help button home button Endocrine Society JCEM JCEM Call for Nominations for EIC
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2006-1225
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
91/12/4995    most recent
Author Manuscript (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Seli, E.
Right arrow Articles by Arici, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Seli, E.
Right arrow Articles by Arici, A.
Related Collections
Right arrow Cardiovascular Endocrinology
Right arrow Female Endocrinology
The Journal of Clinical Endocrinology & Metabolism Vol. 91, No. 12 4995-5001
Copyright © 2006 by The Endocrine Society

Estradiol Increases Apoptosis in Human Coronary Artery Endothelial Cells by Up-Regulating Fas and Fas Ligand Expression

Emre Seli, Ozlem Guzeloglu-Kayisli, Hakan Cakmak, Umit A. Kayisli, Belgin Selam and Aydin Arici

Department of Obstetrics, Gynecology, and Reproductive Sciences, Yale University School of Medicine, New Haven, Connecticut 06520-8063

Address all correspondence and requests for reprints to: Aydin Arici, M.D., Section of Reproductive Endocrinology and Infertility, Department of Obstetrics, Gynecology, and Reproductive Sciences, Yale University School of Medicine, New Haven, Connecticut 06520-8063. E-mail: aydin.arici{at}yale.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Context: In animal models, estrogen inhibits atherogenesis by inhibiting many of the early steps of atherosclerotic plaque formation. However, the lack of cardioprotective effect by postmenopausal hormone replacement therapy and possible increase in cardiovascular events observed during the first year after the initiation of hormone replacement therapy may suggest that once the plaque is formed, estrogen may have additional effects that may counteract its beneficial outcomes. Indeed, the effect of estrogen on plaque stability has not been identified.

Objective: We hypothesized that 17ß-estradiol (E2) may cause increased apoptosis in human coronary artery endothelial cells (HCAECs). This effect would explain an adverse effect on plaque stability in vivo.

Intervention(s) and Main Outcome Measure(s): The effect of E2 on apoptosis, cell proliferation, and expression of proapoptotic molecules Fas and Fas ligand (FasL) in cultured HCAECs was evaluated.

Results: HCAECs in culture treated with E2 showed an increase in DNA strand breaks and nuclear fragmentation indicative of apoptosis. E2 treatment also induced a significant concentration-dependent increase in Fas mRNA and protein expressions in HCAECs. Moreover, the expression of FasL mRNA and secretion of FasL protein by HCAECs were enhanced in response to E2 treatments.

Conclusions: E2 increases the apoptosis in cultured HCAECs. Enhanced Fas and FasL expressions in response to E2 suggest that activation of the Fas/FasL pathway may be a mediator of the proapoptotic effects of E2 in these cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CARDIOVASCULAR DISEASE is the leading cause of death among women in the United States, surpassing all cancers combined (1). The observation that the incidence of cardiovascular disease in premenopausal women is lower than in men, but approaches the same level after menopause (2), led to the investigation of estrogen as a cardioprotective agent. In animal models, estrogen inhibits development of atherosclerosis in a reproducible manner (3, 4). However, randomized prospective clinical trials failed to show benefit from estrogen replacement or combined hormone replacement therapy (HRT) as a cardioprotective measure (5, 6, 7, 8, 9), despite the fact that previous clinical data suggested a decrease in cardiovascular disease in postmenopausal women treated with estrogen (10). Overall, the mechanism of estrogen’s actions on vascular cells is not completely understood and most probably involves multiple pathways.

The primary pathological entity leading to cardiovascular mortality and morbidity is atherosclerosis. The earliest recognizable event in the development of atherosclerosis is an increased recruitment of macrophages into the arterial subendothelium (11, 12). Macrophages first play a protective role by their ability to remove low-density lipoprotein (LDL) from the environment, but when cholesterol is in excess, these cells are converted into foam cells (13, 14). Macrophage foam cells, together with accompanying T cells, form fatty streaks (15) and secrete chemokines and growth factors, causing smooth muscle cells to migrate from the media to the intima and proliferate within the neointima of developing plaques. Thereafter, smooth muscle cell becomes the predominant cell type in the plaque also forming the vast majority of foam cells (12, 16). Once formed, atherosclerotic plaque is separated from the lumen by a fibrous layer called the plaque cap. Plaque cap thinning results from loss of cells and extracellular collagen in the plaque cap and predisposes the plaque to rupture. Plaque rupture with ensuing thrombosis is the most important factor precipitating the lethal consequences of coronary atherosclerosis.

Programmed cell death (apoptosis) has been identified as an important process in a variety of human vascular diseases, including atherosclerosis (17). Early in atherogenesis, apoptotic loss of vascular macrophages and smooth muscle cells may delay the development of the atherosclerotic lesion (18). However, once the plaque is formed, apoptosis can be detrimental for plaque stability and increase the risk of thrombosis (19).

A key mediator of apoptotic cell death is the Fas/Fas ligand (FasL) system. Fas (APO 1, CD95) is a 45-kDa type I transmembrane receptor protein that belongs to the TNF receptor/nerve growth factor receptor family (20). Fas is capable of initiating apoptotic signaling when bound by its ligand, FasL (21, 22). FasL (CD95R) is a 37-kDa type II transmembrane protein that belongs to the TNF and CD40 ligand family of proteins (23). Fas is ubiquitously expressed in a variety of immune and nonimmune tissues, whereas FasL displays a more restricted expression pattern. Interestingly, in addition to activated T cells and natural killer cells, FasL is abundantly expressed in immunoprivileged tissues such as testis and the eye as well as endothelial cells in which it limits extravasation of immune cells into the tissue. FasL expression by normal endothelium is believed to fulfill an atheroprotective function by inhibiting leukocyte extravasation and inducing apoptosis of mononuclear cells invading the vessel wall.

In animal models, estrogen inhibits atherosclerotic plaque formation by inhibiting many of the early steps of atherogenesis, including recruitment of macrophages into the arterial wall by expression of vascular adhesion molecules (24, 25) or monocyte chemotactic factor-1 (26, 27, 28) and vascular smooth muscle cell proliferation (4, 29). The lack of cardioprotection (5, 6, 7, 8, 9) and possible initial increase in cardiovascular events observed with HRT (5, 8, 9) may suggest that, once the plaque is formed (as would be in the age group treated with HRT), estrogen may have additional effects that may counteract its beneficial effects. Indeed, the effect of estrogen and progesterone on plaque stability is not known. Several clinical trials (5, 6, 7, 30) reported a 2- to 4-fold higher risk of venous thromboemboli and pulmonary emboli associated with postmenopausal HRT. Conversely, estrogen replacement with or without progesterone does not result in worsening of coagulation parameters (31). Currently we do not know whether estrogen increases the risk of arterial thrombotic events.

In this study, we hypothesized that estrogen may cause increased apoptosis in human coronary artery endothelial cells (HCAECs). This effect would explain an adverse effect in vivo on plaque stability. To test our hypothesis, we evaluated the effect of 17ß-estradiol (E2) on cultured HCAEC apoptosis, proliferation, and expression of proapoptotic molecules Fas and FasL.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
HCAEC cultures

HCAECs obtained from 48- to 53-yr-old women were purchased from Clonetics (San Diego, CA). Normal HCAEC preparations obtained from two different women were used for experiments. HCAECs were grown in endothelial cell basal medium-2 (Clonetics, BioWhittaker, San Diego, CA) supplemented with human recombinant epidermal growth factor (10 pg/ml), human recombinant basic fibroblast growth factor (4 pg/ml), vascular endothelial growth factor, human recombinant IGF, ascorbic acid, heparin, hydrocortisone (0.4 µg/ml), gentamicin (50 µg/ml), amphotericin B (50 ng/ml), and 5% fetal bovine serum (Clonetics, BioWhittaker). Experiments were conducted at third subcultures at 70–80% confluence. Before each experiment, cells were treated with phenol red-free media prepared with 5% charcoal-stripped calf serum for 24 h. Each experiment was repeated at least three times.

HCAECs were treated with various concentrations of E2 (10–12 to 10–8 M; Sigma-Aldrich, St. Louis, MO) in phenol red-free media prepared with 5% charcoal-stripped calf serum. Controls were treated with the same media alone. Optimum treatment durations and concentrations were determined and used in the following experiments.

Detection of apoptotic activity: terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) and 4'-6-diamidino-2-phenylindole (DAPI) assays for HCAECs in culture

Apoptosis in HCAECs plated on tissue chamber slides was detected by labeling of DNA strand breaks using TUNEL assay. TUNEL assay was performed using a cell death detection kit (Roche, Mannheim, Germany) according to the manufacturer’s instructions. Briefly, chamber slides were fixed in 4% paraformaldehyde for 20 min at 4 C, washed in PBS, and treated with permeabilization solution (0.1% Triton X-100 in 0.1% sodium citrate) for 5 min on ice. After a second wash with PBS, the labeling reaction was performed for 1 h at 37 C using 50 µl TUNEL reagent for each sample, except the negative control, in which reagent without enzyme was added. After PBS washing, slides were incubated with converter reagent for 30 min at 37 C. After washing, color development for localization of cells containing labeled DNA strand breaks was performed by incubating the chambers with Fast Red substrate solution for 10 min. Slides were lightly counterstained with hematoxylin before permanent mounting. Quantitation of apoptotic cells was accomplished by counting the number of cells stained by TUNEL assay per a total of 100 cells, and labeling index was calculated (labeled cells per 100 cells).

DAPI forms fluorescent complexes with natural double-stranded DNA, showing fluorescence specificity for base pairs. When DAPI binds to DNA, its fluorescence is strongly enhanced. Morphological indicators of apoptosis, such as cell shrinkage, nuclear segmentation, and chromatin condensation, were analyzed using the DAPI staining. HCAECs were treated with either vehicle (control) or E2 (10–12 to 10–8 M) for 24 and 72 h. Thereafter, cells were washed with PBS, fixed with 70% ethanol for 20 min at room temperature, and washed again with PBS. Cells were then treated with DAPI (1 µg/ml; Sigma-Aldrich) for 10 min, washed with PBS for 5 min, and mounted with antifade mounting medium (Vector Laboratories, Burlingame, CA). DAPI staining of the cells was evaluated by fluorescence microscopy (32).

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation assay for HCAECs in culture

Cell proliferation was assessed by a colorimetric assay using MTT. MTT assay that detects the formation of dark blue formazan product from MTT in active mitochondria was performed as described previously (33). HCAECs were grown in 96-well plates. Four hours before the end of each experiment, 10 µl of MTT solution was added onto each well of 96-well plates. The optical absorbance at 570 nm was read within 30 min. The last column of each 96-well plate did not contain cells and was used as a blank. Data were expressed in OD units.

Western blot analysis for Fas and FasL in cultured HCAECs

Total protein from treated HCAECs was extracted with T-PER protein extraction reagent (Pierce, Rockford, IL), supplemented with protease inhibitor cocktail (1 mM Na3VO4, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM phenylmethylsulfonylfluoride; Calbiochem, San Diego, CA). Protein concentration was determined by detergent-compatible Bradford protein assay (Bio-Rad Laboratories, Hercules, CA). Western blot analysis was performed as described previously (34). Briefly, 20 µg of protein was loaded into each lane, separated electrophoretically by SDS-PAGE using 10% Tris-HCl Ready Gels (Bio-Rad Laboratories), and electroblotted onto nitrocellulose membrane (Bio-Rad Laboratories). The membrane was blocked with 5% nonfat dry milk in TBS-T buffer (0.1% Tween 20 in Tris-buffered saline) for 1 h to reduce the nonspecific binding. The membrane was then incubated with mouse antihuman Fas or mouse antihuman FasL monoclonal antibodies (1:1000 dilution; Transduction Laboratories, Lexington, KY) for 1 h at room temperature and washed three times with TBS-T for 20 min. Then the membrane was incubated for 1 h with peroxidase-labeled antimouse IgG (1:10,000 dilution; Vector Laboratories) and subsequently washed with TBS-T three times for 20 min. Fas and FasL levels were detected using chemiluminescent detecting reagents (PerkinElmer Life Sciences, Boston, MA) and exposing the membrane to BioMax film (Kodak, Rochester, NY).

Membranes were stripped with stripping solution (Pierce) between Fas and FasL detection and also before reprobing with mouse monoclonal antihuman glyceraldehyde-3-phosphate dehydrogenase antibody (GAPDH; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Immunoblot bands for Fas, FasL, and GAPDH were quantified using a laser densitometer. Each Fas and FasL band was normalized to the value obtained from the corresponding GAPDH band.

Semiquantitative RT-PCR for Fas and FasL in cultured HCAECs

At the end of experimental cell incubations, total RNA was extracted by Trizol reagent (Gibco BRL, Rockville, MD) according to the manufacturer’s instructions. Two micrograms of sample was reverse transcribed in 20 µl of reaction mixture containing 10 mmol/liter each of dATP, dCTP, dGTP, and deoxytocopherol transfer protein; 20 pmol oligo (dT); 40 IU/µl ribonuclease inhibitor, 10 IU/µl avian myeloblastosis virus-reverse transcriptase and buffer (95 C, 5 min; 42 C, 60 min; PerkinElmer model 9600 thermocycler). Semiquantitative RT-PCR was performed using primers specific for Fas, FasL, and glycerol-3-phosphate dehydrogenase (G3PDH), as described previously (32). The primers used for amplification of Fas, FasL, and G3PDH have the following sequences:

Fas primers yielding a 266-bp reaction product, sense, 5'-CAC TAT TGC TGG AGT CAT G-3', antisense, 5'-CTG AGT CAC TAG TAA TGT CC-3'; FasL primers yielding a 311-bp reaction product, sense, 5'-ACA CCT ATG GAA TTG TCC TGC-3', antisense, 5'-GAC CAG AGA GAG CTC AGA TAC G-3'; and G3PDH primers yielding a 788-bp product, sense, 5'-GGT CGG AGT CAA CGG ATT TGG TCG-3', and antisense, 5'-CTT CCG ACG CCT GCT TCA CCA C-3'.

PCR products and molecular weight markers were fractionated in agarose gels containing ethidium bromide (10 mg/ml) and visualized by UV light. The intensity of each band was normalized to its corresponding G3PDH band to compare values semiquantitatively between samples.

ELISA for soluble FasL in HCAEC culture supernatants

After 48- and 72-h treatments, conditioned media were collected from HCAEC cultures and centrifuged at 1800 rpm for 5 min at 4 C to remove cells. Then concentrations of immunoreactive soluble FasL in conditioned media were measured using an ELISA kit according to instructions provided by the manufacturer (Diaclone Research, Cedex, France). The assay sensitivity was less than 12 pg/ml. The intra- and interassay coefficients of variation were 4.5 and 5.6%, respectively. According to the manufacturer, there is no significant cross-reactivity or interference with other known molecules in this assay. Levels of FasL were normalized to the total cell culture protein content as measured by Bradford protein assay (Bio-Rad Laboratories). Briefly, after collecting culture supernatants and washing the monolayers with Hank’s balanced salt solution, the cells were harvested using a cell scraper in cold PBS. After the centrifugation, the cell extraction buffer was added to the cell pellets and sonicated for 5 sec. After the final centrifugation, the supernatant was collected and protein content was measured at 650 nm with a multiwell plate reader.

Statistical analysis

Because the data from TUNEL assay, DAPI staining, MTT cell proliferation assay, and Western blot analysis were normally distributed (as determined by Kolmogorov-Smirnov test), comparisons of samples were analyzed with Student’s t test or one-way ANOVA followed by post hoc Holm-Sidak test, when appropriate. In contrast, the data from semiquantitative RT-PCR and ELISA were not normally distributed and therefore analyzed with nonparametric ANOVA on ranks (Kruskal-Wallis test) followed by post hoc Student-Newman-Keuls test. Statistical calculations were performed using SigmaStat for Windows, version 3.0 (Jandel Scientific Corp., San Rafael, CA). Statistical significance was defined as P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Regulation of apoptosis in cultured HCAECs by estradiol

To investigate the role of estradiol in apoptotic cell death of cultured HCAECs, we first used the TUNEL assay to assess DNA breaks indicative of early stage apoptosis under basal and E2-treated conditions. TUNEL assay showed that treatment of HCAECs with E2 (10–10 and 10–8 M) induced a concentration- and time-dependent increase in the percentage of TUNEL-positive HCAEC percentage after 24- and 72-h treatments (P < 0.05, Fig. 1Go). At the end of 72-h treatment, E2 at 10–8 M enhanced the proportion of TUNEL-positive cells by 2.8 ± 0.2-fold (P < 0.05, mean ± SEM).


Figure 1
View larger version (32K):
[in this window]
[in a new window]

  		FIG. 1. The effect of E2 on HCAEC apoptosis assessed by TUNEL assay and DAPI fluorescence. A, TUNEL assay was used to detect HCAECs with DNA strand breaks indicative of apoptosis. HCAECs treated for 24 and 72 h with vehicle [control (C)] were compared with cells treated with E2 (10–10 and 10–8 M). Inset pictures represent higher magnifications. Treatment of HCAECs with E2 (10–10 and 10–8 M) induced an increase in TUNEL-positive HCAEC ratio when compared with control incubations at 24 and 72 h. B, DAPI staining was used to detect HCAECs with nuclear segmentation and chromatin condensation. HCAECs treated for 24 and 72 h with vehicle [control (C)] were compared with cells treated with E2 (10–8 M). Treatment of HCAECs with E2 induced an increase in HCAECs with nuclear segmentation and cell shrinkage when compared with control incubations at 24 and 72 h. C, E2 treatment at 10–8 M increased the TUNEL-positive HCAECs by 2.8 ± 0.2-fold (mean ± SEM) at 72 h. HCAECs treated with E2 (10–8 M) also showed a 2.2 ± 0.3-fold increase in cell shrinkage and nuclear segmentation after 72 h. *, P < 0.05 vs. control.

 
We then performed DAPI staining to analyze late stage morphological indicators of apoptosis such as nuclear segmentation and chromatin condensation. Similar to the results of TUNEL labeling, E2-treated (10–10 to 10–8 M) HCAECs had significantly more cell shrinkage, nuclear segmentation, and chromatin condensation, compared with control cells at 24 and 72 h (Fig. 1Go, P < 0.05). The proportion of apoptotic cells was increased by 2.2 ± 0.3-fold (mean ± SEM) after a 72-h treatment with E2 at 10–8 M (P < 0.05).

Proliferative activity of HCAECs in response to estradiol

To determine whether E2 (10–12 to 10–8 M) has a proliferative effect on HCAECs, MTT colorimetric assay was performed after 24-, 48-, and 72-h treatments. Estradiol treatment did not result in the proliferation of HCAECs, compared with controls, at any concentration used. However, a decrease that did not reach statistical significance was observed in the total number of cells at 72 h, which was consistent with the observed increase in apoptotic cell death at 72 h (data not shown).

Regulation of Fas protein and mRNA expression by estradiol in cultured HCAECs

To evaluate the regulation of Fas protein expression by E2, HCAECs were incubated with vehicle (control) or various concentrations of E2 (10–12 to 10–8 M) for 12–48 h, and Fas protein levels were analyzed by Western blot analysis. E2 treatments induced a significant concentration-dependent increase in Fas protein expression, compared with controls at all time points investigated (P < 0.05, Fig. 2Go).


Figure 2
View larger version (17K):
[in this window]
[in a new window]

  		FIG. 2. Fas protein expression in HCAECs in culture: concentration-dependent effect of E2. HCAECs in culture were incubated in phenol red-free medium with 5% charcoal-stripped calf serum for 24 h and then were treated with either vehicle [control (C)] or various concentrations of E2 (10–12 to 10–8 M) for 12 (A), 24 (B), and 48 h (C). Total cellular protein was extracted, and Fas protein level was measured by Western blot analysis. E2 treatments induced a significant concentration-dependent increase in Fas protein expression, compared with controls. Representative data were shown. *, P < 0.05 vs. control.

 
To assess whether the up-regulatory effect of E2 on Fas protein expression was secondary to an increase in Fas mRNA transcription, Fas mRNA levels were measured by RT-PCR analysis. HCAECs were incubated with vehicle (control) or various concentrations of E2 (10–12 to 10–8 M) for 8 h. Semiquantitative RT-PCR results corresponded to Western blot analysis findings. Fas mRNA expression was increased by 65 ± 4 and 84 ± 8% with E2 treatment at 10–10 and 10–8 M concentrations, respectively (P < 0.05, Fig. 3Go).


Figure 3
View larger version (37K):
[in this window]
[in a new window]

  		FIG. 3. Effect of E2 on Fas mRNA expression in cultured HCAECs. Cells were incubated in phenol red-free medium with 5% charcoal-stripped calf serum for 24 h and then were treated with either vehicle [control (C)] or various concentrations of E2 (10–12 to 10–8 M) for 8 h. Total RNA was extracted, and Fas mRNA expression was analyzed using RT-PCR. E2 treatments induced a significant concentration-dependent increase in Fas mRNA expression, compared with controls. Representative data were shown. *, P < 0.05 vs. control.

 
Regulation of FasL protein and mRNA expression by estradiol in cultured HCAECs

To determine the regulation of FasL protein expression by E2, HCAECs were incubated with vehicle (control) or various concentrations of E2 (10–12 to 10–8 M) for 48 and 72 h, and Fas protein levels in culture protein lysate and supernatant were analyzed by Western blot analysis and ELISA, respectively. Western blot analysis showed that FasL protein expression in HCAECs was significantly inhibited by E2 in a concentration-dependent manner, compared with controls (P < 0.05, Fig. 4Go). On the other hand, E2 treatments induced a significant concentration-dependent increase in soluble FasL protein levels in culture supernatant, compared with controls at 48 and 72 h (P < 0.05, Fig. 5Go).


Figure 4
View larger version (46K):
[in this window]
[in a new window]

  		FIG. 4. Effect of E2 on FasL protein expression in cultured HCAECs. Cells were incubated in phenol red-free medium with 5% charcoal-stripped calf serum for 24 h and then were treated with either vehicle [control (C)] or various concentrations of E2 (10–12 to 10–8 M) for 48 h. Total cellular protein was extracted, and FasL protein level was measured by Western blot analysis. E2 treatments significantly decreased FasL protein expression, compared with control. Representative data were shown. *, P < 0.05 vs. control.

 

Figure 5
View larger version (16K):
[in this window]
[in a new window]

  		FIG. 5. Effects of E2 on soluble FasL secretion by HCAECs. Cells were incubated in phenol red-free medium with 5% charcoal-stripped calf serum for 24 h and then were treated with either vehicle [control (C)] or various concentrations of E2 (10–12 to 10–10 M) for 48 and 72 h. Soluble FasL levels in culture supernatants were quantified by ELISA and normalized to total cell protein. *, P < 0.05 vs. control at 48 h; **, P < 0.05 vs. control at 72 h.

 
The regulatory effect of E2 on FasL mRNA expression was also evaluated after incubating HCAECs with vehicle (control) or various concentrations of E2 (10–12 to 10–8 M) for 8 h. Semiquantitative RT-PCR demonstrated that FasL mRNA expression was increased by 76 ± 4 and 85 ± 9% with E2 treatment at 10–10 and 10–8 M concentrations, respectively (P < 0.05, Fig. 6Go).


Figure 6
View larger version (38K):
[in this window]
[in a new window]

  		FIG. 6. Effect of E2 on FasL mRNA expression in cultured HCAECs. Cells were incubated in phenol red-free medium with 5% charcoal-stripped calf serum for 24 h and then were treated with either vehicle [control (C)] or various concentrations of E2 (10–12 to 10–8 M) for 8 h. Total RNA was extracted, and FasL mRNA expression was analyzed using RT-PCR. E2 treatments induced a significant increase in FasL mRNA expression, compared with control. Representative data were shown. *, P < 0.05 vs. control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we showed that HCAECs in culture treated with E2 display an increase in DNA strand breaks and nuclear fragmentation indicative of apoptosis. We also detected an increase in HCAEC Fas expression in response to E2, suggesting that activation of the Fas/FasL pathway may be a mediator of the proapoptotic effects of E2 in these cells.

We also found an increase in FasL mRNA in HCAECs treated with E2. FasL protein, however, was decreased in these cells. The evaluation of cell culture medium by ELISA revealed an increase in FasL in response to E2 treatment, suggesting an increase in expression and secretion of FasL. It could be speculated that the FasL released from endothelial cells may lead to apoptosis in an autocrine and paracrine manner by binding Fas. Such a mechanism could lead to apoptosis of endothelial as well as arterial smooth muscle cells in vivo.

The current study was initiated after our in vivo observations in a murine atherosclerosis model consisting of LDL receptor-deficient mice fed a high-cholesterol diet. In ovariectomized LDL receptor-deficient mice, E2 treatment decreased atherosclerotic plaque formation as expected, whereas it caused an increase in the number of TUNEL-positive cells in the vascular wall (35). Our current findings are consistent with our in vivo observations and suggest that E2 may act as a proapoptotic agent in vascular cells.

The cellular response to estrogen is classically mediated by the binding of estrogen to its nuclear receptors, estrogen receptor (ER)-{alpha} and ERß. These receptors function as ligand-dependent transcription factors to modulate gene transcription from promoters by direct binding of the receptor to specific DNA target sequences, designated estrogen response elements (36). Despite the clarity with which the ER has been shown to act as a transcription factor, it has been apparent for several years that not all physiological effects of estrogen are accomplished through a direct effect on gene transcription (36, 37). In many instances, other signaling pathway(s) including phosphatidylinositol 3-kinase/Akt and MAPKs contribute to estrogenic action (38, 39, 40). Although estrogen may modulate the activity of these pathways through ERs, these signaling cascades were also shown to be activated in cells without endogenous ER (41). Moreover, in previous studies, ER antagonist ICI 182,780 failed to block the activation of the signaling pathways including phosphatidylinositol 3-kinase/Akt and MAPK by estrogen (38, 42, 43). Therefore, estrogen may activate these signaling pathways before transcription, and these pathways may enhance genomic actions of estrogen or influence cell function before (or in the absence of) gene transcription. In addition, receptor-independent vascular effects of estrogen are also known (44, 45). In the current study, the proapoptotic effects of E2 were not completely abrogated by ICI 182,780 (data not shown), suggesting that ER-independent signaling pathways may play a role in effects of E2.

The effect of different estrogenic compounds on apoptosis of vascular cells has been previously investigated. Whereas some studies reported a decrease in apoptotic cell death in endothelial cells treated with E2 (46, 47), others showed that E2 induces apoptosis of vascular smooth muscle cells (48). Interestingly, 2-methoxyestradiol, an endogenous metabolite of E2 that arises from the hydroxylation and subsequent methylation at the 2-position, induces apoptosis and Fas expression in cultured endothelial cells (49, 50). LaVallee et al. (51) showed that 2-methoxyestradiol-mediated induction of apoptosis is independent of ER{alpha} and ERß. LaVallee et al. (51) also reported that E2, at doses higher than 10 µM, also induced apoptosis, and that effect is not blocked by ICI 182,780; hence, it is not receptor dependent. We conducted our experiments in cultured HCAECs obtained from women. We believe HCAECs constitute a valuable in vitro system reconstructing the vascular milieu of the most relevant artery for cardiovascular events. Our findings suggest that, at least under certain conditions, E2 may increase endothelial cell apoptosis. Our results are also consistent with recent studies that showed antiproliferative and proapoptotic effects of estrogen on different cell types, including murine mammary epithelium and murine osteoclasts (52), hepatocellular carcinoma cells (53), colonic epithelial cells (54), and prostate epithelium (55).

We speculate that the effect of E2 on apoptotic machinery may have different implications, depending on the stage of the atherosclerotic plaque formation. Initially, E2-mediated increase in FasL leading to a decline in inflammatory cell recruitment into the vascular wall (56), and to an increase in apoptotic death of injured vascular cells, may delay the formation and development of the atherosclerotic plaque. Indeed, in experimental models, an increase in vascular apoptosis leads to a delay in plaque formation (18). However, once the plaque is fully formed, apoptosis in vascular endothelial and smooth muscle cells may disrupt plaque stability and increase the likelihood of cardiovascular events (19). Whereas such a mechanism may play a role in the initial increase in cardiovascular events observed in the recent studies of HRT (5, 7, 8), additional evidence is needed to establish the relevance of this mechanism in vivo.


   Footnotes
 
Disclosure statement: The authors have nothing to disclose.

First Published Online September 26, 2006

Abbreviations: DAPI, 4'-6-Diamidino-2-phenylindole; E2, 17ß-estradiol; ER, estrogen receptor; FasL, Fas ligand; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; G3PDH, glycerol-3-phosphate dehydrogenase; HCAEC, human coronary artery endothelial cell; HRT, hormone replacement therapy; LDL, low-density lipoprotein; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; TBS-T, buffer of Tween 20 and Tris-buffered saline; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling.

Received June 8, 2006.

Accepted September 18, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. American Heart Association 1997 Heart and stroke facts: statistical update. Dallas: American Heart Association
  2. Colditz GA, Willett WC, Stampfer MJ, Rosner B, Speizer FE, Hennekens CH 1987 Menopause and the risk of coronary heart disease in women. N Engl J Med 316:1105–1110[Abstract]
  3. Adams MR, Kaplan JR, Manuck SB, Koritnik DR, Parks JS, Wolfe MS, Clarkson TB 1990 Inhibition of coronary artery atherosclerosis by 17ß estradiol in ovariectomized monkeys. Lack of an effect of added progesterone. Arteriosclerosis 10:1051–1057[Abstract/Free Full Text]
  4. Sullivan TR, Karas RH, Aronovitz M, Faller GT, Ziar JP, Smith JJ, O’Donnell TF, Mendelsohn ME 1995 Estrogen inhibits the response-to-injury in a mouse carotid artery model. J Clin Invest 96:2482–2488[Medline]
  5. Hulley S, Grady D, Bush T, Furberg C, Herrington D, Riggs B, Vittinghoff E 1998 Randomized trial of estrogen plus progestin for secondary prevention of coronary heart disease in postmenopausal women. Heart and Estrogen/progestin Replacement Study (HERS) Research Group. JAMA 280:605–613[Abstract/Free Full Text]
  6. Grady D, Herrington D, Bittner V, Blumenthal R, Davidson M, Hlatky M, Hsia J, Hulley S, Herd A, Khan S, Newby LK, Waters D, Vittinghoff E, Wenger N 2002 Cardiovascular disease outcomes during 6.8 years of hormone therapy: Heart and Estrogen/progestin Replacement Study follow-up (HERS II). JAMA 288:49–57[Abstract/Free Full Text]
  7. Rossouw JE, Anderson GL, Prentice RL, LaCroix AZ, Kooperberg C, Stefanick ML, Jackson RD, Beresford SA, Howard BV, Johnson KC, Kotchen JM, Ockene J 2002 Risks and benefits of estrogen plus progestin in healthy postmenopausal women: principal results from the Women’s Health Initiative randomized controlled trial. JAMA 288:321–333[Abstract/Free Full Text]
  8. Anderson GL, Limacher M, Assaf AR, Bassford T, Beresford SA, Black H, Bonds D, Brunner R, Brzyski R, Caan B, Chlebowski R, Curb D, Gass M, Hays J, Heiss G, Hendrix S, Howard BV, Hsia J, Hubbell A, Jackson R, Johnson KC, Judd H, Kotchen JM, Kuller L, LaCroix AZ, Lane D, Langer RD, Lasser N, Lewis CE, Manson J, Margolis K, Ockene J, O’Sullivan MJ, Phillips L, Prentice RL, Ritenbaugh C, Robbins J, Rossouw JE, Sarto G, Stefanick ML, Van Horn L, Wactawski-Wende J, Wallace R, Wassertheil-Smoller S 2004 Effects of conjugated equine estrogen in postmenopausal women with hysterectomy: the Women’s Health Initiative randomized controlled trial. JAMA 291:1701–1712[Abstract/Free Full Text]
  9. Manson JE, Hsia J, Johnson KC, Rossouw JE, Assaf AR, Lasser NL, Trevisan M, Black HR, Heckbert SR, Detrano R, Strickland OL, Wong ND, Crouse JR, Stein E, Cushman M 2003 Estrogen plus progestin and the risk of coronary heart disease. N Engl J Med 349:523–534[Abstract/Free Full Text]
  10. Stampfer MJ, Colditz GA 1991 Estrogen replacement therapy and coronary heart disease: a quantitative assessment of the epidemiologic evidence. Prev Med 20:47–63[CrossRef][Medline]
  11. Jerome WG, Lewis JC 1984 Early atherogenesis in white carneau pigeons. I. Leukocyte margination and endothelial alterations at the celiac bifurcation. Am J Pathol 116:56–58[Abstract]
  12. Stary HC 1987 Macrophages, macrophage foam cells, and eccentric intimal thickening in the coronary arteries of young children. Atherosclerosis 64:91–108[CrossRef][Medline]
  13. Aviram M 1992 Low density lipoprotein modification by cholesterol oxidase induces enhanced uptake and cholesterol accumulation in cells. J Biol Chem 267:218–225[Abstract/Free Full Text]
  14. Brown MS, Goldstein JL 1983 Lipoprotein metabolism in the macrophage: implications for cholesterol deposition in atherosclerosis. Annu Rev Biochem 52:223–261[CrossRef][Medline]
  15. Ross R 1999 Atherosclerosis-an inflammatory disease. N Engl J Med 340:115–126[Free Full Text]
  16. Katsuda S, Boyd HC, Fligner C, Ross R, Gown AM 1992 Human atherosclerosis. III. Immunocytochemical analysis of the cell composition of lesions of young adults. Am J Pathol 140:907–914[Abstract]
  17. Bennett MR 1999 Apoptosis of vascular smooth muscle cells in vascular remodelling and atherosclerotic plaque rupture. Cardiovasc Res 41:361–368[Abstract/Free Full Text]
  18. Wang BY, Ho HK, Lin PS, Schwarzacher SP, Pollman MJ, Gibbons GH, Tsao PS, Cooke JP 1999 Regression of atherosclerosis: role of nitric oxide and apoptosis. Circulation 99:1236–1241[Abstract/Free Full Text]
  19. Kockx MM, Knaapen MW 2000 The role of apoptosis in vascular disease. J Pathol 190:267–280[CrossRef][Medline]
  20. Itoh N, Yonehara S, Ishii A, Yonehara M, Mizushima S, Sameshima M, Hase A, Seto Y, Nagata S 1991 The polypeptide encoded by the cDNA for human cell surface antigen Fas can mediate apoptosis. Cell 66:233–243[CrossRef][Medline]
  21. Adachi M, Suematsu S, Kondo T, Ogasawara J, Tanaka T, Yoshida N, Nagata S 1995 Targeted mutation in the Fas gene causes hyperplasia in peripheral lymphoid organs and liver. Nat Genet 11:294–300[CrossRef][Medline]
  22. Nagata S 1997 Apoptosis by death factor. Cell 88:355–365[CrossRef][Medline]
  23. Suda T, Takahashi T, Golstein P, Nagata S 1993 Molecular cloning and expression of the Fas ligand, a novel member of the tumor necrosis factor family. Cell 75:1169–1178[CrossRef][Medline]
  24. Caulin-Glaser T, Watson CA, Pardi R, Bender JR 1996 Effects of 17ß-estradiol on cytokine-induced endothelial cell adhesion molecule expression. J Clin Invest 98:36–42[Medline]
  25. Nathan L, Pervin S, Singh R, Rosenfeld M, Chaudhuri G 1999 Estradiol inhibits leukocyte adhesion and transendothelial migration in rabbits in vivo: possible mechanisms for gender differences in atherosclerosis. Circ Res 85:377–385[Abstract/Free Full Text]
  26. Pervin S, Singh R, Rosenfeld ME, Navab M, Chaudhuri G, Nathan L 1998 Estradiol suppresses MCP-1 expression in vivo: implications for atherosclerosis. Arterioscler Thromb Vasc Biol 18:1575–1582[Abstract/Free Full Text]
  27. Seli E, Kayisli UA, Selam B, Seli M, Arici A 2002 Estradiol suppresses vascular monocyte chemotactic protein-1 expression during early atherogenesis. Am J Obstet Gynecol 187:1544–1549[CrossRef][Medline]
  28. Seli E, Pehlivan T, Selam B, Garcia-Velasco JA, Arici A 2002 Estradiol down-regulates MCP-1 expression in human coronary artery endothelial cells. Fertil Steril 77:542–547[CrossRef][Medline]
  29. Foegh ML, Asotra S, Howell MH, Ramwell PW 1994 Estradiol inhibition of arterial neointimal hyperplasia after ballon injury. J Vasc Surg 19:722–726[Medline]
  30. Daly E, Vessey MP, Hawkins MM, Carson JL, Gough P, Marsh S 1996 Risk of venous thromboembolism in users of hormone replacement therapy. Lancet 348:977–980[CrossRef][Medline]
  31. The Writing Group for the PEPI Trial 1995 Effects of estrogen or estrogen/progestin regimens on heart disease risk factors in postmenopausal women. The Postmenopausal Estrogen/Progestin Interventions (PEPI) Trial. JAMA 273:199–208[Abstract]
  32. Kayisli UA, Selam B, Guzeloglu-Kayisli O, Demir R, Arici A 2003 Human chorionic gonadotropin contributes to maternal immunotolerance and endometrial apoptosis by regulating Fas-Fas ligand system. J Immunol 171:2305–2313[Abstract/Free Full Text]
  33. Arici A, Seli E, Zeyneloglu HB, Senturk LM, Oral E, Olive DL 1998 Interleukin-8 induces proliferation of endometrial stromal cells: a potential autocrine growth factor. J Clin Endocrinol Metab 83:1201–1205[Abstract/Free Full Text]
  34. Kayisli UA, Luk J, Guzeloglu-Kayisli O, Seval Y, Demir R, Arici A 2004 Regulation of angiogenic activity of human endometrial endothelial cells in culture by ovarian steroids. J Clin Endocrinol Metab 89:5794–5802[Abstract/Free Full Text]
  35. Seli E, Kayisli UA, Seli M, Guzeloglu-Kayisli O, Arici A, Estrogen decreases vascular wall cellularity in a murine atherosclerosis model. Proc Scientific Program and Abstracts of the 49th Annual Meeting of the Society for Gynecologic Investigation, Los Angeles, CA, 2002, p 234 (Abstract 518)
  36. Segars JH, Driggers PH 2002 Estrogen action and cytoplasmic signaling cascades. Part I: membrane-associated signaling complexes. Trends Endocrinol Metab 13:349–354[CrossRef][Medline]
  37. Hall JM, Couse JF, Korach KS 2001 The multifaceted mechanisms of estradiol and estrogen receptor signaling. J Biol Chem 276:36869–36872[Free Full Text]
  38. Guzeloglu Kayisli O, Kayisli UA, Luleci G, Arici A 2004 In vivo and in vitro regulation of Akt activation in human endometrial cells is estrogen dependent. Biol Reprod 71:714–721[Abstract/Free Full Text]
  39. Guzeloglu-Kayisli O, Kayisli UA, Al-Rejjal R, Zheng W, Luleci G, Arici A 2003 Regulation of PTEN (phosphatase and tensin homolog deleted on chromosome 10) expression by estradiol and progesterone in human endometrium. J Clin Endocrinol Metab 88:5017–5026[Abstract/Free Full Text]
  40. Seval Y, Cakmak H, Kayisli UA, Arici A 2006 Estrogen-mediated regulation of p38 mitogen-activated protein kinase (MAPK) in human endometrium. J Clin Endocrinol Metab 91:2349–2357[Abstract/Free Full Text]
  41. Tsai EM, Wang SC, Lee JN, Hung MC 2001 Akt activation by estrogen in estrogen receptor-negative breast cancer cells. Cancer Res 61:8390–8392[Abstract/Free Full Text]
  42. Singh M, Setalo Jr G, Guan X, Warren M, Toran-Allerand CD 1999 Estrogen-induced activation of mitogen-activated protein kinase in cerebral cortical explants: convergence of estrogen and neurotrophin signaling pathways. J Neurosci 19:1179–1188[Abstract/Free Full Text]
  43. Sylvia VL, Walton J, Lopez D, Dean DD, Boyan BD, Schwartz Z 2001 17ß-estradiol-BSA conjugates and 17ß-estradiol regulate growth plate chondrocytes by common membrane associated mechanisms involving PKC dependent and independent signal transduction. J Cell Biochem 81:413–429[CrossRef][Medline]
  44. Speir E, Yu ZX, Takeda K, Ferrans VJ, Cannon 3rd RO 2000 Antioxidant effect of estrogen on cytomegalovirus-induced gene expression in coronary artery smooth muscle cells. Circulation 102:2990–2996[Abstract/Free Full Text]
  45. Zacharia LC, Gogos JA, Karayiorgou M, Jackson EK, Gillespie DG, Barchiesi F, Dubey RK 2003 Methoxyestradiols mediate the antimitogenic effects of 17ß-estradiol: direct evidence from catechol-O-methyltransferase-knockout mice. Circulation 108:2974–2978[Abstract/Free Full Text]
  46. Alvarez RJ, Gips SJ, Moldovan N, Wilhide CC, Milliken EE, Hoang AT, Hruban RH, Silverman HS, Dang CV, Goldschmidt-Clermont PJ 1997 17ß-estradiol inhibits apoptosis of endothelial cells. Biochem Biophys Res Commun 237:372–381[CrossRef][Medline]
  47. Spyridopoulos I, Sullivan AB, Kearney M, Isner JM, Losordo DW 1997 Estrogen-receptor-mediated inhibition of human endothelial cell apoptosis. Estradiol as a survival factor. Circulation 95:1505–1514[Abstract/Free Full Text]
  48. Mori-Abe A, Tsutsumi S, Takahashi K, Toya M, Yoshida M, Du B, Kawagoe J, Nakahara K, Takahashi T, Ohmichi M, Kurachi H 2003 Estrogen and raloxifene induce apoptosis by activating p38 mitogen-activated protein kinase cascade in synthetic vascular smooth muscle cells. J Endocrinol 178:417–426[Abstract]
  49. Tsukamoto A, Kaneko Y, Yoshida T, Han K, Ichinose M, Kimura S 1998 2-Methoxyestradiol, an endogenous metabolite of estrogen, enhances apoptosis and ß-galactosidase expression in vascular endothelial cells. Biochem Biophys Res Commun 248:9–12[CrossRef][Medline]
  50. Yue TL, Wang X, Louden CS, Gupta S, Pillarisetti K, Gu JL, Hart TK, Lysko PG, Feuerstein GZ 1997 2-Methoxyestradiol, an endogenous estrogen metabolite, induces apoptosis in endothelial cells and inhibits angiogenesis: possible role for stress-activated protein kinase signaling pathway and Fas expression. Mol Pharmacol 51:951–962[Abstract/Free Full Text]
  51. LaVallee TM, Zhan XH, Herbstritt CJ, Kough EC, Green SJ, Pribluda VS 2002 2-Methoxyestradiol inhibits proliferation and induces apoptosis independently of estrogen receptors {alpha} and ß. Cancer Res 62:3691–3697[Abstract/Free Full Text]
  52. Helguero LA, Faulds MH, Gustafsson JA, Haldosen LA 2005 Estrogen receptors {alpha} (ER{alpha}) and ß (ERß) differentially regulate proliferation and apoptosis of the normal murine mammary epithelial cell line HC11. Oncogene 24:6605–6616[CrossRef][Medline]
  53. Huang EJ, Wu CC, Lee SD, Chen JH, Liu JY, Ko JL, Lin JA, Lu MC, Chen LM, Huang CY, Kuo WW 2006 Opposing action of estrogen receptors {alpha} and ß on tumor necrosis factor-{alpha} gene expression and caspase-8-mediated apoptotic effects in HA22T cells. Mol Cell Biochem 287:137–145[CrossRef][Medline]
  54. Wada-Hiraike O, Imamov O, Hiraike H, Hultenby K, Schwend T, Omoto Y, Warner M, Gustafsson JA 2006 Role of estrogen receptor ß in colonic epithelium. Proc Natl Acad Sci USA 103:2959–2964[Abstract/Free Full Text]
  55. Imamov O, Morani A, Shim GJ, Omoto Y, Thulin-Andersson C, Warner M, Gustafsson JA 2004 Estrogen receptor ß regulates epithelial cellular differentiation in the mouse ventral prostate. Proc Natl Acad Sci USA 101:9375–9380[Abstract/Free Full Text]
  56. Amant C, Holm P, Xu SH, Tritman N, Kearney M, Losordo DW 2001 Estrogen receptor-mediated, nitric oxide-dependent modulation of the immunologic barrier function of the endothelium: regulation of fas ligand expression by estradiol. Circulation 104:2576–2581[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J Am Coll CardiolHome page
P. P. Liu and M. Fukuoka
Sex Hormones as Novel Risk Biomarkers for Atherosclerosis in Peripheral Vascular Disease
J. Am. Coll. Cardiol., September 11, 2007; 50(11): 1077 - 1079.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
91/12/4995    most recent
Author Manuscript (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Seli, E.
Right arrow Articles by Arici, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Seli, E.
Right arrow Articles by Arici, A.
Related Collections
Right arrow Cardiovascular Endocrinology
Right arrow Female Endocrinology

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals