title: Zinc finger protein 191 deficiency attenuates vascular smooth muscle cell proliferation, migration, and intimal hyperplasia after endovascular arterial injury.
abstract: OBJECTIVE
  Restenosis engenders surgical vascular intervention failure. Zinc finger protein 191 (ZFP191) is a novel member of the SCAN domain family of Krüppel-like zinc finger transcription factors. Previous work reveals that ZFP191 is a pleiotropic factor that plays important roles in hematopoiesis, brain development, and tumor growth. Here, we sought to determine whether intimal hyperplasia was affected by the activity of ZFP191 and to investigate the molecular mechanisms that may underpin the process.
  
  
  METHODS
  Intimal hyperplasia was induced by guidewire injury in mouse femoral arteries. The arteries were harvested for morphometric assessment and determination of ZFP191 expression. Next, ZFP191 knockdown in cultured mouse aortic vascular smooth muscle cells (VSMCs) was achieved by lentiviral transduction of short-hairpin RNA. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay, [(3)H]thymidine incorporation assay, scratch assay, and transwell migration assay were used to evaluate the effects of ZFP191 knockdown on VSMC growth and migration. In addition, β-catenin, c-myc, cyclin D1, matrix metalloproteinase (MMP) 9, MMP2, and MMP7 were measured by Western blotting in the absence of ZFP191 in vitro and in vivo. Zymography was used to evaluate MMP activity in cell culture-conditioned media. Lastly, artery injury was performed in wild-type (WT) and heterozygous ZFP191 knockout (KO) mice, and morphometric analysis of the arteries was determined.
  
  
  RESULTS
  Guidewire injury was associated with development of intimal hyperplasia, and ZFP191 expression was enhanced by 51% in the injured arteries. Cultured primary VSMCs transfected with lentiviral shZFP191 displayed reduced proliferation and migration compared with controls. Mechanically, ZFP191 knockdown potently decreased the level of β-catenin and its downstream targets c-myc and cyclin D1. ZFP191 knockdown downregulated the expression of MMP9, MMP2, and MMP7, and zymography confirmed that ZFP191 knockdown reduced the activity of MMPs. Consistent with the in vitro data, elevated expression of β-catenin, c-myc, cyclin D1, MMP9, MMP2, and MMP7 accompanied upregulation of ZFP191 after injury in the femoral arteries of mice, and these levels were downregulated in ZFP191 KO vessels. Finally, intimal hyperplasia was greatly blocked in heterozygous ZFP191 KO mice compared with WT mice (intima/media ratio, 0.124 vs 0.412; P < .05).
  
  
  CONCLUSIONS
  ZFP191 played an essential role in aggressive proliferation and migration of VSMCs, which in turn facilitated intimal hyperplasia. Our findings offer the first genetic evidence of ZFP191 as a potential therapeutic target to prevent restenosis.
authors.affiliation: Department of Vascular Surgery, Renji Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Lv
authors.affiliation: Department of Vascular Surgery, Renji Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China.
authors.forename: Jiwei
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Department of Vascular Surgery, Renji Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China.
authors.forename: Peng
authors.initials: P
authors.lastname: Wang
authors.affiliation: Department of Vascular Surgery, Renji Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China.
authors.forename: Qiurong
authors.initials: Q
authors.lastname: Meng
authors.affiliation: Department of Vascular Surgery, Renji Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China. Electronic address: wwxxyyzz@263.net.
authors.forename: Wei
authors.initials: W
authors.lastname: Liang
authors.affiliation: Department of Vascular Surgery, Renji Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China.
authors.forename: Lan
authors.initials: L
authors.lastname: Zhang
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002465:Cell Movement; D049109:Cell Proliferation; D002478:Cells, Cultured; D004195:Disease Models, Animal; D005263:Femoral Artery / Q000293:injuries / Q000378:metabolism / Q000473:pathology; D006965:Hyperplasia; D020782:Matrix Metalloproteinases / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009131:Muscle, Smooth, Vascular / Q000293:injuries* / Q000378:metabolism* / Q000473:pathology; D058426:Neointima; D034622:RNA Interference; D013997:Time Factors; D014161:Transduction, Genetic; D014162:Transfection; D057772:Vascular System Injuries / Q000235:genetics / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control*; D060449:Wnt Signaling Pathway; D051176:beta Catenin / Q000378:metabolism
chemical_list: C495267:CTNNB1 protein, mouse; D002352:Carrier Proteins; C474810:Zfp24 protein, mouse; D051176:beta Catenin; D020782:Matrix Metalloproteinases