Abstract
Purpose :
Blood-retinal-barrier dysfunction is an underlying factor for leading causes of blindness such as diabetic retinopathy and macular edema, often treated with glucocorticoids (GC) to restore retinal epithelial integrity. This study aims to investigate the role of dexamethasone (Dex), a synthetic GC, on ZO1 during normal and migratory conditions in retinal cells. We tested the hypothesis that Dex would upregulate ZO1 in the leading edge of cells during migration while down regulating it under normal conditions.
Methods :
Human Retinal Pigmented Epithelial cells (ARPE-19) were treated with Dex (0 & 1000nM Dex) for 24 hours and wound healing assays were performed. Brightfield images taken at 0, 8 and 24 hours during migration were analyzed with ImageJ to determine the area of wound closed. ZO1 and GR proteins were examined by immunofluorescence (IF) and western blot (WB) in confluent monolayers and in cells at the leading edge of the wound. Bands were quantified using ImageJ and results were normalized to Actin. Student’s t-test was used to calculate statistical significance and a p-value <0.05 was considered significant.
Results :
Dex delayed ARPE-19 migration where Dex treatment healed 69.7% while the control healed over 78.8% of the wound (p<0.05). IF did not show an expected upregulation of ZO1 with Dex when compared to the control, thereby supporting the nature of GCs acting in a cell-type specific manner. Interestingly, cells not subjected to wounding showed a marked downregulation of ZO1 by WB (Control 0.35 a.u. & Dex 0.13 a.u; arbitrary units; p<0.01). However, cells along the leading edge of the wound exhibited Dex-mediated increase in ZO1 protein (Control 0.10 a.u. & Dex 0.26 a.u.; p<0.02).
Conclusions :
Our results demonstrate that GCs differentially regulate ZO1 expression in ARPE-19 cells depending on whether they are under normal conditions or wounded. Future experiments will investigate GC-mediated expression of ZO1 and other genes involved in migratory and barrier functions at the leading edge and in cells grown under normal conditions. Additional aim of this study is to determine the importance of ZO1, by knocking down ZO1, in Dex-mediated migration and epithelial integrity of ARPE-19 cells. Knowledge obtained from this study would have implications on our current understanding of the integrity of the retinal pigment epithelium.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.