ROCK-inhibiter Suppressed Apoptosis of Corneal Endothelial Cells by Inhibiting Membrane Blebbing

Abstract

Purpose: Apoptosis is known to play an important role in the pathological condition of corneal endothelium, such as in Fuchs’ corneal dystrophy or post keratoplasty. We previously reported that Rho kinase (ROCK) inhibitor suppresses the apoptosis of cultured corneal endothelial cells (CECs). At the onset of apoptosis, membrane blebbing is caused by phosphorylation of myosin light chain (MLC) following activation of ROCK. The purpose of this present study was to investigate whether or not ROCK inhibitor suppresses CEC apoptosis by inhibiting blebbing.

Methods: Monkey corneal endothelial cells (MCECs) were treated by ultraviolet (UV) radiation to induce apoptosis, and then treated with 10μM blebbistatin (inhibitor of myosin II) or 10μM Y-27632 ROCK inhibitor. To elucidate the involvement of MLC in apoptosis, apoptotic cells in the MCECs treated by UV radiation were evaluated by Annexin V staining. Cleavage of caspase 3 and Poly (ADP-ribose) polymerase (PARP) were also evaluated by western blotting. Phosphorylation of MLC after UV radiation was evaluated by western blotting and immunostaining. The numbers of blebbing cells was evaluated by phase contrast microscopy.

Results: In the UV-radiated MCECs, blebbistatin and Y-27632 significantly decreased UV-induced Annexin V-positive apoptotic CECs (14.8±2.6% and 7.8±1.4%, respectively) compared to the controls (36.2±2.3%) (p<0.01). In addition, cleavages of caspase 3 and PARP induced by UV radiation were decreased both by blebbistatin and Y-27632. Western blotting and immunostaining showed that MLC phosphorylation was induced by UV radiation and suppressed by blebbistatin and Y-27632. The percentages of blebbing cells among floating cells post UV radiation were 57.5±3.1% in the control, while those in the MCECs treated by blebbistatin and Y-27632 were significantly decreased (26.1±3.6%, 15.2±1.3%, respectively) (p<0.01).

Conclusions: The findings of the present study indicate that ROCK inhibitor suppresses CEC apoptosis by inhibiting blebbing caused by MLC phosphorylation, yet the blebbing independent pathway requires further investigation. Modulation of CEC apoptosis by ROCK inhibitor might be a useful therapeutic tool for corneal endothelial disease.

Keywords: 480 cornea: basic science • 481 cornea: endothelium • 426 apoptosis/cell death