Epo inhibits the fibrosis and migration of Müller glial cells induced by TGF-β and high glucose

• DOI: 10.1007/s00417-016-3290-5
• Publication Date: 2016-02-23T00:16:14Z
• Authors: Wentao Luo; Liumei Hu; Weiye Li; Guotong Xu; Linxinyu Xu; Conghui Zhang; Fang Wang
• Keywords: Blotting, Western; Ependymoglial Cells; Connective Tissue Growth Factor; Enzyme-Linked Immunosorbent Assay; Real-Time Polymerase Chain Reaction; Fibrosis; Actins; Cell Line; Fibronectins; 3. Good health; 03 medical and health sciences; Glucose; 0302 clinical medicine; Matrix Metalloproteinase 9; Cell Movement; Transforming Growth Factor beta; Glial Fibrillary Acidic Protein; Humans; RNA, Messenger; Fluorescent Antibody Technique, Indirect; Erythropoietin

In proliferative diabetic retinopathy (PDR), Müller glial cells (MGCs) acquire migratory ability and exhibit a fibroblast-like phenotype. These activated MGCs contribute to the formation of epiretinal membrane, which will stretch the retina, and cause retinal detachment and vitreous hemorrhage. Erythropoietin (Epo) is now found effective in ameliorating renal fibrosis by inhibiting epithelial-to-mesenchymal transition of tubular epithelial cells. This study is undertaken to determine whether Epo has an effect in inhibiting MGCs activation to attenuate epiretinal membrane formation in PDR.MIO-M1 cell line was used in this study. As a pilot test to determine the most efficient treatment time and concentration of Epo, levels of connective tissue growth factor (CTGF) and transforming growth factor-β (TGF-β) were measured by real-time PCR, after treatment with Epo on MGCs cultured in high glucose. MGCs were cultured in high glucose and normal glucose for 2 days, with or without TGF-β as a pro-fibrogenic cytokine. Epo was introduced at the same time. Immunofluorescence targeting α-smooth muscle actin (α-SMA), fibronectin, and glial fibrillary acidic protein (GFAP) was performed to explore the cell phenotype. Matrix metalloproteinase 9 (MMP9) mRNA level was detected by real-time PCR. Protein levels of CTGF and cytoskeletal proteins like α-SMA and fibronectin were measured by enzyme-linked immunosorbent assay (ELISA) and Western blot respectively. Wound-healing assay was applied to evaluate the migratory ability of MGCs, and actin-tracker green was used to draw the structure of F-actin in MGCs.After being seeded into high-glucose medium containing TGF-β, MGCs expressed a larger amount of MMP9 mRNA as well as α-SMA, fibronectin at protein level. They secreted more CTGF, and their F-actin reorganized in a parallel manner and showed a stronger ability to migrate. In addition, these changes, including mRNA and protein expression, F-actin assembling, and cell migration, could be attenuated significantly by Epo treatment.High glucose together with TGF-β promote MGCs to exhibit a fibroblast-like phenotype and develop a greater migratory ability. These changes can be inhibited by Epo, which therefore may contribute to the controlling of epiretinal membrane formation.