June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
Activation of the Rho/ROCK Signaling Pathway in the Apoptosis of Corneal Endothelial Cells
Author Affiliations & Notes
  • Naoki Okumura
    Biomedical Engineering, Doshisha University, Kyotanabe, Japan
    Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
  • Ai Odajima
    Biomedical Engineering, Doshisha University, Kyotanabe, Japan
    Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
  • EunDuck Kay
    Biomedical Engineering, Doshisha University, Kyotanabe, Japan
  • Wen Chen
    Biomedical Engineering, Doshisha University, Kyotanabe, Japan
  • Morio Ueno
    Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
  • Junji Hamuro
    Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
  • Shigeru Kinoshita
    Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
  • Noriko Koizumi
    Biomedical Engineering, Doshisha University, Kyotanabe, Japan
  • Footnotes
    Commercial Relationships Naoki Okumura, None; Ai Odajima, None; EunDuck Kay, None; Wen Chen, None; Morio Ueno, Senju Pharmaceutical Co (P), Santen Pharmaceutical Co (P); Junji Hamuro, None; Shigeru Kinoshita, Senju Pharmaceutical Co (P), Santen Pharmaceutical Co (P), Otsuka Pharmaceutical Co (C), Alcon (R), AMO (R), HOYA (R); Noriko Koizumi, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 2202. doi:
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      Naoki Okumura, Ai Odajima, EunDuck Kay, Wen Chen, Morio Ueno, Junji Hamuro, Shigeru Kinoshita, Noriko Koizumi; Activation of the Rho/ROCK Signaling Pathway in the Apoptosis of Corneal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2202.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: In the pathological condition of corneal endothelium associated with Fuchs’ dystrophy or post keratoplasty, apoptosis is known to be involved. We previously reported that Rho kinase (ROCK)-inhibitor Y-27632 suppresses the apoptosis of cultured corneal endothelial cells (CECs). The purpose of this present study was to evaluate the involvement of the Rho/ROCK signaling pathway in the apoptosis of CECs and the effect of ROCK inhibitor on modulating apoptosis.

Methods: Monkey corneal endothelial cells (MCECs) were cultured and then exposed to ultra-violet (UV) radiation (100J/m2) to induce apoptosis. To elucidate the involvement of the Rho/ROCK signaling pathway in apoptosis, RhoA-GTPase, myosin light chain (MLC) phosphorylation, and the cleavage of caspase 3 and ROCK 1 was evaluated by western blotting. Anti-blebbing effect of Y-27632 was evaluated after exposure to UV radiation by time-lapse phase contrast microscopy, and actin and MLC phosphorylation were evaluated by immunostaining. Annexin V staining was employed to confirm the anti-apoptotic effect of Y-27632.

Results: UV radiation caused cell death in MCECs in a dose-dependent manner; a significant cell death was observed at 100J/m2and higher dosages. Active GTPase pull-down assay showed that UV radiation activated RhoA of the MCECs. MLC phosphorylation and the cleavage of caspase 3 and ROCK 1 were increased by UV radiation. Compared to the control, Y-27632-treated MCECs exposed to UV radiation showed significantly decreased membrane blebbing (p<0.01). Moreover, actin contraction and MLC phosphorylation were both downregulated by Y-27632. In the human corneal tissue exposed to UV, a significantly decreased number of UV-induced Annexin V-positive apoptotic CECs was found in the Y-27632-treated tissue compared to the control (3.1±0.5% and 20.3±2.0%, respectively) (p<0.01).

Conclusions: The findings of this study indicate that Rho/ROCK signaling activation is involved in the UV-induced apoptosis of CECs. Rho/ROCK signaling might be a potent therapeutic target for the suppression of cell apoptosis in various corneal endothelial diseases. However, further investigation is needed to elucidate the anti-apoptotic mechanisms of ROCK inhibitor.

Keywords: 481 cornea: endothelium • 426 apoptosis/cell death • 503 drug toxicity/drug effects  
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