title: Thrombin induces epithelial-mesenchymal transition and collagen production by retinal pigment epithelial cells via autocrine PDGF-receptor signaling.
abstract: OBJECTIVE
  De-differentiation of RPE cells into mesenchymal cells (epithelial-mesenchymal transition; EMT) and associated collagen production contributes to development of proliferative vitreoretinopathy (PVR). In patients with PVR, intraocular coagulation cascade activation occurs and may play an important initiating role. Therefore, we examined the effect of the coagulation proteins factor Xa and thrombin on EMT and collagen production by RPE cells.
  
  
  METHODS
  Retinal pigment epithelial cells were stimulated with factor Xa or thrombin and the effect on zonula occludens (ZO)-1, α-smooth muscle actin (α-SMA), collagen, and platelet-derived growth factor (PDGF)-B were determined by real-time quantitative-polymerase chain reaction (RQ-PCR), immunofluorescence microscopy, and HPLC and ELISA for collagen and PDGF-BB in culture supernatants, respectively. PDGF-receptor activation was determined by phosphorylation analysis and inhibition studies using the PDGF-receptor tyrosine kinase inhibitor AG1296.
  
  
  RESULTS
  Thrombin reduced ZO-1 gene expression (P < 0.05) and enhanced expression of the genes encoding α-SMA and the pro-alpha1 chain of collagen type-1 (P < 0.05), indicating EMT. Also, ZO-1 protein expression declined on thrombin stimulation, whereas production of α-SMA and collagen increased. In contrast to thrombin, factor Xa hardly stimulated EMT by RPE. Thrombin clearly induced PDGF-BB production and PDGF-Rβ chain phosphorylation in RPE. Moreover, AG1296 significantly blocked the effect of thrombin on EMT and collagen production.
  
  
  CONCLUSIONS
  Our findings demonstrate that thrombin is a potent inducer of EMT by RPE via autocrine activation of PDGF-receptor signaling. Coagulation cascade-induced EMT of RPE may thus contribute to the formation of fibrotic retinal membranes in PVR and should be considered as treatment target in PVR.
authors.affiliation: Department of Immunology, Erasmus MC, Rotterdam, The Netherlands.
authors.forename: Jeroen
authors.initials: J
authors.lastname: Bastiaans
authors.forename: Jan C
authors.initials: JC
authors.lastname: van Meurs
authors.forename: Conny
authors.initials: C
authors.lastname: van Holten-Neelen
authors.forename: Nicole M A
authors.initials: NM
authors.lastname: Nagtzaam
authors.forename: P Martin
authors.initials: PM
authors.lastname: van Hagen
authors.forename: Rachel C
authors.initials: RC
authors.lastname: Chambers
authors.forename: Herbert
authors.initials: H
authors.lastname: Hooijkaas
authors.forename: Willem A
authors.initials: WA
authors.lastname: Dik
mesh_terms: D000199:Actins / Q000235:genetics / Q000378:metabolism; D019898:Autocrine Communication / Q000502:physiology*; D000077214:Becaplermin; D015153:Blotting, Western; D002454:Cell Differentiation; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D003094:Collagen / Q000096:biosynthesis*; D004797:Enzyme-Linked Immunosorbent Assay; D058750:Epithelial-Mesenchymal Transition / Q000187:drug effects*; D015951:Factor Xa / Q000494:pharmacology; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D010766:Phosphorylation; D020574:Proto-Oncogene Proteins c-sis / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D017479:Receptors, Platelet-Derived Growth Factor / Q000037:antagonists & inhibitors / Q000187:drug effects* / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000378:metabolism*; D013917:Thrombin / Q000494:pharmacology*; D020032:Tyrphostins / Q000494:pharmacology; D062826:Zonula Occludens-1 Protein / Q000235:genetics / Q000378:metabolism
chemical_list: C541116:ACTA2 protein, human; D000199:Actins; D020574:Proto-Oncogene Proteins c-sis; C569042:TJP1 protein, human; D020032:Tyrphostins; D062826:Zonula Occludens-1 Protein; C106250:6,7-dimethoxy-3-phenylquinoxaline; D000077214:Becaplermin; D003094:Collagen; D017479:Receptors, Platelet-Derived Growth Factor; D013917:Thrombin; D015951:Factor Xa
keywords: collagen; epithelial-mesenchymal transition (EMT); factor Xa; fibrosis; platelet-derived growth factor (PDGF); proliferative vitreoretinopathy (PVR); retinal pigment epithelium (RPE); thrombin; zonula occludens (ZO)-1; α-smooth muscle actin (α-SMA)