gms | German Medical Science

Purpose: To investigate whether high glucose-induced downregulation of gap junction protein, connexin 43 (Cx43) expression, influences expression of tight junction proteins, occludin and ZO 1.

Methods: Rat retinal endothelial cells (RRECs) were grown in normal (N) medium (5 mM) or high glucose (HG) (30 mM) medium for 7 days. At subconfluency two groups of cells grown in N medium were transfected with either Cx43 antisense oligonucleotides (AS-Cx43 oligos) or random oligonucleotides. The transfected cells along with the N and HG cells were analyzed 72 hours after transfection. Cx43, occludin and ZO-1 protein levels were assessed by Western blot analysis. Similarly, to determine whether HG-induced Cx43 downregulation affects the distribution and localization of occludin and ZO-1, immunostaining for Cx43, occludin and ZO-1 was performed in parallel cultures of cells grown on cover slips.

Results: Western Blot analysis confirmed significant reduction in Cx43 expression in the AS-Cx43 oligos transfected cells compared to the N untransfected cells (67.9±3.61% of control, p<0.005). Cells transfected with random oligonucleotides showed no change. The reduction in Cx43 level by AS-Cx43 oligos transfection was similar to that of HG cells (54.3±9.7% of control, p<0.0005). Interestingly, in the transfected cells, occludin and ZO-1 expression was also downregulated compared to the N untransfected cells (57.58±15.82% of control, p<0.005; 75.18±7.68% of control, p<0.005, respectively). The reduction in the tight junction proteins was similar to that observed in the HG cells (61.49±19.4% of control, p<0.05; 63±5.7% of control, p<0.005, respectively). Cx43 immunostaining was significantly reduced in the AS-Cx43 oligos transfected cells compared to the N untransfected cells (68.24±15.92 of control p<0.05). Cells transfected with random oligonucleotides showed no change. This reduction in Cx43 immunostaining was similar to that of HG cells (61.4±4.8% of control, p<0.0005). Interestingly occludin and ZO-1 immunostaining in the transfected cells was also significantly reduced compared to the N untransfected cells (62.5±14,84% of control, p<0.005; 56.38±18.39% of control, p<0.05, respectively). The reduced occludin and ZO-1 immunostaining was similar to that of HG cells (62.8±6.4% of control, p<0.005, 53.7±22.4% of control, p<0.005).

Conclusions: The findings from this study indicate that the HG-induced reduction in Cx43 expression may contribute to reduction in tight junction protein expression known to compromise blood retinal barrier property in diabetic retinopathy.