C/EBPα-Mediated Transcriptional Activation of PIK3C2A Regulates Autophagy, Matrix Metalloproteinase Expression, and Phenotypic of Vascular Smooth Muscle Cells in Aortic Dissection.

PURPOSE: To investigate the function of C/EBPα in the development of aortic dissection (AD) and the underlying mechanism.

METHODS

Aortic vascular smooth muscle cells (VSMCs) were isolated, cultured, and identified from AD rats. Then, C/EBPα and PIK3C2A were knockdown or overexpressed by siRNA or plasmid transfection, respectively. Rapamycin or 3-MA was utilized to stimulate and restrain autophagy of VSMCs, respectively. Western blot was used to evaluate the expression levels of C/EBPα, PIK3C2A, LC3, Beclin-1, p62, MMP-2, MMP-9, α-SMA, SM-MHC, and OPN. The pathological status of aortic ring was evaluated by stretch stress, and ChIP assay was used to analyze the binding between C/EBPα and PIK3C2A. C/EBPα shRNA was injected into tail vein to observe the effect of C/EBPα knockdown in vivo on phenotype, autophagy of aortic vascular tissue by immunohistochemical staining and Western blot.

RESULTS

The protein levels of C/EBPα, PIK3C2A, MMP-2, MMP-9, and LC3 in the aorta of AD rats were all upregulated significantly. C/EBPα and rapamycin promoted notable upregulation of the synthesized proteins (OPN), PIK3C2A, matrix metalloproteinases, LC3, and Beclin-1 in VSMCs, while suppressed contractile proteins (α-SMA and SM-MHC) and p62. The opposite results were observed in the C/EBPα-knockdown VSMCs, PIK3C2A-knockdown VSMCs, or VSMCs treated with 3-MA. C/EBPα, PIK3C2A, and LC3 were dramatically upregulated by the stimulation of 3 g and 5 g stretch stress. The downregulated contractile proteins, upregulated synthetic proteins, activated autophagy, and aggravated pathological state in 5 g stretch stress-treated aortic rings were significantly reversed by the knockdown of C/EBPα. ChIP results indicated that there was a binding site for C/EBPα in the promoter of PIK3C2A. C/EBPα also downregulated α-SMA level and upregulated OPN levels in AD rats in vivo.

CONCLUSION

Our data indicated that during the development of AD, C/EBPα regulated the transition of VSMC phenotype and extracellular matrix remodeling by activating autophagy through regulating the transcriptional activity of PIK3C2A promoter.