title: Activated phosphatidylinositol 3-kinase/Akt inhibits the transition of endothelial progenitor cells to mesenchymal cells by regulating the forkhead box subgroup O-3a signaling.
abstract: OBJECTIVE
  Endothelial progenitor cells (EPCs) differentiate into mature endothelial cells and may thus be candidates for ischemic disease therapy; however, the transition of EPCs to mesenchymal cells is not fully understood. We explored the role of phosphatidylinositol 3-kinase (PI3K)/Akt signaling in endothelial-to-mesenchymal transition (EndMT) induced by transforming growth factor beta 1 (TGF-β1).
  
  
  METHODS
  Rat bone marrow-derived EPCs were isolated by using Ficoll-Isopaque Plus density-gradient centrifugation. EndMT was induced by TGF-β1 (5 ng/mL). PI3K/Akt signaling was activated by IGF-1 or Lenti-PIK3R2 shRNA. Additionally, FoxO3a expression was suppressed by a lentiviral vector (Lenti-FoxO3a shRNA). Smad3 and FoxO3a co-localization was detected by confocal immunofluorescence microscopy. The expressions of molecules involved in EndMT were exmined by using Western-blot analysis.
  
  
  RESULTS
  EndMT of EPCs was fully developed after TGF-β1 treatment (5 ng/mL) for 7 days. PIK3R2 expression in EPCs was driven by TGF-β1. Lenti-PIK3R2 shRNA blocked alpha-smooth muscle actin (α-SMA) expression in EPCs treated with TGF-β1, drove PI3K/Akt activation, and increased expression of phosphorylated FoxO3a instead of phosphorylated Smad3. The effect of Lenti-PIK3R2 shRNA was reduced by LY294002, a specific inhibitor of PI3K. IGF-1 attenuated α-SMA protein expression in EPCs treated with TGF-β1. Similar to Lenti-PIK3R2 shRNA, IGF-1 also inhibited and elevated the phosphorylation of Smad3 and FoxO3a, respectively. IGF-1 disrupted the co-localization of these proteins in EPCs treated with TGF-β1. Lenti-FoxO3a shRNA transfection of EPCs suppressed expression of FoxO3a as well as that of the mesenchymal markers SM22α and α-SMA.
  
  
  CONCLUSIONS
  Activation of PI3K/Akt signaling by Lenti-PIK3R2 shRNA or by exogenous IGF-1 inhibits EndMT in EPCs via negative regulation of FoxO3a-dependent signaling.
authors.affiliation: Department of Cardiology, No. 3 People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
authors.forename: Zongqi
authors.initials: Z
authors.lastname: Zhang
authors.forename: Tiantian
authors.initials: T
authors.lastname: Zhang
authors.forename: Yaoyao
authors.initials: Y
authors.lastname: Zhou
authors.forename: Xiaowei
authors.initials: X
authors.lastname: Wei
authors.forename: Jianbing
authors.initials: J
authors.lastname: Zhu
authors.forename: Junfeng
authors.initials: J
authors.lastname: Zhang
authors.forename: Changqian
authors.initials: C
authors.lastname: Wang
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D066026:Endothelial Progenitor Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D008297:Male; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism*; D008840:Microfilament Proteins / Q000378:metabolism; D009025:Morpholines / Q000494:pharmacology; D009124:Muscle Proteins / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation / Q000187:drug effects; D021122:Protein Subunits / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D034622:RNA Interference; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000187:drug effects; D051900:Smad3 Protein / Q000378:metabolism; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology
chemical_list: D000199:Actins; D002867:Chromones; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors; D007334:Insulin-Like Growth Factor I; D008840:Microfilament Proteins; D009025:Morpholines; D009124:Muscle Proteins; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D021122:Protein Subunits; D051057:Proto-Oncogene Proteins c-akt; D051900:Smad3 Protein; D053773:Transforming Growth Factor beta1; C499515:FOXO3 protein, rat; C498862:Acta2 protein, rat; C051615:transgelin; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one