title: The effect of connexin43 on the level of vascular endothelial growth factor in human retinal pigment epithelial cells.
abstract: BACKGROUND
  Connexins (Cx) are the basic units of gap junctions and contribute to cellular integrity by promoting intercellular communication. Disruption of the retinal pigment epithelial monolayer may be an early event in the pathogenesis of age-related macular degeneration, a condition in which vascular endothelial growth factor (VEGF) is known to be of importance. This study was designed to assess the effect of connexin43 (Cx43) expression and gap junctional intercellular communication (GJIC) on the expression and secretion of VEGF from the retinal pigment epithelium under normal cell culture and oxidative stress conditions.
  
  
  METHODS
  Stable cell lines of ARPE-19 were produced in which wild-type Cx43 was either over-expressed, down-regulated by targeted shRNA, or functionally inhibited by co-expression of a disease-linked dominant-negative mutant (G21R). Pharmacologic blockade of GJIC was accomplished with flufenamic acid. Oxidant challenge was performed with tert-butyl hydroperoxide (tBH). VEGF gene expression and secretion were assessed by real-time PCR and ELISA respectively.
  
  
  RESULTS
  Over-expression of Cx43 in ARPE-19 cells reduced both gene expression and secretion of VEGF. Down-regulation of Cx43 increased gene expression and secretion of VEGF. Increased secretion of VEGF was also observed in ARPE-19 cells expressing a dominant-negative mutant of Cx43, and when GJIC was blocked. Over-expression of Cx43 reduced tBH-induced secretion of VEGF from ARPE-19 cells.
  
  
  CONCLUSIONS
  These studies show that Cx43 protects against oxidative stress-induced VEGF secretion in ARPE-19 cells, and thus has important implications in understanding the pathogenesis of age-related macular degeneration.
authors.affiliation: Department of Ophthalmology, University of Western Ontario, Ivey Eye Institute, St. Joseph's Hospital, 268 Grosvenor Street, London, ON, Canada.
authors.forename: Cady E
authors.initials: CE
authors.lastname: Pocrnich
authors.forename: Qing
authors.initials: Q
authors.lastname: Shao
authors.forename: Hong
authors.initials: H
authors.lastname: Liu
authors.forename: Mary M
authors.initials: MM
authors.lastname: Feng
authors.forename: Sarah
authors.initials: S
authors.lastname: Harasym
authors.forename: Melissa
authors.initials: M
authors.lastname: Savage
authors.forename: Sarit
authors.initials: S
authors.lastname: Khimdas
authors.forename: Dale W
authors.initials: DW
authors.lastname: Laird
authors.forename: Cindy M L
authors.initials: CM
authors.lastname: Hutnik
mesh_terms: D015153:Blotting, Western; D002460:Cell Line; D018031:Connexin 43 / Q000494:pharmacology*; D004797:Enzyme-Linked Immunosorbent Assay; D005439:Flufenamic Acid / Q000494:pharmacology; D017629:Gap Junctions / Q000187:drug effects; D006801:Humans; D018384:Oxidative Stress; D012333:RNA, Messenger / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D055213:Retinal Pigment Epithelium / Q000187:drug effects* / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics* / Q000378:metabolism*; D020122:tert-Butylhydroperoxide / Q000494:pharmacology
chemical_list: D018031:Connexin 43; D012333:RNA, Messenger; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D005439:Flufenamic Acid; D020122:tert-Butylhydroperoxide