Applied Immunohistochemistry & Molecular Morphology:
Galectin-3 is Overexpressed in Various Forms of Endometriosis
Noël, Jean-Christophe PhD*; Chapron, Charles PhD†; Borghese, Bruno MD†; Fayt, Isabelle MD*; Anaf, Vincent PhD*
*Department of Gynaecopathology, Erasme University Hospital, Université Libre de Bruxelles, Brussels, Belgium
†Service de Gynécologie Obstétrique II and Reproductive Medecine, Université Paris Descartes, CHU Cochin, Saint Vincent de Paul, Paris, France
Reprints: Jean-Christophe Noël, PhD, Service d'Anatomie Pathologique, Hôpital Universitaire Erasme-ULB, 808 route de Lennik, 1070 Brussels, Belgium (e-mail: Jean-Christophe.Noel@erasme.ulb.ac.be).
Received June 10, 2010
Accepted August 4, 2010
Endometriosis is an enigmatic disease of unknown etiology and pathogenesis, which is defined as the presence of endometrial glands and stroma outside the uterus. The most widely accepted theory to explain endometriosis is probably the transplantation of an endometrial fragment during menstruation to ectopic sites, but the development of endometriosis is extremely complex and includes the adherence to the peritoneal surface and secondary invasion of the underlying tissues. In this study, we have investigated the potential role of galectin-3 (gal-3), a member of a group of carbohydrate-binding proteins, which plays a major role in cell adhesion, migration, angiogenesis, and invasion. The expression of gal-3 has been carried out by immunohistochemistry, according to the different phases of cycle in 50 cases of endometriosis (peritoneal endometriosis: n=10; ovarian endometriosis: n=10; deeply infiltrating endometriosis: n=30) and in 34 cases of eutopic endometrium (10 without endometriosis and 24 with endometriosis). In the proliferative and secretory phases of the cycle, the nuclear and membranous gal-3 expression was higher, first in each variant of the endometriosis than in the eutopic endometrium (P<0.05), and second in the eutopic endometrium of women with endometriosis than in eutopic endometrium of women without endometriosis. Our data suggest that gal-3 may have a potential role in the development of endometriosis.
Endometriosis, a disease that affects many women during their reproductive lives, is histologically defined as the presence of endometrial glands and stroma outside the uterus and is associated with various symptoms including chronic pelvic pain, pain during sexual intercourse, and infertility.1–4 There are 3 types of endometriosis: superficial peritoneal endometriosis (PE), ovarian endometrioma (OVE), and deeply infiltrating endometriosis (DIE).5–7 By analogy with cancer growth, which shares common characteristics with DIE, mounting evidence now suggests that invading endometriotic glands and/or stroma interact dynamically with the surrounding microenvironment.8
To this point of view, expression and release of several proteins that regulate cell migration, invasion, angiogenesis, immune functions, and apoptosis have received attention in these last few years, including growth factors, integrins, cadherins, and lectins.9–12
In this study, we have focused on a member of the lectin family, the galectin-3 (gal-3). Gal-3 is a member of a group of carbohydrate-binding proteins that share affinity for β-galactoside-side-containing glycoconjugates and a conserved sequence of the carbohydrate-binding site.13–18 The various functions of this glycoprotein include cell growth, inflammation, angiogenesis, apoptosis, and cancerous transformation.13 In addition, recent data have also suggested that gal-3 upregulated by the neurotrophin nerve growth factor, which is strongly expressed in different variant of endometriosis as we have described earlier.12,19 Gal-3 is mainly a cytosolic protein but can easily translocate to the nucleus with different functions accordingly.13,14
The expression status of gal-3 in endometriosis is poorly understood; therefore, to clarify the issue, in this study, we analyzed the expression of gal-3 in the 3 major forms of endometriosis (PE, OVE, and DIE) by immunohistochemistry, which is actually unknown in correlation with the menstrual cycle phases.
MATERIALS AND METHODS
Fifty cases of endometriosis (PE: n=10, OVE: n=10, DIE: n=30) were prospectively studied in the Gynaecopathology Department from the Erasme University Hospital, Brussels between January 2004 and 2010. All the specimens were obtained by laparoscopy from 50 patients who had stopped hormone treatment for at least 2 months before surgery. The study was carried out according to the criteria required by the local institutional committee. Accurate menstrual dating has been taken according to the last menstrual cycle and corroborated with appropriate histologic dating of the corresponding endometrial biopsy when available. In addition, 20 cases of eutopic endometrium of women without evident endometriosis (10 proliferative and 10 secretory endometrium) were also included as controls. The controls were not under hormonal treatment.
The immunohistochemical demonstration of gal-3 was carried out by using an antigen retrieval method as we have described earlier with the mouse monoclonal antibody NCL-Gal-3 (clone 9C4; Leica Biosystems, Newcastle, UK).20,21 In brief, 4-μm sections were cut sequentially and mounted onto superfrost-treated slides (Menzel-Glasse, Braunschweig, Germany). The slides were then dried overnight at 37°C before deparaffinization in xylene and rehydratation through graded ethanols. For epitope retrieval, the slides were immersed in a waterbath at 99°C for 90 minutes with a citrate buffer, pH 6.0 (S2369; DakoCytomation, Glostrup, Denmark). Then, the slides were cooled in the buffer for 20 minutes at room temperature. Hydrogen peroxide (0, 3% was added to the slides and incubated at room temperature for 30 minutes. The tissues were then incubated for 1 hour with the primary antibody always at room temperature. The Novolink Polymer Detection System (Leica Biosystems) was used for the subsequent steps according to the manufacturer's instructions. Chromogenic development was accomplished using diaminobenzidine hydrogen peroxide. The slides were then slightly counterstained with hematoxylin and dehydrated, and a cover slip was applied. Controls for nonspecific binding of the primary antibody preparations were substituted as the first layer of serial sections. External positive controls consisted of small bowel glands whereas histiocytes/macrophages found at the periphery of endomeriotic lesions served as positive internal controls. Gal-3 immunoreactivity was scored for cytoplasmic/membranous and nuclear staining. Five hundred glandular/epithelial cells and 500 stromal cells were evaluated for each case. We used the semiquantitative H score with a dynamic range of 0 to 300 as described earlier.22,23 This method details the percentage of positive cells showing none, weak, moderate, or strong staining. The score is given as the sum of the percentage of staining multiplied by an ordinal value corresponding to the intensity level (0=none, 1=weak, 2=moderate, 3=strong). With 4 intensity levels, the resulting score ranges from 0 (no staining) to 300 (diffuse intense staining). A score <50 was considered as a negative. The comparison of the data was carried out using Student t test and the Mann-Whitney U test with Bonferroni corrections. A value of P less than 0.05 was considered statistically significant.
The nuclear and cytoplasmic staining results are shown in Tables 1 and 2. In the proliferative and secretory phases the nuclear and the membranous/cytoplasmic gal-3 expression was higher in each type of endometriosis than eutopic endometrium (P<0.05). However, the nuclear or membranous/cytoplasmic gal-3 immunoreactivity was not statistically different in PE, OVE, or DIE (P>0.05). In addition, the expression of gal-3, in proliferative or secretory phases, was statistically higher in the eutopic endometrium of women with endometriosis than in the endometrium of women without endometriosis (P<0.05) (Figs. 1, 2).
Endometriosis is an enigmatic disease of unknown etiology and pathogenesis. Many theories have been suggested to explain the genesis of endometriosis, but the most easily understood and widely accepted mechanism is probably the “transplantation theory,” consisting on transplantation of endometrial fragments during menstruation to ectopic sites.24–26 However, despite evidence that retrograde menstruation occurs in virtually all women, most women do not develop endometriosis, probably because the development of endometriosis is extremely complex, and includes adherence to the peritoneal surface, the establishment of blood supply, and the secondary invasion of the underlying tissues, and vascular structures.4 Therefore, by analogy to the spread of malignant cells, the endometriotic disease can be viewed as a cascade of complex interrelated events and endometriosis is considered by certain authors as a “benign cancer.”8,27,28 To this point of view, the superfamily of cell adhesion proteins and particularly integrins seems to play a major role in the development of endometriosis.9 In this study, we have investigated the potential role of gal-3 that is a member of the large family of lectins, which contain 1 or more carbohydrate recognition domains with affinity of β-galactosides. The unique structure of this protein consisting of a carbohydrate-binding domain, a matrix metalloproteinase-cleavable domain, which enables it to interact with multiple ligands in a carbohydrate-dependent or carbohydrate-independent manner. The multifunctionality of this glycoprotein is, in part, reflected by its wide range of ligands.13–15 Indeed, recent evidence suggests that gal-3 plays a major role in cell adhesion, migration, angiogenesis, invasion, immune functions, and apoptosis in a wide variety of cancers and, in particular, in endometrial carcinomas.15,16,20
The expression of gal-3 in normal endometrium and in endometriosis, however, is poorly understood. In normal endometrium, the expression of gal-3 increases in the glandular and the stromal components in the secretory phase.17 Our findings confirmed these earlier data in normal eutopic endometrium of women without endometriosis but not in eutopic endometrium of women with endometriosis (Tables 1 and 2; Figs. 1, 2).
In this study, we have also clearly shown that in the proliferative or the secretory phases, the nuclear and the membranous/cytoplasmic gal-3 expression is significantly higher in the 3 different variants of endometriosis in comparison with eutopic endometrium (P<0.05). However, the protein expression is not different in PE, OVE, or DIE, suggesting that the protein may play a role in cell-matrix interactions, and that gal-3 could constitute an early trigger signal transduction cascades by cross-linking with the glycan parts of the surface glycoconjugates. In addition, this study confirmed that the expression of gal-3 is more important in the eutopic endometrium of women with endometriosis, than in the eutopic endometrium of women without endometriosis. These data are in accordance with the more developed theory, which suggest that the eutopic endometrium of women with endometriosis is different from the endometrium of women without endometriosis permitting this “modified endometrium” to more easily adhere, grow, and spread outside the uterine cavity when it releases during menstrual regurgitation.4,10,29 Indeed, several investigators have clearly shown that the endometrium of women with endometriosis showed different expression of several proteins incriminated in cell adhesion or invasion, including integrins, cadherins, lectins, or metalloproteinases.11,29,30
Finally, as mentioned earlier, one of the cardinal symptoms of endometriosis is probably the pain.31–33 To this point of view, recent data have suggested that gal-3 may play a role in the development of pain because it was involved in myelin phagocytosis, wallerian degeneration of neurons, and trigger for neuronal apoptosis induction after nerve injury.34–36 In an earlier study, we have clearly shown that there is a close morphologic relationship between nerves and endometriotic foci by means of perineurial and endoneurial invasion, particularly in DIE, which represents the most painful form of the disease. Therefore, gal-3 overexpressed by endometriotic foci could induce nerve degeneration secondarily.3 In addition, the expression of gal-3 is upregulated by the neurotrophin nerve growth factor, which itself strongly expressed in endometriosis.12,19
In conclusion, in thisstudy we have shown that gal-3 is overexpressed in the different variants of endometriosis and in the eutopic endometrium of women with endometriosis suggesting that this protein could play a role in the pathogenesis of this disease. These results may open new areas of research in this field, and may also provide additional tools for therapies. Indeed, some gal-3 antagonists with an acceptable human safety profile such as GCS-100 are now available.37
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