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A. Chronopoulos, S. Roy; High Glucose-Induced Cx43 Downregulation Alters Tight Junction Protein Occludin and ZO-1 Expression in Retinal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5596.
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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.
Rat retinal endothelial cells (RRECs) were grown in normal (N) medium (5mM) 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 (ran-oligos). The transfected cells along with the N and HG cells were analyzed for Cx43, occludin and ZO-1 protein levels by Western blot (WB) 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.
WB analysis confirmed significant reduction in Cx43 expression in the AS-Cx43 oligos transfected cells compared to the N untransfected cells (67.9±3.6% of control, p < 0.005). Cells transfected with ran oligos showed no change in Cx43 protein level. The reduction in Cx43 level by AS-Cx43 oligos transfection was similar to that of HG cells (54.3±9.7% of control, p < 0.001). Interestingly, in the AS-Cx43 oligo transfected cells, occludin and ZO-1 expression was also downregulated compared to the N untransfected cells (57.6±15.8% of control, p < 0.005; 75.2±7.7% of control, p < 0.005, respectively). The reduction in the expression of tight junction proteins was similar to those observed in the HG cells (61.5±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 (68.2±15.9 of control p < 0.05); cells transfected with ran oligos showed no change. This reduction in Cx43 immunostaining was similar to that of HG cells (61.4±4.8% of control, p < 0.001). Interestingly, occludin and ZO-1 immunostaining in the transfected cells was also significantly reduced compared to the N untransfected cells (62.5±14.8% of control, p < 0.005; 56.4±18.4% of control, p < 0.05, respectively). The reduced occludin and ZO-1 immunostaining was similar to those of HG cells (62.8±6.4% of control, p< 0.005, 53.7±22.4% of control, p<0.005).
Findings from this study indicate that HG-induced downregulation of Cx43 expression may contribute to reduced tight junction protein expression known to occur in diabetic retinopathy.
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