Effect of ROCK inhibiter on Apoptosis in Corneal Endothelial Cells

Abstract

Purpose: Apoptosis is reportedly involved in pathological condition of corneal endothelium such as Fuchs dystrophy or post keratoplasty. The purpose of this study is to investigate the feasibility of Rho kinase (ROCK) inhibitorfor modulating apoptosis in corneal endothelial cells.

Methods: Monkey corneal endothelial cells (MCECs) were cultured and were then treated by UV radiation. Cell death was evaluated by trypan blue staining.Apoptosis was evaluated by the quantity of the released cytochrome c and caspase 3 (western blotting), activation level of caspase 3 (CaspaseGlo 3/7 Assay), DNA flagmentation (TUNEL). As an ex vivo apoptosis model, rabbit corneal tissue was treated by either UV (100 J/m²) or H₂O₂ (100 µM). Then, apoptotic cells were evaluated by Annexin Vimmunostaining after 24 hours.

Results: MCECs were exposed to UV radiation ranging from 10 to 1000J/m², there was cell death in a dose-response manner; substantial cell death was observed from 50 J/m².UV radiation caused massive release of cytochrome c into the cytoplasm; and cleavage of caspase 3 was increased with UV radiation. TUNEL positive DNA fragmentationwas detected in the UV radiated cells.ROCK inhibitor significantly decreased UV induced Annexin V-positive apoptotic corneal endothelial cellsin rabbit corneal tissue compared to the control (2.1±0.8% and10.9±3.3%, respectively)(p<0.01). Likewise,ROCK inhibitor significantly decreased H₂O₂ induced apoptotic cells compared to the control (2.3±1.1% and11.6±1.0%,respectively)(p<0.01).

Conclusions: The findings of the present study indicate that UV radiation induces apoptosis in CECs and that ROCK inhibiter could protect corneal endotheliumfrom the apoptosis leading to cell death. Modulation of corneal endothelial apoptosis by ROCK inhibitor might be a useful therapeutic approach for corneal endothelial disease.

Keywords: 481 cornea: endothelium • 480 cornea: basic science • 449 cell survival