The Role And Regulating Mechanism Of EPHB4 In Spirarl Artery Remodeling

Part one.Expression and localization of EPHB4 in pregnancy-related tissues and cellsObjective To investigate the expression of EPHB4 in placental tissue from preeclampsia patients,villous and decidual tissues from first trimester and trophoblast cells(primary EVTs,human extravillous trophoblast cell line HTR-8/SVneo and trophoblastic lines of chorionic carcinoma,JEG-3 and JAR).Methods 1.Detecting EPHB4 expression in the placenta from preeclampsia pregnancy and controls using IHC,q RT-PCR and Western blot.2.Localizing the expression of EPHB4 in villous anddecidual tissues from first trimester by IHC staining.3.Analyzing the expression of EPHB4 gene at m RNA level in primary EVTs,human extravillous trophoblast cell line HTR-8/SVneo and chorioepitheliomacell lines,JAR and JEG-3 by q RT-PCR.Results 1.The EPHB4 expression level of preeclampsia placenta was much higher than controls.2.The trophoblasts in firsttrimester placental villi exhibited relatively strong membrane EPHB4 immunoreactivity.While in deciduas,almost no EPHB4 staining could be observed in EVTs and vascular endothelial cells showed a little slight expression.3.Primary EVT had a lower expression of EPHB4 m RN A compared with trophoblastic lines of chorionic carcinoma and showed no statistically difference with HTR-8/SVneo cells.Conclusion Decreased EPHB4 expression during the differentiation of villous trophoblasts into EVT and different EPHB4 expression between preeclamptic and normal placenta suggested that EPHB4 might modulate EVT biological function,involving in uterine spiral artery remodelling and placentation.Part two.EPHB4 regulated human trophoblast cell line HTR-8/SVneo function in the spiral artery remodellingObjective We aimed to study the effects of EPHB4 up-regulation on HTR-8/SVneo cell biological function and further investigate the role of EPHB4 in spiral artery remodeling by PDC system.Methods 1.HTR-8/SVneo cells were classified into three groups: hyper-expression group(cells transfected with EPHB4 over-expression plasmid),negative control group(cells transfected with empty vector),and control group(cells with non-transfection).2.Applying q RT-PCR and western blotting to testify the expression of EPHB4 at transcriptional level and protein level.3.Examining cell viability by CCK-8.4.Examining cell apoptosis by Annexin V/Propidium Iodide assay and caspase-3 colorimetric assay.5.Examining cell migration and invasion abilities by Trans well models.6.Examining cell epithelial replacement ability by endothelial-trophoblast co-culture model.7.Testifying the effect of EPHB4 up-regulation on spiral artery remodeling by PDC system.Results 1.Over-expression group showed significant improvement in the level of EPHB4 m RNA and protein expression after transfection.2.Increased apoptosis level,decreased cellular activity,increased migration and invasion capacity,and decreased ability for endothelial replacement can be observed in the EPHB4 hyper-expression group.3.The PDC results showed that,compared with negative control group,the uterine spiral artery transformation in EPHB4 super-expression group was at an earlier stage: the number of EVTs in decidua tissue was less and the degree of penetration was more superficial,and the spiral artery structure was not sufficiently damaged,as well.4.The levels of m RN A and protein expression of PI3 K and AKT decreased significantly in EPHB4 superexpression groups.Conclusion EPHB4 significantly inhibited the proliferation,migration,invasion and endothelial replacement and increased apoptosis in HTR-8/SVneo.A similar trend could be observed in PDC results.Therefore,EPHB4 may negatively modulate the biological function of EVT to impair spiral artery remodeling.Part three.HOXA9 transcriptionally regulate d the EPHB4 receptor to modulate trophoblast migration and invasionObjective This study is conducted to investigate the role of HOXA9 and its r elationship with EPHB4 in trophoblast cells.Methods 1.Both m RNA and protein expression levels of HOXA9 and EPHB4 were measured in preeclamptic placenta(n = 15)and normal placenta(n = 15).2.The expression and location of HOXA9 and EPHB4 in first-trimester villi were shown via immunohistochemistry.3.Trophoblast cell line HTR-8/SVneo was used to explore the effect of HOXA9 on EPHB4 expression and trophoblast bioactivity(including migration and invasion)by gainand loss-of function studies.4.Chromatin immunoprecipitation(C h IP)and luciferase assays were conducted to clarify the regulation mechanism of HOXA9 on EPHB4 expression in HTR-8/SVneo.Results 1.HOXA9 and EPHB4 expression were increased in preeclamptic placenta compared with normal placenta.2.HOXA9 and EPHB4 were identified to be strongly expressed in villous trophoblast.3.HOXA9 could promote EPHB4 expression and impaired HTR-8/SVneo cells migration and invasion.4.Ch IP and luciferase assays revealed that HOXA9 could directly bind to EPHB4 promoter and promoted its transcription.Conclusion HOXA9 transcriptionally stimulated EPHB4 expression and further inhibited trophoblasts migration and invasion,which may be an important mechanism of spiral artery remodeling disorder.Part four.EPHB4 me diated the process that IFN-gamma-activated endothelial cells resisted trophoblast invasionObjective Reportedly,IFN-γ-activated endothelial cells resisted trophoblast invasion to impair spiral artery remodeling.This part aimed to illustrate the role of EPHB4 in the pathological process.Methods 1.ICAM-1 was employed as the hallmark of endothelial activation.The serum levels of IFN-γ and the expression of EPHB4 and ICAM-1 were assessed by ELISA,q RT-PCR and WB,respectively.2.HUVECs were added with IFN-γ of different concentrations or for different times to determine the effect of IFN-γ on EPHB4 and ICAM-1 expression.3.Overexpression and sh RNA constructs,chromatin immunoprecipitation(Ch IP)and luciferase assays were conducted to clarify the regulation mechanism of IFN-γ/STAT1 on EPHB4 resulting in HUVECs activation.4.Endothelial–trophoblast co-culture model was used to illustrate the role of EPHB4 in the process of activated endothelial cells resisting trophoblast invasion.Results 1.IFN-γ,EPHB4 and ICAM-1 expression were elevated in preeclampsia.2.IFN-γ induced EPHB4 and ICAM-1expression in a concentration-and time-dependent manner in HUVECs.3.IFN-γ/STAT1 activated HUVECs through promoting EPHB4 expression.C h IP and luciferase assays revealed that IFN-γ promoted EPHB4 transcription by STAT-1 binding to EPHB4 promoter.4.EPHB4 involved in the process that IFN-γ-activated HUVECs showed the resistance to trophoblasts displacement.Conclusion IFN-γ/STAT1 signal pathway induced HUVECs activation and resisted trophoblasts displacement through regulating EPHB4 expression.This part explained the role of EPHB4 in abnormal vascular transformation from the respective of endothelial cells.