The Study Of Endothelial Cells Lacking ZIPK On Fetal Mouse Development And The Regulation Of Endothelial Cell Microfilament Skeleton

Endothelium is an important cell barrier,regulating inflammation and BBB(Blood Brain Barrier)and homeostasis,which control exchange of substance between vessels and tissues and cellular infiltration through changing endothelium permeability.The mechanism of regulating endothelium permeability is essential for studying the development of cardiovascular,lung,brain and other tissues and pathophysiology process of related diseases.Paracellular pathway and transcellular channel are the main patterns which exchange substance between tissues and microvessels.What's more,paracellular pathway which form gaps between cells through altering cytoskeleton remodeling and change endothelium permeability is more important.However,the mechanism of regulating endothelium permeablity is hard to know.Recent research shows that MLC phosphorylation initiate cell contraction,which is essential for changing endothelium permeablity.Zip Kinase(ZIPK)or DAPK3 is a member of DAPK family,which is highly conserved expressed in vertebrate.ZIPK is expressed in kinds of cells including smooth muscles cells and endothelial cells.Now,what we know is that ZIPK is a tumor repressor,which takes part in regulating cell apoptosis.In vitro,ZIPK can phosphorylate MLC in SMCs.However,it is not yet known whether ZIPK possess the same function in endothelial cells.In our study,we use Zipk fl/fl/Tie2-Cre system establishing the ZIPK knockout mouse(KO mouse),which specified deletes ZIPK in endothelial cells.Results show that the KO mouse died in about E17.5.Before embryo 14.5d,there is no obvious difference between the KO mouse and WT one.Besides,we use endothelial cells maker CD31 to show the vessel system of the whole embryo.Results shows that the vessel system of embryo head in KO mouse is normal.Further analysis found that the heart of KO mouse showed abnormal.There is ventricular septal defect(VSD)in the heart of KO mouse.What's more,the edema in the back of the KO mouse is another important feature of VSD.In view of Zipk fl/fl/Tie2-Cre mouse died at embryo stage,we use Zipkfl/fl/E2A-CreERT2 mouse to delete ZIPK in adult by consecutively intraperitoneal injection tamoxifen at a dose of lmg/d for 5 days.Next,we isolate the primary brain microvessels' endothelial cells to study the effect of ZIPK on cell contraction and MLC phosphorylation in vitro.We firstly use thrombin to stimulate the primary cells and detect the expression level of MLC phosphorylation with IF and Western Blotting method.Results shows that in compared with the control,the level of MLC phosphorylation and the ability of cell contraction in the KO cells is significantly reduced.We also found that the ability of cell migration decreased while enhanced cell proliferation.In conclusion,firstly,ZIPK regulate embryo heart development through control the function of endocardium.Secondly,ZIPK control cell contraction by changing MLC phosphorylation.At last,ZIPK decreased the ability of cell migration and enhanced cell proliferation.