Mechanism Of Small Extracellular Vesicles Secreted By Endothelial Cells Treated With AGEs Against Medial Arterial Calcification In Diabetes

Objective:Medial arterial calcification（MAC）is commonly observed in patients with diabetes,chronic kidney disease,and atherosclerosis.Advanced glycation end products（AGEs）are elevated in the circulation of diabetic patients,and circulating AGEs preferentially attack endothelial cells which are the first protective barrier of blood vessel walls.AGEs induce endothelial cell apoptosis to produce extracellular vesicles that have multiple roles in transmitting information and regulating cell function.Nevertheless,it is unclear whether small extracellular vesicles（sEVs）produced by AGEs-treated endothelial cells are involved in regulating diabetic medial arterial calcification.This study aims to investigate the role and mechanism of extracellular vesicles（A-EC/sEVs）produced by AGEs-treated human umbilical vein endothelial cells（HUVECs）in diabetic MAC.Methods:HUVECs were incubated with BSA or AGEs-BSA,and sEVs were collected from the conditioned media,termed A-EC/sEVs and EC/sEVs,respectively.sEVs were identified by detecting their shape,concentration,size,and surface markers using transmission electron microscopy,nanoparticle size analysis,and western blotting,respectively.We observed the internalization of PKH26-labeled sEVs by vascular smooth muscle cells（VSMCs）using confocal laser microscopy,and determined the effect of sEVs on cell viability using CCK8 assay.In the cell model ofβ-glycerophosphate-induced VSMCs calcification,we used A-EC/sEVs and EC/sEVs co-incubation with VSMCs and analyzed the calcification indicators at different time points.Specifically,the expression of osteogenic genes（Runx2 and BMP2）was detected by western blotting 2 days later,alkaline phosphatase（ALP）staining and ALP activity were detected 7 days later,and alizarin red s staining and calcium deposition were detected 21 days later.Furthermore,a co-culture system of HUVECs and VSMCs was constructed to reconfirm that the phenotypes were dependent on sEVs by using the phospholipase inhibitor GW4869.The expression profiles of relevant miRNAs in the two types of sEVs were detected by q RT-PCR,and miRNA-126 was found to be highly enriched in A-EC/sEVs by differential expression analysis of genes.The targeting relationship between miRNA-126 and BMPR1B was verified by bioinformatics and luciferase reporter assays.On the one hand,miRNA-126 mimics were directly transfected into target VSMCs to observe whether the overexpression of miRNA-126 could simulate the anti-calcification effect of A-EC/sEVs and affect the phosphorylation and nuclear translocation of smad1/5/9 that is downstream of BMPR1B.To analyze whether BMPR1B plays a central role in miRNA-126-mediated anti-calcification,we constructed a cell model of BMPR1B silencing and observed whether BMPR1B silencing could reverse the anti-calcification effect of miRNA-126 overexpression and restore its downstream smad1/5/9 signals.On the other hand,we achieved miRNA-126 silencing in sEVs(A-EC^126inhibitor/sEVs)by transfecting miRNA-126 inhibitors in parent cell HUVEC treated with AGEs,to verify whether silencing miRNA-126 could reverse A-EC/sEVs-mediated anti-calcification effect and affect smad1/5/9 signals.In animal experiments,we used streptozotocin and vitamin D to establish a mouse model of diabetic vascular calcification（DVC）.We injected DIR-labeled sEVs into the mice through the tail vein and detected the distribution of sEVs in various tissues by in vivo imaging.We used immunofluorescence staining to observe whether sEVs can reach aortic tissue smoothly.Then,we administered EC/sEVs,A-EC/sEVs,and A-EC^126inhibitor/sEVs systematically through the tail vein and evaluated calcification by detecting ALP activity,expression of key osteogenesis genes,alizarin red staining,and von Kossa staining in thoracic aortic tissue.Results:The sEVs were spherical or ellipsoidal with a diameter of about 30-200 nm and expressed surface markers of extracellular vesicles,including CD9,CD81,and TSG101.The sEVs could be internalized by VSMCs and treatment with A-EC/sEVs and EC/sEVs did not affect cell activity.A-EC/sEVs treatment reduced the levels of Runx2 and BMP2,ALP activity and calcium deposition in VSMCs compared with the EC/sEVs group,but GW4869 reversed these effects.Interestingly,miR-126 was significantly upregulated in A-EC/sEVs,and BMPR1B was confirmed as a specific target gene of miR-126.As expected,overexpression of miR-126 downregulated the expression of BMPR1B,Runx2,and BMP2,ALP activity,calcium deposition,and the phosphorylation and nuclear translocation of smad1/5/9 in VSMCs,while BMPR1B silencing reversed these effects.Consistently,A-EC^126inhibitor/sEVs could reverse the anti-calcification effect of A-EC/sEVs and restore the phosphorylation and nuclear translocation of smad1/5/9.Consistent with in vitro results,DIR-labeled sEVs successfully reached the aortic tissue,and A-EC/sEVs administration reduced the expression of BMPR1B,p-smad1/5/9,Runx2,and BMP2,the activity of ALP,along with calcium deposition in the aorta,thereby alleviating medial arterial calcification in DVC mice,while A-EC^{126 inhibitor}/sEVs counteracted these effects.Conclusion:This study for the first time validates that sEVs derived from AGEs-treated HUVECs inhibit medial arterial calcification in diabetic mice by the miR-126-BMPR1B-smad1/5/9 axis,deepening the understanding of the pathological mechanism in MAC of diabetes and providing a potential target for the prevention and treatment of diabetic arterial calcification.