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A.–F. Li, T. Sato, S. Roy; Decreased Gap Junction Intercellular Communication Activity and Apoptosis in Microvascular Endothelial Cells . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3219.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To identify whether reduced GJIC activity by downregulation of Connexin 43 plays a role in the development of apoptosis in rat microvascular endothelial cells (RMEC). Methods: To specifically downregulate Cx43 expression, RMECs grown to subconfluency were transfected with 0.1uM antisense Cx43 phosphorothiorate oligonucleotides (AS oligos) in the presence of 8 uM lipofectin and subjected to analysis 48 h after transfection. Random oligos used as control was similarly transfected in these cells. In parallel studies, RMECs grown in normal glucose medium (5 mM) or high glucose medium (30mM) for 7 days were analyzed accordingly. Cx43 protein level was determined by Western blot analysis. GJIC activity was assessed using Scrape Load Dye Transfer (SLDT) technique. Apoptosis were determined by TUNEL staining, acridine orange/ethidium bromide staining, and DNA ladder assay. Results: Cx43 protein level was reduced by AS oligos (76.6±12.8% of control, p=0.0039) as well as GJIC activity (59.4±9.3% of control, p=0.012). Apoptosis detected by TUNEL staining, DNA ladder gel electrophoresis, and acridine orange/ethidium bromide staining in AS oligos–treated cells showed increased proportion of apoptotic cells (191.7±17.7% of control, p=0.006, 137.9±16% of control, p=0.003, and 148.3±13% of control, p=0.001, respectively). Similar to cells transfected with AS–Cx43oligos, cells grown in high glucose medium showed significantly decreased GJIC activity, (63±19% of control, p<0.001) and increased apoptosis (151.6±19% of control, p=0.003) compared to cells grown in normal medium. Random oligos showed no effect on Cx43 protein level, GJIC activity, or apoptosis. Conclusion: Decreased GJIC activity in RMECs by high glucose condition or antisense Cx43 oligos was associated with increased apoptosis. Disruption of cell–cell communication may contribute to premature vascular cell death in diabetic retinopathy.
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