title: Epo inhibits the fibrosis and migration of Müller glial cells induced by TGF-β and high glucose.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Ophthalmology, Shanghai Tenth People's Hospital, Tongji University, Shanghai, China.
authors.forename: Wentao
authors.initials: W
authors.lastname: Luo
authors.affiliation: Department of Ophthalmology, Shanghai Tenth People's Hospital, Tongji University, Shanghai, China.
authors.forename: Liumei
authors.initials: L
authors.lastname: Hu
authors.affiliation: Department of Ophthalmology, Drexel University College of Medicine, Philadelphia, PA, USA.
authors.forename: Weiye
authors.initials: W
authors.lastname: Li
authors.affiliation: Department of Ophthalmology of Shanghai Tenth People's Hospital and Tongji Eye Institute, Tongji University School of Medicine, Shanghai, China.|Department of Regenerative Medicine and Stem Cell Research Center, Tongji University School of Medicine, Shanghai, China.|Institute for Nutritional Sciences, Tongji University School of Medicine, Shanghai, China.
authors.forename: Guotong
authors.initials: G
authors.lastname: Xu
authors.affiliation: Department of Ophthalmology, Shanghai Tenth People's Hospital, Tongji University, Shanghai, China.
authors.forename: Linxinyu
authors.initials: L
authors.lastname: Xu
authors.affiliation: Department of Ophthalmology, Shanghai Tenth People's Hospital, Tongji University, Shanghai, China.
authors.forename: Conghui
authors.initials: C
authors.lastname: Zhang
authors.affiliation: Department of Ophthalmology, Shanghai Tenth People's Hospital, Tongji University, Shanghai, China. wangfangopth@163.com.|Department of Ophthalmology, Shanghai Tenth People's Hospital, Tongji University School of Medicine, 301 Yanchang Road, 15th floor, Building 1, Shanghai, 200071, China. wangfangopth@163.com.
authors.forename: Fang
authors.initials: F
authors.lastname: Wang
mesh_terms: D000199:Actins / Q000378:metabolism; D015153:Blotting, Western; D002460:Cell Line; D002465:Cell Movement / Q000187:drug effects*; D055513:Connective Tissue Growth Factor / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D063928:Ependymoglial Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D004921:Erythropoietin / Q000494:pharmacology*; D005353:Fibronectins / Q000378:metabolism; D005355:Fibrosis / Q000517:prevention & control; D019084:Fluorescent Antibody Technique, Indirect; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D005947:Glucose / Q000494:pharmacology*; D006801:Humans; D020780:Matrix Metalloproteinase 9 / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000502:physiology*
chemical_list: C541116:ACTA2 protein, human; D000199:Actins; C526317:CCN2 protein, human; D005353:Fibronectins; D005904:Glial Fibrillary Acidic Protein; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta; D004921:Erythropoietin; D055513:Connective Tissue Growth Factor; C579270:MMP9 protein, human; D020780:Matrix Metalloproteinase 9; D005947:Glucose
keywords: Epiretinal membrane formation; Erythropoietin; Müller glial cells; Phenotype; Proliferative Diabetic Retinopathy