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Thomas Tien, Tetsuya Muto, Sayon Roy; Effects of High Glucose-Induced Cx43 Downregulation on Tight Junction Protein Expression and Tight Junction Barrier Characteristics in Retinal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):2415.
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© ARVO (1962-2015); The Authors (2016-present)
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.
Rat retinal endothelial cells (RRECs) were grown in normal medium (N; 5mM), high glucose medium (HG; 30 mM), HG transfected with Cx43 siRNA, or HG transfected with scrambled siRNA. Western blot (WB) analysis was performed to assess Cx43, occludin, and ZO-1 protein expression in the four groups of cells. In parallel, cells grown in above conditions 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 (76.1±13.4% of control). Cells grown in HG and transfected with Cx43 siRNA showed significant reduction in Cx43 expression (61.5±5.0% of control), whereas scrambled siRNA used as control showed no effect on Cx43 protein expression. Interestingly, compared to cells grown in HG alone, cells grown in HG and transfected with Cx43 siRNA showed further reduction in occludin and ZO-1 expression (79.6±5.6% of control, 80.81±13.3% of control, respectively vs. 72.0±7.3% of control, 41.2±8.0% of control, respectively). Similarly, IVP assay showed increased permeability in cells grown in HG compared to cells grown in N medium (116.8±4.8% of control). Importantly, compared to cells grown in HG alone, cells grown in HG and transfected with Cx43 siRNA showed greater increase in monolayer permeability (131.0±8.5% of control). Notably, cells grown in N and transfected with Cx43 siRNA resulted in decreased ZO-1 expression (70.5±13.9% of control) and increased cell monolayer permeability (113.1±7.9% of control).
Findings from this study indicate that HG-induced downregulation of Cx43 expression may influence tight junction protein expression and contribute to increased vascular permeability associated with diabetic retinopathy.
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