NCOA4-Mediated Ferritinophagy Induces Ferroptosis and Enriches Ferritin-Containing EVs via Ferritin Phase Separation to Promote Mechanical Ventilation-Induced Pulmonary Fibrosis.

INTRODUCTION: Mechanical ventilation (MV) is essential for treating respiratory failure but can paradoxically lead to pulmonary fibrosis. The mechanisms of MV-induced pulmonary fibrosis (MVPF) remain poorly understood. Ferritinophagy, a novel autophagic process, regulates ferroptosis and the release of ferritin-containing extracellular vesicles (EVs), both of which may contribute to MVPF. OBJECTIVES: This study aimed to investigate the mechanism of ferritinophagy, as well as how ferritin-containing EVs contribute to intercellular communication during MVPF progression. METHODS: A mouse MVPF model was established using high tidal volume ventilation. Lung tissues were analyzed via single-cell RNA sequencing (scRNA-seq). Mechanical stretch (MS) was applied to alveolar epithelial cells (AECs) in vitro. Fluorescence recovery after photobleaching (FRAP) analysis was used to capture ferritin phase separation in live cells. Ferritinophagy and ferroptosis were assessed via key molecular markers. Chloroquine and AAV-mediated knockdown of AGTR1 and NCOA4 were used to inhibit ferritinophagy. EVs were isolated by ultracentrifugation and evaluated by immunoblotting and uptake assays. RESULTS: scRNA-seq revealed iron metabolism dysregulation and downregulation of ferroptosis-suppressor genes (Gpx4 and Fth) in AECs after MV. The ANG II/AGTR1 axis initiated ferritinophagy, leading to iron overload and subsequent ferroptosis. NCOA4-mediated ferritin phase separation under MS promoted ferritinophagy in AECs.Inhibition of ferritinophagy effectively reduced ferroptosis and alleviated MVPF. Moreover, ferritin-containing EVs released from injured AECs due to ferritinophagy can be assimilated by fibroblasts, resulting in fibroblast activation and extracellular matrix (ECM) accumulation through iron overload. CONCLUSION: MV induces ANG II/AGTR1-mediated ferritinophagy and ferroptosis in AECs. NCOA4-driven ferritin phase separation promotes ferritinophagy under mechanical stress. Ferritin-containing EVs from damaged AECs activate fibroblasts, exacerbating MVPF. Our findings underscore the pivotal role of iron metabolism dysregulation in biomechanically induced programmed cell death and intercellular communication, and reveal potential therapeutic targets for the prevention and treatment of MVPF.