The Efficacy of Atelocollagen to Inhibit Fibrotic Proliferation in Tenon Tissue: in vitro study

Abstract

Introduction: To evaluate the safety and efficacy of atelocollagen in preventing the fibrotic change of human tenon tissue induced by transforming growth factor β1 (TGFβ1) Methods: Primary cultured human Tenon’s fibroblasts (HTFs) were incubated with TGFβ1 alone, and with a various concentrations of atelocollagen respectively. Cell viability was measured by cell counting kit-8 (CCK8). The mRNA levels of α-smooth muscle actin (α-SMA), vimentin, fibronectin, zonular occludens scaffolding protein (ZO-1), cellular communication network factor 2 (CCN2) and interleukin-6 (IL-6) were measured by quantitative reverse transcription polymerase chain reaction (RT-PCR), western blot and immunofluorescence analysis. Wound healing assay and collagen contraction assay were additionally evaluated for identifying the inhibitory effect of atelocollagen in HTFs. To elucidate the mechanism by which atelocollagen affects HTFs proliferation, the phospho-extracellular-signal-regulated kinases (pERK)/total-extracellular-signal-regulated kinases (tERK), phospho-focal adhesion kinase (pFAK)/total-focal adhesion kinase (tFAK), and pSmad3/tSmad3 protein expression ratios were measured by Western blot. Results: The safety of atelocollagen in HTF was identified by CCK8 analysis. The expression of α-SMA and vimentin in HTFs treated with 0.023% and 0.046% atelocollagen significantly decreased at both mRNA and protein levels, while that of ZO-1 in 0.046% atelocollagen increased compared with TGFβ1-treated cells. The protein expression of fibronectin, CCN2, and IL-6 in HTFs treated with 0.023% and 0.046% atelocollagen significantly decreased. Immunofluorescence microscopy of α-SMA and ZO-1 showed results similar to those of the western blot. In the wound scratch assays, cell migration was significantly attenuated in HTFs treated with 0.005% atelocollagen. Atelocollagen at 0.005, 0.011, and 0.023% significantly inhibited the gel contraction induced by TGFβ1 at both 24 h and 48 h. The increase in pERK/tERK and pSmad3/tSmad3 protein expression ratios in TGFβ1-treated HTFs significantly decreased after treatment with 0.023 and 0.046% atelocollagen. Conclusion: Since atelocollagen gel effectively suppresses the proliferation of HTFs in TGFβ1 - induced transdifferentiation, it may be a potential therapeutic agent in glaucoma surgery.