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

BACKGROUND AND AIMS: 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.