HIF-1 alpha Stabilization Enhances Angio-/Vasculogenic Properties of SHED

The outcome of regenerative procedures could be augmented by enhancing the biological performances of stem cells prior to their transplantation. The current study aimed to investigate whether hypoxic preconditioning through stabilization of hypoxia-inducible factor 1 alpha (HIF-1 alpha) could enhance the angio-/vasculogenic properties of stem cells from human exfoliated deciduous teeth (SHED). HIF-1 alpha expression in SHED under normoxia was stabilized by silencing the expression of prolyl hydroxylase domain-containing protein 2 (PHD2) via lentiviral small hairpin RNA. This in turn significantly increased the expression of an angiogenic factor: vascular endothelial growth factor. Conditioned medium of HIF-1 alpha-stabilized SHED increased the migration and proliferation of human umbilical vein endothelial cells (HUVECs), indicating enhanced paracrine signaling of SHED following PHD2 knockdown (P < 0.05). Furthermore, the coculture of HIF-1 alpha-stabilized SHED with HUVECs directly and in fibrin beads demonstrated significantly longer vascular sprouts through juxtacrine and paracrine effects (P < 0.05). When HIF-1 alpha-stabilized SHED were added to a preformed HUVEC vascular tube network on Matrigel, it not only stabilized the vessels, as shown by the increased thickness (P < 0.05) and junctional area (P < 0.01) of tubes, but also gave rise to new sprouting (P < 0.01). This observation, with the morphologic changes and increased CD31 expression, suggested that HIF-1 alpha stabilization enhanced the endothelial differentiation capacity of SHED through autocrine signaling. In vivo Matrigel plug assay demonstrated that HIF-1 alpha-stabilized SHED alone could give rise to a vasculature that was significantly higher than that of control SHED +/- HUVECs and similar to that of HIF-1 alpha-stabilized SHED + HUVECs. In addition to vasculogenesis by endothelial differentiation, HIF-1 alpha-stabilized SHED recruited host blood vessels into the implant by exerting a significant paracrine effect. Taken together, our results confirmed that HIF-1 alpha-stabilized SHED could replace the function of HUVECs and act as the sole cell source of vascularization. Thus, targeting PHD2 to stabilize HIF-1 alpha expression is an appealing strategy that enables the use of a single cell source for achieving vascularized tissue regeneration.