1 Instituto de Biología y Medicina Experimental (IBYME-CONICET), Vuelta de Obligado 2490, (C1428DNA) and 2 Instituto de Ginecología y Fertilidad (IFER), M.T. de Alvear 2259, (C1122AAI) Buenos Aires, Argentina
3 To whom correspondence should be addressed. e-mail: meresman{at}dna.uba.ar
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Abstract |
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Key words: apoptosis/endometrial cell/endometriosis/GnRH agonist/IL-1/VEGF
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Introduction |
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The vascular endothelial growth factor (VEGF) family of angiogenic molecules is involved in both physiological angiogenesis and a number of pathological conditions that are characterized by excessive angiogenesis (Liu et al., 2000; Bachelder et al., 2001
; Harris et al., 2002
). Increasing evidence suggests that VEGF may also be involved in the aetiology and maintenance of endometriosis (Donnez et al., 1998
; McLaren, 2000
; Smith, 2001
).
VEGF is a highly specific mitogen for vascular endothelial cells (Neufeld et al., 1999). Several reports have demonstrated that VEGF not only induces angiogenesis, but also works as a survival factor for tumour and endothelial cells, protecting them from apoptosis (Liu et al., 2000
; Harmey and Bouchier-Hayes, 2002
). We recently reported on the increased survival capability of the eutopic endometrium from patients with endometriosis, possibly regulated by an elevated expression of Bcl-2, as well as on the impact of short-term oral contraceptives in reversing this effect (Meresman et al., 2000
, 2002).
An immunological basis, mediated through various factors, has long been considered to be important in the pathogenesis of endometriosis (Sidell et al., 2002). Interleukin-1
(IL-1
) is a pleiotrophic cytokine involved in the inflammatory immune response (Dinarello, 1997
) and is known to act as a growth factor. In addition to its mitogenic effects, IL-1
induces the protection of different cells from apoptotic death (Markstrom et al., 2002
; Simonart et al., 2002
).
Both, IL-1 and VEGF are produced by endometrial cells and are critical factors involved in the pathogenesis of endometriosis (Donnez et al., 1998
; Lebovic et al., 2000
). Inflammatory cytokines, such as IL-1
and IL-6, were found to be elevated in the peritoneal fluid of women with endometriosis (Ho et al., 1997
). In endometriotic cells, as in other tissues, the expression of VEGF was found to be potentiated by a variety of cytokines, especially IL-1
(Lebovic et al., 2000
; Jung et al., 2001
; Dias et al., 2002
).
Among the most widely used hormonal treatments for endometriosis are the GnRH analogues (Rice et al., 2002). Leuprolide acetate (LA) is a GnRH agonist commonly prescribed for the treatment of endometriosis that acts primarily on the anterior pituitary, inducing initially an undesirable transient rise in gonadotrophin release. With continued administration, LA causes pituitary desensitization, leading to a complete suppression of gonadal function (Plosker et al., 1994
). In addition, there is growing evidence supporting a direct action of GnRH agonists on endometrial growth, since both GnRH and GnRH receptors have been isolated from eutopic and ectopic endometrium (Borroni et al., 2000
). It was suggested that GnRH may act as a direct regulator of this growth.
We recently showed that GnRH agonists have a beneficial effect on endometriosis, by diminishing cell proliferation and enhancing apoptosis in eutopic endometrium (Meresman et al., 2003). Nevertheless, the effect of GnRH agonists on the release of regulatory cytokines and angiogenic factors by the eutopic endometrium from patients with endometriosis, as well as the direct effect of GnRH antagonists on the same tissue, is as yet unknown.
Based on these data, the objectives of this work are (i) to study and compare the direct effect of a GnRH agonist and a GnRH antagonist on apoptosis in epithelial cells from eutopic endometrium obtained from endometriosis and control patients; and (ii) to evaluate the effect of a GnRH agonist and antagonist on the release of IL-1 and VEGF by the same endometrial cells in culture.
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Materials and methods |
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This study was approved by the Ethics and Research Committee of the Biology and Experimental Medicine Institute, and all subjects included in the study signed informed consent.
Epithelial endometrial cell culture
The tissue was immediately placed into culture medium and processed within 60 min of collection. Epithelial cells were enzymatically separated, isolated by successive centrifugation, and primary cultures established for in-vitro studies on implantation using a modification of the method described by Bongso et al. (1988).
Briefly, the explant was minced, washed and placed in basic medium (MEM D-Val, Gibco, Paisley, UK) containing 100 IU/ml penicillin, 100 µg/ml streptomycin and 25 µg/ml amphotericin B (Gibco) with 1 mg/ml collagenase (Gibco, type I). After a 2 h incubation at 37°C in an atmosphere of 5% CO2, the resulting suspension was centrifuged at 100 g for 5 min. The pellet containing glands was resuspended in nutrient medium and spun again at 100 g for 5 min. The final pellet mainly contained epithelial cells. After a 1 h incubation, the medium containing enriched epithelial cells was removed and plated in fresh medium. The cells were cultured in quadruplicate for each patient, with 10% fetal bovine serum (FBS; Gibco) before the experiments.
Immunocytochemical evaluation for cytokeratin was done as described in our previous study, and the cells were identified as epithelial based on their intense labelling (Meresman et al., 2003).
Apoptosis assay
Cultures of epithelial cells were plated in 4-well culture dishes (Nalge Nunc International, NY) and left undisturbed at 37°C for 2 days. Prior to the addition of the different agents, the medium was changed to a low serum medium (MEM D-Val supplemented with 2.5% FBS). Cultures were treated with LA (Lupron; Abbot, Buenos Aires, Argentina) 100 ng/ml, as GnRH agonist, antide (ANT) (Sigma, St Louis, MO) at a concentration of 107 mol/l, as GnRH antagonist, or a combination of ANT and LA, adding LA 100 ng/ml 3 h after the supplementation with ANT 107 mol/l (ANT + LA). For each patient, one culture with four experimental treatments (basal, LA 100 ng/ml, ANT 107 mol/l and LA + ANT) were assessed in quadruplicate.
The percentage of apoptotic cells was assessed by the acridine orangeethidium bromide technique in endometrial cultures, under basal conditions and after exposure to the different agents (GnRH agonist, GnRH antagonist or both).
Acridine orange is a vital dye that is excluded from viable cells. It is specific for apoptotic forms of cell death and does not significantly label cells undergoing necrotic death caused by injury (Abrams et al., 1993). After addition of the acridine orange (1 mg/l)ethidium bromide (250 mg/l) mix, the cells were viewed by a fluorescence microscope and the apoptotic cells were counted manually as a percentage of the total number of cells by two independent observers. Each observer viewed 30 randomly selected fields in a blind manner. There was no significant difference in results between the two observers (P = 0.63).
Quantification of IL-1 and VEGF
Epithelial cells cultures were left undisturbed at 37°C for 2 days. Subsequently, the cells were washed and incubated with basic medium supplemented with 2.5% FBS for an additional 48 h under basal conditions, after the addition of LA at 100 ng/ml, ANT at 107 mol/l and ANT 107 mol/l + LA 100 ng/ml. The conditioned medium was collected and assayed for IL-1 and VEGF using commercial enzyme-linked immunoassay (ELISA) kits (Cytimmune Sciences Inc., MD, USA). The sensitivity level for the IL-1
ELISA was 0.87 pg/ml and for the VEGF ELISA was 18.6 pg/ml.
The intra-assay variability for IL-1 and VEGF was ±7.9 and 8.9% respectively, while the inter-assay variability was ±11.4 and 11.1% for IL-1
and VEGF respectively. All samples were assessed in triplicate.
Statistics
Statistical comparisons were performed by KruskalWallis non-parametric ANOVA test, followed by Dunn's multiple comparison test. Regardless of the statistical test, only a P-value 0.05 was considered significant.
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Results |
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Endometrial cell cultures from endometriosis patients and controls treated with ANT at 107 mol/l by itself did not show any significant difference compared with the levels of IL-1 found under basal conditions (Table I).
It is of interest that there was no difference in the release of IL-1 between the endometrial cell cultures from endometriosis and control patients.
Effects of GnRH agonist and antagonist on VEGF release by epithelial endometrial cell cultures
Exposure to LA significantly decreased the levels of VEGF from 283.6 ± 83.9 to 134.5 ± 37.3 pg/ml (P < 0.04), and from 250.9 ± 60.8 to 96.0 ± 11.0 pg/ml (P < 0.03), in conditioned media from endometrial cell cultures in endometriosis patients and controls respectively (Figure 3).
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Endometrial cell cultures from endometriosis patients and controls treated with ANT at 107 mol/l alone, did not show any significant difference in the levels of VEGF compared with basal conditions.
In addition, we found no differences in VEGF release between endometrial cell cultures from endometriosis patients and controls (Table I).
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Discussion |
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In this study, epithelial cell cultures of human endometrium were used as a model to evaluate apoptosis, as well as IL-1 and VEGF release, in response to the addition of a GnRH agonist, a GnRH antagonist or both to the cultures. Although the cells employed in this investigation were not derived from endometriotic implants, and therefore their in-vitro response may not be identical, the rationale for the use of endometrial cells in short-term culture as a model for endometriotic implants is scientifically acceptable and has been documented in the literature (Surrey and Halme, 1990
; Meresman et al., 2003
).
In our study, we found that LA increased the apoptotic rate in eutopic endometrial cells from controls and endometriosis patients. This finding is in agreement with the report by Andreu et al. (1998), showing that incubation with a GnRH agonist stimulated apoptosis in rat granulosa cells, as well as with a recent study reporting that a GnRH agonist triggered apoptosis in a single suspension of stromal and glandular epithelial endometrial cells from patients with endometriosis (Imai et al., 2000
). These findings, as well as the data contained in the present study, seems to confirm that GnRH agonists may be effective in reducing the growth of endometrial cells not only through an indirect effect (via causing hypoestrogenism), but also through a direct effect on the endometrial tissue.
VEGF is an important angiogenic factor and IL-1 is an important pleiotrophic factor that appear to be involved in the establishment and progression of endometriosis (Lebovic et al., 2000
). The human endometrium and endometriotic tissue were found to express significant levels of VEGF and IL-1
(Donnez et al., 1998
; Bergqvist et al., 2001
). In addition, their concentrations were found to be elevated in peritoneal fluid from patients with endometriosis (Mori et al, 1992
; McLaren et al., 1996
) and it was suggested that IL-1
could induce VEGF expression in endometriotic cells (Lebovic et al., 2000
).
In the present study, we showed for the first time that VEGF and IL-1 content in the conditioned media from endometrial cell cultures exposed to a GnRH agonist were significantly lower than the concentrations encountered under basal conditions in the same cultures. Our results are in agreement with those of Kupker et al. (1998
) suggesting that after GnRH agonist therapy, endometriosis patients showed a significant drop in the mean concentrations of VEGF in the peritoneal fluid. VEGF expression was found to be elevated in the endometrium from patients with endometriosis and in the endometriotic tissue itself (Donnez et al., 1998
; McLaren, 2000
). We propose that GnRH agonist therapy seems to regulate the angiogenic activity of the endometriotic lesions by modulating the secretion of angiogenic factors.
The same was true for IL-1; Taketani et al. (1992
) found that the levels of this cytokine was significantly higher in the peritoneal fluid from women with active endometriosis, compared with patients without the disease, but that the levels were extremely low in women with endometriosis who had undergone medical treatment with either danazol or buserelin. Based on these data, and on our own results, the possibility remains that GnRH agonists modulate the production of specific cytokines, and in turn regulate the growth of the endometriotic tissue.
In our study, the GnRH antagonist ANT by itself did not produce any significant effects on epithelial endometrial cells in culture, but it was able to prevent the enhanced effect of the GnRH agonist on programmed cell death, and reversed the inhibition by LA of VEGF and IL-1 release. These findings combined strongly suggest that the direct effects of GnRH agonists on endometrial cells in vitro are probably mediated by homologous GnRH receptors.
We did not find significant differences in the rate of apoptosis or in VEGF and IL-1 release between endometrial cell cultures from endometriosis patients and controls; this finding of a similar degree of apoptosis in these two different groups of patients is in contrast to the results observed in whole endometrial sections in our previous study (Meresman et al., 2000
). We could speculate that after 4 days in culture of the glandular fraction, the epithelial endometrial cells altered their initial conditions, and therefore we could not detect maintenance of their apoptotic characteristics in comparison with when the entire tissue was used.
In conclusion, GnRH agonists appear to have a direct local effect on endometrial cells in culture; this effect is manifested by the enhancement in the percentage of apoptotic cells as well as by the significant inhibition observed in the release of pro-mitogenic cytokines such as IL-1 and VEGF. Furthermore, we were unable to observe a significant direct effect of a GnRH antagonist on any of the parameters studied, on the same tissue cultures, although it was able to reverse the effects caused by the GnRH agonist.
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Acknowledgements |
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Submitted on February 17, 2003; accepted on May 13, 2003.