HIF-1α stabilization in SHED enhances angiogenic properties in-vitro and in-vivo

Abstract

Objectives: To examine the effects of HIF-1α stabilization on angiogenic properties of stem cells from human exfoliated deciduous teeth (SHED). Methods: Stabilized expression of HIF-1α in SHED under normoxia was achieved by knocking-down of prolyl-hydroxylase domain-containing protein-2 (PHD2) using target shRNA lentiviral particles. Knockdown of PHD-2 and subsequent HIF-1α stabilization were confirmed by quantitative-PCR and western blotting. Expression of vascular endothelial growth factor (VEGF) mRNA and protein was measured by quantitative-PCR and ELISA assays, respectively. Vessel morphogenesis by endothelial cells was examined using conditioned media from HIF-1α-stabilized-SHED and control-SHED in Matrigel assay and in a direct co-culture system. Stabilization of vascular structures by HIF-1α-Stabilized-SHED was assessed in fibrin bead and Matrigel assays. In-vivo angiogenic potential of these cells was examined by Matrigel-plug assay performed on severe-combined-immunodeficient mice. Briefly, cells mixed with Matrigel was injected subcutaneously, retrieved after 3- or 7-days and examined for histology and immunohistochemistry. Results: HIF-1α stabilization resulted in significantly increased secretion of VEGF in PHD-2 knocked-down SHED under normoxia compared to that of control group as shown by PCR, western blotting and ELISA assays (p<0.05). Endothelial cells seeded on Matrigel and cultured in conditioned media of HIF-1α-stabilized-SHED formed a network of capillary-like structures. Further, vascular initiation was observed when endothelial cells were co-cultured directly with HIF-1α-stabilized-SHED whereas no such vessels were observed in the control co-culture. When HIF-1α-stabilized-SHED were seeded on to the pre-formed vascular network on Matrigel, it significantly increased the number, thickness and junctional area of vascular branches (p<0.05). Fibrin bead assay showed longer and more stable spouting in HIF-1α-stabilized-SHED incorporated group compared to that of the control group (p<0.05). Histological assessment of in-vivo Matrigel plugs showed significantly enhanced vasculature in HIF-1α-stabilized-SHED group compared to that of control-SHED. Conclusions: Stabilization of HIF-1α significantly enhances angiogenic properties of SHED and precondition the cells to face in-vivo hypoxic microenvironment.