June 2015
Volume 56, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2015
High glucose-induced decrease in claudin-5 expression is facilitated by connexin 43 (Cx43) downregulation in retinal endothelial cells.
Author Affiliations & Notes
  • Shruti Amin
    Boston University School of Medicine, Boston, MA
    American University of Antigua College of Medicine, New York, NY
  • Haares Mirzan
    Boston University School of Medicine, Boston, MA
  • Dongjoon Kim
    Boston University School of Medicine, Boston, MA
  • Sayon Roy
    Boston University School of Medicine, Boston, MA
  • Footnotes
    Commercial Relationships Shruti Amin, None; Haares Mirzan, None; Dongjoon Kim, None; Sayon Roy, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 4278. doi:
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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)

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Abstract

Purpose: 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).

Methods: 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.

Results: 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.

Conclusions: 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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