1 Institute of Anatomy and 3 Department of Gynaecology, University Hospital Essen, 45122 Essen and 2 Female Health Care Research, Schering AG, 13342 Berlin, Germany
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Abstract |
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Key words: angiogenesis/ectopic endometrium/endometriosis/NOD-SCID mouse/nude mouse
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Introduction |
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To design new strategies for therapy, the mechanisms for proliferation, neoangiogenesis and hormonal and immunological responsiveness need to be analysed. Since occurrence of spontaneous endometriosis is dependent on menstruation, the development of this disease is restricted to humans and subhuman primates. However, monkeys which could be used as a model (Sillem et al., 1996) have limited value. For this reason, animal models have been developed in recent years to investigate the pathomechanism and to validate therapeutic concepts of endometriosis. Several studies reported that human endometrial tissue transplanted into nude or severe combined immune deficiency (SCID) mice seems to be a valid and appropriate model to study the pathophysiology of endometriosis (Zamah et al., 1984
; Bergqvist et al., 1985; Aoki et al., 1994
; Bruner et al., 1997
; Awward et al., 1999). These authors have shown that human endometrial tissue or cells grafted into the peritoneal cavity of immunodeficient mice have the ability to implant and to develop lesions with well preserved endometrial glandular tissue. Recently, it was reported (Tabibzadeh et al., 1999
) that adhesion and implantation of the endometrial cells is increased in this model when peritoneal fluid from endometriotic patients was used and was even more effective when human blood lymphocytes were injected into the peritoneal cavity. It could be shown that such ectopic endometrial tissues grown in nude mice developed a vascular network combined with a high vascular endothelial growth factor content within these ectopic fragments (Nisolle et al., 2000
).
The present study describes the temporal pattern of adhesion and development of the grafted endometrial fragments. We could show that fragments keep their hormonal receptors when transplanted into cycling mice indicating hormonal responsiveness and that neoangiogenesis seems to be a major event in early development of endometriotic lesions. These investigations confirm that implantation of human endometrium into the peritoneal cavity of athymic mice is a convincing model for the study of endometriosis.
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Materials and methods |
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Some of the fragments were cultured in petri dishes on semi-solid agar medium [2% agar solution in 50% aqua dest., 25% Dulbecco's modified Eagle medium (DMEM), Dulbecco, 25% Ham's F12, Gibco] covered with culture medium [DMEM + Ham's F12 (1:1) + 10% FCS] supplemented with oestradiol-17ß (109 mol/l) for 24 h prior to transplantation into nude mice.
Transplantation of endometrial fragments into mice
Animals
Athymic nude mice (Han:NMRI nu/nu) and non-obese diabetic (NOD)-SCID mice (Shultz et al.1995) were maintained in a barrier unit in a well-controlled pathogen-free environment with regulated cycles of light/dark (12 h/12 h). All equipment and food entering the barrier was autoclaved. Mice had free access to food and water ad libitum.
Transplantation of fragments and tissue processing
Fragments of human endometrium were implanted into the peritoneal cavity of nude mice and NOD-SCID mice and cultivated up to 21 and 28 days, respectively. For each time point a minimum of three mice were transplanted with 10 fragments each. Endometrial fragments were placed by laparotomy into the endometrial cavity of nude and NOD-SCID mice in the region of gut, lateral abdominal wall, liver and mesenterium. In another set of experiments three fragments per mouse were fixed with surgical sutures (Ethicon 6/0, Johnson and Johnson, Belgium) to the outer gut wall, the mesenterium and the muscle layer of the lateral abdominal wall of nude mice, one fragment at each location.
At the end point of each experiment, mice were killed by cervical dislocation. Implanted endometrial lesions were dissected by laparatomy and were either frozen directly in liquid nitrogen or fixed in 10% formalin and routinely embedded in paraffin for morphological and immunohistochemical analysis.
Hormonal treatment
Nude mice were bilaterally ovariectomized and left untreated for at least 14 days. 17-ß-oestradiol (oestradiol; Sigma, Germany), was dissolved in benzyl-benzoate and 0.2 µg/mouse were administered s.c. in a volume of 200 µl arachis oil. Each animal received one s.c. oestradiol injection per day from the time of transplantation onwards during the course of the experiment. Vehicle only was injected into animals of the control groups using the same experimental design.
Immunohistochemistry
Immunostaining for panendothelial antigen was performed on ethanol-fixed cryostat sections from freshly frozen tissues, all other immunohistochemical reactions were performed on paraffin sections. All sections were rinsed in phosphate buffered saline (PBS) containing 0.5% bovine serum albumin (BSA) to reduce nonspecific antibody binding, endogenous peroxidase activity was blocked with 3% hydrogen peroxide. After washing in PBS, the sections were incubated for 1 h at room temperature with the following primary antibodies: Endothelia of mouse vessels were stained with a rat anti-mouse panendothelial cell antigen (MECA-32; Pharmingen, Hamburg, 1:100), vessels of human origin with anti-von-Willebrand Factor (A082; Dako Denmark, 1:200). Staining of human endometrial epithelium and stroma was performed using anti-human cytokeratin (M0821, Dako, Denmark, 1:100) and anti-human vimentin (M0725; Dako, Denmark, 1:100), respectively. Proliferation of endometrial glandular epithelial cells was evaluated using a monoclonal anti- Ki-67 antibody (MIB-1; Dia505, Dianova, Germany, 1:100) and human steroid hormone receptors by mouse monoclonal anti-human oestrogen alpha receptor (MU272-UC; Bio Genex, 1:50) and anti-human progesterone receptor (PR88; Bio Genex, undiluted). The following secondary antibodies were used: biotinylated rabbit anti-rat antibody (E0468; Dako, Denmark) for panendothelial cell antigen, biotinylated goat anti-rabbit immunoglobulin (E0432; Dako, Denmark) for anti-von-Willebrand Factor, LSAB plus kit (K0690; Dako, Denmark) for Ki-67, anti-cytokeratin and anti-vimentin, and a biotinylated anti-mouse IgG (HK3305K; Bio Genex) for steroid hormone receptors.
The chromogenic reaction was carried out by incubating the sections with the peroxidase substrate 3,3'-diaminobenzidine for 5 min; sections were rinsed in PBS, dehydrated in ascending ethanol concentrations and coverslipped. To demonstrate specificity of the staining, consecutive sections were stained with the same protocol, except that the primary antibody was omitted. To show the tissue morphology and to confirm both orientation and localization parallel sections were stained with haematoxylin-eosin. Results were recorded with a Zeiss Axiophot photomicroscope.
Proliferation studies
For each experimental approach percentage of the Ki-67 positive fraction of 36 endometrial glands of three different endometrial fragments was analysed. Statistical analysis was performed using the ANOVA with Scheffé's test. Differences were considered significant when P 0.05.
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Results |
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Lymphocyte infiltration was found within the endometriotic lesions grown in nude mice to a different extent but was not greater in fragments which had been fixed by suturing and was not decreased by in-vitro pre-culturing of the fragments.
Proliferation and differentiation markers
Expression of cytokeratin and vimentin
Immunohistochemical staining using antibodies specific for human cytokeratin and vimentin led to a clear distinction between tissue of mouse and human origin, respectively. Intense cytokeratin staining of the glandular epithelial lining was observed exclusively in the human endometrial implants (Figure 1d,e) and maintained even after 14 days of culturing in nude mice (Figure 1f,g
). Vimentin staining was found in the stromal but not the epithelial compartment of endometriotic fragments which had been cultured in nude mice for up to 9 days (Figure 1h
). After 14 days of culturing, however, epithelial cells expressed vimentin in addition to cytokeratin (Figure 1i
), indicating a dedifferentiation process of the epithelium. This expression of the additional intermediate filament vimentin in glandular epithelium was accompanied by a reorganisation of the stroma indicated by a decrease in the number of surrounding stromal cells and increase in intercellular matrix and fibrils. In those regions with a high amount of stromal cells indicated by a strong vimentin staining, glandular epithelial cells obviously maintained their differentiation status even after culturing for more than 14 days and revealed no vimentin staining (Figure 1k
).
Expression of steroid hormone receptors
Oestrogen receptors were expressed in the glandular epithelia and in stromal cells of endometriotic lesions cultured for up to 14 and 28 days in cycling nude mice (Figure 2c) and NOD-SCID-mice (Figure 2a
), respectively, whereas progesterone receptors could be detected mainly in glandular epithelial cells but only sparsely in the stromal compartment (Figure 2b
). Steroid hormone receptors were preserved for a longer time period in endometrial tissue cultured in NOD-SCID mice compared to using nude mice as a host (Table II
). Endometriotic lesions grown in untreated or oestrogen-treated ovariectomized nude mice revealed no staining for either oestrogen nor progesterone receptors (Table II
).
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Angiogenesis
After 1 week of culturing in nude mice, numerous vessels could be observed in haematoxylin-eosin stained paraffin sections throughout the endometrial fragments (Figure 3a) as well as crossing the tunica muscularis of the gut in direction to the human endometrial tissue (Figure 3b
). Staining of cryostat sections with an anti-mouse panendothelial antibody revealed that those vessels were of mouse origin. Whereas no staining for mouse endothelial cells could be detected in endometriotic lesions after 2 days of culturing (Figure 3c
), neoangiogenesis into the endometrial tissue attached to the liver (Figure 3d
), the adipose fat (Figure 3e
), and the gut (Figure 3f
) appeared from day 4 post-implantation onwards. The extent of angiogenesis, however, seemed not to be correlated to the localization of the endometriotic lesions within the peritoneal cavity.
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Discussion |
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In contrast to previous observations (Awwad et al., 1999; Tabibzadeh et al., 1999
; Nisolle et al., 2000
) which describe adhesion of endometrial fragments mainly at the side wall and abdominal fat tissue, this study in addition observed implanted endometrial fragments at the gut and the liver. The difference in the implantation sites between the human (endometriotic lesions mainly in the region of the Pouch of Douglas attached for example to the peritoneum of the bladder, ovary, rectum, or oviduct) and the mouse (attachment of endometrial fragments mainly to the liver, intestine and abdominal side wall) can be explained by the different direction of gravity action between the quadruped mouse and the upright position of humans.
The comparable high survival rate of endometrial fragments described in this study may be due to the use of fresh, untreated human endometrial tissue without enzymatic pre-treatment in contrast to investigations performed previously, injecting endometrial tissue suspended with collagenase (Tabibzadeh et al., 1999). However, it has to be taken into account that the high adhesion rate of fragments could also be a result of the exposure of the mesothelial lining to air and surgical manipulations that lead to damage of the mesothelium and by this facilitate attachment of the endometrial tissue. A clear improvement in the adhesion rate could be achieved by fixing the fragments without impairment in morphology. Thus, this method may be very well suited for investigations of drug effects on progression of endometriosis. In contrast to the findings of other studies using precultured endometrial fragments (Bruner et al., 1997
; Nisolle et al., 2000
), short-time culture of endometrial tissue compromised the ability of the fragments to establish viable ectopic lesions in our study.
The implantation rate as well as preservation of morphology of endometrial lesions was not dependent on the availability of ovarian hormones. This corresponds to previous results describing that the growth of human endometrium was unaffected by the oestrous stage of the animal (Aoki et al., 1994) and that no difference could be observed in the development of necrosis of the transplants in unsupplemented versus oestradiol-supplemented mice (Zamah et al., 1984
). Treatment of cycling mice with additional progesterone has been described to be associated with larger implants and the presence of fluid-filled endometrial glands referring to the preservation of secretory capacity of these implants (Awwad et al., 1999
), a phenomenon which we could observe in endometrial fragments grown in untreated cycling nude mice for the same time period. Substitution of oestrogen alone maintained proliferation of glandular epithelium in the ectopic human tissue for up to 28 days corresponding to observations in eutopic endometrial epithelium (Kimura et al., 1978
; Gerschenson et al., 1984
).
An influence of ovarian hormones could also be observed in correlation with the preservation of steroid hormone receptors. Expression of oestrogen receptor alpha and progesterone receptor was maintained for up to 28 days in endometriotic lesions grown in cycling but not in ovariectomized mice. Thus proliferation as well as expression of hormone receptors is reduced in untreated ovariectomized mice while rate of persistence of the lesions seems to be similar. This is in agreement with clinical observations that the extent of lesions in patients treated with GnRH analogue decreases while the maintenance of the lesions is not affected (Schindler et al.1994). This survival of the ectopic endometrial lesions might explain the high recurrence rate in patients with endometriosis.
Expression of cytokeratin in the glandular epithelium indicated that the human endometrium conserved specific structural cellular patterns throughout the process of implantation. Interestingly, after 14 days of culture, some of the glands in nude mice exhibited vimentin in addition to cytokeratin staining. This phenomenon could be observed only in parallel with a decrease in surrounding stromal cells. Obviously, the preservation of specific endometrial stroma is necessary for the maintenance of the normal expression pattern of the glandular epithelium, confirming the importance of the interaction between the stromal and the epithelial compartment (Cooke et al., 1997). These results confirm previous observations that epithelial cells keep their original morphology only in areas where the stroma was highly developed whereas the glandular epithelium revealed a flattened morphology when surrounded by sparsely developed stroma (Nisolle et al., 2000
).
The establishment of a new blood supply is essential for the survival of the endometrial implants and the development of endometriosis. Recent investigations in humans confirmed the importance of angiogenesis for the pathogenesis of endometriosis (Donnez et al., 1998; Abulafia and Scherer 1999; Fujishita et al., 1999
). Angiogenesis of mouse vessels into the endometrial fragments occurred from day 4 onwards, independent of the localization of the ectopic lesions. It has been suggested that the expression of vascular endothelial growth factor (VEGF) might provoke an increase of the subperitoneal vascular network and facilitate implantation and viability of endometriotic lesions (Shifren et al., 1996
; Fujimoto et al., 1999
). A network of capillaries has been demonstrated in the stromal compartment of endometrial fragments grafted into the peritoneal pouch of nude mice after 3 weeks of cultivation in combination with VEGF positive stained cells in the glandular epithelium, and to a lesser extent in the stromal compartment of the endometrial grafts (Nisolle et al., 2000
). Excessive angiogenesis suggests novel medical treatments for endometriosis aimed at the inhibition of angiogenesis, and further studies are needed to analyse the cell biological mechanisms and the role of angiogenesis, e.g. by inhibitors of angiogenesis for the development and persistence of endometriosis.
In conclusion, the nude mouse model is a model well suited to the study of endometriosis, as endometrial implants retain morphological characteristics and differentiation parameters for up to 14 days of intraperitoneal culture in nude mice, and for up to at least 28 days in NOD-SCID mice. This model allows investigation of the very early events of the cell biological mechanisms involved in the implantation of endometrial fragments, since attachment occurs 2 days after implantation and in-growth of numerous blood vessels can be observed after 4 days. For longer term experiments exceeding 3 weeks the NOD-SCID model provides advantages since endometriotic fragments reveal a better morphological preservationpossibly due to a reduced immunological responseas well as a good maintenance of proliferation rate and steroid hormone receptor status.
Thus, human endometrial tissue grown in the peritoneal cavity of nude mice is a very valuable tool to test the effect of compounds such as different (anti-) hormones or anti-angiogenic factors and to develop new therapeutic concepts in endometriosis.
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Acknowledgements |
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Notes |
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References |
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Submitted on January 19, 2001; accepted on April 26, 2001.