title: Effect of Robo4 on retinal endothelial permeability.
abstract: OBJECTIVE
  To examine the role of Roundabout 4 (Robo4) in retinal endothelial permeability and analyze the structural events that lead to barrier disruption.
  
  
  METHODS
  Small interfering RNA (siRNA) technology was used to knockdown Robo4 expression to study its effects on the permeability of human retinal vascular endothelial cells (HRVECs) in vitro. The endothelial cell permeability was detected by measuring the flux of rhodamine B isothiocyanate (RITC)-dextran across the HRVEC monolayers. The impact of Robo4 siRNA on the expression of tight junction (TJ) proteins and the activation of LIM-kinase (LIMK)/cofilin pathway were measured by western blotting. The change of actin cytoskeleton was detected using indirect immunofluorescence.
  
  
  RESULTS
  Robo4 siRNA increased the permeability of HRVEC monolayers. The expression levels of TJ-associated proteins occludin and zonula occludens (ZO)-1 were suppressed in Robo4-depleted cells. In addition, there was rearrangement of F-actin in HRVECs. These processes were induced through increased activity in the LIMK/cofilin pathway which coincided with a disruption in the barrier properties of retinal endothelial monolayers.
  
  
  CONCLUSIONS
  Knockdown of Robo4 expression in HRVECs induced endothelial hyperpermeability associated with the downregulation of ZO-1, occludin, and the rearrangement of F-actin and that LIM-kinase 1 (LIMK1)/cofilin signal transduction system may be involved in the modulating process.
authors.affiliation: Department of Ophthalmology, Second Hospital of Jilin University, Changchun, Jilin, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Cheng
authors.forename: Zaoxia
authors.initials: Z
authors.lastname: Liu
authors.forename: Shurong
authors.initials: S
authors.lastname: Wang
authors.forename: Chenguang
authors.initials: C
authors.lastname: Wang
authors.forename: Shounan
authors.initials: S
authors.lastname: Qi
authors.forename: Jinsong
authors.initials: J
authors.lastname: Zhao
authors.forename: Rui
authors.initials: R
authors.lastname: Tian
authors.forename: Guanfang
authors.initials: G
authors.lastname: Su
mesh_terms: D000199:Actins / Q000378:metabolism; D015153:Blotting, Western; D002463:Cell Membrane Permeability / Q000502:physiology; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D008840:Microfilament Proteins / Q000378:metabolism; D034741:RNA, Small Interfering / Q000096:biosynthesis; D011956:Receptors, Cell Surface / Q000096:biosynthesis*; D012169:Retinal Vein / Q000166:cytology / Q000378:metabolism*; D019108:Tight Junctions / Q000378:metabolism; D000097905:Roundabout Proteins
chemical_list: D000199:Actins; D008840:Microfilament Proteins; D034741:RNA, Small Interfering; D011956:Receptors, Cell Surface; C462951:ROBO4 protein, human; D000097905:Roundabout Proteins