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The Anatolian Journal of Cardiology

[Anatol J Cardiol]
Anatol J Cardiol. 2016; 16(6): 385-391 | DOI: 10.14744/AnatolJCardiol.2015.6426  

Everolimus (RAD001) inhibits the proliferation of rat vascular smooth muscle cells by up-regulating the activity of the p27/kip1 gene promoter

Boli Ran, Minfeng Li, Yeqing Li, Yang Lin, Weimin Liu, Qiulin Luo, Yongxin Fu, Qianmei Tang, Ya Yang, Yunfei Pu
Department of Cardiology, Third People's Hospital of Chongqing, Chongqing-P. R. China

Objective: We investigated whether the inhibitory effect of the immunosuppressant everolimus (RAD001) on vascular smooth muscle cell (VSMC) proliferation is mediated by p27/kip1 gene promoter activity.
Methods: In this experimental study, cultured rat VSMCs were transiently transfected with a recombinant plasmid (pXp27) containing p27/kip1 gene promoter sequence and a chloramphenicol acetyltransferase (CAT) reporter gene. After stimulation with the mitogen platelet-derived growth factor (PDGF-BB, 10 ng/mL) in the presence or absence of RAD001 (10 nM), the promoter activity, mRNA expression, and protein expression of p27/kip1 were examined by CAT assay, RT–PCR, and immunoblotting, respectively. Cell cycle–related changes were detected by flow cytometry. DNA synthesis was determined using 3H-TdR incorporation.
Results: Compared with the non-stimulation group, PDGF-BB stimulation induced a significant proliferative response in the VSMCs as indicated by decreased p27/kip1 gene promoter activity, decreased p27/kip1 mRNA and protein expression, increased S-phase and G2/M-phase cells, and increased DNA synthesis. RAD001 intervention increased p27/kip1 gene promoter activity 3.5-fold, promoted p27/kip1 mRNA and protein expression, increased G0-phase cells, reduced DNA synthesis, and, overall, inhibited PDGF-BB–stimulated cell proliferation.
Conclusion: RAD001 inhibits PDGF-BB–stimulated proliferation of cultured VSMCs by upregulating p27/kip1 gene promoter activity and increasing p27/kip1 mRNA and protein expression. (Anatol J Cardiol 2016; 16: 385-91)

Keywords: p27/kip1, everolimus, promoter, vascular smooth muscle cell, proliferation


Boli Ran, Minfeng Li, Yeqing Li, Yang Lin, Weimin Liu, Qiulin Luo, Yongxin Fu, Qianmei Tang, Ya Yang, Yunfei Pu. Everolimus (RAD001) inhibits the proliferation of rat vascular smooth muscle cells by up-regulating the activity of the p27/kip1 gene promoter. Anatol J Cardiol. 2016; 16(6): 385-391

Sorumlu Yazar: Yunfei Pu, China


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