A Cross-Species Hydroquinone Model Replicates Smoking-Related Corneal Endothelial Oxidative Injury and Enables...

To establish an in vitro model of smoking-associated oxidative injury to the corneal endothelium and evaluate an injury-mitigating factor.

Primary human and porcine corneal endothelial cells (CEnCs) were exposed to hydroquinone (HQ; 0-150 µM, 48 hours). Outcomes included viability (ATP-based assay), reactive oxygen species (ROS), and apoptosis (TUNEL). Bulk RNA sequencing (RNA-seq) profiled HQ (Nrf2 and Hippo-Yap pathways) and HQ ± fibroblast growth factor 10 (FGF10) responses under moderate stress (cadherin/Wnt programs). FGF10 dose-response (1-100 ng/mL) identified 30 ng/mL for subsequent HQ ± FGF10 studies. Western blotting provided protein validation.

HQ caused dose-dependent loss of viability with increased ROS and apoptosis (EC50: 191.2 µM human; 151.2 µM porcine). RNA-seq showed induction of Nrf2 antioxidant/detoxification genes with attenuation of Hippo-Yap signaling. At 150 µM HQ, total Yap decreased and the pYap/Yap ratio increased, consistent with Yap inactivation. FGF10 improved viability across doses (1-100 ng/mL; plateau 30-100 ng/mL) and required cotreatment during HQ exposure. Using 30 ng/mL, FGF10 improved viability across HQ doses, reduced ROS at 100 to 150 µM, and decreased apoptosis at 50 to 100 µM. At 50 µM HQ, FGF10 increased cadherin/Wnt-related transcripts, while N-cadherin protein decreased and β-catenin remained stable, consistent with junctional remodeling rather than full endothelial-mesenchymal transition. Key injury and rescue phenotypes were consistent across species.

Our study supports HQ as a translatable in vitro model of smoking-related oxidative stress in CEnCs. FGF10 shows injury-mitigating and pro-regenerative effects, supporting further preclinical evaluation to preserve endothelial integrity and potentially lessen reliance on transplantation in corneal endothelial dystrophies.