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