Overexpression of Atg5 Inhibits Osteogenic Differentiation of VICs by Upregulating Autophagy Levels
CHEN Bin, ZHANG Menghao, FAN Mengtian, WU Jinghong, WANG Yue, HUANG Qin, SHI Qiong*
Our study was to investigate the effect of autophagyrelated protein Atg5 on autophagy and osteogenic differentiation of aortic valve interstitial cells, which was order to provide new methods and ideas for the study of the calcified aortic valve disease. Primary valve interstitial cells were isolated from the porcine aortic valve. Cells phenotype identification was used by immunofluorescent staining. The expression of Runx2, OPN, p62 and LC3B-I/II was detected by Western blot to identified the relationship between autophagy and osteogenesis in valvular interstitial cells. The plasmid pAdTrack-ATG5 overexpressing Atg5 was constructed and transfected into VICs. After transfecting with control plasmid or pAdTrack-ATG5, the VICs were cultured with osteogenic medium. The osteogenic differentiation ability of valve interstitial cells was detected by Western blot, alkaline phosphatase staining and alizarin red S staining. For immunofluorescent staining, Alpha -SMA and Vimentin were positive and vWF was negative in VICs. Compared with the CM group, the expression of Runx2, OPN and p62 protein in the OM group was increased but LC3B-II/I decreased. The expression of Atg5 and LC3B-II/I in cells transfeced with pAdTrack- ATG5 was raised but p62 was decreased compared with the negative control. The expression level of Runx2 and OPN in the OM+Atg5 group was lower than that in the OM+NC group. Alkaline phosphatase staining and alizarin red S staining were also showed that the osteogenic differentiation ability in the OM+Atg5 group was weaker than the OM+NC group. Take together, overexpression of Atg5 inhibits osteogenic differentiation of valvular interstitial cells by upregulating autophagic activity.
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