Abstract Rationale Patients with acute respiratory distress syndrome require ventilatory support with hyperoxia. Work from our group in mouse models suggests that acute exposure to hyperoxia prior to mechanical ventilation amplifies the severity of lung injury compared to either insult alone. We previously reported that hyperoxia promotes F-actin stress fiber formation in lung epithelial cells, leading to cellular stiffening and increased susceptibility to damage during mechanical stretch. Studies from other groups demonstrate that endothelial cells exhibit a similar increase in stress fibers in response to hyperoxia. Piezo1, a mechanosensitive channel which mediates calcium influx in response to mechanical cues, has been implicated in vascular permeability and endothelial dysfunction in several organs, but its role in hyperoxia- and/or ventilator-induced lung injury remains unclear. We therefore tested the hypothesis that hyperoxia enhances Piezo1 expression and activity, contributing to endothelial barrier dysfunction and lung injury. Methods C57BL/6J mice were exposed to 90% oxygen for 24 hours, followed by high-tidal volume mechanical ventilation (25 mL/kg, 4.5 hours, 90% FiO2). Lung injury was assessed by histopathology, lung mechanics (FlexiVent), and bronchoalveolar lavage (BAL) protein and interleukin-6 quantification via western blot and ELISA, respectively. Piezo1 protein expression in whole-lung homogenates was determined by western blot. Piezo1 transcript expression in human lungs was examined using single-cell RNA sequencing datasets from the LungMAP consortium and the Human Lung CellRef (v1) ShinyCell Browser. Primary human pulmonary artery endothelial cells (HPAECs, passages 3-8) were exposed to hyperoxia (90% O2) for 72 hours, and Piezo1 expression, cytoskeletal organization, and junctional protein levels were evaluated by immunofluorescence and western blot. Endothelial barrier function was quantified by transendothelial electrical resistance (TEER). To determine the contribution of Piezo1 activity to hyperoxia-induced effects, cells were treated with the pharmacologic inhibitor GsMTx4. Results We observed a significant increase in expression of Piezo1 in our in vivo model of ventilator-induced lung injury, which correlated with lung injury severity. Analysis of the LungMAP dataset revealed that Piezo1 is highly expressed in the human lung endothelium. Hyperoxia upregulated Piezo1 expression in human pulmonary artery endothelial cells and promoted cytoskeletal remodeling. Pharmacologic inhibition of Piezo1 prevented hyperoxia-induced actin remodeling. Additionally, hyperoxia decreased intercellular junctional protein expression and reduced TEER, indicating impaired endothelial barrier integrity. Inhibition of Piezo1 prevented changes in TEER under hyperoxic conditions. Conclusion These findings identify Piezo1 as a critical mediator of hyperoxia-induced endothelial barrier disruption and suggest it as a potential therapeutic target in lung injury. This abstract is funded by: Supported by NIH grant HL151419 (CMW), AHA Predoctoral Fellowship 25PRE1363542 (EEH).
Huffman et al. (Fri,) studied this question.