The Study Of The Role And Activation Mechanism Of EphrinB2 And Angiogenesis Mediated By It In The Development Of Dural Arteriovenous Fistula Induced By Venous Hypertension

Background Complex dural arteriovenous fistula(DAVF)is associate with high morbidity and mortality rates,and difficult to cure.Venous hypertension is regarded as a major cause of the formation because of the tissue hypoxia and elevated shear stress in the venous system,while the mechanism is unknown.High level expression of the hypoxia-inducible factor(HIF)-1α and the downstream angiogenesis-related factors in rats with intracranial venous hypertension has been observed in our previous study,suggesting that HIF-1α and subsequent angiogenesis take the important part in the formation of DAVF,while this is not the unique signaling pathway.Some report found that EphrinB2 was highly expressed and related with angiogenesis.EphrinB2 will be detected in the brain and dura of venous hypertension rats and the micro vessel density and the induced ratio of DAVF will be detached after lentivirus vectors are administrated to alter the expression level of EphrinB2 in rats.Human umbilical vein endothelial cells will be cultured in deferent shear stress level and HIF expression level to reveal the relationship between EphrinB2 and shear stress.The research project will clarify the role and mechanism of the upregulation of EphrinB2 in the formation of DAVF induced by venous hypertension.Purpose 1.Establish rat model with different stress of venous and detect the spatial and temporal expression of EphrinB2 and VIII angiogenesis factors in the dura mater to confirm the relation between the activation of EphrinB2 and venous hypertension.Then,by regulating the expression level of EphrinB2,the level of VEGFR2 and angiogenesis were detected and observed in different mater of the cerebrum.Finally,the interaction between EphrinB2 and VEGFR2 was determined by immunocoprecipitation.2.HIF-1α and EphrinB2/Eph B4 under different shear stress conditions in vitro experiments would be detected to confirm that whether shear stress can play a role through Ephri B2/Eph B4 pathway.Then,detect EphrinB2/Eph B4 pathway expression level after the regulation of HIF-1α expression level in endothelial cells,then clarify that the activation of EphrinB2/Eph B4 pathway activated by venous hypertension may be related to the shear stress-induced HIF-1α expression in endothelial cells.Methods 1.Establish the rat model with venous hypertension.120 male SD rats were randomly divided into three groups: sham operation group,venous sinus occlusion group and venous hypertension group.The expression of EphrinB2 and VEGFR2 protein in dura mater was detected by Western blot.Immunohistochemistry was used to detect the phosphorylation of EphrinB2 and VEGFR2 in occipital cortex,torcular and dura mater at 1,3 and 14 days.The transcription levels of EphrinB2 and VEGFR2 were detected by RT-PCR at 1,3,7 and 14 days after the operation respectively.2.Construct EphrinB2 sh RNA lentivirus and confirm the infection efficiency in vitro,112 male SD rats were divided into three groups: sham operation group,venous high pressure group,venous high pressure combined with venous sinus blank lentivirus infection group,venous high pressure combined with venous sinus EphrinB2 sh RNA lentivirus carrier infection group.The transcription levels of EphrinB2 and VEGFR2 were detected by RT-PCR at 1 day,1 week,and 4 weeks after operation,respectively.The expression of EphrinB2 and VEGFR2 in the dura mater was detected by Western method.The levels of EphrinB2,VEGFR2 and VIII phosphorylation in the occipital cortex,torcular and dura mater were detected by immunohistochemistry.3.Construct EphrinB2 lentivirus and confirm the efficiency of infection in vitro.112 male SD rats were divided into sham operation group,venous sinus occlusion group,venous sinus occlusion with blank lentivirus infection group and venous sinus occlusion with recombinant lentivirus infection group.The transcription levels of EphrinB2 and VEGFR2 were detected by RT-PCR at 1 day,1 week,and 4 weeks after operation,respectively.The expression of EphrinB2 and VEGFR2 in the dura mater was detected by Western method.The levels of EphrinB2,VEGFR2 and VIII phosphorylation in the occipital cortex,torcular and dura mater were detected by immunohistochemistry.4.Based on the principle of specific binding between antibody and antigen,it is confirmed whether the two proteins can interact with each other.5.Human umbilical vein endothelial cells(HUVECs)were cultured under shear stress of 4 and 10 dyn/cm2 for 24 hours respectively,and the endothelial cells cultured at rest(shear stress = 0)were used as control.The expression of HIF-1α and EphrinB2/Eph B4 and were detected by RT-PCR and Western blot,and the phosphorylation level of EphrinB2/Eph B4 was determined by immunohistochemistry.6.Construct HIF-1α sh RNA lentivirus to confirm the efficiency of infection in vitro,and then group them according to 10 dyn/ m2 shear stress environment,10 dyn/cm2 shear stress environment with blank lentivirus infection,10 dyn/cm2 shear stress environment with HIF-1α sh RNA lentivirus infection.The expression of HIF-1α and EphrinB2/Eph B4 were detected by RT-PCR and Western blot.Results 1.Compared with the control group,the expression of EphrinB2 and VEGFR2 in the dura mater of the rats in the venous sinus occlusion group and the venous hypertension group increased significantly.Compared with the control group,EphrinB2,Eph B4 and VEGFR2 were significantly expressed in the sinus confluence and dural tissue,and the expression site of the sinus confluence was mainly in the vascular endothelial layer.2.Compared with venous high pressure with venous sinus blank lentivirus infection group,the expression of EphrinB2 and VEGFR2 decreased in venous high pressure combined with EphrinB2 sh RNA lentivirus infection group.This phenomenon was founded in torcular and dura mater,not founded in occipital cortex.3.Compared with the venous sinus occlusion with blank lentivirus infection group,the expression of EphrinB2 and VEGFR2 increased in venous sinus occlusion with recombinant lentivirus infection group.This phenomenon was founded in torcular and dura mater,not founded in occipital cortex.4.EphrinB2 ccould interact with VEGFR2 in the cell in the immune coprecipitation reaction.5.The expression of EphrinB2/Eph B4 in HUVECs increased under 4,10 dyn / cm2 shear stress compared with that in the static culture group.The expression of HIF-1α in HUVECs increased under 10 dyn/cm2 shear stress,not increased under 4 dyn / cm2.6.The expression of EphrinB2/Eph B4 had no significant change after HIF-1 α was silenced under 10 dyn/cm2 shear stress compared with the control group.Conclusion 1.EphrinB2 could interact with VEGFR2 specifically.The activation of Ephrin B is related to venous hypertension.EphrinB2 can activate VEGFR,then initiate angiogenesis on dura mater.2.The activation of EphrinB2/Eph B4 pathway is related to the high shear stress environment in the vein.The activation of EphrinB2/Eph B4 pathway by high shear stress under venous hypertension is not related to the activation of HIF-1α in endothelial cells.