title: Protein kinase Cδ and c-Abl kinase are required for transforming growth factor β induction of endothelial-mesenchymal transition in vitro.
abstract: OBJECTIVE
  The origin of the mesenchymal cells responsible for the intimal fibrosis in systemic sclerosis (SSc) has not been fully identified. The present study was undertaken to investigate whether subendothelial mesenchymal cells may emerge through transdifferentiation of endothelial cells (ECs) into myofibroblasts via endothelial-mesenchymal transition (EndoMT) in vitro and to explore the signaling pathways involved in this process.
  
  
  METHODS
  Primary mouse pulmonary ECs isolated by immunomagnetic methods with sequential anti-CD34 and anti-CD102 antibody selection were cultured in monolayers. Cell morphology and diacetylated low-density lipoprotein uptake assays confirmed their EC characteristics. The induction of EndoMT was assessed by determination of α-smooth muscle actin (α-SMA), type I collagen, and VE-cadherin expression, and the expression of the transcriptional repressor Snail-1 was analyzed. The signaling pathways involved were examined using small-molecule kinase inhibitors and RNA interference.
  
  
  RESULTS
  Transforming growth factor β1 (TGFβ1) induced α-SMA and type I collagen expression and inhibited VE-cadherin. These effects were mediated by a marked increase in Snail-1 expression and were abolished by treatment with either the c-Abl tyrosine kinase inhibitor imatinib mesylate or the protein kinase Cδ (PKCδ) inhibitor rottlerin. The inhibitory effects of imatinib mesylate and rottlerin were mediated by inhibition of phosphorylation of glycogen synthase kinase 3β at residue Ser(9). These observations were confirmed in experiments using small interfering RNA specific for c-Abl and PKCδ.
  
  
  CONCLUSIONS
  These results indicate that c-Abl and PKCδ are crucial for TGFβ-induced EndoMT and that imatinib mesylate and rottlerin or similar kinase inhibitor molecules may be effective therapeutic agents for SSc and other fibroproliferative vasculopathies in which EndoMT plays a pathogenetic role.
authors.affiliation: Thomas Jefferson University, Philadelphia, Pennsylvania, USA.
authors.forename: Zhaodong
authors.initials: Z
authors.lastname: Li
authors.forename: Sergio A
authors.initials: SA
authors.lastname: Jimenez
mesh_terms: D000818:Animals; D054338:Cell Transdifferentiation / Q000187:drug effects* / Q000502:physiology; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism; D051379:Mice; D058628:Myofibroblasts / Q000187:drug effects / Q000378:metabolism; D051745:Protein Kinase C-delta / Q000378:metabolism*; D016315:Proto-Oncogene Proteins c-abl / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
chemical_list: D016212:Transforming Growth Factor beta; D016315:Proto-Oncogene Proteins c-abl; D051745:Protein Kinase C-delta