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Akiko Futakuchi, Toshihiro Inoue, Tomokazu Fujimoto, Miyuki Mochita Inoue, Hidenobu Tanihara; Effects of ripasudil (K-115), a Rho kinase inhibitor, on the activation of human conjunctival fibroblasts. Invest. Ophthalmol. Vis. Sci. 2016;57(12):2929.
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© ARVO (1962-2015); The Authors (2016-present)
Ripasudil, a selective Rho kinase inhibitor, is an ophthalmic solution which was approved in Japan for the twice-daily treatment of glaucoma and ocular hypertension in 2014. The purpose of this study is to assess the effects of ripasudil on the activation of human conjunctival fibroblasts.
Human conjunctival fibroblasts were pretreated with or without different concentrations of ripasudil (25 and 50 μM) for 1 hour and subsequently stimulated with 5 ng/ml TGF-β2 for 48 hours. The effects of ripasudil on α-smooth muscle actin (α-SMA) expression and extracellular matrix (ECM) expression were analyzed by Western blot analysis. Contractile activity was evaluated by collagen gel contraction assay. Cell viability and cytotoxicity were assessed using WST-8 assay and Hoechst 33342/propidium iodide (PI) dual staining, respectively. The human monocytic cell line THP-1 were differentiated into M1- and M2-like macrophages, and fibroblasts were treated with conditioned medium derived from these macrophages in the presence or in the absence of 50 μM ripasudil to quantify the α-SMA expression level.
TGF-β2-treated fibroblasts showed a significant increase (p < 0.0001), 38 ± 4-fold in the expression of α-SMA compared with controls without TGF-β2 stimulation. When fibroblasts were pretreated with ripasudil before TGF-β2 stimulation, α-SMA expression was significantly decreased in a dose-dependent manner (25 and 50 μM, respectively, p < 0.01). Ripasudil pretreatment significantly attenuated TGF-β2-induced fibronectin expression (p < 0.0001). Ripasudil pretreatment significantly attenuated TGF-β2-induced collagen gel contraction (p < 0.0001). TGF-β2 increased the proliferation of fibroblasts (p < 0.0001), and the effect was significantly attenuated by ripasudil (p < 0.0001). Ripasudil did not cause any cellular toxicity at the tested doses. Conditioned medium from M2-like macrophages induced a significant increase (p < 0.001), 3.4 ± 0.7-fold in the expression of α-SMA, and ripasudil suppressed this upregulated expression (p < 0.01).
Ripasudil attenuated the activation of human conjunctival fibroblasts. Our results suggest that ripasudil might have a therapeutic potential in the prevention of excessive scarring after glaucoma filtration surgery.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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