The Role Of MMP-3 In The Formation And Development Of Brain Arteriovenous Malformation

Objective:Detect the expression levels of MMP-3 in human brain arteriovenous malformations tissues, and explore the potential mechanism of MMP-3 in the pathogenesis of BAVM in vitro and in vivo.Materials and methods:MMP-3 is a key member of the matrix metalloproteinase family. It plays an important role in the mediation and regulation of other MMPs, which show abnormal expression in brain arteriovenous malformation tissues and may involve in the formation and development of brain arteriovenous malformations. Firstly, we will carry out qualitative and quantitative analysis on the MMP-3 expression levels by immunohistochemistry and RT-PCR in the samples of human brain arteriovenous malformation from 3 individual patients and 1 control brain tissues. During the following experiment, we construct the plasmids for MMP-3 overexpression and gene silencing, followed by selecting stable HBMEC cells2, determine the regulating effect of MMP-3 on the proliferation, migration and invasion ability of HBMEC via CCK8 experiment, scratch assay and transwell assay, and study the effect of MMP-3 overexpression or silecening on the capillary-like tube formation by establishing matrigel vessel formation model, aiming to investigate the potential mechanism of MMP-3 in the pathogenesis of BAVM in vitro. In the final experiment in vitro, the cerebral venous hypertension rat model was firstly established by common carotid artery-internal carotid artery anastomosis, and then the MMP-3-overexpressing HBMEC cells were stereotacticly injected to the para-sagittal sinus brain tissues to overexpress MMP-3 locally in the area, to mimick the cerebral microenvironment in the pathogenesis of BAVMs. Finally, we studied the changes of the cerebral vessels through immunohistochemistry and injecting a fluorescent dye, lectin, to the peripheral vascular.Results:The MMP-3 expression is significantly higher in human BAVM tissues than that in the contrast in vascular endothelial cells and basement membrane which is further confirmed by RT-PCR. During the following experiment in vitro, the plasmids for MMP-3 over-expression and gene silencing are constructed and MMP-3 stably-expressing HBMEC are selected. CCK8 experiment, scratch assay and transwell assay revealed that overexpression of MMP-3 promoted the proliferation, migration and invasion ability of HBMEC, and capillary-like tube formation model showed MMP-3 was important for the number and phenotype of capillary-like tube formation in vitro. Cerebral venous hypertension rat model with local overexpression of MMP-3 is successfully established, and in this model, we found that over-expression of MMP-3 increased the cerebral vascular permeability and changed the structure of the vessels. We also observed that the coloring matter lectin diffused into the rat brain tissue in earlier time (4w),but were differentially ingested by the astrocytes in later time (8w).Conclusion:MMP-3 levels are upregulated in human brain arteriovenous malformations. MMP-3 may play important role in the pathogenesis of BAVM via directly and/or undirectly influencing the proliferation, migration, invasion and vessel formation ability of HBMEC to influence the cerebral angiogenesis and inducing the material exchange between astrocytes and cerebral vessels to change the function and structure of cerebral vessels.