Suppression of transforming growth factor-beta effects in rabbit subconjunctival fibroblasts by activin receptor-like kinase 5 inhibitor

Suppression of transforming growth factor-beta effects in rabbit subconjunctival fibroblasts by activin receptor-like kinase 5 inhibitor

Sapitro J; Dunmire J J; Scott S E; Sutariya V; Geldenhuys W J; Hewit M; Yue Byjt; Nakamura H

Molecular Vision

2010

2010-09

Purpose: Transforming growth factor-beta (TGF-beta) activity has been implicated in subconjunctival scarring in eyes following glaucoma filtration surgery (GFS). The purpose of this study is to determine whether an inhibitor for activin receptor-like kinase (ALK) 5 (also known as TGF-beta receptor type I) could suppress TGF-beta activity and thereby promote filtering bleb survival after GFS in a rabbit model. Methods: An ALK-5 inhibitor, SB-505124, was used. A docking study was performed to investigate the interaction between the inhibitor and the receptor. Immunofluorescence for connective tissue growth factor (CTGF) and alpha-smooth muscle actin (alpha-SMA) was performed in cultured rabbit subconjunctival fibroblasts. Immunoblotting for phosphorylated Smad2 (pSmad2), CTGF, and alpha-SMA was also performed. In an in vivo rabbit GFS model, SB-505124 was delivered in a lactose tablet during surgery. Eyes were examined by slit-lamp and intraocular pressure (IOP) was measured until the time of bleb failure or up to 28 days after surgery. Tissue sections on day 5 after surgery were histologically evaluated after staining with hematoxylin and eosin. The sections were also immunostained for CTGF and alpha-SMA. In addition, cell outgrowth from dissected subconjunctival tissues placed in a cell culture flask with media was investigated. Results: The docking study indicated hydrogen bond interactions between SB-505124 and amino acids His-283 and Ser-280 ALK-5. Suppression of pSmad2, CTGF, and alpha-SMA by SB-505124 was observed in cultured fibroblasts. Filtering blebs in the GFS with SB-505124 group were maintained for more than 10 days, and the period of bleb survival was significantly longer than that in controls. IOP levels after surgery seemed to be related to bleb survival. Histologically, subconjunctival cell infiltration and scarring at the surgical site in the GFS with SB-505124 and mitomycin C (MMC) groups were much subsided compared to controls. Suppression of CTGF and alpha-SMA by SB-505124 was also observed by immunofluorescence. Cell outgrowth from explants dissected from eyes to which SB-505124 was applied during GFS was robust while outgrowth was poor from those treated with MMC. Conclusions: The ALK-5 inhibitor SB-505124 was efficacious both in vitro and in vivo in suppressing the TGF-beta action. The inhibitor may provide a novel therapy for preventing ocular inflammation and scarring.

anti-scarring agent; Biochemistry & Molecular Biology; choroidal; extracellular-matrix; fibrosis; glaucoma; identification; in-vivo; mitomycin-c; neovascularization; Ophthalmology; Surgery; tgf-beta

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Journal Article

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1880-1892

204

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