). Immunocytochemical localization of eNOS protein in ER-transfected cells also revealed an increase in eNOS protein expression compared to GFP-transfected cells (data not shown). Hence, ER gene transfer in BAEC results in functionally coupled upregulation of eNOS gene expression, suggesting that the overexpressed ER is capable of inducing genomic effects.

View larger version (38K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 eNOS gene expression in ER- and GFP-transfected BAEC. Confluent cells were transfected with adenoviral vectors containing ER GFP genes and the total cellular RNA or cell lysates were prepared as described in the Methods. Panel (A) shows a representative autoradiogram demonstrating that ER gene transfer upregulated eNOS protein levels by Western blot analysis. Panel (B) shows the densitometric analysis of data, mean±SEM (n=3). There were no significant differences in the eNOS protein levels in the presence and absence of estradiol for both GFP- and ER-transfected cells. Panel (C) shows a representative autoradiogram demonstrating that ER gene transfer upregulated eNOS mRNA levels by northern blot analysis. Ethidium-bromide stained 18s RNA was used to compare RNA loading in different lanes. Panel (D) shows the densitometric analysis of data, mean±SEM (n=6–8). * Indicates a significant increase (P<0.05) in eNOS protein or mRNA/18s RNA ratio compared to control GFP-transfected cells.

 
To further confirm the effect of overexpressed ER on eNOS gene expression, eNOS enzyme activity was measured using total cell homogenates by the conversion of L-[³H]-arginine to L-[³H]-citrulline. The L-[³H]-citrulline production was significantly increased (P<0.05) in ER-transfected cells compared to GFP-transfected cells (Fig. 3(A)). The NOS activity was calcium dependent (Fig. 3(A)). In addition, nitrite production in culture media was evaluated spectrophotometrically. Since NO is produced in endothelial cells in response to bradykinin that increases cytosolic calcium concentration [35], bradykinin was used. ER- and GFP-transfected cells were stimulated with 1 µM bradykinin for 60 min and the nitrite level in the media was measured using the Griess reagent. Nitrite production in response to bradykinin was significantly (P<0.05) increased in ER-transfected cells compared to GFP-transfected cells while basal nitrite production in the absence of bradykinin was not different between GFP- and ER-transfected cells (Fig. 3(B)).

View larger version (13K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Effect of ER gene transfer on eNOS enzyme activity. Panel (A) show eNOS activity measured by citrulline production in ER- and GFP-transfected cells. Data are given as mean±SEM (n=9). Transfected cells were homogenized and total cell homogenate used for citrulline assay as described in the Methods. * Denotes a significant increase (P<0.05) in citrulline production in ER-transfected cells compared to GFP-transfected cells. Panel (B) shows eNOS activity by nitrite accumulation in the culture medium in response to bradykinin in ER- and GFP-transfected cells. Cells grown in 100 mm dishes were washed twice with serum-free and phenol red-free M199 media containing 0.15% BSA, and incubated in 2 ml of the same media for 15 min for equilibration. Bradykinin (BK, 1 µmol/l) was then added into the culture medium and cells were further incubated for 60 min at 37°C. Nitrite levels in the culture medium were measured by nitrite assay as described in the Methods. Results represent mean±SEM (n=9). * Denotes a significant increase (P<0.05) in nitrite production in ER-transfected cells compared to GFP-transfected cells.

 
3.3 Effect of ICI 182780 on ER-mediated increase in eNOS expression
To confirm that increase in eNOS expression in ER gene transfected cells is due to transactivation of eNOS gene by ER, we tested the effect of ER antagonist ICI 182780. Recently it has been shown in transient transfection assays that ICI 182780 (10 µM) inhibits expression of reporter plasmid carrying estrogen response element driving luciferase gene (ERE-Luc) and mVEGF-Luc reporter gene expression in endothelial and VSM cells [36]. Therefore, we used 10 µM ICI 182780 to demonstrate the specificity of overexpressed ER on eNOS gene expression. ER-induced eNOS expression was significantly inhibited by ICI 182780 (Fig. 4(A) and (B)). In GFP-transfected cells ICI 182780 slightly inhibited the eNOS activity and protein levels, but the differences were not significant (Fig. 4(A) and (B)). These results would suggest that an increase in eNOS expression in ER gene transfected BAEC is due to overexpressed ER that function in the absence of a ligand.

View larger version (17K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Effect of estrogen antagonist ICI 182780 on ER gene transfer-mediated increase in eNOS enzyme activity. Cells were transfected with GFP or ER vectors as described in the Methods. ICI 182780 was added at the time of gene transfer and maintained for 48 h after gene transfer. Control cells were incubated with equivalent concentration of DMSO (solvent for ICI 182780). Cell lysates were collected 48 h after gene transfer for western blotting and citrulline assay as described in the Methods. Panel (A) shows a representative Western blot for eNOS protein levels measured in ER- and GFP-transfected cells. Similar results were obtained in two other experiments. Panel (B) shows eNOS activity measured by citrulline assay in different treatment groups. Data are given mean±SEM (n=3). (a) Comparison between GFP and ER, (b) comparison between ER and ER plus ICI 182780. * Denotes a significant difference between treatment groups. ER gene transfer increased eNOS activity and protein levels by 2-fold compared to GFP-transfected cells, and ICI 182780 significantly inhibited ER-mediated increase in eNOS protein levels and enzyme activity.

 
3.4 Effect of ER gene transfer on BAEC migration
To study the effect of overexpressed ER on endothelial cell behaviors in vitro, we performed the cell migration assay using the Transwell migration apparatus. Basic FGF-induced BAEC migration was significantly inhibited (P<0.05) in ER-transfected cells compared to GFP-transfected cells (Fig. 5). The presence of estradiol did not affect bFGF-induced BAEC migration in both ER- and GFP-transfected cells.

View larger version (31K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Effect of ER gene transfer on BAEC migration. The cells were transfected with ER- or GFP-containing vectors and the migration assay was performed as described in the Methods. The results represent the mean±SEM (n=4). * Denotes that overexpression of ER significantly (P<0.05) inhibited bFGF-induced BAEC migration compared to GFP-transfected cells. There were no significant differences in bFGF-induced BAEC migration in the presence and absence of estradiol for both GFP- and ER-transfected cells.

 
3.5 Effect of ER gene transfer on tyrosine phosphorylation of FAK
Treatment of BAEC with 10 ng/ml bFGF gave rise to multiple protein bands containing phosphotyrosine as detected by anti-phosphotyrosine immunoblotting in both GFP- and ER-transfected cells. The most prominent phosphorylated band was FAK. To confirm the phosphorylation of FAK, PVDF membranes carrying separated proteins were first immunoblotted with anti-phosphotyrosine antibody, then stripped and re-blotted for FAK. In GFP-transfected cells the tyrosine phosphorylation of FAK in response to bFGF stimulation peaked at 15 min and then gradually returned to the pre-stimulation level, while in ER-transfected cells bFGF-stimulated tyrosine phosphorylation of FAK showed a slow and gradual increase (Fig. 6).

View larger version (31K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 Effect of ER gene transfer on bFGF-induced tyrosine phosphorylation of focal adhesion kinase (FAK). Confluent cells were transfected with ER- or GFP-containing adenoviral vectors as described in the Methods. Then bFGF was added into the culture medium at the final concentration of 10 ng/ml and incubated at 37°C for indicated time periods. Western blot analysis was performed as described in the Methods using anti-phosphotyrosine and anti-FAK antibodies. Panel (A) gives a set of representative autoradiograms showing FAK tyrosine phosphorylation and FAK protein levels in GFP- and ER-transfected cells. Panel (B) shows the densitometric analysis of data for FAK tyrosine phosphorylation expressed as the mean±SEM (n=3).

 
3.6 Effect of ER gene transfer on BAEC adhesion to extracellular matrix proteins
To examine whether ER gene transfer affects BAEC adhesion to various ECM proteins, GFP- and ER-transfected BAEC were seeded in 24-well dishes precoated with 7.5 µg/cm² of either laminin, fibronectin, type I collagen or type IV collagen. The cells were allowed to adhere for 4 h. ER gene transfer significantly (P<0.05) increased the cell attachment to all these ECM proteins (Fig. 7).

View larger version (17K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 7 Effect of ER gene transfer on BAEC attachment to various extracellular matrix proteins. GFP- and ER-transfected cells were lifted with trypsin/EDTA and resuspended in serum-free, steroid-free, and phenol red-free medium. The cells were seeded onto ECM protein-coated 24-well dishes at a concentration of 10 000 cells/well and were incubated for 4 h at 37°C. The cells were washed, trypsinized and counted using a Coulter cell counter. Data are given as the mean±SEM (n=8). * Denotes a significant increase (P<0.05) in the cell number in ER-transfected cells compared to GFP-transfected cells for each ECM protein.

 

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
The present study demonstrates that (1) adenovirus vectors are efficient tools for overexpressing ER gene in vascular endothelial cells; (2) ER gene transfection in BAEC increases eNOS gene expression at mRNA and protein levels as well as NOS enzyme activity, and this increase is inhibited by estrogen antagonist ICI 182780; (3) overexpression of ER gene inhibits bFGF-induced BAEC migration and tyrosine phosphorylation of FAK; (4) ER gene transfer increases endothelial cell attachment to various ECM proteins. These results suggest that ER is capable of inducing eNOS gene expression in endothelial cells.

Immunocytochemical studies have demonstrated that the expression of ER is much lower in atherosclerotic vessels than in normal arteries, suggesting a role for the ER or its deficiency in atherosclerosis [25]. It has been suggested that estrogen may alter arterial function by increasing NO production by the endothelial cells [4,37]. However, studies testing the effect of estrogen on eNOS gene expression in cultured endothelial cells have been inconsistent. A few studies have demonstrated estrogen-mediated upregulation of eNOS gene in cultured endothelial cells [9,11]. In contrast, several investigators have failed to show estrogen-dependent induction of eNOS gene expression in cultured endothelial cells [6,7,10], which is consistent with our observation that estradiol fails to induce eNOS gene expression in BAEC. It has been reported that the expression of ER decreases in endothelial cells with increasing passage number in culture [5]. Thus, it is possible that these discrepancies could be due to the lack of or very low levels of ER in cultured cells.

In the present study, we were unable to detect ER gene expression by Northern and Western analyses, suggesting that cultured BAEC may lack or express very low levels of ER. This allowed us to directly test whether ER can induce eNOS gene expression in endothelial cells by overexpressing ER in BAEC. Our results show that overexpression of ER in BAEC results in a marked (2–3-fold) increase in eNOS gene expression estimated by an increase in mRNA level, protein level, enzyme activity and nitrite production. The upregulation of eNOS gene expression in BAEC expressing ER unequivocally demonstrates that ER is capable of inducing endogenous gene expression.

We observed that the addition of estradiol to ER-transfected cells did not further increase eNOS gene expression, indicating that the overexpressed estrogen receptor may be activated by ligand-independent mechanisms. Similar to our findings Karas et al. [36] have shown estrogen-independent transcriptional activation of the ER promoter in transient assay in arterial cells using reporter plasmids ERE-Luc as well as ERE containing VEGF reporter plasmid mVEGF-Luc. Activation of ERE containing reporter plasmids was dependent on AF-1 domain of ER in endothelial and VSM [36]. In addition, it has been shown that ER can be activated independent of estrogen by a variety of other extracellular factors including okadaic acid, dopamine, cyclic adenosine monophosphate (cAMP), insulin-like growth factor-I, and tumor necrosis factor [38,39]. Our results confirm and extend these studies and show for the first time that transfected ER gene can act as transactivating factor for important endothelial cell genes like eNOS, in the absence of estrogen.

Therefore, ER gene overexpression in BAEC offers a unique opportunity to test the molecular mechanisms of ER-mediated regulation of genes containing ERE in cultured endothelial cells. Further, this model will be very valuable in identifying estrogen responsive genes other than eNOS gene in endothelial cells, and to investigate the interaction of EREs with other promoter regulatory elements like AP-1, and NF-1 [8]. Moreover, transfection of ER in atherosclerotic coronary arteries may have important clinical potential for atherosclerosis that has been postulated to be due to endothelial cell dysfunction, especially loss of NO production. Transfer of ER gene in atherosclerotic artery endothelium may increase NO production at the site of lesion by inducing eNOS gene in endothelial cells.

Our results also show that overexpression of ER inhibited bFGF-induced BAEC migration. Similar to our results, another study reported estradiol-mediated inhibition of bFGF-stimulated endothelial cell proliferation [15]. Cell migration requires a dynamic interaction between the cell, its substrate, and the cytoskeleton-associated motile apparatus [40] and cell migration is regulated by level of attachment strength [41]. In the present study, we have demonstrated that ER gene transfer enhanced BAEC adhesion to extracellular matrix proteins including laminin, fibronectin, type I and type IV collagens. The enhanced cell adhesion may in part underlie the inhibitory effect of ER gene transfer on BAEC migration.

Basic FGF has been shown to stimulate the tyrosine phosphorylation of FAK in endothelial cells, suggesting that the regulation of FAK tyrosine phosphorylation may be involved in the signaling pathway of bFGF [20,42]. We have demonstrated that ER gene transfection significantly inhibited bFGF-stimulated tyrosine phosphorylation of FAK. These data would suggest that the inhibitory effect of ER overexpression on bFGF-induced BAEC migration might be due to inhibition of bFGF stimulation of FAK tyrosine phosphorylation. In support of these contentions, it has been shown that transfection of the C-terminal of FAK cDNA producing dominant negative inhibition of FAK was found to inhibit cell-spreading [43]. Similarly, cells from FAK knockout mice showed decreased cell migration, whereas overexpression of FAK resulted in increased cell migration [44,45].

In summary, our results conclusively demonstrate that ER is capable of inducing eNOS gene expression in endothelial cells. In addition, we have shown that ER overexpression inhibits endothelial cell migration and bFGF-stimulated tyrosine phosphorylation of FAK, but increases endothelial cell attachment to extracellular matrix proteins. These results suggest that the atheroprotective effects of estrogen may in part be mediated by ER-induced upregulation of eNOS gene expression and maintenance of endothelial cell function and integrity.

Time for primary review 24 days.

	Acknowledgements

 
This research was supported by grants HL-51735 and HL-14388 from the National Institutes of Health and a grant from Iowa affiliate of the American Heart Association.

	Notes

 
¹ Present address: Cardiovascular Division, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Thorn 1319, 75 Francis St, MA, 02115, USA.

	References

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 

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