Inhibition Of Autophagy Protects Human Vascular Smooth Muscle Cells From Age-induced Apoptosis And Calcification

Objective:Diabetes mellitus?Diabetes mellitus,DM?is a worldwide metabolic disease,the pathogenesis and mechanisms of DM are unclear,caused a heavy burden to the society and the patients.Vascular calcification?VC?is the key pathological change of cardiovascular diseases caused by type 2 diabetes mellitus?T2DM?,and is also the main cause of its death and amputation.Advanced glycation end-products?AGEs?play an important role in the process of T2DM caused vascular calcification.Therefore,in this study,human aortic smooth muscle cells?HASMCs?were cultured with AGEs to induce its calcification in vitro,and explore whether osteoblastic transformation,autophagy,apoptosis and activation of relevant intracellular signals occurred in HASMCs during this process.Furthermore,using Atg7 knockout HASMCs,autophagy inhibitor Wortmannin?WM?cultured with AGEs,to confirm the role of autophagy in the pathological process of AGEs-induced HASMCs calcification and apoptosis.Methods:1.3-8 generations cultured HASMCs were selected as the study object.The cells were co-cultured with AGEs?0mg/L,25 mg/L,50 mg/L,100mg/L?in a special medium.The expression of osteopontin?OPN?in different concentrations of AGEs?0mg/L,25 mg/L,50 mg/L,100mg/L?were observed by Western blot and Immunofluorescence staining.In addition,Western blot was used to detect the expression of AGEs receptor?RAGE?,?-catenin,runt-related transcription factor?Runx2?and osteoblast-related proteins?OPG?,matrix metalloproteinase 7?MMP7?and bone morphogenetic protein 2?BMP2?in HASMCs after AGEs treatment at different concentrations for 24 h.2.To observe the effect of AGEs on the autophagy of HASMCs,HASMCs were co-cultured with different concentrations of AGEs for 24 h,and Western blot was used to detect the expression of autophagy-related proteins such as LC3-II,beclin1,p62 and p-m TOR.In addition,Immunofluorescence was used to observe the expression of LC3-point structures in HASMCs treated with AGEs.3:To observe whether AGEs induced apoptosis in HASMCs cells,HASMCs were co-cultured with AGEs at different concentrations for 24 h,and the expressions of apoptotic proteins caspase-3,caspase-9,Bax and Bak were detected by Western blot.Furthermore,Annexin v-PI flow cytometry was used to further clarify that AGEs induced the apoptosis of HASMCs cells.4:On the basis of HASMCs pretreatment with autophagy inhibitor wortmannin?WM??1M?for 1 h,and then co-culture with 100mg/L AGEs for 24 h,Western blot was used to detect the expression of autophagy marker proteins LC3-II,,Beclin1,P62,p-m TOR and apoptosis-related proteins,such as Bak,Bax,caspase-3,caspase-9 and?-SMA.Immunofluorescence staining was used to detect the expression and localization of F-actin and LC3-point structure in cells.5.After WM?1 M?pretreatment of HASMCs for 1 h and co-culture with 100mg/L AGE for 24 h,Von Kossa staining and FLIPR calcium 6 staining were used to observe the calcium levels inside and outside of HASMCs cells.6:Constructing Atg7 knocked out HASMCs,then the genotype and wild-type HASMCs were treated with 100mg/L AGE for 24 h,respectively.Western blot was used to detected the expression levels of autophagy-related proteins:of LC3-I,LC3-II,Atg7,calcification-related proteins:?-catenin,OPN,BMP2,Runx2,and apoptosis-related proteins:caspase 9,caspase 3,Bax,and Bak.Results:1.Von Kossa and Immunofluorescence staining showed that AGEs increased Ca2+deposition and OPN expression in HASMCs in a dose-dependent way.In addition,the expression levels of RAGE,?-catenin,OPG,MMP7,and BMP2 in HASMCs after treatment with 25 mg/L,50 mg/L,and 100mg/L AGEs were significantly higher than the blank control?0mg/L AGEs??P<0.05?.2.Immunofluorescence staining suggested that AGEs increased the expression of LC3-point structures in HASMCs in a dose-dependent manner.In addition,the expression levels of autophagy-related proteins LC3-II and beclin1 in HASMCs treated with 25 mg/L,50 mg/L and100mg/L AGEs were significantly increased compared with the blank control?0mg/L AGEs??P<0.05?,while the expressions of p62 and p-m TOR in HASMCs treated with 25 mg/L,50 mg/L and 100mg/L AGEs were significantly lower than those in the blank control?P<0.05?.3:the expression levels of apoptosis-related proteins caspase-3,caspase-9,Bax and Bak in HASMCs after treatment with 25 mg/L,50 mg/L and 100mg/L AGEs were obviously increased compared with the blank control HASMCs?0mg/L AGEs??P<0.05?.In addition,flow cytometry apoptosis staining exhibited that AGEs increased the apoptosis rate of HASMCs in a dose-dependent manner,and HASMCs apoptosis rates significantly increased after 25 mg/L,50 mg/L,and100mg/L AGEs treatments in comparison to control group?P<0.05?.4.WM pretreatment significantly inhibited AGEs-induced autophagy and apoptosis.Before co-culture AGEs with HASMCs for 24 h,compared with HASMCs without WM,WM pretreatment significantly reduced the expression of LC3-II and Beclin1 and increased the expression of p62 and p-m TOR?P<0.05?.Similarly,Immunofluorescence staining suggested that WM significantly inhibited AGEs-induced up-regulation of LC3-II expression?P<0.05?.In addition,AGEs-induced up-regulation of apoptosis-related proteins caspase-3,caspase-9,Bax and Bak were also significantly inhibited by WM?P<0.05?.5:WM pretreatment significantly inhibited AGEs-induced differentiation of HASMCs and calcification.Before co-cultured with AGEs for 24 h,HASMCs pretreated with WM significantly decreased the expression of?-SMA??-catenin?OPN?BMP2 and Runx2compared with HASMCs without WM pretreatment?P<0.05?.In addition,Immunofluorescence,Von Kossa and FLIPR calcium 6 staining suggested that WM significantly inhibited AGEs-induced Ca²⁺deposition in HASMCs and down-regulation of F-actin?P<0.05?.6:after 100mg/L AGEs treatment,autophagy-related proteins LC3-I,LC3-II,Atg7,calcification-related proteins including?-catenin,OPN,BMP2,Runx2,and apoptosis-related proteins caspase 9,caspase 3,Bax,and Bak were significantly reduced in Atg7 knockout HASMCs compared with wild-type HASMCs?P<0.05?,indicating that inhibition of autophagy can protect HASMCs from AGEs-induced apoptosis and calcification.Conclusion:1.AGEs-induced calcification of vascular smooth muscle cells,the relevant mechanisms involved include AGEs-induced autophagy,apoptosis and differentiation into osteoblast.2.In the process of autophagy,apoptosis and osteoblastic transformation induced by AGEs,the initiating factor may be AGEs-induced autophagy,which further leads to apoptosis and calcification of vascular smooth muscle cells.3.Autophagy inhibitor or autophagy-related gene Atg7 knockout can inhibit AGEs-induced autophagy,calcification and apoptosis in HASMCs,further confirming that AGEs-induced autophagy may caused the calcification and apoptosis in HASMCs,which also indicates that inhibition of autophagy can prevent AGEs-induced calcification and apoptosis in vascular smooth muscle cells.