Purchase this article with an account.
Kevin Barrette, Lucky Challyandra, Sayon Roy; Connexin 43 (Cx43) Upregulation Protects Retinal Endothelial Cells Against High Glucose Insult. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2689.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
To determine if upregulation of Cx43 prevents high glucose-induced apoptosis and excess monolayer permeability in rat retinal endothelial cells (RRECs).
RRECs were grown in normal (5 mM glucose; N) or high glucose (30 mM; HG) medium for 7 days. In parallel, cells grown in either N or HG medium were transfected with plasmid pEGFPN1 containing full-length rat Cx43 cDNA conjugated to green fluorescent protein (GFP) using a lipofectamine reagent. To select stable colonies, transfected cells were grown in G418. To determine Cx43 expression in the transfected cells, Cx43 immunofluorescence microscopy (IF) and Western blot analysis were performed. Additionally, cells were assessed for changes in gap junction intercellular communication (GJIC) using a scrape loading dye transfer assay (SLDT). To identify cells undergoing apoptosis, differential staining with acridine orange/ethidium bromide was performed, and an in vitro permeability assay (IVP) was used to assess changes in cell monolayer permeability.
Western blot analysis confirmed a significant increase in Cx43 expression in cells transfected with pEGFPN1 compared to untransfected cells (152±13.1% of N; p<0.05), and IF identified 80% of cells as containing the GFP conjugated Cx43 from the plasmid. Importantly, Western blot analysis showed that the negative effect of HG on Cx43 expression was abrogated in transfected cells grown in HG medium (114±9.6% of N; p<0.05). Similarly, SLDT analysis indicated that HG-induced decrease in cell-cell communication (51.2±5.3% of N) was returned to near normal levels in cells transfected with pEGFPN1, even under HG condition (88.7±8.8% of N). As expected, cells grown in HG medium showed a significant increase in the number of apoptotic cells (225±16.8% of N). Interestingly, normalization of Cx43 expression protected 50% of the HG cells from apoptosis compared to cells grown in HG medium only (140.2±9.7 of N), and similarly reduced cell monolayer permeability by 50% in cells grown in HG medium (263.2±12.4% of N vs. 131.6±6.7%).
Findings from this study indicate that upregulation of Cx43 protects cells from HG-induced apoptosis by improving GJIC, and reduces cell monolayer permeability in RRECs grown in high glucose condition.
This PDF is available to Subscribers Only