Research On Pathogenic Mechanism Of Venous Malformation Related Mutation TIE2-R849W Based On Zebrafish Model

PURPOSE: A TIE2 mutation causing arginine-to-tryptophan substitution at residue 849(TIE2-R849W)is commonly identified in heredofamilial venous malformation.The aim of study is to confirm the pathogenic mechnism of TIE2-R849 W via establishing an in vivo zebrafish model.METHODS:1.Based on humanized TIE2 cDNA,TIE2-R849 W plasmid was constructed via PCR-mediated site-directed mutagenesis.Mutation site was identified by DNA sequencing.2.After transcription of TIE2-R849 W plasmid,fertilized one-celled fli1a: EGFP zebrafish embryos were injected with m RNA.3.Embryos larvae were analyzed with fluorescence microscope.The tissues were embedded in paraffin,and stained with hematoxylin and eosin.4.Total RNA was extracted and ananlysed by quantitative real-time PCR(qRT-PCR).The microarray data GSE46684 was acquired in the National Center for Biotechnology Information(NCBI)Gene Expression Omnibus(GEO).Statistical analysis,and visualization of the microarray data were carried out by using Hem I.RESULTS:1.Based on normal humanized TIE2 sequence,plasmid encoding mutant TIE2 was constructed via PCR-mediated site-directed mutagenesis.Target nucleotide T in control group had successfully transformed to C in mutant TIE2 group.2.TIE2-R849 W significantly induces significant caudal vein plexus(CVP)defect in zebrafish,displaying reduced number and area of loops.3.TIE2-R849 W also induces maxillofacial malformations,including eye abnormalities,forebrain formation perturbations,and mandibular malformation.Histologically,these phenotypes accompany aphakia,confused retina plexiform layer,abnormal mandibular cartilage,ectopic myelencephalon proliferation and aberrant location of neurogliocytes.4.According to qRT-PCR,except for high expression of egfl7,the other CVP-related genes cd146,nr2f1 a,and s1pr1 are not significantly different from control.TIE2-R849 W also induced upregulation of the wnt signaling pathway.5.Gene array in vitro shows that under the effect of TIE2-R849 W,consistent with high expression of pik3 and foxo1,high levels of egfl7,wnt9 a,lrp5 and dkk1 were partly confirmed.CONCLUSIONS:1.This in vivo model directly identifies the venous-related pathogenic role of TIE2-R849 W.2.Under up-regulation of TIE2-R849 W,egfl7 could be considered a potential reason for venous defects.3.The wnt pathway may perform an important role as a key trigger for head multi-malformations.