Vascular endothelial growth factor and matrix metalloproteinase-2 expedite formation of endometriosis in the early stage ICR mouse model

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1 Vascular endothelial growth factor and matrix metalloproteinase-2 expedite formation of endometriosis in the early stage ICR mouse model Xiu-E Lu, M.D., Wei-Xuan Ning, M.D., Min-Yue Dong, M.D., Ai-Xia Liu, M.D., Fan Jin, M.D., and He-Feng Huang, M.D. Department of Reproductive Endocrinology, Women s Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China Objective: To establish a mouse model for endometriosis and to evaluate roles of vascular endothelial growth factor (VEGF) and matrix metalloproteinase-2 (MMP-2) in the formation of disease. Design: Experimental laboratory study. Setting: A women s hospital in China. Patient(s) and Animal(s): Ten women with endometriosis and 10 control women, as well as ICR mice. Intervention(s): Endometrial fragments were transplanted in the peritoneal cavities of mice at minilaparotomy. Transplants were observed and then removed for the assessment of morphology and immunohistochemical staining of VEGF and MMP-2. Main Outcome Measure(s): Observation of transplants, expression of VEGF and MMP-2. Result(s): On days 1 and 2, glandular and stromal cells were viable at the margins of transplants. On day 3, the transplants were surrounded by mesothelial cells, and the endometrial glands and stromal cells were clearly viable at the interface. The scores of VEGF and MMP-2 of viable glandular cells of transplants were increased compared with the ones before transplantation. The scores of VEGF and MMP-2 of transplants from women with endometriosis were higher than those of control women. Conclusion(s): Endometrial transplants from the patients with endometriosis express more VEGF and MMP-2 than endometrium in control women, suggesting that VEGF and MMP-2 may expedite the formation of endometriosis in its early stage. (Fertil Steril 2006;86(Suppl 3): by American Society for Reproductive Medicine.) Key Words: Endometriosis, ICR mice, vascular epithelial growth factor (VEGF), matrix metalloproteinase 2 (MMP2) Endometriosis is a common disease in women of reproductive age and is closely associated with infertility. Despite 50 years of research, cause and pathogenesis of the disease remain elusive, and treatment options are limited. The most accepted theory was proposed by Sampson (1), who suggested that endometriosis results from retrograde menstruation. After retrograde menstruation, endometrial tissue fragments attach to and invade the epithelium of peritoneum and become vascularized to form endometriotic lesions. Retrograde menstruation occurs in 70% 90% of reproductive women; however, depending on the diagnostic criteria used and the populations studied in asymptomatic women, the estimated prevalence of endometriosis ranges from 2% to 22%. In women with dysmenorrhea, the incidence of endometriosis ranges from 20% to 60%, and it range from 20% to 30% in women with subfertility (2, 3). This indicates that Received May 30, 2005; revised and accepted December 26, Supported by the Science and Technology Bureau of Zhejiang Province, Hangzhou, China (grant 2002C23034). Authors X.-E.L. and W.-X.N. contributed equally to the work and both should be considered to be the first author. Reprint requests: He-Feng Huang, M.D., Department of Reproductive Endocrinology, Women s Hospital, Zhejiang University School of Medicine, 2 Xueshi Road, Hangzhou, , China (FAX: ; huanghefg@hotmail.com). retrograde endometrial fragment is the key factor in the pathogenesis of endometriosis. Other factors must be involved to allow the dislocated endometrial tissue to implant and develop into endometriotic lesions. It has been shown that eutopic endometrium shares alterations with ectopic tissue that are not found in the eutopic endometrium of disease-free women. One proposition states that the primary defects in endometriosis may be located in the eutopic endometrium of these women (4, 5). Although the cause effect relationship has yet to be determined, alterations in immune factors of peritoneal cavity have been well established in endometriosis, and their roles have long been hypothesized (6). The degradation of extracellular matrix and neovascularization of implanted endometrial fragments are important processes of disease evolution and development. After attachment, endometrial cells invade extracellular matrix in a process affected by matrix metalloproteinases (MMPs). Matrix metalloproteinases are a group of enzymes that is responsible for the control of extracellular matrix turnover, and this group also is believed to participate in the pathogenesis of the disease by facilitating endometrial cells to invade peritoneal surface. After invasion of the epithelium of the /06/$32.00 Fertility and Sterility Vol. 86, Suppl 3, October 2006 doi: /j.fertnstert Copyright 2006 American Society for Reproductive Medicine, Published by Elsevier Inc. 1175

2 peritoneum, neovascularization is required to support the invading endometrial cells (7 10). Vascular endothelial growth factor (VEGF) is a key mediator of angiogenesis, and it is suggested to contribute to the development by promoting neovascularization of endometrial cells that attach to and invade the surface of the peritoneum (11, 12). Our current understanding of the pathophysiology and cause of endometriosis is limited. Because of limitations on experiments in the human model, animal models provide an invaluable tool in elucidating the mechanisms underlying this disease and make possible the study of the in vivo mechanisms that are involved in the attachment of endometrial cells and the early evolution of lesions through examination of the morphology of transplanted tissue at different time intervals. Primate and nonprimate animal models have been established to use their advantages. Nude mice are well accepted as a model for this disease. However, T-lymphocyte immunity is not taken into account because of thymus deficiency. On the contrary, ICR mice are advantageous in their abundance and in the presence of immune response to the transplanted endometrium (13). The ICR mice have normal immune function. Humoral immunity reaction occurs within 7 10 days of antigen stimulation, whereas cellular immunity reaction takes place after 9 12 days (13). Thus, it is a useful model for studying early events in the lesion development because of the timeline of immune response. The purposes of the current investigation were to establish a mouse model for endometriosis to study the in vivo mechanisms involved. This model would allow the exploration of any differences in the prognosis of transplanted eutopic endometrium of women with and without endometriosis and allow us to evaluate the roles of VEGF and MMP-2 in the pathogenesis of the early evolution of lesions. MATERIALS AND METHODS The current research was conducted according to the protocol approved by the Institutional Review Committee of the Zhejiang University School of Medicine. Written informed consents were obtained from all patients who provided samples. Animal care followed the guidelines recommended by the committee on animal research. Endometrium Late-secretory endometrial tissues were applied because retrograde endometrial tissues during menses are from the late-secretory phase. The samples were obtained from reproductive-aged women (10 patients with endometriosis and 10 with other benign gynecological disease; age range, years) who underwent hysterectomy. Endometrial tissue samples were collected in cold sterile phosphatebuffered saline, washed twice, and cut into 2- to 3-mm 3 pieces. One part of the biopsy was fixed in neutrally buffered formalin solution and was embedded in paraffin for histological examination. Tissue sections were stained with either Gomori s trichrome for histological confirmation of the menstrual phase or specific antibodies (CD10, CK-LWM, VEGF, and MMP2) for immunohistochemical studies. Transplantation into Mice Mature female ICR mice, weighting g, were used in the present study (13). They were kept in a regular cycle of light and darkness, and fed both laboratory chow and water ad libitum. The mice were anesthetized with an IM injection of ketamine (0.07 ml). On day 0, minilaparotomy (3 mm) was performed on the ventral midline just caudal to the umbilicus to place endometrial tissue in the peritoneal cavity. For each individual mouse, one transplant was deposited without any suture or damage to the peritoneal surface. Reduction to air exposure was performed by keeping the duration of the opening of the peritoneal cavity to 1 minute. During this short experimental period, no hormonal therapy was administered to these mice. Three distinct samples from an individual patient were separately transplanted into three mice, and those mice were observed on days 1, 2, and 3, respectively. Immunohistochemical Study of VEGF and MMP2 Mouse monoclonal antibodies against human CD10 and CK-LWM were obtained from Zhongshan Biotechology Co., Ltd (Beijing, China), and rabbit antibodies against human VEGF and MMP-2 were obtained from Boster Biological Technology (Wuhan, China). Tissues were fixed in neutrally buffered formalin. Serial 4- m sections of tissue were sectioned and stained with either Gomori s trichrome for histological observation or with specific antibodies (CD10, CK-LWM, VEGF and MMP2) for immunohistochemical studies. Staining intensity was assigned with a semiquantitative H score, which was calculated with the following equation: H score (P i i) where i intensity of staining with a value of 0, 1, 2, or 3 (null, weak, moderate, or strong, respectively) and P i is the percentage of stained epithelial cells for each intensity, varying from 0 to 100%. The pathologists who read the slides were blind to the grouping information. Statistical Analysis Variables are normally distributed, as demonstrated by Kolmogorov-Smirnov s test. Data are expressed as mean SEM. The H scores for antigens were compared between different days and different groups using two-way analysis of variance or Student s t test. Differences were considered statistically significant when P.05. RESULTS Morphological Study After Transplantation On day 1 after transplantation, all transplants were free in peritoneal cavities, but one in the control group was attached 1176 Lu et al. VEGF and MMP-2 expedite formation of endometriosis Vol. 86, Suppl 3, October 2006

3 FIGURE 1 Human endometrial transplant on day 1. The margin of transplant was clear. The structure of endometrial glands and stroma were clear and cells were viable at the margin of transplants. Only nuclei were present in the middle (magnification, 100). FIGURE 3 Human endometrial transplant on day 3. Transplants were attached to the peritoneum. Blood vessels were present at the junction between the transplant and mesothelium (magnification, 200). to the peri-intestinal fatty tissues. From a microscopical aspect, at the margin of transplants, the structures of endometrial glands and stroma were clear and viable. However, the interior portion appeared degenerative and necrotic, and only nuclei were present (Fig. 1). On day 2, all transplants attached to the peritoneum or peri-intestinal fatty tissues, with the exception of one sample from the control group. On day 3, all transplants expressed firm attachment (Figs. 2 and 3) and could not be FIGURE 2 Human endometrial transplant on day 2. Transplants were attached to the peritoneum. The endometrial glandular cells and stromal cells were clearly viable at the transplant peritoneum interface (magnification, 200). separated from mesothelium gently by tweezers. Mesothelial cells wrapped up the transplants. The endometrial glands and stromal cells clearly were viable at the transplant peritoneum interface. In some cases, blood vessels were present at the junction between the transplants and mesothelium (Fig. 3). Cytokeratin Expression and CD10 CD10 is a sensitive immunohistochemical marker of endometrial stromal cell, whereas CK-LMD (low-molecularweight keratin) is a marker of nonsquamous endometrial epithelium cells. Mouse monoclonal antibodies are specific to human CD10 and CK-LWM and do not react with the mouse counterpart. Human endometrial epithelium cells labeled with CK-LMD antibody were identified adjacent to human endometrial stromal cells and mice tissues (Fig. 4). Under the same condition, stromal cells stained with CD10 antibody were different from epithelium cells and mice tissues (Fig. 5). Vascular Endothelial Growth Factor and MMP2 Expression Vascular endothelial growth factor and MMP-2 were localized to the cytoplasm of endometrial glandular epithelium and stromal cells (Figs. 6 and 7). On days 1, 2, and 3, the H scores of VEGF and MMP-2 of viable glandular cells in transplants were higher than those in the preoperation sample (P.05). Meanwhile, H scores of VEGF and MMP-2 in viable glandular cells of transplants from the patients with endometriosis were higher than those from patients without endometriosis (P.05, Table 1). Fertility and Sterility 1177

4 FIGURE 4 Immunohistochemical staining of CK-LMD. Localization of CK-LMD was found in the human endometrial epithelium cells adjacent to human endometrial stromal cells and mice tissues (magnification, 200). FIGURE 6 Immunohistochemical staining of VEGF. Endometrial epithelium glandular cells and stromal cells were positive for VEGF. The positive staining was of cytoplasm type (magnification, 200). DISCUSSION In the current research, we investigated the adherence to the peritoneal surface and MMP-2 and VEGF expression of the endometrial tissue that was transplanted into the peritoneal cavity of ICR mice. Observation revealed that the transplants expressed easy adhesion to the epithelium of peritoneum, that endometrial cells are viable, that MMP-2 and VEGF expression in transplanted endometrial tissue are increased, and that MMP-2 and VEGF expression are significantly higher in transplants from women with endometriosis than in those without. On days 2 and 3 after transplants, observations indicated that all endometrial transplants attached to mesothelium of ICR mice and that both stromal cells and endometrial glands clearly were viable at the transplant peritoneum interface. A few studies have been conducted on the evaluation of the initial adhesion of endometrium to the peritoneum. Witz et al. (14, 15) evaluated the initial adhesion of proliferative and secretory endometrium to peritoneum in vitro. Their results indicated that endothelium rapidly adhered to the intact peritoneal mesothelium and that both stromal and epithelial cells attached to the mesothelium. Nisolle et al. (16) evaluated the implantation of menstrual endometrium FIGURE 5 Immunohistochemical staining of CD10. The endometrial stromal cells were positive for CD10 (magnification, 200). FIGURE 7 Immunohistochemical staining of MMP-2. The endometrial epithelium and stromal cell were positive for MMP-2 (magnification, 200) Lu et al. VEGF and MMP-2 expedite formation of endometriosis Vol. 86, Suppl 3, October 2006

5 TABLE 1 H scores of VEGF and MMP2 in the epithelium grandular cells before and after transplant into ICR mice. Before Day 1 Day 2 Day 3 EM Control EM Control EM Control EM Control Variable No. of mice No. of transplants Implant transplant 0/10 1/10 10/10 9/10 10/10 10/10 rate VEGF H-score a,b a a,b a a,b a MMP2 H-score a,b a a,b a a,b a Note. EM endometriosis. a H scores of VEGF and MMP2 in viable glandular cells were higher than those in preoperation on days 1, 2, and 3 after transplant (P.05). b H scores of VEGF and MMP2 in viable glandular cells of transplants from the patients with endometriosis were higher than those from the control women on days 1, 2, and 3 after transplant (P.05). on the peritoneal mesothelium and evaluated the early stages of evolution of the endometriotic lesion in nude mice. Their studies confirmed that human menstrual endometrium implanted the intact mesothelium. Discoveries by Nisolle et al. (16) indicated that stromal and glandular cells had two distinct roles: stromal cells were involved in the attachment process, whereas glandular cells were responsible for growth of the endometriotic lesion. Nude mice were frequently used as the model of experimental endometriosis. However, the role of T-lymphocyte response was not taken into account. To observe the attachment of endometrial cells to peritoneal mesothelium under normal immune response, ICR mice were used. Observations indicated that the process of attachment in ICR mice was similar to that in the observations made by Nisolle et al. (16) in nude mice and by Witz et al. (14, 15) in vitro. In addition, in our experiment, endometrial tissues taken from both women with endometriosis and control-group women were transplanted into the peritoneal cavity of ICR mice, which was contrary to the procedures conducted by Witz et al. (14, 15) and Nisolle et al. (16). Our experiments indicated that the attachment behavior and morphological appearance of transplanted endometrium demonstrated a fair amount of similarities between women with and without endometriosis. Studies indicate that MMPs may play a critical role in matrix degradation that takes place at time of menstruation. The establishment of endometriotic lesions in the peritoneal cavity involves a break in the basal membrane by endometrial cells (9). Other studies (4) have suggested that both MMP-2 and tissue inhibitors of MMPs (TIMPs) are involved in the pathogenesis of endometriosis by affecting the invasive properties of endometrial cells. Matrix metalloproteinase-2 showed a higher enzymatic activity concerning the degradation of collagen IV, which is the main constituent of the basement membrane, compared with other subtypes of MMP. Eutopic and ectopic endometrium from women with endometriosis express significantly lower levels of TIMP-3 mrna than do samples from women without endometriosis. Ectopic endometrium expresses higher levels of MMP-9 and a higher ratio of MMP-9 to TIMP-3 compared with eutopic endometrium (4, 5). Uterine endometrium from women with endometriosis expressed higher levels of MMP-2 and lower levels of TIMP-2 than did endometrium from normal women. The altered balance of MMPs and TIMP-1 might favor progression and invasiveness of endometriosis through altered tissue remodeling. Thus, it is considered an important pathology of the disease (17). Because VEGF production in the endometrium of women with retrograde menstruation may promote vascularization and survival of the ectopic tissue, studies have examined the role of VEGF in the pathogenesis of endometriosis. Reports by Donnez et al. (18) stated that during the late-secretory phase, VEGF protein content was significantly higher in the endometrial glandular epithelium of women with endometriosis compared with healthy women. The peritoneal fluid of Fertility and Sterility 1179

6 patients with endometriosis exhibits stronger angiogenic activity, and it also displays increased levels of VEGF when comparisons are made to the control group (19, 20). In previous studies, patients with endometriosis exhibit a high level of MMP-2 in sera and peritoneal fluids (21). Eutopic endometrium of women with endometriosis had higher expression levels of VEGF messenger RNA and lower expression levels of TSP-1 messenger RNA than that of women without endometriosis (22). The increased expression of VEGF facilitates survival of ectopic tissue in the peritoneal cavity and the development of endometriotic lesions. Thus it may be associated with the pathogenesis of endometriosis (23). Proposals regarding the roles of higher levels of MMP-2 and VEGF expression in the pathogenesis of disease have been developed. However, there remain controversial results, such as those of Li et al. (24), whose observation suggests that the expression of MMP-2 was similar in the eutopic endometrium of women with and without endometriosis, and those of Gescher et al. (25), who reported that the expression of VEGF in the endometrial fragments of women with endometriosis and in control women displayed little difference of significance. Those experiments support our findings of similar expression of MMP-2 and VEGF in eutopic endometrium of women with and without endometriosis. Therefore, it is safe to state that there are no differences between the two groups of women in the potential to form endometriotic lesions. Although the behaviors of adherence to the peritoneal cavity of mice are not significantly different, as demonstrated in the current investigation, this does not necessarily mean that endometrial tissues from women with and without endometriosis have the same potential to develop endometriotic lesions. One key datum is that transplanted endometria from women with endometriosis have a significantly higher level of MMP-2 and VEGF than do those from women without endometriosis. Furthermore, the quantity of MMP-2 and VEGF stains in transplanted endometrial tissues was greater than before transplantation. Increased MMP-2 production enhances the invasive property of the tissue. A higher level of VEGF facilitates vascularization and thus the survival of transplanted tissues (4, 18). Taking the putative roles of MMP-2 and VEGF in the pathogenesis of the disease into account, the current results indicate that MMP-2 and VEGF are associated with the evolution and development of endometriotic lesion and that endometrial tissue from women with endometriosis presents a higher probability of development of endometriotic lesions as a result of the higher levels of MMP-2 and VEGF. The presence of immunological abnormalities in patients with endometriosis has been well documented. It is possible that retrograde endometrial debris may become more invasive and is prone to peritoneal implantation and vascularization, even though the causal relationship between immunological abnormalities and endometriosis has never been determined (6). The ICR-mouse model for endometriosis that is established in the present study has normal T-lymphocyte immunity, contrary to the case of nude mice, which are deficient in T-lymphocyte immunity. There has been a great deal of interest regarding the exploration of the longterm fate of endometrial tissue transplanted into the peritoneal cavity of ICR mice. Determination of alterations in peritoneal immunity would offer insight into the pathogenesis of endometriosis and assist understanding of the causal relationship between immune abnormalities of peritoneal cavity and endometriosis. In summary, a mouse model with normal T-lymphocyte immunity for endometriosis was established. Endometrial transplants easily adhered to peritoneum and then transplanted in peritoneal cavities. Endometrial transplants from the patients with endometriosis expressed more VEGF and MMP-2 than do those from control women, suggesting that VEGF and MMP-2 may participate in the early-stage evolution of endometriosis. REFERENCES 1. Sampson JA. Peritoneal endometriosis due to menstrual dissemination of endometrial tissue into the pelvic cavity. Am J Obstet Gynecol 1927;14: Farquhar CM. Extracts from the clinical evidence. Endometriosis. BMJ 2000;320: Blumenkrantz MJ, Gallagher N, Bashore RA, Tenckhoff H. Retrograde menstruation in women undergoing chronic peritoneal dialysis. Obstet Gynecol 1981;57: Chung HW, Wen Y, Chun SH, Nezhat C, Woo BH, Lake Polan M. Matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-3 mrna expression in ectopic and eutopic endometrium in women with endometriosis: a rationale for endometriotic invasiveness. 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