Pabelick, C.M.

Abstract Rationale: While survival of premature infants has improved with NICU interventions, prematurity remains a risk for developing chronic lung diseases. Oxygen supplementation in the NICU is unfortunately necessary, and clinical practice has shifted towards use of moderate hyperoxia (<60% O2) to minimize detrimental effects of very high O2 concentration (80-90%) such as bronchopulmonary dysplasia. However, even moderate hyperoxia increases the risk of bronchial airway diseases and wheezing/asthma with airway hyperreactivity and remodeling. We previously found that moderate hyperoxia induces cellular senescence in human fetal airway smooth muscle cells (fASM) and fibroblasts. However, the mechanisms by which hyperoxia enhances cellular senescence in fASM are not known. We investigated the role of endoplasmic reticulum (ER) stress and mitochondrial dysfunction, using fASM cells exposed to 21% (normoxia) vs. 50% O2 (hyperoxia). Methods: fASM isolated from lungs of 18-20 week aborted fetuses (Mayo IRB exempt; period of rapid bronchial airway growth) were exposed to normoxia, hyperoxia or etoposide (10 µM; positive control for senescence induction). Cells and lysates were harvested at days 2, 3 or 7 to assess markers of senescence markers, ER stress and mitochondrial morphology, and levels of cytosolic and mitochondrial ROS, using real-time fluorescence imaging, Western blotting, immunofluorescence, and a novel immuno-FISH staining of telomere associated foci (TAF). Mitochondria-specific antioxidant MitoQ [100 nM], ER stress inhibitor Salubrinal [12.5 µM] or mitochondrial fission inhibitor Mdivi-1 [10 µM] were used to assess their effects on senescence markers and interactions between ER stress and mitochondria in inducing senescence. Results: Hyperoxia enhanced cellular senescence via upregulation of p21, PAI-1, and Cav-1; downregulation of LMNB1 and Ki-67, with increased γH2A.X- and TAF-positive cells. Hyperoxia increased cytosolic and mitochondrial ROS, and markers of the three ER stress pathways (ATF6, IRE1α, XBP1s, PDI, GADD34). Hyperoxia altered mitochondrial morphology with increased fission protein Drp1 and fusion proteins Mfn1/2. Inhibition of Drp1 with Mdivi-1 blocked mitochondrial fragmentation, but not senescence. However, hyperoxia-induced p21 was reduced by the PERK pathway inhibitor Salubrinal and MitoQ. Conclusions: Hyperoxia induces senescence in fASM via mitochondrial ROS and ER stress: pathways that offer new opportunities to blunt detrimental effects of oxygen in developing lung via mitochondrial targeted antioxidants and/or ER stress inhibition.