Abstract Abnormal repair following traumatic airway injury, characterized by fibroblast hyperactivation and excessive extracellular matrix (ECM) deposition, frequently results in airway stenosis, a condition with high morbidity and limited therapeutic options. This study elucidates a critical protective mechanism mediated by Annexin A1 (ANXA1). We integrated clinical sample analysis, in vitro cellular models, and in vivo animal studies. Histological analysis of injured human and rat airway tissues confirmed that ANXA1 expression is significantly downregulated, correlating inversely with ECM accumulation, elevated oxidative stress (ROS), and pronounced mitochondrial fragmentation. In vitro, using a TGF-β1-stimulated fibroblast model, we demonstrated that ANXA1 overexpression (OE) effectively suppressed fibroblast migration, proliferation, and myofibroblast differentiation (activation). Conversely, ANXA1 knockdown (KD) exacerbated these pro-fibrotic phenotypes, which was associated with mitochondrial hyperfission, diminished ATP synthesis, and ROS bursts, confirmed by electron microscopy and functional assays. Mechanistic investigations using co-immunoprecipitation (CO-IP) and mass spectrometry identified macrophage migration inhibitory factor (MIF) as a key downstream effector. We revealed that ANXA1 directly interacts with MIF and the scaffold protein RACK1. ANXA1 competitively binds to RACK1, thereby disrupting the RACK1-MIF complex. This disruption destabilizes MIF, promoting its degradation via the ubiquitin-proteasome pathway. The subsequent reduction in MIF levels attenuated the activation of the downstream ERK/DRP1 signaling cascade, leading to decreased DRP1 phosphorylation and inhibition of pathological mitochondrial fission. The centrality of this pathway was validated by rescue experiments: both the mitochondrial fission inhibitor Mdivi-1 and the MIF inhibitor 4-IPP successfully reversed the mitochondrial dysfunction and pro-fibrotic activation induced by ANXA1 KD. Finally, in vivo studies using a rat airway injury model demonstrated that adenovirus-mediated local OE of ANXA1 significantly alleviated granuloma formation and airway stenosis. In contrast, ANXA1 KD aggravated stenosis, an effect that was effectively mitigated by the administration of 4-IPP. In conclusion, this study unveils the “ANXA1-RACK1-MIF-mitochondrial dynamics” axis as a novel regulatory pathway in airway repair. Our findings establish ANXA1 as a crucial regulator of fibroblast behavior by modulating MIF stability and mitochondrial homeostasis, presenting the ANXA1/MIF pathway as a promising therapeutic target for preventing traumatic airway stenosis. This abstract is funded by: none
Liao et al. (Fri,) studied this question.