The Role Of Tissue Factor And Protease Activated Receptor2in Endometriosis And Enmd-1068, Protease Activated Receptor-2Antagonist, Inhibit The Development Of Endometriosis In Nude Model

BackgroundEndometriosis is a benign disease characterized by the presence of endometrialtissue outside of the uterine cavity causing infertility and pelvic pain inapproximately6%-10%of reproductive aged women. The incidence of endometriosisis on the rise, however, the etiology and physiopathology of the disease are stillelusive. Currently, therapies mainly rely upon surgical intervention andpharmacologic treatment with a poor effect and it ’s high recurrent rate was up to40%. Therefore, the further study of Gynecologic and Reproductive field will focuson the investigations of treatment strategies.Some studies found that tissue factor (TF) is not only a cellular receptor toinitiate the blood coagulation cascade, but also important for mediating intracellularsignal. The signal mainly involved with its downstream receptor, proteinase activatedreceptors (PAR)-2, participates in the development and progression of many types ofmalignancy tumors. In recent years, there are some studies showed that theimmunoreactivity of TF is highly upregulated in ectopic and eutopic endometrium with endometriosis and, the immunoreactivity of PAR-2is also highly upregulated ineutopic endometrium with endometriosis. Krikum G, et al proposed that TF/PAR-2signal might be participate the pathogenesis of endometriosis. Moreover, PAR-2deficient mice model could significantly inhibited the development of endometriosis.Though there are many researches on TF/PAR-2signal in oncology, there are stillabsent in the study of TF/PAR-2expression in endometriosis and whether PAR-2antagonists could inhibit the development in endometriosis? Our study investigatedthe expression of TF and PAR-2in ectopic and eutopic endometrium withendometriosis through a clinical study and to address a question whether PAR-2presents a new target for the treatment of endometriosis by an PAR-2antagonist toaim at find a new antibody curative method.ObjectiveTo investigate the expression of TF and PAR-2in ectopic and eutopicendometrium with endometriosis and their relationship with the menstrual cycle by aclinical study, we hope to find out the pathologic role of TF/PAR-2signal inendometriosis. To prepare for the clinical theory basis and develop a new target andspecific antibody therapy on endometriosis, we established human RFPendometriosis model in nude mouse to explore the therapeutic effect of PAR-2antagonist on human endometriosis mice model and study the possible treatmentmechanism. Part1. The role of Tissue factor and protease activated receptor2in endometriosisMethods1. Sample collectionFrom September2010to July2011,42women with endometriosis and20control subjects were recruited in the study. Endometriosis was diagnosed atoperative laparoscopy and postoperative confirmed by pathology. The patientswhose surgical diagnose were simple ovarian cyst excluding endometriosis wererecruited as control subjects. All patients’ ectopic endometrium, along with20patients’ homologous eutopic endometrium with endometriosis and all the controlsubjects’ normal endometrium were collected through curettage. None of women ineither group had been diagnosed for fibroids, adenomyosis or had received hormonestreatment or placed the IUD for a least6months prior to surgery(eg gonadotropin-releasing hormone agonist, oral contraceptives). All patients had signed an informedconsent and the research protocol had been approved by the institutional ethical andreview board of the Third Affiliated Hospital of Guangzhou Medical College andZhuJiang Hospital of Southern Medical University.2. TF and PAR-2expression in each group detectionAll the samples were assessed for TF and PAR-2protein location usingimmunohistochemistry and for relative TF and PAR-2mRNA expression using real-time florescent quantitative polymerase chain reaction(FQ-PCR).Results1. TF and PAR-2were detected mainly locate at membrane and cytoplasm ofglandular epithelial cells and in the stroma found in some cases. Total protein andmRNA expression of TF and PAR-2were significantly higher in ectopic and eutopicendometrium of women with endometriosis when compared to controls(p<0.05).However, both mRNA and immunoreactivity expression of TF and PAR-2showedno statistical difference between ectopic and eutopic endometrium tissues(p>0.05). 2. The expression of TF protein and mRNA in ectopic and eutopic endometriumand, PAR-2protein and mRNA expression in ectopic endometrium weresignificantly increased through the whole menstrual cycle(p<0.05). However, ineutopic endometrium with endometriosis, PAR-2expression only in secretory phasewas higher than its cycle-matched normal controls(p<0.05). There is no suchdifference in the proliferative phase(p>0.05). No variation of TF and PAR-2expression were seen between phases in the same group(p>0.05).Conclusions1. The abnormal co-upregulated expression of TF and PAR-2in eutopic andectopic endometrium may affect the development and growth of endometrioticlesions and they may be the ideal targets of interventional therapy.2. The co-upregulated expression of TF and PAR-2protein and mRNA ineutopic endometrium in secretory phase highlighted the pathologic role of TF andPAR-2in eutopic endometrium in endometriosis.Part2. ENMD-1068, protease activated receptor-2antagonist,inhibit the development of Endometriosis in nude ModelMethods1. Sample correction18women with eutopic endometrium with endometriosis in secretory phasewere corrected and the inclusion criteria of patients were the same as above.Explanted endometrial tissue was cut into fragments of1mm3~2mm3in diameterunder sterile conditions after rinsed by PBS.2. Preparation of endometrial fragments and transduction with LV-CMV-RFPHuman endometrium fragments were infected in vitro by lentivirus particlescarrying a strong CMV promotor driving expression of a bright mutant of enhancedred fluorescent protein recombination (LV-CMV-RFP) were used for RFP genetransfer into human endometrial fragments(5pieces each well of a48-well plate;1× 108PFU/mL of end concentration). Transduction of endometrial fragments wasperformed by incubation at37℃,5%CO2. After a24-hour incubation with lentivirusparticles, endometrial fragments were harvested and washed with PBS.3. Establishment and observation of human endometrial nude mice with RFP-expressing.(1) Six-to eight-week-old female nude mice (nu/nu-BABL/C. SPF) received aintra-abdominal planting10~12pieces of endometrial fragments with LV-CMV-RFPlentivirus transduction. The endometriosis mice model were given subcutaneousinjection of estradiol benzoate (0.02mg/d) and penicillin(1000U/d) for3consecutivedays. after8~10days later, the fluorescence or macroscopic lesions were observed.(2) Nine nude mice for tumorigenic pre-test. The establishment mice modeldivided into3groups randomly: the control A group=intraperitoneal injection ofnormal saline(NS)0.2ml/d; E0.5group: ENMD-10680.5mg/d;E1.0group:ENMD-10681.0mg/d. On days1,3,5after drugs intraperitoneal injection, thelesions of one mice per group were corrected and the mouse were killed throughcervical dislocation. We preliminary judged whether a tumor formed and the drug’scurative effect. The pre-test group were3mouse per group and points cage raising.(3) Twenty five nude mice for experimental test(one failed): The establishmentmice model were divided into3groups randomly and8mice per group. Thetreatment dose per day is similar to pre-test: NS group=NS0.2ml/d×5d; E0.5group=ENMD-10680.5mg/d×5d;E1.0group=ENMD-10681.0mg/d×5d. The nudemouse daily activities were observed.4. On days6after drugs intraperitoneal injection(days16~18after endometrialimplantation), we killed the mouse and measured the ectopic lesions volume andcorrected them into0.5ml sterile, free-antibiotic M199culture solution(pH=7.4) andincubated by37℃for2hours. The culture solution interleukin(IL)-6, monocytechemoattractant protein-1(MCP-1) is prepared for ELISA detection.5. Vascular endothelial growth factor (VEGF), nuclear factor-kappa B(NF-kB),Ki-67of ectopic lesions were detected by immunochemistry analysis.6. TUNEL cell apoptosis detection. 7. Histopathologic examination of heart, liver, spleen, lung, kidney, uterine andovarian displayed no pathological abnormalities in treated ENMD-1068animals.Results1. Establishment of dynamic endometriosis nude mouse model(1) Twenty five female nude mice were received intraperitoneal implantation ofhuman endometrium: implantation efficiencies24mouse(96%)(24mouse, each onewas proved successful by image of RFP-expressing human endometrial tissue andpathology examination), survived rate were100%. Twelve mouse were found brightRFP-expressing lesions on days8~10after intraperitoneal implantation ofendometrial fragments(48%)(Fig.2-2), Twelve mouse were detected the lesions bylaparotomy(48%), no fluorescence and even without naked eye lesions as modelingfailure(4%).(2) There are1~3lesions in NS group and E0.5group which mainly located informer, side peritoneal, fat(near the bladder), deep pelvic and bowel surface(Fig.2-3D). The lesions owing rich blood vessels showed strong adhesion withabdominal tissues. It’s not easy to strip the adhesions except the mild adhesions withbowel(Fig.2-3C). E1.0group ectopic lesions were shrink into flat plaques or roughsurface trace and without significant adhesion(Fig.2-3E).(3) Compared to the NS group, histopathology examination showed E0.5group(Fig.2-4B) and E1.0(Fig.2-4C) has less glands as sparse shrinking state, and theglands lumen narrowed and accompany with inflammation and necrosis in stroma.2. Ectopic lesions volume comparison: the average volume of ectopic lesions innude models with E0.5, E1.0group is significant lower than NS group (p<0.05), andas the increased doses, there is a significant difference among the three groups(p<0.05)(Fig.2-5).3. IL-6, MCP-1release from ectopic endometrium(pg/mg/2h) weredetected by Enzyme linked immunosorbent assay(ELISA).There was ansignificant decreased in the release of IL-6, MCP-1from the E0.5andE1.0group than NS group(p<0.05). Additionally, the concentration of IL-6in E1.0group is lower than E0.5group(p<0.05)(Fig.2-6).4. The expression of VEGF, NF-kB and Ki-67in ectopic lesions of E0.5, E1.0group is lower than NS group(p<0.05), and the difference in NF-kB and Ki-67expression was doses-dependent(p<0.05)(Fig.2-7,2-8).5. The cell apoptosis of E0.5and E1.0group was significantly higher than thatof NS group(p<0.05), however, the difference between E0.5and E1.0group showedno difference(p>0.05)(Fig.2-9,2-10).6. Histological aspect of vital organs of nude mouse such as heart, liver, spleen,lung, kidney, uterine and ovarian showed their morphology is normal(Fig.2-11).Conclusions1. The code LV-CMV-RFP transfected human endometrium wereintraperitoneal implanted into the abdominal of nude mouse. The noninvasivevisualization of RFP-expression lesions in living animals provided an effective toolsfor the treatment of endometriosis.2. ENMD-1068could significant inhibit the development of ectopic lesions inendometriosis mouses model and it significant inhibit the expression of VEGF,NF-kB, Ki-67, IL-6and MCP-1. The difference in volume of ectopic lesions, NF-kB,Ki-67and IL-6expression was doses-dependent. The efficient treatment mechanismof ENMD-1068on endometriosis mouses models may involved with the inhibitoryeffect of angiogenesis and inflammation in ectopic lesions, inhibited the ectopicendometriotic cell proliferation and promoted the cell apoptosis.