Effect Of Down-regulation Of GLP-1R Expression On The Function Of Endothelial Progenitor Cells In Mice And Its Mechanism

Background: Diabetes mellitus is an important risk factor of cardiovascular disease.Ischemic vascular disease caused by diabetes mellitus has become one of the major diseases harmful to human health.Endothelial progenitor cells(EPCs)are an important cellular biological basis for repairing vascular injury and improving angiogenesis,which brings a new light to solve this problem.Recently,glucagon-like peptide-1 receptor agonist(GLP-1RA)has been shown to exert beneficial effects on cardiovascular disease through other mechanisms besides glycemic control.However,whether the glucagon-like peptide-1 receptor(GLP-1R)regulates EPCs functional activity remains unclear.This study aims to investigate the effect of down-regulation of GLP-1R expression on the functional activity of EPCs in mice,and to preliminarily explore its possible mechanism,and to provide a new theoretical basis for EPC-mediated cell therapy.Aim : To investigate the effects of altered glucagon-like peptide-1 receptor(GLP-1R)gene expression on migration,adhesion and senescence of endothelial progenitor cells(EPCs)derived from bone marrow in mice and its possible mechanism.Methods : Bone marrow-derived endothelial progenitor cells from healthy C57BL/6J mouse were isolated and cultured by density gradient centrifugation.Using adenovirus-mediated sh RNA to downregulate EPCs glucagon-like peptide-1 receptor(GLP-1R)gene expression,The EPCs were transfected with recombinant adenovirus carrying sh RNA targeting GLP-1R and expressing green fluorescent protein(GFP),Ad-GLP-1R-sh RNA,and the control adenovirus carrying nonsence sh RNA and expressing GFP,Ad-scramble-sh RNA.The expression of GLP-1R in the EPCs was measured by real-time fluorescent quantitative PCR(RT-q PCR)and Western blot.Transwell assay was used to detect the cell migration capacity.The EPCs adhesion and senescence were observed in different groups by adhension assay andβ-galactosidase staining,respectively.The protein expression of AKT、p-AKT,eNOS and p-eNOS in EPCs were measured by Western blot.The EPCs were divided into Normal group(using EGM-2 instead of adenovirus for transfection),Control group(the EPCs were transfected with the empty adenovirus expressing green fluorescent protein alones)and Transfection group(the EPCs were transfected with recombinant adenovirus targeting GLP-1R).Results:Compared with the Control group,the GLP-1R m RNA and protein expression level were significantly decreased in GLP-1R Transfection group(P<0.01).Additionally,a significant decreased number of migrated cells were observed in Transfection group compared with the Control group(P<0.01).Further study showed attenuated adhesion and anti-senescence ability of EPCs in Transfection group(P<0.01).Western Blot results showed that the expression level of p-AKT and p-eNOS was significantly decaresed in Transfection group(P<0.01).Conclusions: GLP-1R changes in gene expression can affect the functional activity of EPCs in mice.Knock-down of GLP-1R expression leads to inhibited EPCs migration,adhesion and induced cell senescence from healthy C57BL/6J mouse,and its main mechanism may be related to down-regulation of AKT/eNOS signaling pathways.