Abstract Rationale Premature infants exposed to supplemental oxygen are at increased risk of developing airway diseases such as bronchopulmonary dysplasia (BPD) and asthma, making it important to understand how oxygen detrimentally impacts developing airways. Even in moderation, hyperoxia promotes bronchial airway hyperresponsiveness (AHR) through effects on fetal airway smooth muscle (fASM), a cell type involved in impaired bronchodilation and remodeling. Iron dysregulation has been implicated in adult airway diseases such as asthma, COPD, and hyperoxic lung injury (HLI). Immature antioxidant systems in neonates and their increased susceptibility to iron-induced oxidative stress may lead to ferroptosis, contributing to pathologies observed in subsequent airway disease. The role of impaired iron metabolism, mediated by hyperoxia, remains largely unexplored in fetal airways. This study investigated the effects of increasing oxygen levels on antioxidant systems, iron metabolism, and lipid peroxidation in human fASM. Methods fASM from 18-20 week gestational age (canalicular stage; Mayo IRB exempt) were isolated and exposed to normoxia (21% O2) or increasing levels of hyperoxia (50%, 70%, and 90% O2) and treated with iron chelator, deferoxamine (DFO) 100uM or reactive oxygen species (ROS) scavenger ferrostatin (Fer-1) 10uM for 48h. Levels of intracellular iron (Fe2+) were determined using ferroOrange. Cell lysates were examined for protein expression of antioxidant markers (GPX4, SLC7A11, CBS, NRF2, KEAP1, and FSP1) and iron metabolism markers (TfR1, FTH1, and NCOA4). 4-HNE (lipid radical) expression was determined by immunofluorescence staining. Results Hyperoxia decreased SLC7A11, CBS, NRF2, KEAP1 (redox regulators), and iron storage (FTH1) at all concentrations. Iron influx (TfR1) increased under 50% and 70% O2 but not 90% O2. Cytosolic iron levels increased under hyperoxia in a dose-dependent manner and decreased with iron chelator, DFO. DFO further decreased FTH1 expression under 90% O2. 4-HNE (lipid radical) increased under 90% O2 and was reduced by ROS scavenger, Fer-1. Conclusion Our findings suggest that hyperoxia simultaneously impairs antioxidant systems and iron metabolism in fASM. Specifically, hyperoxia dose-dependently increases the labile iron pool which exhausts anti-ferroptotic mechanisms: effects which can be reduced by DFO and Fer-1. This implies the presence of a redox threshold tightly regulating iron accumulation in fASM. Overall, antioxidant impairment and iron metabolism dysfunction synergistically can contribute to pediatric airway disease: mechanisms which allow for the development of targetable strategies to blunt the impact of supplemental oxygen in the premature lung. This abstract is funded by: National Institutes of Health
Ortiz et al. (Fri,) studied this question.