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Shruti Amin, Haares Mirzan, Dongjoon Kim, Sayon Roy; High glucose-induced decrease in claudin-5 expression is facilitated by connexin 43 (Cx43) downregulation in retinal endothelial cells.. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4278.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate whether high glucose (HG)-induced Cx43 downregulation alters claudin-5 expression and affects monolayer permeability and barrier characteristics in rat retinal endothelial cells (RRECs).
Rat retinal endothelial cells (RRECs) were grown in normal (N; 5 mM) or high glucose (HG; 30 mM) medium for 7 days, and in parallel, cells grown in N medium were transfected with either Cx43 siRNA or scrambled siRNA as control. Western blot analysis was performed to assess Cx43 and claudin-5 protein expression in the four groups of RRECs. Additionally, to determine Cx43 and claudin-5 association, total protein isolated from cells grown in N or HG medium was subjected to co-immunoprecipitation (co-IP) using Cx43 antibody, followed by Western blot analysis using Cx43 and claudin-5 primary antibodies.
Western blot analysis indicated significant reduction in Cx43 protein level in cells grown in HG medium (64% of control; p<0.05), and as expected, cells transfected with Cx43 siRNA and grown in N medium showed reduced Cx43 protein level (61% of control; p<0.05). Claudin-5 expression was significantly reduced in cells grown in HG medium (75% of control; p<0.05). Importantly, cells transfected with Cx43 siRNA showed significant downregulation in claudin-5 expression (77% of control; p<0.05). The presence of claudin-5 after co-IP with Cx43 antibody was detected. Furthermore, co-IP results indicated that claudin-5 level was reduced in the HG cells (80% of control; p<0.05) at least in part due to HG-induced reduction in Cx43 and also its physical association with Cx43.
HG-induced downregulation of Cx43 decreases claudin-5 level and compromises tight junction barrier characteristic in RRECs. Cx43 upregulation may not only improve gap junction intercellular communication activity, but also ultimately prevent HG-induced excess vascular permeability associated with diabetic retinopathy.
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