Deciphering the optimal small molecule activators of HIF-1α stabilization in dental stem cells

Abstract

Dental stem cell-based tissue regeneration is a promising strategy for promoting oral health. However, the engineered cellular constructs are immediately exposed to a hypoxic microenvironment after transplantation, inducing harmful effects due to a lack of oxygen and nutrients. Hypoxic preconditioning, achieved through the stabilization of hypoxia-inducible factor-1α (HIF-1α), primarily via hypoxia chambers, hypoxia-inducible gene therapy, and hypoxia mimetic agents, has demonstrated a significant enhancement of protective effects on cells and tissues. However, the cost and unstable efficacy of hypoxia chambers, along with the safety concerns regarding viral or non-viral vectors in gene therapy, limit their clinical application. Hypoxia mimetic agents have been increasingly explored as safer and clinically applicable strategies for stabilizing HIF-1α in cell-based therapies. Currently, the agents applied in dental cells are associated with low efficacy and high cytotoxicity. Therefore, deciphering the optimal small molecules and their dosages for applying the hypoxic preconditioning phenomenon in dental stem cells is essential.

TW-37, ML228, and ciclopirox (CPX) were identified as the top three potential hypoxia mimetic agents by RNA sequencing and bioinformatics analysis of PHD2 knockdown stem cells from human exfoliated deciduous teeth (SHED). The effects of the three drugs on cell viability, HIF-1α expression, and vascular endothelial growth factor (VEGF) were then examined, followed by in-vitro and in-vivo Matrigel assays. The results revealed that all three compounds could activate HIF-1α expression and upregulate VEGF secretion levels in a dose-dependent manner in SHED. Additionally, SHED preconditioned with 10 μM TW-37, 1 μM ML228, or 7 μM CPX showed significantly enhanced in-vitro vascular network formation and sprouting of human umbilical vein endothelial cells, resulting in an increased number of blood vessels by recruiting host vasculature into the in-vivo Matrigel implants. Interestingly, 10 μM TW-37 applied to SHED for 24 hours demonstrated a superior effect on VEGF secretion, cell viability, and in-vivo blood vessel formation compared to 1 μM ML228 or 7 μM CPX.

Then, the differential gene expression of TW-37-treated SHED compared to untreated SHED was analyzed using RNA sequencing, and hub genes were identified to explore the mechanism of TW-37 on HIF-1α activation. Bioinformatics analysis of the data revealed that the HIF-1 signaling pathway was the most significantly enriched, and adenylate kinase 4 (AK4) was identified as a hub protein downstream of TW-37 induction. Silencing of AK4 expression via target small interfering RNA verified that TW-37 stabilized the expression of HIF-1α in-vitro and promoted the vasculogenic properties of SHED in-vivo via AK4 activation.

In conclusion, TW-37, ML228, and CPX were identified as optimal hypoxia mimetic agents that significantly stabilize HIF-1α and increase VEGF secretion in a dose-dependent manner in SHED. Pretreatment with 10 μM TW-37, 1 μM ML228, or 7 μM CPX for 24 hours was determined to be a safe and effective dosage for hypoxic preconditioning of SHED, enhancing both angiogenic properties in-vitro and in-vivo. Among those compounds, TW-37 was reported for the first time as a hypoxia mimetic agent and verified as the optimal activator of HIF-1α stabilization in SHED by regulating AK4, thereby enhancing post-implantation vascularization in-vivo.