title: Effects of high glucose-induced Cx43 downregulation on occludin and ZO-1 expression and tight junction barrier function in retinal endothelial cells.
abstract: OBJECTIVE
  To investigate whether high glucose (HG)-induced downregulation of connexin 43 (Cx43), a gap junction protein, alters ZO-1 and occludin expression and cell monolayer permeability.
  
  
  METHODS
  Rat retinal endothelial cells (RRECs) were grown in normal (N; 5 mM) medium, high glucose (HG; 30 mM) medium, N medium transfected with Cx43 siRNA, or N medium transfected with scrambled siRNA. To determine Cx43, occludin, and ZO-1 protein expression, Western blot (WB) analysis and immunostaining were performed. Gap junction intercellular communication (GJIC) was determined using scrape load dye transfer (SLDT) assay. In parallel, cell monolayer permeability was assessed in the four groups of cells, and in cells transfected with Cx43 plasmid or dominant negative Cx43 plasmid.
  
  
  RESULTS
  Connexin 43 protein expression was significantly reduced in cells grown in HG (67 ± 15% of control), and a significant reduction in Cx43 was achieved when cells grown in N medium were transfected with Cx43 siRNA (76 ± 12% of control), with concomitant decrease in GJIC activity. Cells grown in HG showed significant reduction in occludin (77 ± 9% of control) and ZO-1 (80 ± 11% of control) protein level compared with cells grown in N media. Importantly, cells transfected with Cx43 siRNA and grown in N medium showed significant downregulation in occludin (78 ± 8% of control) and ZO-1 (81 ± 6% of control) expression, and exhibited increased cell monolayer permeability. Furthermore, Cx43 upregulation protected cells against HG-induced excess cell monolayer permeability.
  
  
  CONCLUSIONS
  Our findings indicate that HG-induced downregulation of Cx43 expression and GJIC may contribute to the breakdown of endothelial barrier tight junctions associated with diabetic retinopathy.
authors.affiliation: Departments of Medicine and Ophthalmology, Boston University School of Medicine, Boston, Massachusetts.
authors.forename: Thomas
authors.initials: T
authors.lastname: Tien
authors.forename: Kevin F
authors.initials: KF
authors.lastname: Barrette
authors.forename: Argyrios
authors.initials: A
authors.lastname: Chronopoulos
authors.forename: Sayon
authors.initials: S
authors.lastname: Roy
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002450:Cell Communication / Q000187:drug effects / Q000235:genetics; D002478:Cells, Cultured; D018031:Connexin 43 / Q000096:biosynthesis / Q000187:drug effects / Q000235:genetics*; D003921:Diabetes Mellitus, Experimental; D003930:Diabetic Retinopathy / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D015536:Down-Regulation / Q000187:drug effects*; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D005947:Glucose / Q000494:pharmacology*; D062793:Occludin / Q000096:biosynthesis / Q000187:drug effects / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics*; D051381:Rats; D012160:Retina / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D019108:Tight Junctions / Q000187:drug effects; D062826:Zonula Occludens-1 Protein / Q000096:biosynthesis / Q000187:drug effects / Q000235:genetics*
chemical_list: D018031:Connexin 43; D062793:Occludin; D012333:RNA, Messenger; C569044:Tjp1 protein, rat; D062826:Zonula Occludens-1 Protein; D005947:Glucose
keywords: connexins; gap junctions; tight junctions