Hyperoxia and iron homeostasis in developing human airway smooth muscle

Premature infants exposed to supplemental oxygen (O 2 ) are at increased risk of developing airway diseases such as asthma, hyperoxic lung injury (HLI), and bronchopulmonary dysplasia (BPD). Therefore, it is important to understand how O 2 detrimentally impacts developing airways. Previous studies found that severe (80-90%) O 2 exposure increases reactive oxygen species (ROS) and lipid peroxidation, inducing ferroptosis in models of HLI. However, the impact of clinically-relevant moderate (<60%) O 2 exposure is less understood. Recognizing the importance of smooth muscle in airway dysfunction, the present study uses human fetal airway smooth muscle (fASM) as a model to investigate whether hyperoxia contributes to establishment of a ferroptotic phenotype. fASM pretreated with or without Deferoxamine (DFO) (100 µM) or Ferrostatin (Fer-1) (10 µM) were exposed for 48h to normoxia (21% O 2 ) vs. moderate to severe hyperoxia (50%, 70% or 90% O 2 ). Hyperoxia’s effect on antioxidant systems, iron metabolism, and lipid peroxidation, and alleviating effect of DFO or Fer-1 were examined. Moderate hyperoxia impaired antioxidant systems involved in preventing ferroptosis and dysregulated iron metabolism. Interestingly, only severe hyperoxia (90% O 2 ) induced negative effects on downstream mechanisms involving early onset of ferroptosis such as increased labile iron and lipid peroxidation. DFO and Fer-1 showed no rescue effect on antioxidant systems. However, DFO decreased cytosolic iron and Fer-1 decreased lipid peroxidation byproduct. Together, these data highlight the impact of supplemental oxygen on premature airways and introduce the concept of a dose-dependent effect of hyperoxia in the context of iron metabolism, lipid peroxidation, and ultimately ferroptosis.