Minli Wang1, Jiayi Wei1, Hui Li1, Fang Wang1,2. 1. Department of Ophthalmology, Shanghai Tenth People's Hospital, Tongji University School of Medicine Shanghai 200072, China. 2. Department of Ophthalmology, Shanghai Bright Eye Hospital Shanghai 200336, China.
Abstract
OBJECTIVE: This study aimed to investigate the changes in transepithelial electrical resistance (TEER) and ion concentrations, and their relationship in TGF-β-induced epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells. METHODS: RPE cell line ARPE-19 was employed and treated with 10 ng/ml TGF-β1 and TGF-β2 to establish the EMT model in vitro. The EMT markers fibronectin, N-cadherin, occludin, zona occludens 1(ZO-1) and claudin-19 were investigated by western blot and immunofluorescence. CellZscope system was used to monitor the TEER values. Fluorescent probe, flow cytometry and automatic microplate reader were employed to detect the changes of Ca2+, Mg2+, Zn2+, Na+ and K+ in ARPE-19 cells. RESULTS: The TGF-β1-induced EMT of ARPE-19 cells was marked by the disruption of the distribution of occludin, ZO-1, and claudin-19. The development of TEER was significantly disturbed in both TGF-β1 and TGF-β2 treatment groups. Also, the time course of the maximum slope indicated that the fastest decrease in TEER values occurred after 36 hours. The concentrations of Ca2+, Mg2+, Zn2+, and K+ increased in TGF-β1- and TGF-β2-treated ARPE-19 cells, while the concentration of Na+ decreased. Significant inverse correlations were detected between the concentrations of Ca2+, Mg2+, Zn2+, and K+ and TEER values in ARPE-19 cells treated with TGF-β1. The Na+ concentration and TEER values showed a positive correlation. Similar results were observed in the TGF-β2 treatment group. The time-effect analysis showed that the concentrations of Ca2+, Mg2+, Zn2+ and K+ increased and peaked after 72, 72, 48, and 72 h, respectively, with the extension of TGF-β1 treatment time. In the TGF-β2 treatment group, the Ca2+, Mg2+, Zn2+, and K+ concentrations were also upregulated and reached their highest after 72, 72, 72, and 36 h, respectively. In contrast, the concentration of Na+ decreased and reached the lowest after 48 h in the TGF-β1 treatment group and after 72 h in the TGF-β2 treatment group. CONCLUSION: TGF-β1 and TGF-β2 disrupted the ARPE-19 cell monolayer, disturbed TJs integrity, downregulated TEER values, and changed intracellular ion permeability. These findings might help further understand the EMT of RPE cells during PVR. AJTR
OBJECTIVE: This study aimed to investigate the changes in transepithelial electrical resistance (TEER) and ion concentrations, and their relationship in TGF-β-induced epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells. METHODS: RPE cell line ARPE-19 was employed and treated with 10 ng/ml TGF-β1 and TGF-β2 to establish the EMT model in vitro. The EMT markers fibronectin, N-cadherin, occludin, zona occludens 1(ZO-1) and claudin-19 were investigated by western blot and immunofluorescence. CellZscope system was used to monitor the TEER values. Fluorescent probe, flow cytometry and automatic microplate reader were employed to detect the changes of Ca2+, Mg2+, Zn2+, Na+ and K+ in ARPE-19 cells. RESULTS: The TGF-β1-induced EMT of ARPE-19 cells was marked by the disruption of the distribution of occludin, ZO-1, and claudin-19. The development of TEER was significantly disturbed in both TGF-β1 and TGF-β2 treatment groups. Also, the time course of the maximum slope indicated that the fastest decrease in TEER values occurred after 36 hours. The concentrations of Ca2+, Mg2+, Zn2+, and K+ increased in TGF-β1- and TGF-β2-treated ARPE-19 cells, while the concentration of Na+ decreased. Significant inverse correlations were detected between the concentrations of Ca2+, Mg2+, Zn2+, and K+ and TEER values in ARPE-19 cells treated with TGF-β1. The Na+ concentration and TEER values showed a positive correlation. Similar results were observed in the TGF-β2 treatment group. The time-effect analysis showed that the concentrations of Ca2+, Mg2+, Zn2+ and K+ increased and peaked after 72, 72, 48, and 72 h, respectively, with the extension of TGF-β1 treatment time. In the TGF-β2 treatment group, the Ca2+, Mg2+, Zn2+, and K+ concentrations were also upregulated and reached their highest after 72, 72, 72, and 36 h, respectively. In contrast, the concentration of Na+ decreased and reached the lowest after 48 h in the TGF-β1 treatment group and after 72 h in the TGF-β2 treatment group. CONCLUSION: TGF-β1 and TGF-β2 disrupted the ARPE-19 cell monolayer, disturbed TJs integrity, downregulated TEER values, and changed intracellular ion permeability. These findings might help further understand the EMT of RPE cells during PVR. AJTR
Authors: Emily E Brown; Alexander J DeWeerd; Cristhian J Ildefonso; Alfred S Lewin; John D Ash Journal: Redox Biol Date: 2019-04-20 Impact factor: 11.799