The Role Of Transforming Growth Factor-beta1 In The Pathogenesis Of Endometriosis And Its Mechanism In The Condition Of Hypoxia

Background: Endometriosis (Ems) is a common gynecological disorder in infertility women with a prevalence rate of 25%-40%. Although endometriosis is one of the most frequent benign gynecological diseases, it has strong invasion, metastasis and recurrence capability which commonly characterized as malignent tumor. However, its pathogenic mechanism remains unclear.It has been widely accepted that endometriosis originates from retrograde menstruation and eutopic endometrium determinism is the complement and development.Due to the abnormal contraction of uterine smooth muscle and spiral artery during premenstrual and menstrual periods, the endometrium is in different degrees of hypoxia in the whole processing before and after stripping, countercurrent flow to the abdominal cavity, and ectopic adhesion, invasion and angiogenesis.TGF-?1 is responsible for regulating cell proliferation, differentiation and angiogenesis, etc. In tumor tissue, TGF-?1 up-regulated by hypoxia could increase cell capability of invasion,angiogenesisand immune repression. TGF-?1 expression is high in endometriosis lesions, abdominal fluid and blood of endometriosis patients. However, its role in hypoxic conditions is not clear.We aimed to determine whether TGF-?1 affects endometriosis development and progression and its related mechanisms in hypoxic conditions. It provides the experimental basis for the diagnosis of the disease and the new treatment ideas.Objective: We aimed to investigate whether TGF-?1 affects endometriosis development and progression and its related mechanisms in hypoxic conditions.Methods: Endometriosis tissue was obtained from women with and without endometriosis and the expression of TGF-?1, hypoxia-inducible factor 1-a (HIF-la) and vascular endothelial growth factor (VEGF) were detected. Endometriosis cells were cultured and then exposed to hypoxia and TGF-?1 or TGF-?1 inhibitors. The mRNA and protein expression levels of TGF-?1,VEGF,and HIF-1? were measured. Bioinformatics software was used for prediction of transcription factor binding sites in the promoter region of VEGF gene. A dual-luciferase reporter assay was employed to examine the SBE binding site of TGF-?1 signaling pathway.ChIP assay was used to detect the binding of HIF-1? and Smad in this region. Establishment of NOD-SCID and C57BL/6 mouse Ems model was used for observing the tumor growth after dealing with hypoxia or TGF-?3. Cell proliferation, apoptosis, angiogenesis and expression of related factors were detected by MTT, flow cytometry, Western Blot, quantitative RT-PCR, immunofluorescence and immunohistochemistry.Results: 1. TGF-?1, VEGF, HIF-1? mRNA, and protein expression were significantly higher in Ems tissue than in normal endometrial tissue and the expression of HIF-1? was positively correlated with the expression of TGF-?1, HIF-la and VEGF. 2. Endometriosis primary cultured cells exposed to hypoxia(1%) within 16h expressed lower proliferation and higher apoptosis rate. HIF-1? and VEGF mRNA and protein levels increased in response to hypoxia over 16h , whereas the expression of TGF-?1 had no obvious change.The proliferation ability of cells after hypoxia 24-48h is beginning to stabilize while decrease over 72h. 3. Cell proliferation, apoptosis and HIF-la, VEGF expression was not significantly affected by TGF-?i whereas cell proliferation, apoptosis and the expression of mRNA and protein of VEGF and HIF-1? were significantly increased under combined treatment of hypoxia/TGF-(31. 4. Increased cell viability, reduced apoptosis, and increased expression of HIF-1? and VEGF after hypoxia in 1%02 24h then normoxia. 5. The upstream of VEGF gene promoter SBE1 and HRE are respectively the sites of action of TGF-?1 and HIF-1?. ChIP experiments showed that VEGF gene expression was regulated by TGF-?1 and HIF-1?. 6. In NOD-SCID mice model and C57BL/6 mice model, lesion presented peripheral neovascularization and up-regulated expression of HIF-la and VEGF after continuous administration of TGF-?1. In hypoxia group, lesion presented rapidly growth after 2-3 weeks, neovascularization network formation and increasing expression of HIF-1? and VEGF. Joint action of HIF-1? and TGF-?1 presented rapidly growth and abundant glandular and stromal cells and vessels. Expression of VEGF, HIF-1? and TGF-?1 were up-regulated.Conclusions: 1. TGF-?3, VEGF, HIF-1? mRNA, and protein expression were significantly higher in Ems tissue than in normal endometrial tissue. 2. Hypoxia, HPC and HIF-1?/TGF-?1 joint action could increase endometrial cell growth, increasing expression of HIF-la and VEGF, decreasing GLUT 1 and increasing LDHA,PDK1. With the extension of hypoxia time, the apoptosis increased and the viability decreased. After hypoxia over 16h, cell ability of resistance to hypoxia and viability incerased and the strongest ability of hypoxia tolerance was observed hypoxia for 24h. Regulation of VEGF expression by hypoxia and TGF-(31 occurs at the transcriptional level and hypoxia achieve its function through HRE in -975 to -968 area. HIF-1? and Smad combine with HRE and SBE region DNA sequences. In the foundation of hypoxia, TGF-?1 could affect Ems cells.TGF-?1 could promote affection of hypoxia on Ems through VEGF. Hypoxia has stronger effect than TGF-?1 on VEGF.HIF-la/TGF-?1 combination treatment could promote NOD-SCID mice and C57BL/6 mice model lesion peripheral neovascularization. An additive effect of TGF-?1 and hypoxia is taking place in this processing.