Angiopoietin-like 4 is Upregulated by Hypoxia and the Hypoxia Mimetic Agent L-Mimosine in Oral Cells

Objectives: Angiopoietin-like 4 is a key player in angiogenesis and resorption of hard tissue. However, the role of angiopoietin-like 4 in oral tissues comprising the dentin-pulp complex and the periodontium is unclear. We hypothesized that angiopoietin-like 4 production is increased by hypoxia and hypoxia mimetic agents in oral tissues such as dental pulp, gingiva, and periodontal ligament.
Methods: Monolayer and spheroid cultures of primary human cells derived from dental pulp, gingiva, and periodontal ligament were exposed to hypoxia or the hypoxia mimetic agent L-mimosine (L-MIM). Furthermore, tooth slice cultures were treated with hypoxia or L-MIM. Untreated cultures under normoxia served as control. Angiopoietin-like 4 mRNA levels were measured with reverse transcription qPCR. Angiopoietin-like 4 protein levels in culture supernatant were assessed with ELISA. Echinomycin was applied to investigate the involvement of hypoxia inducible factor-1 alpha (HIF-1alpha).
Results: Angiopoietin-like 4 mRNA and protein levels were increased upon treatment with hypoxia and L-MIM in monolayer cultures of cells derived from dental pulp, gingiva, or periodontal ligament. Echinomycin prevented the impact of hypoxia and L-MIM on angiopoietin-like 4 production. The response of spheroids depended on the cell type. In tooth slice cultures the dental pulp tissue produced and released angiopoietin-like 4 under normoxia, hypoxia, and in the presence of L-MIM. However, the results of monolayer and spheroid cultures were not paralleled in tooth slice cultures. Trends for an increase in angiopoietin-like 4 mRNA levels and a decrease in the protein levels of the culture supernatants were seen, which did not reach the level of significance. Exposure of oral cells to recombinant angiopoietin-like 4 did not modulate proliferation, differentiation, or mineralization.
Conclusions: Hypoxia and the hypoxia mimetic agent L-MIM can stimulate angiopoietin-like 4 production in cells derived from dental pulp, gingiva, and periodontal ligament via HIF-1alpha signaling. Interestingly, the response of oral cells depends on the culture model.