Low‐Dose 6‐Bromoindirubin‐3′‐oxime Induces Partial Dedifferentiation of Endothelial Cells to Promote Increased Neovascularization

A bstract Endothelial cell (EC) dedifferentiation in relation to neovascularization is a poorly understood process. In this report, we addressed the role of Wnt signaling in the mechanisms of neovascularization in adult tissues. Here, we show that a low‐dose of 6‐bromoindirubin‐3′‐oxime (BIO), a competitive inhibitor of glycogen synthase kinase‐3β, induced the stabilization of β‐catenin and its subsequent direct interaction with the transcription factor NANOG in the nucleus of ECs. This event induced loss of VE‐cadherin from the adherens junctions, increased EC proliferation accompanied by asymmetric cell division (ACD), and formed cellular aggregates in hanging drop assays indicating the acquisition of a dedifferentiated state. In a chromatin immunoprecipitation assay, nuclear NANOG protein bound to the NANOG‐ and VEGFR2‐ promoters in ECs, and the addition of BIO activated the NANOG ‐promoter‐luciferase reporter system in a cell‐based assay. Consequently, NANOG ‐knockdown decreased BIO‐induced NOTCH‐1 expression, thereby decreasing cell proliferation, ACD, and neovascularization. In a Matrigel plug assay, BIO induced increased neovascularization, secondary to the presence of vascular endothelial growth factor (VEGF). Moreover, in a mouse model of hind limb ischemia, BIO augmented neovascularization that was coupled with increased expression of NOTCH‐1 in ECs and increased smooth muscle α‐actin + cell recruitment around the neovessels. Thus, these results demonstrate the ability of a low‐dose of BIO to augment neovascularization secondary to VEGF, a process that was accompanied by a partial dedifferentiation of ECs via β‐catenin and the NANOG signaling pathway. S tem C ells 2014;32:1538–1552