Hypoxic Preconditioning Restores Mitochondrial Dynamics and Endothelial Function

Introduction: Endothelial dysfunction drives cardiovascular disease, with acute stressors like high glucose (HG) or intraluminal pressure (IILP) by reducing nitric oxide (NO) and increasing hydrogen peroxide (H 2 O 2 ). Mitochondrial fission amplifies ROS, while fusion mitigates it. Hypoxic preconditioning (HPC) may shift mitochondrial dynamics from fission to fusion under stress, potentially restoring endothelial function and reducing damage. METHOD: Endothelial cells (HUVECs) were exposed to HG (22 mM) or normal glucose (NG; 5.5 mM) for 15–20 hours. Human arterioles from healthy adipose tissue were isolated for video microscopy. Internal diameters were measured after increases in intraluminal pressure gradients (flow), with/without NOS inhibitor (L-NAME, 100 μM) or H 2 O 2 scavenger (Peg-Catalase, 500 U/mL). HPC was performed on separate groups of cells (prior to HG;2-hour hypoxia/normoxia cycles) and arterioles, (Prior to IILP three 30-minute cycles of hypoxia -5% O 2 and normoxia -21% O 2 ). Controls underwent no gas changes. Mitochondrial networks were imaged via confocal microscopy, fragmentation quantified (MFC; particles/pixel x 1000). Drp-1 (S616) and MFN-2 were analyzed by western blot. Molecular data are mean ± SEM; vascular data as % dilation; significance set at P < 0.05. Result: Exposure to HG significantly increases the MFC (n=2, p<0.05) compared to NG. HPC effectively preserve the mitochondrial phenotype. Western blot analysis revealed a significant reduction in Drp-1 pS616 levels, while MFN-2 levels were significantly increased (p<0.05) in cells subjected to HPC prior to high glucose treatment, compared to NG-treated cells. HG exposure elevated Drp-1 pS616, and reduced MFN-2 protein expression, while exposure to HPC reversed these effects ( insert values and or p-values ). Exposure to IILP was associated with a switch in FMD from NO to H2O2, while HPC prevented this switch (p<0.05)). Inhibition of MFN-2 via siRNA (50 nM) prevented the restorative effects of HPC, maintaining H2O2-mediated dilation following IILP ((p<0.05)). Conclusion: This study shows that acute stressors induce mitochondrial fission and microvascular dysfunction, while HPC preserves mitochondrial fusion and endothelial function. Funded by R01-HL133029-05 (AMB), R01-HL157025-01 (DDG), and R01-HL135901-04 (DDG), K99-HL161491 (WEH) This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.