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Rhonda Sue Silva, Sayon Roy; Effects of High Glucose-Induced Cx43 Downregulation on Claudin-5 Expression and Cell Monolayer Permeability in Retinal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):4903.
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To investigate whether high glucose (HG) induced downregulation of connexin 43 (Cx43), a gap junction protein, alters claudin-5 expression and cell monolayer permeability
Rat retinal endothelial cells (RRECs) were grown in normal medium (N; 5mM), high glucose medium (HG; 30 mM), N transfected with Cx43 siRNA, or N transfected with scrambled siRNA for 7 days. Western blot (WB) analysis was performed to assess Cx43 and claudin-5 protein expression in the four groups of cells. In parallel, cells grown in above conditions and a fifth group of cells grown in N medium transfected with claudin-5 siRNA were subjected to in vitro permeability (IVP) assay to assess cell monolayer permeability.
Cx43 protein expression was significantly reduced in cells grown in HG medium compared to cells grown in N medium as indicated by WB analysis (64±13% of control, p<0.05). Cells grown in N medium and transfected with Cx43 siRNA showed as expected significant reduction in Cx43 expression (62±5% of control, p<0.05), whereas scrambled siRNA used as control showed no effect on Cx43 protein expression. Interestingly, compared to cells grown in N medium alone, cells grown in N medium and transfected with Cx43 siRNA showed significant reduction in claudin-5 expression (73±6% of control, p<0.05) but not actin expression. Similarly, IVP assay showed increased permeability in cell monolayers grown in HG compared to cells grown in N medium (127±5% of control, p<0.05). Interestingly, compared to cells grown in N medium alone, cells grown in N medium and transfected with Cx43 siRNA or claudin-5 siRNA showed increased monolayer permeability (129±6% of control, 147±5, p<0.05).
Findings from this study indicate that HG-induced downregulation of Cx43 expression can reduce claudin-5 expression and thereby contribute to increased vascular permeability associated with diabetic retinopathy.
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