The Role of Nanog in Wnt Signaling-Mediated Endothelial Cell Dedifferentiation and Neovascularization

Cardiovascular disease is the leading cause of death worldwide, attributing to seven million deaths per year. Coronary artery bypass grafts (CABG) using the saphenous vein (SVG), internal mammary artery, or radial artery is the current method of treatment. These grafts carry risks of atherosclerosis, atrophy, restenosis, and fatality. The SVG is the least invasive; however it maintains the highest occlusion rates as veins contain thinner basement membranes and are not surrounded by smooth muscle cells to allow for the resistance to sheer stress and pulsatile blood flow required of the coronary artery. Wingless (Wnt) signaling is associated with epithelial-mesenchymal transition (EMT), endothelial-mesenchymal transition (EnMT), self-renewal of pluripotent stem cells, and transcriptional activation of genes involved in cell proliferation, migration, embryogenesis, and wound healing. The objective of this study is to determine if Wnt pathway activation can dedifferentiate venous endothelial cells (ECs) towards an arterial phenotype to increase the patency, while decreasing the atherosclerosis and atrophy associated with CABG such as the SVG as a mechanism to treat cardiovascular disease. Here, I used Wnt3a and 6-bromoindirubin-3’-oxime (BIO) to activate canonical Wnt signaling. Accordingly, I demonstrate that BIO induced the interaction of beta-catenin and NANOG within the nucleus of ECs, resulting in increased expression of mesenchymal and hemangioblastic cell markers NANOG, BRACHYURY, CD133, OCT4, and VEGFR2, while decreased the expression of mature EC markers vWF and CD31. Bromodeoxyuridine (BrdU) incorporation coupled with microscopy provided evidence of asymmetric cell division (ACD) in a subset of ECs upon BIO treatment; however NANOG-knockdown inhibited BIO’s ability to induce ACD. Moreover, microscopic analyses of Matrigel plugs containing venous ECs treated with BIO showed increased alpha-SMA, NOTCH-1, HEY2, and EphB2 staining suggesting acquisition of dedifferentiated EC phenotype, compared with control vascular ECs. Importantly, analyses of hind limb ischemia revealed the ability of BIO to induce increased neovascularization. Altogether, these results show that BIO not only induced dedifferentiation of mature ECs, but also neovascularization through the activation of NANOG gene networks.