Authors: Guo Feng, Zhu Xiaoyan, Chen Xia, Zhang Yong, Tan Yan, Wu Xiaofeng, Liu Rui, Li Lei, Xian Guangjun, Xie Lin
Researches showed that transforming growth factor-β2 (TGF-β2) promotes the activity of human Tenon capsular fibroblasts (TFs), which plays a role in the scarring of filtering blebs after anti-glaucoma surgery.However, its mechanism is not fully clear.Lysyl oxidases (LOXs) are important extracellular matrix proteases which can catalyze the cross-linking of collagen and elastin.Investigating the impact of TGF-β2 on the expression of LOXs has a great significance for the understanding of the pathogenesis of filtering bleb scarring and its prevention.
This study was to investigate the effect of TGF-β2 on the expression of LOXs in cultured human TFs.
The TFs at 4-8 generations were divided into normal control group and different concentrations of TGF-β2 treated-groups, and 100, 200, 400, 800 μl of TGF-β2 with the final concentration of 2, 4, 8 and 16 ng/ml was added into the medium to treat human TFs respectively for 24 hours.The LOXs in the cells were detected by Western blot to determine the optimal dose of TGF-β2.The 4 ng/ml TGF-β2 (200 μl) was used to treat human TFs for 6, 12, 24 and 48 hours respectively, and the change of LOXs expression in the cells over time was assayed by Western blot.The expression and distribution of LOX protein in the normal cells and TGF-β2-treated cells was detected by using immunofluorescence technique.This study was approved by Daping Hospital of Third Military Medical University Ethic Commission.The guardians of the patients who offered the specimen knew the purpose of the study and signed informed consent.
Western blot assay showed that the expressions of LOX, LOXL1, LOXL2, LOXL3 and LOXL4 in the cells were gradually elevated from the normal control group and 2, 4, 8, 16 ng/ml TGF-β2-treated groups, showing significant differences among the groups (F=37.338, 13.438, 31.067, 11.767, 15.167, all at P<0.01). The expression of LOXL2 protein in the cells was 0.68±0.07, 1.09±0.10, 1.32±0.07, 1.50±0.06 and 1.89±0.12 in the normal control group and 6-, 12-, 24- and 48-hour groups respectively after 4 ng/ml TGF-β2 treatment, with a significant increase over time (F=82.832, P=0.000). The expression of LOX was weak in the normal cultured TFs, while the fluorescence intensity of LOX expression was evidently enhanced in the cytoplasm of the cells in the TGF-β2-treated group.
TGF-β2 upregulates the expressions of LOXs in human TFs in a dose- and time-dependent manner, which probably offers a basis for the further study on the prevention of filtering bleb scarring after glaucoma surgery.