title: Fasudil inhibits platelet-derived growth factor-induced human pulmonary artery smooth muscle cell proliferation by up-regulation of p27kip¹ via the ERK signal pathway.
abstract: BACKGROUND
  RhoA/Rho kinase (ROCK) pathway is involved in pulmonary arterial hypertension (PAH) and pulmonary artery smooth muscle cell (PASMC) proliferation. Inhibition of ROCK has been proposed as a treatment for PAH. But the mechanism of RhoA/ROCK pathway and its downstream signaling in proliferation of human PASMCs is unclear. We investigated the effect of fasudil, a selective ROCK inhibitor, on platelet-derived growth factor (PDGF) induced human PASMC proliferation, and the possible association between RhoA/ROCK and extracellular signal-regulated kinase (ERK), p27(Kip1).
  
  
  METHODS
  Human PASMCs were cultured with the stimulation of 10 ng/ml PDGF, and different concentrations of fasudil were added before the addition of mitogen. Cell viability and cell cycle were determined with MTT and flow cytometry respectively. ROCK activity, ERK activity and protein expression of proliferating cell nuclear angigen (PCNA) and p27(Kip1) were measured by immunoblotting.
  
  
  RESULTS
  By MTT assay, PDGF significantly increased the OD value that represented human PASMC proliferation, and pretreatment with fasudil significantly reversed this effect in a dose-dependent manner. After PDGF stimulation, the percentage of cells in S phase increased dramatically from 15.6% to 24.3%, while the percentage in G(0)/G(1) phase was reduced from 80.6% to 59%. And pretreatment with fasudil reversed the cell cycle effect of PDGF significantly in a dose-dependent manner. PDGF markedly induced ROCK activity and ERK activity with a peak at 15 minutes, which were significantly inhibited by fasudil. In addition, fasudil significantly inhibited PDGF-induced PCNA expression and fasudil also upregulated p27(Kip1) expression in human PASMCs, which decreased after PDGF stimulation.
  
  
  CONCLUSIONS
  RhoA/ROCK is vital for PDFG-induced human PASMC proliferation, and fasudil effectively inhibited PDGF-induced human PASMC proliferation by up-regulation of p27(Kip1), which may be associated with inhibition of ERK activity.
authors: Department of Pediatric Cardiac Surgery Center, Fuwai Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100037, China.
authors: Ai-Jun
authors: AJ
authors: Liu
authors: Feng
authors: F
authors: Ling
authors: Dong
authors: D
authors: Wang
authors: Qiang
authors: Q
authors: Wang
authors: Xiao-Dong
authors: XD
authors: Lü
authors: Ying-Long
authors: YL
authors: Liu
mesh_terms: D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives* / Q000494:pharmacology; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000378:metabolism*; D006801:Humans; D020935:MAP Kinase Signaling System / Q000502:physiology*; D009131:Muscle, Smooth, Vascular / Q000166:cytology*; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011651:Pulmonary Artery / Q000166:cytology*; D015854:Up-Regulation
chemical_list: D010982:Platelet-Derived Growth Factor; D047428:Protein Kinase Inhibitors; D050760:Cyclin-Dependent Kinase Inhibitor p27; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; C049347:fasudil