High Glucose-Induced Upregulation of Collagen Type IV Expression Modulates Connexin 43 Expression in Microvascular Endothelial Cells

Abstract

Purpose:: To determine if modulation of collagen type IV (ColIV) expression with antisense oligonucleotides against ColIV (AS-Col oligos) alters connexin 43 (Cx43) expression and localization in rat microvascular endothelial cells (RMEC) grown in high glucose (HG) medium.

Methods:: RMECs were grown in normal (5mM) or high (30mM) glucose medium for 7 days, transfected with AS-Col oligos for 3 days, and then harvested. Cells grown in high glucose media were transfected with 0.4µM AS-Col oligos, in the presence of 8µM lipofectin and Optimem. After transfection, cells were reincubated for 72 hours in HG medium and then harvested for protein isolation or immunofluorescent microscopy. Western blot analysis was performed to determine the protein expression of ColIV and Cx43. To determine whether HG induced changes in Cx43 localization and distribution, cells were grown on cover slips and examined by immunofluorescent microscopy. Digital images were photographed and Cx43 "dot-like" punctate fluorescence were counted in cells grown in HG medium or in cells grown in HG medium and transfected with AS-Col oligos.

Results:: Cells grown in HG medium showed significant increase in ColIV expression (136±27% of control, p<0.01) and significant decrease in Cx43 expression(60.4±12% of control, p<0.01). When cells grown in HG medium were transfected with AS-Col oligos, ColIV protein level was reduced (95.2±17%, p<0.05) with a corresponding increase in Cx43 expression (92±6%, p<0.05). Likewise Cx43 punctate fluorescence count was significantly increased in cells grown in HG medium and transfected with AS-Col oligos (58±3, p<0.01) compared to those grown in HG medium only (33±5).

Conclusions:: The AS-Col oligos strategy successfully reverted the HG-induced upregulation in ColIV expression as well as downregulation in Cx43 expression. The findings indicate that HG-induced upregulation of ColIV expression plays a role in inhibiting Cx43 expression and localization in RMECs.

Keywords: diabetic retinopathy • extracellular matrix • gap junctions/coupling