HIF-1[alpha] stabilization promotes pulp regeneration by enhancing angio-/vasculogenesis and survival of dental stem cells

Abstract

Stem cell-based dental pulp regeneration is a promising approach for restoring tooth function and preventing further damage. However, two main goals are yet to be achieved for efficient pulp-dentin regeneration: 1) improving the poor post-implantation cell survival in hypoxic/ischemic environment, 2) timely vascularization of the engineered construct. Hypoxia-inducible factor 1-alpha (HIF-1α) is a transcriptional factor that plays a critical role in cell adaptation to low oxygen levels by regulating angiogenesis, apoptosis, reactive oxygen species (ROS) homeostasis, and metabolism. In normoxia, HIF-1α is rapidly degraded via the ubiquitin-dependent proteasomal pathway after being hydroxylated by prolyl hydroxylase domain protein 2 (PHD2). Therefore, I hypothesised that inhibition of PHD2 activity could stabilize HIF-1α in normoxia, which would precondition cells to be more resistant to an in-vivo hostile environment. In this study, firstly, the role of HIF-1α in stem cells from human exfoliated deciduous teeth (SHED) was examined by inhibiting its expression using either silencing RNA (siRNA) or chemical inhibitor (YC-1). The results revealed that suppression of HIF-1α reduced the cell survival in hypoxia, which was related to the affected metabolic adaptations by downregulated pyruvate dehydrogenase kinase 1 (PDK1), hexokinase-2 (HK2), and glucose transporter 1 (Glut1) expression. Moreover, the number of blood vessels was noticeably reduced in HIF-1α- suppressed SHED, which could be attributed to the diminished secretion levels of vascular endothelial growth factor (VEGF) . With these findings in sight, the effects of HIF-1α stabilization in SHED were further determined by PHD2 knockdown using lentiviral small hairpin RNA. HIF-1α stabilization significantly increased the expression of VEGF and in turn promoted the endothelial differentiation of SHED through autocrine signaling and increased the proliferation, migration, and vascular tube formation of human umbilical vein endothelial cells (HUVECs) through paracrine effects. In addition to vasculogenesis by endothelial differentiation, HIF-1α- stabilized SHED recruited host blood vessels into the in-vivo Matrigel implant by exerting a significant paracrine effect. Taken together, our results confirmed that HIF-1α-stabilized SHED could replace the function of HUVECs and act as the sole cell source of vascularization. Consecutively, HIF-1α-stabilized SHED were used in a full-length pulp-dentin regeneration mouse model. The results revealed that HIF-1α stabilization increased post-transplantation SHED survival with lower DNA damage and higher cell proliferation, augmented vascularization, and boosted dentin formation during pulp-dentin regeneration in-vivo. In unravelling the mechanism, it was found that HIF-1α-stabilized SHED activated PI3K/AKT signaling pathway, which inhibited downstream caspase-3 activation leading to lower cell death in hypoxia. HIF-1α stabilization also regulated intracellular ROS levels in hypoxia through upregulating PDK1 expression which suppressed the mitochondrial Krebs cycle and inhibited ROS production. Simultaneously, HIF-1α stabilization facilitated the energy demands by upregulating HK2 and Glut1 to promote shift towards anaerobic glycolysis and glucose transport. Furthermore, Smad7 was identified as a downstream hub protein of HIF-1α, which played an essential role in HIF-1α induced HK2 and Glut1 upregulation. In conclusion, HIF-1α plays an indispensable role in post-implantation survival and angio- /vasculogenic properties of SHED. Stabilization of HIF-1α ex-vivo is a promising approach to enhance post-transplantation stem cell survival, vascularization, and dentin formation during pulp-dentin regeneration in-vivo.