title: Targeting of integrin-linked kinase with small interfering RNA inhibits VEGF-induced angiogenesis in retinal endothelial cells.
abstract: BACKGROUND
  The pathological angiogenesis in the retina is a major cause of vision loss at all ages. Vascular endothelial growth factor (VEGF) has been reported as the most potent inducer of retinal neovascularization. We previously demonstrated that integrin-linked kinase (ILK) regulates retinal vascular endothelial proliferation, migration and tube formation. However, little is known about the existence of cross-talk between ILK and VEGF signaling in retinal vascular endothelial cells and the probable regulatory role of ILK during VEGF-induced retinal endothelial cell migration. The purpose of this work was to investigate the role of ILK in VEGF-induced retinal neovascularization.
  
  
  METHODS
  Cultured retinal endothelial cells (RF/6A) were knocked down for ILK using a small interfering RNA (siRNA). For this, cellular ILK expression was quantified by real-time quantitative PCR, Western blot analysis and immunocytochemical assay, and cytotoxicity of transfection was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. ILK siRNA-transfected RF/6A cells were induced by VEGF, and cell proliferation was determined by the MTT assay, cell migration was measured by cell counting in modified Boyden chambers and cell spreading and tube formation assays were performed. Furthermore, the impact of ILK-specific siRNA on VEGF-induced VEGF receptor 2 (VEGFR-2) phosphorylation and activation of downstream signal pathways were tested by Western blot analysis.
  
  
  RESULTS
  Both ILK mRNA and protein levels were virtually undetectable after transfection with ILK siRNA, and blocking the expression of ILK by siRNA significantly inhibited VEGF-induced retinal endothelial cell proliferation, attachment, spreading, migration and tube formation. Knockdown of ILK effectively suppressed VEGF-induced p38 mitogen-activated protein kinase (MAPK) and Akt phosphorylation, but had no effects on VEGFR-2, extracellular signal-regulated protein kinase and Jun N terminus kinase phosphorylation.
  
  
  CONCLUSIONS
  We conclude that knockdown of ILK with siRNA effectively inhibited VEGF-induced retinal endothelial cell attachment, spreading, migration and tube formation. p38 MAPK and Akt are downstream signaling pathways of the ILK that regulated VEGF-induced retinal neovascularization. Targeting ILK may be a potentially useful therapeutic approach for treating ocular neovascularization.
authors.affiliation: Department of Ophthalmology, Peking University People's Hospital, Beijing, China.
authors.forename: Wankun
authors.initials: W
authors.lastname: Xie
authors.forename: Min
authors.initials: M
authors.lastname: Zhao
authors.forename: Weiyan
authors.initials: W
authors.lastname: Zhou
authors.forename: Lili
authors.initials: L
authors.lastname: Guo
authors.forename: Lvzhen
authors.initials: L
authors.lastname: Huang
authors.forename: Wenzhen
authors.initials: W
authors.lastname: Yu
authors.forename: Xiaoxin
authors.initials: X
authors.lastname: Li
mesh_terms: D015153:Blotting, Western; D002465:Cell Movement; D049109:Cell Proliferation; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism; D020868:Gene Silencing; D006801:Humans; D007150:Immunohistochemistry; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000502:physiology; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology*; D060888:Real-Time Polymerase Chain Reaction; D015861:Retinal Neovascularization / Q000517:prevention & control*; D055213:Retinal Pigment Epithelium; D042461:Vascular Endothelial Growth Factor A / Q000037:antagonists & inhibitors*; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism; D000099200:Scaffold Protein ILK
chemical_list: D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D042461:Vascular Endothelial Growth Factor A; D048051:p38 Mitogen-Activated Protein Kinases; D000099200:Scaffold Protein ILK