The Role Of Platelets In The Pathogenesis And Progression Of Endometriosis

Background:Endometriosis is a common gynecologic disorder in women of reproductive age. Its symptoms includes dysmenorrhea, chronic pelvic pain, and infertility, impacting negatively on women’s life quality and productivity and exerting a heavy economic burden on the society. Its prevalence is reportedly increased in recent years, yet its pathogenesis is still poorly understood. As a result, its clinical management is still challenging. Although surgery and anti-estrogen therapy may reduce pain and relieve other associated symptoms, however, the relief is often short-term and the recurrence risk is high. Wound healing is a highly dynamic and complicated process, in which platelets play a critical role in hemostasis, immune responses, and tissue remodeling. While the ectopic endometrium undergoes cyclic bleeding and tissue repair process along with the patient’s menstrual cycle, the platelets would be activated and aggregated in and around lesions, therefore the endometriotic lesion effectively becomes a wound that repeatedly injured and never healed. Our study is to investigate the role of platelets in the development and progression of endometriosis. Part Ⅰ The expression of platelets and associated molecules in EndometriosisObjective:To compare the platelets staining and expression of their related molecules in endometriosis. Methods:Sixty-seven ectopic endometrial and37eutopic endometrial tissue samples from women with laparoscopically diagnosed endometriosis (ovarian endometriotic cyst) were used in this study. In addition, normal endometrial tissue samples were obtained from60cycling women who underwent surgery for benign gynecological disorders but no endometrial abnormalities, endometriosis, adenomyosis and uterine myoma. The localization and abundance of platelets and their related molecules were evaluated by immunohistochemistry staining. Platelets and microvessels co-localization was investigated by immunofluorescent staining. Tissue fibrosis of ectopic endomerium was determined by Masson’s Trichrome staining. Results:Compared with the normal and eutopic endometrium, the immunoreactivity to CD41, CD68, MPO, HIF1-α, VEGF, CD31(marker of microvessel density or MVD), fibronectin and collagen I was significantly higher in the ectopic endometrium (p<0.01). PCNA expression was also seen in the ectopic endometrium. The immunofluorescent staining showed that the platelets infiltrated the stroma of the ectopic endometrium and were proximal to the blood vessels. The degree of tissue fibrosis was highly associated with lesion color, a proxy of lesion age. Conclusions:Platelets are activated and aggregated in the ectopic endometrium and may play a critical role in the pathogenesis and progress of endometriosis, including angiogenesis, immune responses, vessel growth and tissue fibrosis/remodeling.Part II The effect of activated platelets on ectopic endometrial stromal cells (ESC), and the effect of ectopic ESC and peritoneal environment on the activation/aggregation of platelets in vitroObjectives:To investigate the effect of activated platelets on ectopic endometrial stromal cells (ESC), and the effect of ectopic ESC and peritoneal environment on the activation/aggregation of platelets in vitro. Methods:Twenty cases of ectopic endometrium from women with laparoscopic diagnosed endometriosis (ovarian endometriotic cyst) and twelve cases of normal endometrium were collected and primarily cultured. Eightteen cases of peritoneal fluid from women with laparoscopic diagnosed endometriosis and twenty cases of control peritoneal fluid were also collected in this study. Ectopic ESCs were co-cultured with platelets or human thrombin activated platelets, then the COX-2, VEGF and MMP-9protein expression levels of the ectopic ESCs were evaluated by WB. The cell cycles of the ectopic ESCs were analyzed by FACS. The TF protein expression levels of normal or ectopic ESCs were evaluated by WB and ELISA. The normal or ectopic ESCs were treated with different dose of IL-1β, COX-2expression of both group ESCs were evaluated by WB, the concentrations of TXB2(TXA2degradation product) and6-keto PGF1α in the cell culture media of both group were detected by ELISA. The concentrations of TF and human thrombin in EMs or control peritoneal fluid were detected by ELISA. The platelets were treated by EMs or control peritoneal fluid, and their activation rate and whether it could be inhibited by huridin were analyzed by FACS. Results:The ESCs protein expression levels of COX-2, VEGF and MMP-9were up-regulated in the platelet treatment group and even up-regulated in the activated platelet treatment group compared with the control and thrombin treatment group (P<0.01). The percentage of ESCs in cycle S and G2were increased by the platelet treatment, especially increased by the activated platelet treatment. TF expression level of primary cultured ectopic ESCs is significantly higher than normal ESCs (p<0.05). Treatment of either normal or ectopic group of ESCs with IL-1β, we convinced that ESCs COX-2expression levels in both groups were up-regulated dose-dependently, meanwhile, the concentrations of TXB2and6-keto PGF1α of ESCs culture media were both up-regulated dose-dependently in both ectopic and normal groups(P<0.05). Platelets treated by EMs PF caused higher activation rates than treated by control PF (P<0.01), and the activation rates of platelets in both groups could be inhibited by huridin up to15.3%-67.1%. Conclusions:Activated platelets could up-regulate COX-2, VEGF and MMP-9protein expression levels of the ectopic ESCs, and could also stimulate the proliferation of ectopic ESCs. Meanwhile, ectopic ESCs and the EMs peritoneal environment may promote the activation/aggregation of platelets to the local lesion. Therefore, each side works together and contributes to the progress of endometriosis.Part III The impact of anti-platelets therapy, immunological depletion or infusion of platelets, P-selectin treatment and P-selectin knockout on the lesion growth and hyperalgesia of mouse model for endometriosisObjectives:To investigate the impact of anti-platelets therapy, immunological depletion or infusion of platelets, P-selectin treatment and P-selectin knockout on the lesion growth and generalized hyperalgesia of mouse model for endometriosis. Methods:After the first hot plate latency test was administrated, C57bl mice were received a endometriosis-inducing surgery. Two weeks after the surgery, the second hot plate latency test was administrated, then each group were received different treatments including anti-platelets therapy, immunological depletion or infusion of platelets, soluble P-selectin protein treatment and sterile saline or non-immune IgG treatment. After two weeks’treatment, the final hot plate latency test was administrated before sacrifice, then the number and mean size of the endometriotic lesions in all groups were evaluated. The expression of studying molecules of the lesion were evaluated by the immunohistochemistry staining. Tissue fibrosis was evaluated by Masson’s Trichrome staining. C57bl-WT background P-selectin-/-mice were used in this study, that received a endometriosis-inducing cross surgery with C57bl-WT mice (Group WW, PW, WP, PP). After receiving or not receiving GFP-expressing platelets, each group of mice were sacrificed, then the number and mean size of the endometriotic lesions were evaluated. Lesion samples were frozen embedded, slides were examined by immunofluorescent staining to detect the degree of the GFP-expressing platelets infiltrated the lesion. Results: Each anti platelets aggregation treatment relieved generalized hyperalgesia, and reduced the lesion growth(P<0.05). The expression of CD41, F4/80, VEGF, CD31-MVD, PCNA, p-NF-kB, COX-2, collagen I and a-SMA was reduced (P<0.05), and the degree of tissue fibrosis was also reduced. Platelet infusion did not increase the lesion growth any more (P>0.05). P-selectin-/-restricted the lesion growth (P<0.05). The GFP-expressing platelets were present in the stroma of the total four various groups of lesions, but showed apparently higher density in Group WW-G and PW-G, however, lower density in Group WP-G and even less in Group PP-G. Conclusions:Platelets play a critical role in generalized hyperalgesia and lesion growth of mouse model for endometriosis. P-selectin could mediate the platelets adhesion to the endometriotic lesion so that promote the lesion growth. Anti-platelets therapy, or soluble P-selectin protein treatment could relieve generalized hyperalgesia, and reduce lesion growth, seem to be the potent and promising methods for treating endometriosis.Part IV The impact of immunological depletion of plateletsor peritoneal macrophages at different lesion growth stage on the peritoneal macrophage activation, lesion growth and hyperalgesia of mouse model for endometriosisObjectives:To investigate the impact of immunological depletion of platelets or peritoneal macrophages at different lesion growth stage on the peritoneal macrophage activation, the lesion growth and generalized hyperalgesia of mouse model for endometriosis. Methods:Two days before the endometrium peritoneal transfer, a baseline hotplate latency test was administrated to all Balb/c recipient mice, then they were started to receive the rat anti mouse F4/80depleting mAb (Group MD), the rat anti mouse platelet depleting Ab (Group PD), or rat anti mouse non-immune Ab (Group N), or platelets infusion (Group PI).12days after the endometrium transfer, the second hot plate latency test was administrated before sacrifice, then peritoneal cells (majorly were macrophages) were collected, lesions excised and evaluated by assessing the dry weight, and processed for disease assessment by immunohistochemistry staining, HE staining, and Masson’s Trichrome staining. The areas of stroma and epithelium of the lesions were calculated by the ipp6.0soft and then the stroma/epithelium component ratios were obtained. The percentages of alternatively activated (M2) macrophages in total peritoneal macrophages were analyzed by FACS. The same model was repeated, but different treatment at different lesion growth stage instead:the rat anti mouse platelet depleting Ab was received two days before endometrium transfer (at day-2, Group EPD), or was received four days after endometrium transfer (at day4,9, Group LPD). The rat anti mouse non-immune Ab was given to Group n (at day-2,4,9). Group EPDLMD mice were intravenously given the rat anti mouse platelet depleting Ab two days before endometrium transfer (at day-2), and the rat anti mouse F4/80depleting mAb was intraperitoneally injected at day4,6,8,10after endometrium transfer. Four groups were all sacrificed12days after the endometrium transfer. Results:In Group MD, PD, EPD, LPD and PDMD, generalized hyperalgesia was lighter, the lesion growth was restricted (P<0.05), and the percentages of alternatively activated (M2) macrophages in total peritoneal macrophages were reduced (P<0.05). The expressions of CD41, F4/80, VEGF, CD31-MVD, TGFF-β1, collagen I and a-SMA were reduced (P<0.05), the stroma/epithelium component ratios as well as the degree of tissue fibrosis was also reduced. While in Group PI, generalized hyperalgesia was heavier, and the lesion growth was promoted (P<0.05). Conclusions: Platelets play a critical role both in the early endometrium adhesion, lesion growth stage and in the late lesion fibrosis stage in mouse model for endometriosis. Platelet depletion or macrophage depletion could reduce the peritoneal alternatively activated (M2) macrophages, reduce TGF-β1production, and relieve lesion fibrosis.