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Keita Fujii, Naoki Okumura, Ai Odajima, Morio Ueno, Shigeru Kinoshita, Noriko Koizumi; Rho-associated protein kinase inhibitor suppresses corneal endothelial cell apoptosis by suppressing cell contraction via inhibiting myosin light chainphosphorylation . Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2579.
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© ARVO (1962-2015); The Authors (2016-present)
In the pathology of corneal endothelium in Fuchs' corneal dystrophy or endotheliitis, apoptosis is involved in corneal endothelial damage. We previously reported that Rho-associated protein kinase (ROCK) inhibitor suppresses the apoptosis of cultured corneal endothelial cells (CECs), and are now developing an eye-drop treatment for corneal endothelial dysfunction. The purpose of this present study was to investigate the mechanism by which ROCK inhibitor suppresses apoptosis.
Monkey CECs (MCECs) were isolated from cynomolgus monkey corneas and then cultured. The cultured MCECs were then subjected to ultraviolet (UV) radiation (100 J/m2) to induce apoptosis. The MCECs were then treated with EGTA (3 mM) for 24 hours to induce detachment from the substrate. ROCK-inhibitor Y-27632, myosin-II-inhibitor blebbistatin, and caspase-inhibitor Z-VAD-FMK were then added to the culture medium to evaluate the ROCK-inhibitor effect and myosin light chain (MLC) activation. MLC phosphorylation was then investigated by immunofluorescence staining. To evaluate the effect of ROCK signaling inhibition on anoikis, cleavage of caspase 3 and poly (ADP-ribose) polymerase (PARP) were evaluated by western blotting.
Phase contrast images showed that Y-27632 and blebbistatin significantly suppressed UV-induced cell contraction (22.8±0.6% and 53.2±1.2%, respectively). Y-27632 also significantly suppressed UV-induced MLC phosphorylation (p<0.01). Western blotting showed that Y-27632 and blebbistatin suppressed UV-induced caspase 3 cleavage and suppressed cell detachment from the substrate due to EGTA-related cell contraction, yet Z-VAD-FMK did not suppress cell detachment. Immunostaining showed that MLC phosphorylation was induced by EGTA (50.9±1.1%), yet significantly suppressed by Y-27632 and blebbistatin (0.4±0.3% and 3.8±2.1%, respectively). MLC phosphorylation was not suppressed by Z-VAD-FMK (45.2±6.1%). Western blotting demonstrated that Y-27632 and blebbistatin decreased EGTA-induced cleavage of caspase 3 and PARP.
Our results indicate that Y-27632 suppresses apoptosis by suppressing cell detachment from the substrate via inhibiting MLC phosphorylation, and that it could be further developed as a therapeutic agent to modulate CEC apoptosis.
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