Purchase this article with an account.
Jessica Moon, Hilary P. Knack, Sayon Roy; Effect of Inhibiting High Glucose-induced Upregulation of ECM on Connexin 43 (Cx43) Expression in Retinal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3554.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Apoptotic vascular cell death in the retina is a characteristic of early diabetic retinopathy (DR). Previous studies have shown that high glucose upregulates basement membrane (BM) component expression and downregulates gap junction protein (Cx43) expression; however, it is unclear whether a link exists between the two expressions. In this study, we determined whether HG-induced overexpression of extracellular matrix (ECM) components, fibronectin (FN) and collagen IV (Coll IV), regulates Cx43 expression/gap junction intercellular communication (GJIC) in retinal endothelial cells.
Rat retinal endothelial cells were grown in normal (5mM) glucose or high glucose (HG; 30mM) medium for 8 days. In parallel, cells grown in HG were transfected with FN siRNA, Coll IV siRNA, or scrambled siRNA as control. Protein isolated from these 5 groups of cells was analyzed for FN, Coll IV, and Cx43 expression using Western blot (WB) analysis. In parallel, Scrape Load Dye Transfer (SLDT) assays were conducted to determine GJIC. TUNEL assays were performed to determine whether reduction of the abnormally high FN and coll IV expression improved GJIC and subsequently rescued cells from apoptosis. To determine the effects of altered ECM production on Cx43 localization and distribution, cells were immunostained for FN, Coll IV, and Cx43.
WB analysis indicated that FN and Coll IV expression was significantly increased in cells grown in HG medium compared to those grown in normal medium. When cells grown in HG medium were transfected with FN siRNA or Coll IV siRNA, a significant reduction in their protein levels was observed; scrambled siRNA had no effect. As expected, HG cells showed decreased Cx43 expression. Interestingly, the HG cells transfected with FN siRNA or Coll IV siRNA, showed an upregulation of Cx43 expression and improved GJIC. This finding was supported by immunofluorescence studies in which Cx43 immunostaining was increased in HG cells transfected with FN siRNA or Coll IV siRNA. TUNEL assays indicated that HG cells transfected with FN siRNA or Coll IV siRNA exhibited reduced number of apoptotic cells.
Results from this study indicate that HG-induced overexpression of BM components may reduce Cx43 expression and promote apoptosis in the retinal endothelial cells. This finding provides a better understanding of the association between ECM upregulation and Cx43 downregulation in the pathogenesis of DR.
This PDF is available to Subscribers Only