The Regulatory Effect Of VEGF-Ax On Rat Bone Marrow Mesenchymal Stem Cells Angioblastic Differentiation And Its Pro-angiogenic Ability

Background:Vascular endothelial growth factor A(VEGF-A)plays a key role in the regulation of angiogenesis.VEGF-A includes multiple subtypes.At first,it was thought that different VEGF-A subtypes were produced by alternative splicing of VEGF-A mRNA,and these subtypes play different regulatory roles in the process of angiogenesis,mainly including the opposite effects in promoting angiogenesis and anti-angiogenesis.So it is named VEGFxxxa or VEGFxxxb,including VEGF165b,VEGF121a and so on.Where "xxx" represents the number of amino acids present in the final protein sequence,and "a/b" represents different fragments of exon 8 of the VEGF-A gene:CDKPRR/SLTRKD.Recent studies have found that the mRNA programmed translational readthrough(PTR)can generate a new subtype of VEGF-A.This subtype uses a non-classical stop codon to generate a 22-amino acid segment at the carboxy terminus of VEGF-A.And it is therefore named VEGF-Ax,where x stands for "extended".Interestingly,the function of VEGF-Ax is unclear,especially the conclusion that VEGF-Ax regulates angiogenesis is contradictory.Two teams concluded that VEGF-Ax has "pro-" and "anti-" angiogenic effects.Methods:We first extracted rat bone marrow mesenchymal stem cells(BMMSCs),and performed identification of stem cell multipotential differentiation and surface-specific markers.We then designed an over-expressing lentivirus against rat VEGF-Ax.After over-expressing VEGF-Ax in BMMSCs,FCM was used to detect cell apoptosis and cycle,CCK-K and EdU staining was used to detect cell proliferation level,wound healing test and transwell test were used to detect cell migration ability,and Western blot was used to detected the expression of MMP-2,MMP-9,E-cadherin and N-cadherin.Tubule formation experiments,flow cytometry,and subcutaneous angiogenesis experiments in nude mice were used to detect the effect of BMMSCs angiogenesis,and Western blot was used to detect the expression of CD31,VEGFR2,vWF,cdh5.At the same time,the expression of CD31,VEGFR2,vWF in the subcutaneous neonatal tissues of nude mice was detected by immunohistochemistry.On the other hand,we used endoplasmic reticulum stress(ERS)agonists to induce BMMSCs apoptosis,then used FCM to detect cell apoptosis,CCK-K and EdU staining to detect cell proliferation,and Western blot to detect the expression levels of ERS-related proteins and apoptosis-related proteins.Results:Our experimental results show that VEGF-Ax can promote the proliferation and migration of BMMSCs,stimulate the differentiation of BMMSCs into endothelial cell(EC)-like cells,upregulate endothelial cell-related markers,and promote angiogenesis in vivo and in vitro.And protect BMMSCs from endoplasmic reticulum stress(ERS)-induced apoptosis.Conclusions:Our study first explored the effects of VEGF-Ax on the proliferation,migration and vascular differentiation of rat BMMSCs cells.We also investigated the protective effect of VEGF-Ax on BMMSCs by stimulating ERS-induced apoptosis.These studies have demonstrated for the first time that VEGF-Ax can stimulate the differentiation of BMMSCs into endothelial(EC)-like cells.We also investigated the regulatory effects of VEGF-Ax on the proliferation and migration of BMMSCs via the Wnt/?-catenin pathway,and demonstrated that VEGF-Ax protects BMMSCs from ERS-induced apoptosis.The long-term prospect of this study is to provide evidence for the role of VEGF-A functional groups.