Optimal Small Molecule Activators of HIF-1α Stabilization in SHED

Objectives: This study aimed to identify the optimal small molecules (SMs) and their dosages for applying the hypoxic preconditioning phenomenon in stem cells from human exfoliated deciduous teeth (SHEDs).
Methods: Two SMs, ciclopirox (CPX) and ML228, involved in HIF-1α stabilization in SHED were shortlisted based on a connectivity map database via bioinformatics analysis of RNA sequencing and the unique signature profile of a drug. CoCl₂ and DFO are used as the positive controls. The expression of HIF-1α in SHED was explored by western blot under different concentrations of CPX and ML228 and the VEGF levels were assessed by ELISA. CCK-8 assay and Live/Dead staining were done to examine the effect of CPX and ML228 on the proliferation of SHED. To evaluate the function of SMs on the angio-/vasculogenic properties, Matrigel and fibrin sprouting assays of HUVECs were conducted using conditioned media (CM) collected from SHED treated with CPX and ML228.
Results: Western blot results showed that CoCl₂ and DFO required considerably high dose to stabilize HIF-1α in SHED (p<0.05), whereas CCK-8 assay indicated significant cytotoxicity of these chemical concentrations to SHED (p<0.05). Both CPX and ML228 could efficiently enhance the expression of HIF-1α in SHED at 6h and 24h in a dose-depending manner (p<0.05). As detected by ELISA, the secretory VEGF levels were significantly increased in the CPX/ML228-induced SHED (p<0.05). CCK-8 results indicated that CPX and ML228 could inhibit the proliferation of SHED at 24h, 48h and 72h (p<0.05). However, no dead cells were detected in Live/Dead staining at 24h, 48h and 72h, which suggested that CPX and ML228 affect cell proliferation without imparting cellular death. Matrigel and fibrin sprouting assays demonstrated that the CM could promote the tube formation and sprouting ability of HUVECs (p<0.05).
Conclusions: CPX and ML228 induce HIF-1α stabilization, enhance VEGF secretion and promote the angio-/vasculogenic properties of SHED.