title: Tissue factor induces VEGF expression via activation of the Wnt/β-catenin signaling pathway in ARPE-19 cells.
abstract: The purpose of the present study was to investigate the potential signal mechanism of tissue factor (TF) in the regulation of the expression of vascular endothelial growth factor (VEGF) in human retinal pigment epithelial (ARPE-19) cells.
  
  
  
  An in vitro RPE cell chemical hypoxia model was established by adding cobalt chloride (CoCl2) in the culture medium. The irritative concentration of CoCl2 was determined with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay kit. VEGF production in ARPE-19 cells was measured with enzyme-linked immunosorbent assay (ELISA) and western blotting. The Wnt signaling pathway-associated molecules, including phospho-glycogen synthase kinase 3β (p-GSK3β), GSK3β, p-β-catenin and β-catenin, were detected with western blotting. pEGFP-N3-hTF was constructed and verified with digestion of the restriction enzyme and sequencing analysis. Human TF overexpression and silencing plasmids were transfected into the ARPE-19 cells to clarify the causal relationship between TF and VEGF expression. The Transwell coculture system of ARPE-19 cells and RF/6A rhesus macaque choroid-retinal endothelial cells was performed to evaluate cell invasion and tube formation ability.
  
  
  
  Our anoxic model of ARPE-19 cells showed that TF expression was upregulated in accordance with variations in hypoxia-inducible factor 1-alpha (HIF-1α) and VEGF levels. Silencing and overexpression of TF decreased and increased VEGF expression, respectively. The Wnt/β-catenin signaling pathway played an important role in this effect. Results from the ARPE-19 cell and RF/6A cell coculture system showed that the enhancement of TF expression in the ARPE-19 cells led to significantly faster invasion and stronger tube-forming ability of the RF/6A cells, while siRNA-mediated TF silencing caused the opposite effects. Pharmacological disruption of Wnt signaling IWR-1-endo inhibited the effects compared to the TF-overexpressing group, indicating the importance of the Wnt/β-catenin signaling pathway in the process of TF-induced VEGF expression and angiogenesis.
  
  
  
  Involvement of the activation of the Wnt/β-catenin signaling pathway is an important mediator for TF-induced VEGF production during the process of angiogenesis. Thus, our findings may ascertain the molecular regulation of TF in neovessel formation and show significant therapeutic implications.
authors: Eye Institute, Affiliated Hospital of Nantong University, Nantong, Jiangsu, China.
authors: Ying
authors: Y
authors: Wang
authors: Eye Institute, Affiliated Hospital of Nantong University, Nantong, Jiangsu, China.
authors: Aimin
authors: A
authors: Sang
authors: Eye Institute, Affiliated Hospital of Nantong University, Nantong, Jiangsu, China.
authors: Manhui
authors: M
authors: Zhu
authors: Eye Institute, Affiliated Hospital of Nantong University, Nantong, Jiangsu, China.
authors: Guowei
authors: G
authors: Zhang
authors: Eye Institute, Affiliated Hospital of Nantong University, Nantong, Jiangsu, China.
authors: Huaijin
authors: H
authors: Guan
authors: Eye Institute, Affiliated Hospital of Nantong University, Nantong, Jiangsu, China.
authors: Min
authors: M
authors: Ji
authors: Eye Institute, Affiliated Hospital of Nantong University, Nantong, Jiangsu, China.
authors: Hui
authors: H
authors: Chen
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002452:Cell Count; D015687:Cell Hypoxia; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D003035:Cobalt / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D000071679:Glycogen Synthase Kinase 3 beta / Q000378:metabolism; D006801:Humans; D008253:Macaca mulatta; D010957:Plasmids; D055213:Retinal Pigment Epithelium / Q000378:metabolism*; D013925:Thromboplastin / Q000502:physiology*; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*; D060449:Wnt Signaling Pathway / Q000502:physiology*; D051176:beta Catenin / Q000378:metabolism
chemical_list: C495270:CTNNB1 protein, human; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D051176:beta Catenin; D003035:Cobalt; D013925:Thromboplastin; D000071679:Glycogen Synthase Kinase 3 beta; C018021:cobaltous chloride