An-Fei Li1, Sayon Roy. 1. Department of Ophthalmology, University School of Medicine, Boston, Massachusetts 02118, USA.
Abstract
PURPOSE: This study aims to determine whether high glucose-induced inhibition of connexin 43 (Cx43) expression and reduced gap junction intercellular communication (GJIC) promote microvascular endothelial cell loss. METHODS: To downregulate Cx43 protein expression in rat microvascular endothelial cells (RMECs), the cells were grown in high (30 mM) glucose medium for 7 days, or transfected with antisense-Cx43 (AS-Cx43) oligonucleotides. Western blot analysis confirmed significant inhibition of Cx43 protein expression. Scrape load dye transfer (SLDT) assay showed significant reduction in GJIC activity in these cells compared to cells grown in normal medium or transfected with random oligonucleotides. In parallel, Cx43 immunostaining showed significant decrease in number of Cx43 plaques in cells with reduced Cx43 expression. DNA ladder assay, TUNEL assay, and differential staining were performed to identify cells undergoing apoptosis. RESULTS: DNA ladder analysis, TUNEL assay, and differential staining indicated a significant increase in the number of apoptotic cells when Cx43 protein expression was reduced in both high-glucose cells or cells transfected with AS-Cx43 oligonucleotides with concomitant downregulation of GJIC activity. Additionally, DNA fragmentation, which was evident in cells with reduced Cx43 expression, suggested early phases of apoptosis. CONCLUSIONS: These results provide the first evidence that high glucose-induced downregulation of Cx43 expression is an early trigger for inducing apoptosis in microvascular endothelial cells. This finding may have important implications toward breakdown of vascular homeostasis and initiation of apoptosis in diabetic retinopathy.
PURPOSE: This study aims to determine whether high glucose-induced inhibition of connexin 43 (Cx43) expression and reduced gap junction intercellular communication (GJIC) promote microvascular endothelial cell loss. METHODS: To downregulate Cx43 protein expression in rat microvascular endothelial cells (RMECs), the cells were grown in high (30 mM) glucose medium for 7 days, or transfected with antisense-Cx43 (AS-Cx43) oligonucleotides. Western blot analysis confirmed significant inhibition of Cx43 protein expression. Scrape load dye transfer (SLDT) assay showed significant reduction in GJIC activity in these cells compared to cells grown in normal medium or transfected with random oligonucleotides. In parallel, Cx43 immunostaining showed significant decrease in number of Cx43 plaques in cells with reduced Cx43 expression. DNA ladder assay, TUNEL assay, and differential staining were performed to identify cells undergoing apoptosis. RESULTS: DNA ladder analysis, TUNEL assay, and differential staining indicated a significant increase in the number of apoptotic cells when Cx43 protein expression was reduced in both high-glucose cells or cells transfected with AS-Cx43oligonucleotides with concomitant downregulation of GJIC activity. Additionally, DNA fragmentation, which was evident in cells with reduced Cx43 expression, suggested early phases of apoptosis. CONCLUSIONS: These results provide the first evidence that high glucose-induced downregulation of Cx43 expression is an early trigger for inducing apoptosis in microvascular endothelial cells. This finding may have important implications toward breakdown of vascular homeostasis and initiation of apoptosis in diabetic retinopathy.