Abstract
Purpose:
To investigate whether high glucose (HG) induced downregulation of connexin 43 (Cx43), a gap junction protein, alters claudin-5 expression and cell monolayer permeability
Methods:
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.
Results:
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).
Conclusions:
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.
Keywords: 499 diabetic retinopathy •
498 diabetes