High Glucose-Induced Downregulation of Connexin 43 Expression Promotes Apoptosis in Microvascular Endothelial Cells

¹ Medicine, Boston University School of Medicine, Boston, Massachusetts, United States

^* To whom correspondence should be addressed. E-mail: sayon{at}bu.edu.

	Abstract

PURPOSE. To determine whether high glucose-induced inhibition of connexin 43 (Cx43) expression and reduced gap junction intercellular communication (GJIC) promotes 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, and scrape load dye transfer (SLDT) assay showed significant reduction in GJIC activity in these cells compared to cells grown in normal medium or cells transfected with random oligonucleotides. In parallel, Cx43 immunostaining showed significant decrease in number of Cx43 plaques in cells with reduced Cx43 expression. To identify cells undergoing apoptosis, DNA ladder assay, TUNEL assay, and differential staining were performed in these cells. RESULTS. DNA ladder analysis, TUNEL assay, and differential staining technique 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 findings provide the first evidence that high glucose-induced downregulation of Cx43 expression is an early trigger for inducing apoptosis in microvascular endothelial cells that may have important implications towards breakdown of vascular homeostasis and possibly initiation of apoptosis in diabetic retinopathy.

Key Words: diabetic retinopathy, apoptosis, Connexin 43, Gap Junction

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