June 2015
Volume 56, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2015
Rho-associated protein kinase inhibitor suppresses corneal endothelial cell apoptosis by suppressing cell contraction via inhibiting myosin light chainphosphorylation
Author Affiliations & Notes
  • Keita Fujii
    Biomedical Engineering, Doshisha University, Kyotanabe, Japan
  • Naoki Okumura
    Biomedical Engineering, Doshisha University, Kyotanabe, Japan
    Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
  • Ai Odajima
    Biomedical Engineering, Doshisha University, Kyotanabe, Japan
  • Morio Ueno
    Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
  • Shigeru Kinoshita
    Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
  • Noriko Koizumi
    Biomedical Engineering, Doshisha University, Kyotanabe, Japan
  • Footnotes
    Commercial Relationships Keita Fujii, None; Naoki Okumura, JCR Pharmaceuticals Co (P), Senju Pharmaceutical Co (P); Ai Odajima, None; Morio Ueno, None; Shigeru Kinoshita, Alcon (R), AMO (R), HOYA (R), Otsuka Pharmaceutical Co (C), Santen Pharmaceutical Co (P), Senju Pharmaceutical Co (P); Noriko Koizumi, JCR Pharmaceuticals Co (P), Senju Pharmaceutical Co (P)
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 2579. doi:
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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)

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Abstract

Purpose: 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.

Methods: 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.

Results: 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.

Conclusions: 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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