Hypoxia-Induced Ferroptosis Resistance Drives Orbital Fibrosis in Thyroid Eye Disease

To determine whether hypoxia promotes a ferroptosis-resistant phenotype in patient-derived thyroid eye disease (TED) orbital fibroblasts (OFs), and whether hypoxia-associated metabolic changes (lactate) relate to profibrotic activation readouts, as well as to test whether modulating ferroptosis alters these readouts.

Primary OFs from TED patients and controls were exposed to graded hypoxia (21% to 1% O₂). Ferroptosis sensitivity was assessed by reactive oxygen species, mitochondrial membrane potential, viability, and SLC7A11/GPX4/ACSL4. The profibrotic phenotype was assessed by COL1A1/α-SMA/fibronectin, immunofluorescence, and scratch assays. We perturbed metabolism with exogenous lactate and 2-deoxy-D-glucose, induced ferroptosis with RSL3, and inhibited ferroptosis with liproxstatin-1.

TED fibroblasts exhibited baseline ferroptosis resistance, marked by higher GPX4 and SLC7A11 and lower ACSL4 than controls, which intensified under hypoxia. Hypoxia alone was sufficient to increase fibrotic marker expression and further augmented TGF-β-evoked fibrotic responses. Under hypoxia, RSL3 at 50 to 100 nM reduced profibrotic marker expression in TED OFs, an effect lost at 150 nM where viability declined; liproxstatin-1 attenuated the RSL3-associated changes. Hypoxia increased intracellular lactate; lactate alone upregulated GPX4 and promoted fibrotic activation, whereas 2-deoxy-D-glucose decreased lactate, restored RSL3 sensitivity, and diminished fibrotic readouts.

Our data suggest that hypoxia-associated lactate accumulation contributes to GPX4-linked ferroptosis resistance and amplifies profibrotic activation in TED OFs. Mechanism-guided intervention combining metabolic inhibition with calibrated ferroptosis induction warrants further investigation as a rational strategy to restrain fibrotic remodeling.