title: Ephrin-a4 is involved in retinal neovascularization by regulating the VEGF signaling pathway.
abstract: OBJECTIVE
  Retinal neovascularization (NV) is a major cause of blindness. Recent research suggests that factors other than VEGF participate in this process. This study aimed to determine the role of ephrin-A4 in retinal NV.
  
  
  METHODS
  The expression and effect of ephrin-A4 was investigated in a mouse model of oxygen-induced retinopathy (OIR) and the RF/6A retina endothelial cell line. Ephrin-A4 expression and VEGF signaling pathway phosphorylation were determined by PCR, immunohistochemistry, and western blot analyses. ShRNA was used to silence ephrin-A4 in vitro and in vivo. Retinal flat mounts and tube formation assays were performed to evaluate ephrin-A4 function in the NV process in vivo and in vitro.
  
  
  RESULTS
  Ephrin-A4 was overexpressed in the retina of OIR mice and in RF/6A and RPE cells after CoCl₂ stimulation. In vitro, Ephrin-A4/Fc treatment significantly increased the tube number of RF/6A cells on a membrane preparation and the phosphorylation levels of VEGR2, Akt1, and ERK1/2 in RF/6A cells. Moreover, ephrin-A4 knockout markedly suppressed pathologic neovascularization in vivo and inhibited the proliferation and tube formation capacity of RF/6A cells in vitro. Furthermore, in the absence of ephrin-A4, the phosphorylation of VEGFR2, Akt1, and ERK1/2 was defective under VEGF₁₆₅ stimulation, and the proangiogenic function of VEGF₁₆₅ was also compromised.
  
  
  CONCLUSIONS
  This study suggests that ephrin-A4 plays an important role in retinal NV and is a potential target against retinal NV. The proangiogenic function of ephrin-A4 may be linked to its crucial role in the VEGF signaling pathway.
authors.affiliation: Key Laboratory of Vision Loss and Restoration, Ministry of Education, Department of Ophthalmology, Peking University People's Hospital, Beijing, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Du
authors.forename: Wenzhen
authors.initials: W
authors.lastname: Yu
authors.forename: Lvzhen
authors.initials: L
authors.lastname: Huang
authors.forename: Min
authors.initials: M
authors.lastname: Zhao
authors.forename: Xiaoxin
authors.initials: X
authors.lastname: Li
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D015153:Blotting, Western; D002478:Cells, Cultured; D004195:Disease Models, Animal; D036385:Ephrin-A4 / Q000096:biosynthesis / Q000235:genetics*; D005500:Follow-Up Studies; D018507:Gene Expression Regulation, Developmental; D007150:Immunohistochemistry; D051379:Mice; D008810:Mice, Inbred C57BL; D012313:RNA / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D015861:Retinal Neovascularization / Q000378:metabolism* / Q000473:pathology; D055213:Retinal Pigment Epithelium / Q000378:metabolism / Q000473:pathology*; D015398:Signal Transduction / Q000235:genetics; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis / Q000235:genetics*
chemical_list: D036385:Ephrin-A4; D042461:Vascular Endothelial Growth Factor A; C467480:vascular endothelial growth factor A, mouse; D012313:RNA