The effects of ripasudil (K-115), a Rho kinase inhibitor, on activation of human conjunctival fibroblasts

• Published in: Experimental eye research Vol. 149; pp. 107 - 115
• Main Authors: Futakuchi, Akiko, Inoue, Toshihiro, Fujimoto, Tomokazu, Inoue-Mochita, Miyuki, Kawai, Motofumi, Tanihara, Hidenobu
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.08.2016
• Subjects: Actins - biosynthesis; Blotting, Western; Cell Differentiation; Cell Division; Cells, Cultured; Cicatrix - metabolism; Cicatrix - pathology; Cicatrix - prevention & control; Conjunctiva - drug effects; Conjunctiva - metabolism; Conjunctiva - pathology; Culture Media, Conditioned; Fibroblasts - drug effects; Fibroblasts - metabolism; Fibroblasts - pathology; Filtering Surgery - adverse effects; Glaucoma; Glaucoma - surgery; Human conjunctival fibroblasts; Humans; Immunohistochemistry; Isoquinolines - pharmacology; Macrophage; Macrophages - metabolism; Macrophages - pathology; Myofibroblasts; Rho kinase inhibitor; rho-Associated Kinases - antagonists & inhibitors; Ripasudil (K-115); Signal Transduction; Sulfonamides - pharmacology; TGF-beta; Transforming Growth Factor beta2 - metabolism; Wound healing; Glaucoma; TGF-beta; Wound healing; Rho kinase inhibitor; Ripasudil (K-115); Human conjunctival fibroblasts; Myofibroblasts; Macrophage
• ISSN: 0014-4835; 1096-0007
• DOI: 10.1016/j.exer.2016.07.001

The most common cause of glaucoma surgery failure is scar formation induced by activation of wound-healing responses and resultant fibrosis at the surgical site. We investigated the effects of ripasudil, a Rho kinase inhibitor, on activation of human conjunctival fibroblasts (HConF). HConF were pretreated with different concentrations of ripasudil for 1 h before addition of transforming growth factor (TGF)-β2, followed by incubation for 48 h. TGF-β2-treated fibroblasts exhibited a significant increase in expression of α-smooth muscle actin (α-SMA), a marker of fibroblast-to-myofibroblast differentiation, and this increase was significantly suppressed, in a dose-dependent manner, by pretreatment with ripasudil. Ripasudil pretreatment also significantly attenuated TGF-β2-induced fibronectin production and collagen gel contraction. TGF-β2 increased both the number of viable cells and the number of cells in the G2/M phase of the cell cycle; these effects were attenuated by pretreatment with ripasudil. In addition, we explored the effects of ripasudil on stimulation of HConF by activated macrophages. Human monocytic cell line THP-1 cells were differentiated into M1 or M2 macrophage-like cells, and HConF were treated with conditioned media derived from these macrophages in the presence or absence of ripasudil. Conditioned medium from M2 macrophage-like cells induced a significant increase in α-SMA expression, viable cell numbers, and gel contraction, all of which were significantly suppressed by ripasudil. Thus, overall, ripasudil attenuated activation of human conjunctival fibroblasts. Ripasudil may be of therapeutic utility, preventing excessive scarring after glaucoma filtration surgery. •Ripasudil attenuated TGF-β- or macrophage-induced differentiation of HConF.•Collagen gel contraction or fibronectin production was inhibited by ripasudil.•Cell proliferation was suppressed by ripasudil with little cytotoxity.•Ripasudil may be useful for modulating post-surgical wound healing.