Ye Liu1, Xiao-Jing Zhao2, Xiao-Shuo Zheng2, Hui Zheng2, Lei Liu3, Ling-Bin Meng4, Qin Li2, Yang Liu2. 1. Department of Pathology, the Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai 519000, Guangdong Province, China. 2. Department of Ophthalmology, the Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai 519000, Guangdong Province, China. 3. Department of Ophthalmology, the First Hospital of Jilin University, Jilin 130021, Jilin Province, China. 4. Department of Internal Medicine, Florida Hospital, Orlando, Florida 32803, USA.
Abstract
AIM: To determine if tranilast affects human corneal fibroblast (HCFs) contraction. METHODS: HCFs cultured in a three-dimensional type I collagen gel were treated with or without transforming growth factor beta (TGF-β) or tranilast. Gel diameter was measured as an indicator for collagen contraction. Immunoblot was performed to evaluate myosin light chain (MLC) and paxillin phosphorylation. Confocal microscopy was employed to examine the focal adhesions and actin stress fiber formation. Immunoblot analysis and gelatin zymography were performed to detect tissue inhibitors of metalloproteinases and matrix metalloproteinases (MMPs) in supernatant. RESULTS: The inhibitory effect of tranilast on HCFs-mediated collagen gel contraction induced by TGF-β was dose-dependent. The significant effect of tranilast was started from 100 µmol/L and maximized at 300 µmol/L. The peak effect of 300 µmol/L tranilast also relied on the duration of treatment, which showed statistical significance from day 2. TGF-β-induced paxillin and MLC phosphorylation, stress fiber formation, focal adhesions, and MMP-1, MMP-2, and MMP-3 secretion in HCFs were also inhibited by tranilast. CONCLUSION: Tranilast suppresses the HCFs-cultured collagen gel contraction induced by TGF-β. It attenuates actin stress fibers formation, focal adhesions, and the secretion of MMPs, with these actions likely contributing to the inhibitory effect on HCF contractility. By attenuating the contractility of corneal fibroblasts, tranilast treatment may inhibit corneal scarring.
AIM: To determine if tranilast affects human corneal fibroblast (HCFs) contraction. METHODS: HCFs cultured in a three-dimensional type I collagen gel were treated with or without transforming growth factor beta (TGF-β) or tranilast. Gel diameter was measured as an indicator for collagen contraction. Immunoblot was performed to evaluate myosin light chain (MLC) and paxillin phosphorylation. Confocal microscopy was employed to examine the focal adhesions and actin stress fiber formation. Immunoblot analysis and gelatin zymography were performed to detect tissue inhibitors of metalloproteinases and matrix metalloproteinases (MMPs) in supernatant. RESULTS: The inhibitory effect of tranilast on HCFs-mediated collagen gel contraction induced by TGF-β was dose-dependent. The significant effect of tranilast was started from 100 µmol/L and maximized at 300 µmol/L. The peak effect of 300 µmol/L tranilast also relied on the duration of treatment, which showed statistical significance from day 2. TGF-β-induced paxillin and MLC phosphorylation, stress fiber formation, focal adhesions, and MMP-1, MMP-2, and MMP-3 secretion in HCFs were also inhibited by tranilast. CONCLUSION: Tranilast suppresses the HCFs-cultured collagen gel contraction induced by TGF-β. It attenuates actin stress fibers formation, focal adhesions, and the secretion of MMPs, with these actions likely contributing to the inhibitory effect on HCF contractility. By attenuating the contractility of corneal fibroblasts, tranilast treatment may inhibit corneal scarring.
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