April 2011
Volume 52, Issue 14
Free
ARVO Annual Meeting Abstract  |   April 2011
ROCK Inhibition Regulates the Cell Adhesion of Corneal Endothelial Cells
Kyoko Kumagai; Noriko Koizumi; Naoki Okumura; Kenta Yamazaki; Morio Ueno; Yuji Sakamoto; Nagahisa Yoshimura; Junji Hamuro; Shigeru Kinoshita
Author Affiliations & Notes
  • Kyoko Kumagai
    Ophthalmology, Kyoto university, Kyoto, Japan
  • Noriko Koizumi
    Biomedical Engineering, Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe, Japan
  • Naoki Okumura
    Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
  • Kenta Yamazaki
    Biomedical Engineering, Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe, Japan
  • Morio Ueno
    Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
  • Yuji Sakamoto
    Biomedical Engineering, Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe, Japan
  • Nagahisa Yoshimura
    Ophthalmology, Kyoto university, Kyoto, Japan
  • Junji Hamuro
    Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
  • Shigeru Kinoshita
    Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
  • Footnotes
    Commercial Relationships  Kyoko Kumagai, None; Noriko Koizumi, None; Naoki Okumura, None; Kenta Yamazaki, None; Morio Ueno, None; Yuji Sakamoto, None; Nagahisa Yoshimura, None; Junji Hamuro, None; Shigeru Kinoshita, None
  • Footnotes
    Support  The Adaptable and Seamless Technology Transfer Program through Target-driven R & D (grant number: AS2111180G) from the Japan Science and Technology Agency.
Investigative Ophthalmology & Visual Science April 2011, Vol.52, 6465. doi:
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      Kyoko Kumagai, Noriko Koizumi, Naoki Okumura, Kenta Yamazaki, Morio Ueno, Yuji Sakamoto, Nagahisa Yoshimura, Junji Hamuro, Shigeru Kinoshita; ROCK Inhibition Regulates the Cell Adhesion of Corneal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):6465.

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Abstract

Purpose: : We previously reported that the Rho kinase (ROCK) inhibitor Y-27632 promotes cell adhesion of corneal endothelial cells (CECs) and showed that cell injection into the anterior chamber combined with ROCK inhibitor enabled the transplantation of cultivated CECs without a substrate (ARVO, 2009). This current study was conducted to examine how the ROCK signaling regulates cell adhesion of corneal endothelial cells in vitro.

Methods: : CECs of cynomolgus monkeys were cultured. ROCK-I or ROCK-II genes were knocked-down by siRNA and cells were then seeded at a density of 3.0×103/cm2. Random siRNA was used as a control. The numbers of attached cells were evaluated by CellTiter-Glo® Luminescent Cell Viability Assay (Promega Corp., Madison, Wisconsin) after 24 hours. Next, cultured CECs without knockdown by siRNA were seeded with medium containing 10µM of cytochalasin D, a potent inhibitor of actin polymerization. CECs seeded without cytochalasin D were as a control. The numbers of attached cells were evaluated after 24 hours to examine the involvement of actin polymerization in the cell adhesion of CECs.

Results: : The mean numbers of attached cells were 420.3±79.1% in the ROCK-I siRNA group and 115.5±7.0% in the ROCK-II siRNA group as a ratio of the control siRNA group. The CellTiter-Glo® assay revealed that ROCK-I siRNA significantly enhanced cell adhesion of CECs (p<0.01). Inhibition of actin polymerization by cytochalasin D also demonstrated that cell adhesion was significantly enhanced (199.3±18.6% as a ratio of control cells; p<0.01).

Conclusions: : The findings of this study demonstrate that activation of ROCK-I negatively regulates cell adhesion of CECs, possibly via the actin polymerization. We theorize that the regulation of ROCK activity enables cell injection therapy without the use of a substrate for the treatment of corneal endothelial dysfunction.

Keywords: cornea: endothelium • cornea: basic science • cell adhesions/cell junctions 
© 2011, The Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Permission to republish any abstract or part of an abstract in any form must be obtained in writing from the ARVO Office prior to publication.
Copyright © 2015 Association for Research in Vision and Ophthalmology.
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