Oxidative Stress-Induced Age-Related Bullous Keratopathy in Corneal Endothelial Cells With Lipid Droplets and...

We previously demonstrated that intracellular oxidative stress reduces corneal endothelial cells in Tet-mev-1 mice that genetically overproduce mitochondrial superoxide anions. This study aimed to elucidate the physiological processes underlying oxidative stress-induced corneal endothelial cell loss.

Primary cultures of mouse corneal endothelial cells were established. Intracellular lipid droplets were detected using Lipi-Green, and autophagosomes in live cells were visualized using DAPGreen. Autophagy was assessed by immunostaining and Western blot analysis using microtubule-associated protein 1 light chain 3 (LC3). Furthermore, autophagic flux was evaluated by LC3 and p62/SQSTM1 levels with bafilomycin A1. DNA damage was assessed by γH2AX pSer139 immunostaining.

Corneal endothelial cells formed a paving stone-like monolayer and expressed endothelial markers. Tet-mev-1 cells, which were mitochondrial oxidative stress-overproduced mouse cells, exhibited senescent-like cells with abnormal lipid droplet accumulation, vacuolation, and DNA damage compared with wild-type cells. Quantitative analyses confirmed increased lipid droplets, autophagosomes, DNA damage, and vacuolated cells. In young Tet-mev-1 cells, autophagy-related responses were relatively preserved under oxidative stress conditions. In contrast, aged Tet-mev-1 cells showed attenuated responses to lysosomal inhibition, suggesting a decline in autophagic degradation capacity with premature aging by oxidative stress.

Intracellular oxidative stress is associated with enhanced autophagic activity in young corneal endothelial cells, whereas progressive endothelial cell loss with aging is accompanied by alterations in autophagy-related processes. This shift contributes to the accumulation of lipid droplets, vacuolations, and DNA damage. These findings suggest that maintenance of lipid homeostasis through autophagy-related pathways may play an important role in delaying age-related corneal endothelial dysfunction.