The Role Of Delta-like 4 Ligand/Notch4-ephrin-B2 Cascade In Placental Vascular Endothelial Injury By Regulating Functions Of Endothelial Progenitor Cell

Part oneThe role of ephrin-B2 in the pathogenesis of preeclampsia by regulating EPC functionsPurposeExploring the expression of ephrin-B2 in EPCs and placental tissue from preeclampsia patients and studying the role of ephrin-B2 in the pathogenesis of preeclampsia by regulating EPC functions.MethodsThe ephrin-B2 expression was detected in the placenta and EPCs from preeclampsia pregnancy and controls using qRT-PCR and Western Blot.Then ephrin-B2 was silenced or over-expressed to estimate its role of on EPC functions.The proliferation,differentiation,migration and HUVEC-tube formation of EPC were severally tested by CCK-8,transwell assay,differentiation assay and in vitro HUVEC-tube formation assay.Results1.The ephrin-B2 expression level of preeclampsia placenta and EPCs was much higher than controls.2.p-regulation of ephrin-B2 damaged EPCs' proliferation and differentiation.The migration and HUVEC-tube formation were also inhibited by ephrin-B2 over-expression.However,ephrin-B2 silence contributed to the opposite effects on EPCs.ConclusionEphrin-B2 might play a part in the pathogenesis of preeclampsia by damaging the EPC-mediated vasculogenesis.Part twoThe role of D114/Notch signaling on EPC biofunctions by increasing the expression of ephrin-B2PurposeThe role of D114/Notch signaling on ephrin-B2 expression and EPCs' functions were explored.MethodsWe used D114 or DAPT to activate or block Notch signaling,respectively.Normal EPCs were incubated with sD114 or DAPT for 24 h.Then,we detected ephrin-B2 expression by employing qRT-PCR and Western Blot.In addition,the biological functions of EPC were respectively tested by CCK-8,transwell assay,differentiation assay and in vitro HUVEC-tube formation assay.Results1.Dll4/Notch activation resulted in increased ephrin-B2 expression and simultaneously depressed of EPCs' capactivity.2.Block of Notch signaling contributed to the opposite effects.ConclusionD114/Notch signaling impairs EPC bioactivities by increasing the expression of ephrin-B2.The Dll4/Notch-ephrin-B2 cascade is confirmed in EPCs.Part threeNotch4 signaling controls ephrin-B2-induced EPC dysfunctionPurposeThere are two specific receptors combined with D114 signaling,Notchl and Notch4.However,it is not very clear that which receptor is appropriate for EPCs'D114/Notch-ephrinB2 cascade.Therefore,a series of experiments were performed to explore it.MethodsThe expression of ephrinB2 was detected under Notch4 or Notch 1 silence in EPCs,with or without sD114 treatment,employing qRT-PCR and Western Blot.After that the role of Notch4 on EPC activity were examined by CCK-8,transwell assay,differentiation assay and in vitro HUVEC-tube formation assay.Results1.Ephrin-B2 expression was decreased when Notch4 silence in EPCs,and then kept the same level when D114/Notch activated.In contrast,Notchl down-regulation in EPCs led to a slight increased expression of ephrin-B2,which further augmented in the activation of Dll4/Notch.2.Notch4 silence contributed to an enhancement of EPC function which was similar to ephrin-B2 down-regulation.ConclusionThese findings suggest that Notch4,not Notchl,is the specific receptor for the Dll4/Notch-ephrin-B2 cascade.Notch4 signaling controls ephrin-B2-induced EPC dysfunction in preeclampsia.Part fourTransplantation of EPCs repairs the damaged placental vascular endothelium in preeclampsia ratsPurposeTo investigate whether transplantation of ephrinB2-downregulated EPCs can enhance placental vasculogenesis and improve pregnancy outcome.Methods1.The SD rat model of preeclampsia was induced with L-NAME.2.Rat-EPCs were harvested from the marrow and cultured for 7 days.Then EPCs were transfected with lentivirus vector to inhibit the expression of ephrin-B2.s3.The pregnant rats were divided into four groups:1)normal control group(normal pregnancy);2)preeclampsia group;3)treatment group(transplantation of ephrinB2-downregulated EPCs to the placenta of preeclampsia rats);4)treatment control group(transplantation of normal EPCs to the placenta of preeclampsia rats).4.Blood pressure was checked at 17 and 19 days gestation.5.At 20 days gestation,pregnant rats were killed.Frozen sections of placenta were observed by fluorescence microscope to study EPCs migration in vivo;the ephrin-B2 expression level of the placenta were detected by qRT-PCR and Western Blot.6.The gestational outcome was observed by measurement of the weight and size of rat fetus and placenta.7.The placental micro vascular were marked with CD34 antibody shown by immuno histo c he mistry.Results1.EPCs with green fluorescence were alive along the placental vascular distribution trend under the fluorescent microscope.2.The blood pressure of two control groups and treatment group were both significantly reduced compared to preeclampsia group.The blood pressure of treatment group was also decreased compared with treatment control group.3.In preeclampsia group,the weight of placentas and fetus were significantly reduced compared to both two control groups and treatment group,the size of placenta was also decreased in preeclampsia group compared with control while improved in treatment group.4.In control group,the extensive vasculature network was positive staining with CD34 antibodies.In contrast,vasculature in placenta labyrinth was narrowed and the number of positive blood vessels was reduced in preeclampsia group.However,in treatment group,microvessel density and the blood flow were all increased.5.The size,weight and microvessel density of placentas were greater in treatment group compared with treatment control group.ConclusionTransplantation of EPCs can repair and restore the damaged epithelium in preeclampsia placenta and may have a great potential for clinical application.Compared with normal EPCs,transplantation of ephrinB2-downregulated EPCs can repair and restore more epithelium and have better pregnancy outcomes.