The Role And Mechanism Of Exosomes Derived From Endothelial Cells In Vascular Smooth Muscle Cells Senescence/Calcification Under High Glucose

Objective: Vascular aging/calcification is a crucial feature of diabetic macro vasculopathy,resulting in serious cardiovascular events with great harm and tough prophylaxis and treatment.Exosomes(Exo)can mediate information communication between cells and participate in a variety of pathophysiological processes.The aim of this study is to elucidate the role and mechanism of exosomes derived from endothelial cells(ECs)in vascular smooth muscle cells(VSMCs)senescence/calcification under high glucose and provide a new train of thought for the prevention and treatment of diabetic vascular aging/calcification.Methods: Exosomes were isolated from normal glucose(NG)and high glucose(HG)stimulated human umbilical vein endothelial cell(HUVEC)by ultracentrifugation.Transmission electron microscopy(TEM),nanoparticle tracking analyzer(NTA),and western blot were used to identify the Exo.Proteomic analysis was conducted to analyze the difference of protein expression profiles between NG-HUVEC-Exo and HG-HUVEC-Exo.Bioinformatics database and literature review were used to identify the target protein as MFGE8.Fluorescence microscope showed that the Di D-labeled Exo could carry MFGE8 to VSMCs.The RGD disease network was used to preliminarily identify SIRT1 as the key gene in regulating MFGE8.Quantitative real-time polymerase chain reaction(q RT-PCR)was used to detect the m RNA levels of SIRT1 and MFGE8.Chromatin immunoprecipitation(Ch IP)was carried to detect the binding of SIRT1 to the MFGE8 promoter region.After the intervention of HUVEC by SIRT1 agonist SRT2104 and inhibitor Selisistat,the HUVEC was co-cultured with VSMCs to detect the senescence/calcification indexes of VSMCs and verify the regulation of SIRT1 on VSMCs senescence/calcification.The differentially expressed genes in VSMCs treated by NG-HUVEC-Exo and HG-HUVEC-Exo were analyzed by microarray.Inflammatory cytokines were detected by enzyme linked immunosorbent assay(ELISA).Results: The TEM and NTA showed that the Exo exhibited a cup-shaped morphology with an average diameter of 140.3 nm and 131.2nm of NG-HUVEC-Exo and HG-HUVEC-Exo,respectively.WB detected the presence of Exo markers including CD63 and CD9.HG-HUVEC-Exo promoted VSMCs senescence/calcification,which is characterized by the increase of SA-β-gal staining positive cells,the formation of mineralized nodules,and the increased expression of ALP and Runx2.Besides,the role of HG-HUVEC pre-treated with Exo secretion inhibitor GW4869 on VSMCs senescence/calcification was almost abrogated.The expression of MFGE8 was increased in HG-HUVEC-Exo by proteomics and western blot analysis.The fluorescence microscope verified that Di D-labeled Exo could carry MFGE8 into VSMCs.HUVEC treated with MFGE8 overexpressing(MFGE8-OV)promoted VSMCs senescence/calcification.The effect of HG-HUVEC-Exo on VSMCs senescence/calcification was significantly reduced after MFGE8 silencing(MFGE8-si RNA)by si RNA.RGD disease network database analysis found that SIRT1 is one of the cross genes of diabetes,vascular disease,diabetic vascular disease and hyperglycemia.In HG-HUVEC,the m RNA and protein levels of SIRT1 decreased,while the m RNA and protein levels of MFGE8 increased.When SIRT1 was activated by SRT2104,the m RNA and protein expression of MFGE8 decreased.Further experiments confirmed that SIRT1 could bind to the MFGE8 promoter region and participate in the regulation of VSMCs senescence/calcification.Microarray combined with bioinformatics analysis showed that MFGE8 and inflammatory factors,including interleukin(IL)-1β,IL-6 and IL-8 were up-regulated in VSMCs treated by HG-HUVEC-Exo.Gene ontology biological process(GO-BP)enrichment analysis showed that up-regulated differential genes were partially enriched in the inflammatory response pathway.ELISA showed that the expressions of IL-1β,IL-6 and IL-8 were up-regulated in VSMCs treated with HG-HUVEC-Exo,while the expressions of IL-1β,IL-6 and IL-8 were down-regulated by MFGE8 silencing.Conclusion: SIRT1 is down-regulated in HUVECs under high glucose,leading to the up-regulation of MFGE8 in exosomes,which in turn promotes VSMCs senescence/calcification through inflammatory pathway.