The Study Of The Role Of Endothelial Cells Specificity Molecular-2(ECSM2) In Angiogenesis

Objects:To investigate the functional sites of endothelial cell specific molecule ECSM2, the crosstalk between ECSM2and other specific molecules, the function of ECSM2in endothelial cell and the expression of ECSM2in tissues of angiogenesis diseases.Methods:1. The bioinformatic software CBS Prediction Server was used to predict the functional sites of ECSM2. Mutation of the extracellular54th tyrosine to alanine (Y54A) was obtained via a site-specific mutation and expression vectors of pECSM2Y54A-GFP and pECSM2Y54A-3flag were constructed.2. Expression plasmid of ECSM2wild type and Y54A mutation were transfected into293T cells. Immumofluorescence and co-immunoprecipitation were performed to determine the location and phosphorylation sites of ECSM2. Wound healing assay and transwell assay were used to detect cell mobility.3. The combination of ECSM2with VE-cadherin in VEGF-stimulated HUVEC was detected by western blot and co-immunoprecipitation.4. HUVEC cells were seeded in Matrigel. Real-Time PCR was applied to detect the ecsm2expression levels of mRNA in the progress of angiogenesis in vitro. The siRNA interference was used to inhibit the expression of ecsm2genes. The tube formation assay and in vitro permeability assay were used to observe angiogenesis in vitro and effects on the stability of blood vessels.5. The expression of ECSM2in liver tissue from46cases of hepatocellular carcinoma was detected with immunohistochemistry and the SAS9.2statistical software was used to perform the statistical analysis.Results: 1. There are1potential phosphorylation site (Y54, tyrosine),12serine/threonine phosphorylation sites,1N-glycosylation site (N96, asparagine),23O-glycosylation sites in extracellular domain of ECSM2, and7serine phosphorylation sites in intracellular domain of ECSM2.2. The54th tyrosine residue was identified as the phosphorylation site of ECSM2and its mutation did not influence its membrane location, however, abrogated the inhibition effect of ECSM2on cell mobility.3. The combination of ECSM2with VE-cadherin in HUVEC was enhanced after VEGF stimulation.4. The transcription levels of ECSM2molecules was reduced in the progress of angiogenesis in vitro and are associated with maintainance of vascular stability.5. The expression of ECSM2in hepatocellular carcinoma was increased.Conclusions:The54th tyrosine residue is tyrosine phosphorylation site. Its mutation does not influence the membrane location of ECSM2, but abrogates the inhibition of ECSM2on cell mobility. The expression and combination of ECSM2with VE-cadherin is increased in VEGF stimulated HUVEC cells. Functionally, ECSM2participates in maintaining vascular stability and permeability. The expression of ECSM2is increased in angiogenesis diseases, such as hepatocellular carcinoma.