Optimal Hypoxia Mimetic Small Molecules for Enhancing Angiogenic Properties of Stem Cells From Human Exfoliated...

Hypoxic preconditioning of cells holds promise for regenerative therapies, yet identifying effective and safe methods for clinical application remains challenging. We aimed to determine optimal hypoxia-mimetic small molecules (SMs) that stabilize hypoxia-inducible factor-1α (HIF-1α) and their dosages for hypoxic preconditioning in stem cells from human exfoliated deciduous teeth (SHED), and to examine their effects on SHED angiogenic properties.

A systematic approach integrating transcriptomics, bioinformatics, and experimental validation was employed. RNA sequencing data from prolyl-hydroxylase domain 2-knockdown SHED were queried against the Connectivity Map to identify the top 3 SMs that most closely mimic this hypoxic signature. To establish causality and mechanism, the selective Bcl-2 inhibitor venetoclax and HIF-1α siRNA were used. Candidate compounds and conventional agents (CoCl₂, deferoxamine) were tested for HIF-1α expression, vascular endothelial growth factor (VEGF) secretion, and cell viability. Pro-angiogenic functionality was evaluated by in vitro Matrigel and spheroid-sprouting assays. In vivo angiogenic potential and safety (TUNEL/Ki67) were assessed using a Matrigel plug assay in SCID mice. Additional RNA sequencing compared TW-37- and CoCl₂-treated SHED.

CoCl₂ and deferoxamine increased HIF-1α and VEGF but showed substantial cytotoxicity at effective doses. TW-37, ML228, and CPX stabilized HIF-1α at non-toxic doses (10 μM, 1 μM, and 7 μM, respectively), and sustained VEGF secretion. SM-treated SHED-CM boosted endothelial tube formation and sprouting in vitro, with TW-37 showing the strongest pro-angiogenic effect, confirmed to be HIF-1α-dependent and separate from Bcl-2 inhibition. SM-pretreated-SHED (24 h) yielded persistent VEGF release and robust in vitro angiogenic activity. In vivo Matrigel plug assays revealed markedly increased vessel density in plugs containing SHED pretreated with TW-37, ML228, or CPX. In particular, TW-37-pretreated SHED showed increased vessel density, enhanced Ki67-positive proliferation, and no increase in apoptosis (TUNEL). RNA sequencing revealed that TW-37 upregulated genes associated with angiogenesis, metabolic adaptation, and odontogenesis, indicating a more favourable profile than CoCl₂.

TW-37 (10 μM), ML228 (1 μM), and CPX (7 μM) are promising hypoxia-mimetic regimens in this proof-of-concept model. TW-37 acts through a specific, reversible HIF-1α-dependent mechanism, demonstrating a favorable balance between angiogenic enhancement and preliminary safety in regenerative endodontics.