title: Retinal pigment epithelium and endothelial cell interaction causes retinal pigment epithelial barrier dysfunction via a soluble VEGF-dependent mechanism.
abstract: OBJECTIVE
  To investigate the effect of endothelial cells (EC) on the barrier function of the retinal pigment epithelium (RPE).
  
  
  METHODS
  Primary bovine RPE were grown in solo culture or in coculture with bovine EC. Culture media of RPE were varied to develop a monolayer with stable barrier properties determined by transepithelial electrical resistance (TER) and permeability to sodium fluorescein. The effect of EC on the barrier properties of RPE was tested in contacting and non-contacting cocultures of RPE and EC. The conditioned media of cocultures were analysed for soluble vascular endothelial growth factor (VEGF) by ELISA. A neutralizing antibody to VEGF(165) was added to cocultures of RPE and EC and the TER was measured.
  
  
  RESULTS
  RPE had maximal barrier properties (high TER, low permeability, positive staining for barrier proteins) at day 10 that persisted until day 20. Compared to solo RPE culture, cocultivation of RPE with EC reduced RPE barrier function significantly and led to a greater release of soluble VEGF into the conditioned media (p<0.05). Neutralizing VEGF with antibody led to partial recovery of barrier properties in the coculture conditions (p<0.03).
  
  
  CONCLUSIONS
  Coculture of RPE with EC reduces RPE barrier properties and the reduction is, in part, mediated by soluble VEGF. EC-RPE contact-induced disruption of barrier properties occurs in ocular pathologies such as choroidal neovascularization, where EC move through Bruch's membrane and contact the RPE, leading to further exacerbation of the already compromised blood-retinal barrier.
authors.affiliation: Department of Ophthalmology, University of North Carolina, 5109D Bioinformatics Building, 130 Mason Farm Road, CB #7040, Chapel Hill, NC 27599-7040, USA. maryelizabeth_hartnett@med.unc.edu
authors.forename: M Elizabeth
authors.initials: ME
authors.lastname: Hartnett
authors.forename: Alexandra
authors.initials: A
authors.lastname: Lappas
authors.forename: Diane
authors.initials: D
authors.lastname: Darland
authors.forename: Janet R
authors.initials: JR
authors.lastname: McColm
authors.forename: Stephanie
authors.initials: S
authors.lastname: Lovejoy
authors.forename: Patricia A
authors.initials: PA
authors.lastname: D'Amore
mesh_terms: D000818:Animals; D001813:Blood-Retinal Barrier / Q000502:physiology*; D002417:Cattle; D002450:Cell Communication; D002478:Cells, Cultured; D020256:Choroidal Neovascularization / Q000503:physiopathology*; D018920:Coculture Techniques; D017077:Culture Media, Conditioned; D004730:Endothelium, Vascular / Q000166:cytology / Q000503:physiopathology*; D010857:Pigment Epithelium of Eye / Q000503:physiopathology*; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis / Q000502:physiology*
chemical_list: D017077:Culture Media, Conditioned; D042461:Vascular Endothelial Growth Factor A