Effects Of Exosomes Derived From Mesenchymal Stem Cells On AGEs-BSA Induced Vascular Smooth Muscle Cells Calcification

BackgroundVascular calcification(VC),which is characterized by the inappropriate deposition of calcium phosphate crystals in the vasculature,is associated with an increased risk of cardiovascular events and death.The pathophysiology of VC is a complex biological process,some of which involving the phenotypic transition of vascular smooth muscle cells(VSMCs)into osteoblast-like cells.Advanced gly cation end products(AGEs),which are generated by a non-enzymatic reaction between proteins and sugar residues,have been reported to promote osteogenic differentiation and calcification of VSMCs via oxidative stress and inflammation.Previous studies have demonstrated that VC is highly prevalent in diabetes population and usually accompanied by osteoporosis.Since VC and osteoporosis share some common pathophysiological mechanisms,illuminating the regulation mechanism of AGEs on bone metabolism may be helpful for the prevention of diabetic vascular calcificationMesenchymal stem cells(MSCs)and their exosomes have made significant progress in the field of cardiovascular diseases therapy.Exosomes is considered as a mechanism of intercellular signaling.Researches observed that the content of exosomes changed based on the type of cellular stress.Studies have shown that AGEs can inhibit MSC osteogenesis,which suggested that AGEs exposure may cause the release of exosomes containing osteogenic inhibitors from MSCsmiR-146a is recognized as an important regulator of inflammation and oxidative stress.Several studies have reported that the decreased expression of miR-146a is tightly associated with the diabetic complications.In mesenchymal stem cells(MSCs),miR-146a is reported to inhibit the osteogenic differentiation of MSCs,whereas pathological upregulation of miR-146a results in the progression of osteoporosis AGEs-BSA are regarded as capable of expression of miR-146a in some cell types and may be involved in the inhibition of MSC osteogenic differentiation.Therefore,dysregulation of miR-146a in VSMC and MSC induced by AGEs-BSA may be an important factor in diabetic VC and abnormal bone metabolism.TXNIP,an oxidative stress inducer,is predicted as the potential targeted gene of miR-146a on the miRDB databaseBased on the above descrnption,The current study proposes to demonstrate the following hypothesis:1.miR-146a is an inhibitor of osteogenic differentiation in MSC:2.AGEs-BSA affects the osteoblastic differentiation in both VSMCs and MSCs via regulating the expression of miR-146a;3.AGEs-BSA up-regulates the expression of miR-146a in the MSC exosomes.which can be shuttled into VSMC and inhibit VSMC calcification;4.miR-146a inhibits the phenotypic transition of VSMCs through the TXNIPIROS pathwayMethods1.Primary VSMC and MSC were isolated from the aorta and bone marrow of SD rats respectively.Exosomes were obtained from MSC culture supernatants by ultracentrnfugation.MSC-derived exosomes were characterized by transmission electron microscopy and subjected to western blot analyses of CD9,Alix,and TsglOl Nanosight analysis was performed to measure the particle size2.Cells were treated with different concentration of AGEs-BSA for 48 h and the cell viability was assessed using CCK-8 assay kit.Western blot and RT-PCR were used to evaluate the levels of protein and mRNA of Runx2,BMP-2,TXNIP,and miRNA-146a.ROS probe was used to measure the ROS production by flow cy tometty.The M DA content and SOD activity in VSM C were measured by the MDA and SOD detection kit3.Condition medium of MSCs cultured with and without 200 μ/mL AGEs-BSA for 48 h are termed MSC/CM and A-MSC/CM,respectively;exosomes isolated from MSC/CM or A-MSC/CM are termed MSC/exo and A-MSC/exo respectively exome-free supernatants are termed MSC/CM(-exo)and A-MSC/CM(-exo).RT-PCR was used to measure the mRNA levels of miRNA-146a of CM and exosomes above VSMCs were treated with:1.200 μg/mL BSA;2.200 μg/mL AGEs-BSA;3 AGEs-BSA+MSC/CM;4.AGEs-BSA+A-MSC/CM;5.AGEs-BSA+MSC/exo;6 AGEs-BSA+A-M SC/exo;7.AGEs-BSA+M SC/CM(-exo);8 AGEs-BSA+A-MSC/CM(-exo);Western blot and RT-PCR were used to measure the protein and mRNA levels of Runx2,BMP-2,TXNIP,and miRNA-146a expression respectively4.MSCs were transfected with pre-miR-146a(miR-146a/MSCs)or a plasmid harboring an NTC sequence(NTC/MSCs)VSMCs,with or without addition of AGEs-BSA,were cocultured for 48 h in transwells with miR-146a/MSCs or NTC/MSCs,and the expression level of miR-146a was confirmed by qPCR.Next miR-146a/MSCs were treated with the exosome-release inhibitor GW4869 for 12 h before coculture.Western blot and RT-PCR were used to measure the protein and mRNA levels of Runx2,BMP-2,TXNIP,and miRNA-146a expression5.VSMCs were treated with 1.200 μh/mL BSA;2.200 μg/mL AGEs-BSA;3200 μg/mL AGEs-BSA+10mmol/L β-GP;4.200 μg/mL AGEs-BSA+10mmol/L β-GP+ 146a/exo(5、10、20 μg/mL).Western blot and RT-PCR was used to measure the protein and mRNA levels of Runx2,BMP-2,TXNIP and miRNA-146a expressions.ROS probes were used to measure the ROS production MDA and SOD detection kit was used to measure the M DA content and SOD activity in VSMC.The extent of matrix mineralization in cultured VSMCs was assayed by Alizarin Red S staining.6.VSMC was transfected with TXNIP-overexpressed plasmid,then treated with 1.200 μg/mL BSA;2.200 μg/mL AGEs-BSA;3.AGEs-BSA+β-GP+146a/exo.ROS probes wrer used to measure the ROS production MDA and SOD detection kit was used to measure the MDAcontent and SOD activity in VSMC7.Luciferase reporter assays were performed to prove that the TXNIP 3’UTR sequence containing the seed sequence for miR-146a recognitionResults1.More than 95%of VSMCs showed positive staining for both a-SMA and SM22a.More than 95%of MSC were positive for CD73,CD90,and were negative for CD45,CD34.Size distribution between 50-200nm.Western blot results confirmed the presence of exosome markers CD9,Alix,and Tsg101.2.There was no significant change of cell viability when treated with 0-200μg/mL AGEs-BSA.0-200 μg/mL AGEs-BSA unregulated the mRNA and protein expression of Runx2,Bmp-2,TXNIP,while decreased the expression of miR-146a what’s more,the ROS production and MDA content were increased while the SOD activity was suppressed by AGEs-BSA3.The miR-146a level of A-MSC/CM and A-MSC/exo were higher than any other groups.The mRNA and protein expression of Runx2,BMP-2,and TXNIP were increased in AGEs-BSA group compared to the normal controls,whereas the expression of miR-146a showed the opposite trend.The mRNA and protein expression of Runx2,BMP-2,and TXNIP were decreased,whereas miR-146a was increased after treated with A-M SC/CM and A-M SC/exo4.The increased expression of miR-146a,as well as the decreased expression of Runx2,BMP-2,and TXNIP,were observed in VSMC following 146a/MSC coculture whereas this effect was suppressed by GW48695.The expression of Runx2,BM P-2,and TXNIP,ROS production,M DA content ALP activity,and alizarin red color intensity were decreased in β-GP+AGEs-BSA group by addition with 146a/exo,accompanied with the unregulated SOD activity6.The expression of Runx2,BMP-2,TXNIP are increased in while of miR-146a is less in TXNIP/VSMC than in NTCANSMC;Overexpression of TXNIP by plasmid transfection increased the levels of ROS and MDA in VSMCs,whereas SOD activity was decreased.146a/exo administration had no effect on Runx2,BMP-2 expression and ROS level,MDAcontent and SOD activity in TXNIP/VSMC7.The fluorescence intensity in VSMC transfected with while type TXNIP-3’UTR/plasmid and miR-146a mimic was decreased than others groupsConclusionsIn the present study,the results indicated that miR-146a is an important osteogenesis inhibitor.AGEs-BSA upregulated the miR-146a expression in the condition medium and exosomes of MSC.The exosomal miR-146a can shuttle into VSMC and inhibit TXNIPIROS signaling pathway in receptor cells,resulting in decreased VSMC calcification.MSC exosomes can be used as effective vectors for miRNA intercellular transportation.Therefore,this study provides critical information about possible therapeutic applications in vascular calcification.