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

Authors

  • Erin E. Kohler,

    1. Department of Pharmacology, University of Illinois at Chicago, Chicago, Illinois, USA
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  • Jugajyoti Baruah,

    1. Department of Pharmacology, University of Illinois at Chicago, Chicago, Illinois, USA
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  • Norifumi Urao,

    1. Department of Pharmacology, University of Illinois at Chicago, Chicago, Illinois, USA
    2. Department of Cardiology, University of Illinois at Chicago, Chicago, Illinois, USA
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  • Masuko Ushio-Fukai,

    1. Department of Pharmacology, University of Illinois at Chicago, Chicago, Illinois, USA
    2. Department of Cardiology, University of Illinois at Chicago, Chicago, Illinois, USA
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  • Tohru Fukai,

    1. Department of Pharmacology, University of Illinois at Chicago, Chicago, Illinois, USA
    2. Department of Cardiology, University of Illinois at Chicago, Chicago, Illinois, USA
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  • Ishita Chatterjee,

    1. Department of Pharmacology, University of Illinois at Chicago, Chicago, Illinois, USA
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  • Kishore K. Wary

    Corresponding author
    1. Department of Pharmacology, University of Illinois at Chicago, Chicago, Illinois, USA
    • Correspondence: Kishore Wary, Ph.D., Department of Pharmacology, University of Illinois at Chicago, 835 S. Wolcott Avenue, Room E403, Chicago, Illinois 60612, USA. Telephone: 312-413-9582; Fax: 312-996-1225; e-mail: kkwary@uic.edu

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Abstract

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. Stem Cells 2014;32:1538–1552

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