title: Everolimus (RAD001) inhibits the proliferation of rat vascular smooth muscle cells by up-regulating the activity of the p27/kip1 gene promoter.
abstract: 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.
  
  
  
  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.
  
  
  
  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.
  
  
  
  RAD001 inhibits PDGF-BB-stimulated proliferation of cultured VSMCs by upregulating p27/kip1 gene promoter activity and increasing p27/kip1 mRNA and protein expression.
authors.affiliation: Department of Cardiology, Third People's Hospital of Chongqing, Chongqing-P. R. China.
authors.forename: Boli
authors.initials: B
authors.lastname: Ran
authors.affiliation: Department of Cardiology, Third People's Hospital of Chongqing, Chongqing-P. R. China.
authors.forename: Minfeng
authors.initials: M
authors.lastname: Li
authors.affiliation: Department of Cardiology, Third People's Hospital of Chongqing, Chongqing-P. R. China.
authors.forename: Yeqing
authors.initials: Y
authors.lastname: Li
authors.affiliation: Department of Cardiology, Third People's Hospital of Chongqing, Chongqing-P. R. China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Lin
authors.affiliation: Department of Cardiology, Third People's Hospital of Chongqing, Chongqing-P. R. China.
authors.forename: Weimin
authors.initials: W
authors.lastname: Liu
authors.affiliation: Department of Cardiology, Third People's Hospital of Chongqing, Chongqing-P. R. China.
authors.forename: Qiulin
authors.initials: Q
authors.lastname: Luo
authors.affiliation: Department of Cardiology, Third People's Hospital of Chongqing, Chongqing-P. R. China.
authors.forename: Yongxin
authors.initials: Y
authors.lastname: Fu
authors.affiliation: Department of Cardiology, Third People's Hospital of Chongqing, Chongqing-P. R. China.
authors.forename: Qianmei
authors.initials: Q
authors.lastname: Tang
authors.affiliation: Department of Cardiology, Third People's Hospital of Chongqing, Chongqing-P. R. China.
authors.forename: Ya
authors.initials: Y
authors.lastname: Yang
authors.affiliation: Department of Cardiology, Third People's Hospital of Chongqing, Chongqing-P. R. China. 12944048@qq.com.
authors.forename: Yunfei
authors.initials: Y
authors.lastname: Pu
mesh_terms: D000818:Animals; D049109:Cell Proliferation / Q000187:drug effects*; D002478:Cells, Cultured; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000187:drug effects / Q000378:metabolism*; D000068338:Everolimus / Q000494:pharmacology*; D009131:Muscle, Smooth, Vascular / Q000187:drug effects* / Q000378:metabolism; D032389:Myocytes, Smooth Muscle / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar
chemical_list: C493962:Cdkn1b protein, rat; D050760:Cyclin-Dependent Kinase Inhibitor p27; D000068338:Everolimus