| Objective: Establish a rat model of arteriovenous malformation(AVM)to explore the biological characteristics of vascular endothelial cells in rats with arteriovenous malformation,as well as the expression of EphB4 and ephrinB2,so as to lay a foundation for further study of the angiogenesis mechanism in cerebral arteriovenous malformation(cAVM).Methods: The left side of the common carotid artery in 6-week-old male Sprague-Dawley rats was anastomosed end-to-end with the proximal end of the external jugular vein to establish a rat model of arteriovenous malformation.A total of 50 rats were modeled for subsequent experiments.Digital subtraction angiography(DSA),electron microscopy,HE staining,CD31,anti-smooth muscle antibody(SMA)immunohistochemical staining were performed to verify the model.Normal rats were used as the control group.At cellular level,VWF,CD31,VE-Cadherin staining were performed by immunofluorescence staining to identify the original generation of cultivation of endothelial cells.MTT assay,cell scratch test,cell apoptosis test,tube formation experiment and VEGF ELISA determination were applied to observe the cell proliferation,migration,apoptosis,tube formation,and the difference of VEGF secretion between AVM endothelial cells(AV-ECs)and normal vein endothelial cells(NV-ECs).The expression of EphB4 and ephrinB2 was observed by immunofluorescence staining first,and the gene expression was detected by Reverse transcription PCR.Finally,the expression of EphB4 and ephrinB2 was quantitatively analyzed by Western blot.Results: Of the 50 rats who received the model,42 were successful,with a success rate of 84%.There were morphological differences between the normal group and the AVM model group,and the AVM model group had pathological changes of cerebral arteriovenous malformation.The proliferation of endothelial cells in the AVM model group detected by MTT was stronger than that in the normal group,and the p values at the points of day 1,3 and 5 were p = 0.019,p = 0.00009,and p = 0.039,respectively.The migration ability of endothelial cells in the AVM model group was higher than that in the normal group(p<0.05).In the tube formation experiment,the tube formation ability of endothelial cells in the AVM model group was greater than that of the normal group,and the p values at 6h,12 h and 24 h were p = 0.099,p = 0.003,and p = 0.001,respectively,indicating that there was a difference in the tube formation ability between the two groups after 6hours.The apoptosis rate of vascular endothelial cells in the AVM model group was lower than that in the normal group.There was more VEGF secretion in the AVM model group,which may indicate that there is a relationship between increased VEGF secretion and increased proliferation and migration in the AVM model group.Immunofluorescence staining of EphB4/ephrinB2 suggested that the expression of two proteins might increase in the AVM group.The result of RT-PCR of EphB4/ephrinB2 showed that the expression of both genes were increased in the model group,indicating that the AVM model of rats established by surgical modeling may have simultaneous genetic changes due to hemodynamic changes.Western blot results showed that the expression of EphB4/ephrinB2 proteins was indeed higher in the AVM group than that in the normal group.Conclusion: AVM rat model and human c AVM have similar vascular pathological structure.In the model group,stronger proliferation,migration,tube formation capacity,increased VEGF secretion and change in apoptosis indicated that the model changed the biological characteristics of vascular endothelial cells,which were the basis for the development of c AVM.The increased expression of EphB4 and ephrinB2 in the model group suggested that EphB4 and ephrinB2 proteins might play a regulatory role in the development of cerebral arteriovenous malformation.Using this model,we could further study the angiogenesis in c AVM,and the regulatory effects of EphB4 and ephrinB2 on blood vessels need to be further studied. |
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