6-bromoindirubin-3--oxime and Ischemia

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.

Topics: Angiogenesis Inducing Agents; Animals; beta Catenin; Cell Aggregation; Cell Dedifferentiation; Cell Division; Cell Movement; Cell Nucleus; Cell Proliferation; Disease Models, Animal; Dose-Response Relationship, Drug; Endothelial Cells; Fetal Proteins; Hindlimb; Homeodomain Proteins; Human Umbilical Vein Endothelial Cells; Humans; Indoles; Ischemia; Mice; Nanog Homeobox Protein; Neovascularization, Physiologic; Oximes; Phenotype; Pluripotent Stem Cells; Promoter Regions, Genetic; Protein Binding; Protein Stability; T-Box Domain Proteins; Vascular Endothelial Growth Factor A