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Naoki Okumura, Shinichiro Nakano, EunDuck P. Kay, Ryohei Numata, Aya Ota, Yoshihiro Sowa, Toshiyuki Sakai, Morio Ueno, Shigeru Kinoshita, Noriko Koizumi; Involvement of Cyclin D and p27 in Cell Proliferation Mediated by ROCK Inhibitors Y-27632 and Y-39983 During Corneal Endothelium Wound Healing. Invest. Ophthalmol. Vis. Sci. 2014;55(1):318-329. doi: 10.1167/iovs.13-12225.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate the molecular mechanism of Rho-associated kinase (ROCK) inhibitors Y-27632 and Y-39983 on corneal endothelial cell (CEC) proliferation and their wound-healing effect.
The expression of G1 proteins of the cell cycle and expression of phosphorylated Akt in monkey CECs (MCECs) treated with Y-27632 were determined by Western blotting. The effect of Y-39983 on the proliferation of MCECs and human CECs (HCECs) was evaluated by both Ki67 staining and incorporation of BrdU. As an in vivo study, Y-39983 was topically instilled in a corneal-endothelial partially injured rabbit model, and CEC proliferation was then evaluated.
Investigation of the molecular mechanism of Y-27632 on CEC proliferation revealed that Y-27632 facilitated degradation of p27Kip1 (p27), and promoted the expression of cyclin D. When CECs were stimulated with Y-27632, a 1.7-fold increase in the activation of Akt was seen in comparison to the control after 1 hour. The presence of LY294002, the PI 3-kinase inhibitor, sustained the level of p27. When the efficacy of Y-39983 on cell proliferation was measured in a rabbit model, Y-39983 eye-drop instillation demonstrated rapid wound healing in a concentration range of 0.095 to 0.95 mM, whereas Y-27632 demonstrated rapid wound healing in a concentration range of 3 to 10 mM.
These findings show that ROCK inhibitors employ both cyclin D and p27 via PI 3-kinase signaling to promote CEC proliferation, and that Y-39983 may be a more potent agent than Y-27632 for facilitating corneal endothelium wound healing.
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