PhD Student The University of Hong Kong, Hong Kong
Abstract: The objective of this study is to elucidate the presence and directionality of mitochondria transfer between dental pulp stem cells (DPSCs) and human umbilical vein endothelial cells (HUVECs) under normoxic and hypoxic conditions and test whether they are transferred through intercellular tunneling nanotubes (TNTs), as well as its roles in HUVECs survival and apoptosis. Mitochondria were labeled by MitoTracker within either DPSCs or HUVECs, then cocultured under normoxia or hypoxia (1% oxygen) for 12 or 24 hours. Mitochondria transferred to recipient cells were observed using laser scanning confocal microscopy (LSCM), and its transfer rate was quantified through flow cytometry. Intercellular TNT structures were visualized via F-actin immunofluorescent staining. After TNT formation was disrupted via Boyden chamber indirect coculture assay or the F-actin polymerization inhibitor Cytochalasin B, the mitochondria transfer rate was quantified through flow cytometry. Rotenone and Antimycin A (ROT/AA) were used to inhibit mitochondrial respiration of DPSCs before cocultured with HUVECs; its effects on HUVEC proliferation and apoptosis were assessed through Ki67 and AnnexinV/7-AAD staining. Besides, MitoSOX and MitoProbe TMRM staining was performed to detect mitochondrial superoxide levels and mitochondrial membrane potential. Our results found a significantly higher mitochondria transfer rate from DPSCs to HUVECs (p < 0.05), with a significantly higher transfer rate under hypoxia (p < 0.05). Mitochondria transferring via TNTs was successfully observed, and Boyden chamber indirect coculture and Cytochalasin B treatment significantly decreased mitochondria transfer rate from DPSCs to HUVECs (p < 0.05). Compared with the control group, ROT/AA treatment of DPSCs has no significant effects on Ki67, MitoSOX, and MitoProbe TMRM expression in HUVECs. However, it significantly decreased the apoptosis rate of HUVECs (p < 0.05) under hypoxia. In conclusion, mitochondria preferentially transfer from DPSCs to ECs, with an increased transfer rate under hypoxia. Mitochondria transfer from DPSCs to ECs was partially mediated via TNTs, and it rescued apoptotic ECs under hypoxia, indicating a critical role of mitochondria transfer in DPSC-EC interactions.
Funding Source: This research was supported by the Research Grants Council, Hong Kong (General Research Fund; 17119524)
