Mobilization and differentiation of bone marrow-derived endothelial progenitor cells by GSK-3β inhibition

Abstract

An imbalance between vascular injury and repair gives rise to endothelial dysfunction that causes damage inflicted by traditional cardiovascular risk factors and this situation is mitigated by reparative endothelial progenitor cells (EPCs). Therefore, administration of EPCs has been shown to be an effective strategy in reendothelialization and adult neovascularization with granulocyte colony-stimulating factor (G-CSF). However, the functional role of G-CSF treatment on neointimal formation and vascular function after vascular injury remains unknown or controversial. This study focuses on the mobilization and differentiation of progenitor cells by changing cell population and improving reendothelialization in the damaged blood vessels. A population of progenitor cells in peripheral blood changed after injection of 0.2 mg/kg SB216763 ((3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione), a glycogen synthase kinase-3β (GSK-3β) inhibitor, in vivo. Especially, mobilization of progenitor cells expressing CD31, CD45, Von Willebrand factor (vWF), and Flk1, a receptor for vascular endothelial growth factor (VEGF), was significantly increased in the SB216763-treated group compared to the control group. The cells expressing CD31/c-Kit or Flk-1/c-kit mobilized from bone marrow (BM) in endosteal niche into peripheral blood. To examine the mobilization of progenitor cells, matrix metalloproteinase-9 (MMP-9) activation and nitric oxide release were detected in buffy coats from the peripheral blood. Mobilized progenitor cells expressing integrin-related protein (VE-cadherin co-stained with Tie-2 or αvβ3 co-stained with vWF) for powerful adhesion function were increased in peripheral blood, whereas expression of integrin and endothelial phenotype was decreased in bone marrow. Especially, an increase of CD31 and Tie-2 positive cell population showed greater endothelial character with SB216763 treatment. In in vivo analysis to validate endothelial function, neointimal hyperplasia was diminished; however, endothelial lining and reendothelialization was both dramatically increased in a balloon injury (BI) rat model treated with SB216763. Furthermore, primary cilium related to mechanotransduction in endothelial cells was detected in the SB216763-treated group compared to the BI group. To explore the underlying mechanism for the change of cell phenotype and function, we used microRNAs (miRNAs). MiRNAs are short non-coding RNAs and key regulators in various biological functions that negatively regulate gene expression. BM mesenchymal stromal cells (BMMSCs) transfected with miR-26a had a strong potential to induce endothelial-like cells. It was also found in BMMSCs that miR-26a is a possible miRNA targeting GSK-3β, regulating β-catenin downstream signal. These findings suggest that regulation of GSK-3β signal in BM-derived progenitor cells may be a remarkable strategy for cell mobilization and fate regulation, and be exploited to provide effective therapy for vascular regeneration.