UNASSIGNED:探讨C/EBPα在主动脉夹层(AD)发展中的作用及其机制。
未经证实:分离主动脉血管平滑肌细胞(VSMC),培养,并从AD大鼠中鉴定。然后,通过siRNA或质粒转染敲低或过表达C/EBPα和PIK3C2A,分别。雷帕霉素或3-MA用于刺激和抑制VSMC的自噬,分别。Westernblot用于评估C/EBPα的表达水平,PIK3C2A,LC3,Beclin-1,p62,MMP-2,MMP-9,α-SMA,SM-MHC,OPN。通过拉伸应力评估主动脉环的病理状态,和ChIP分析用于分析C/EBPα与PIK3C2A之间的结合。尾静脉注射C/EBPαshRNA,观察体内C/EBPα敲低对表型的影响,通过免疫组织化学染色和Westernblot检测主动脉血管组织的自噬。
未经证实:C/EBPα的蛋白质水平,PIK3C2A,AD大鼠主动脉中MMP-2、MMP-9和LC3均显著上调。C/EBPα和雷帕霉素促进合成蛋白(OPN)的显着上调,PIK3C2A,基质金属蛋白酶,VSMC中的LC3和Beclin-1,同时抑制收缩蛋白(α-SMA和SM-MHC)和p62。在C/EBPα敲低的VSMC中观察到相反的结果,PIK3C2A敲低VSMC,或用3-MA处理的VSMC。C/EBPα,PIK3C2A,3g和5g拉伸应力的刺激显着上调了LC3。下调的收缩蛋白,上调合成蛋白质,激活自噬,通过C/EBPα的敲低,可以明显逆转5g拉伸应激处理的主动脉环的加重病理状态。ChIP结果表明在PIK3C2A的启动子中存在C/EBPα的结合位点。在AD大鼠体内,C/EBPα也下调α-SMA水平和上调OPN水平。
UNASSIGNED:我们的数据表明,在AD的发展过程中,C/EBPα通过调控PIK3C2A启动子的转录活性,激活自噬,从而调控VSMC表型转变和细胞外基质重塑。
UNASSIGNED: To investigate the function of C/EBPα in the development of aortic dissection (AD) and the underlying mechanism.
UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.