Abstract
Purpose :
An intact tight junction barrier is crucial for maintenance of retinal homeostasis. The outer blood retinal barrier (oBRB) is formed by retinal pigment epithelial cells and oBRB breakdown occurs in several ocular disorders including Age related Macular Degeneration (AMD). In AMD low grade inflammation may play a role in the pathogenesis. In addition, AMD is associated with accumulation of lipid and lipid oxidative deposits including the pro-inflammatory oxysterol 7ketocholesterol. In this study we report the effects of proinflammatory cytokines and oxysterols on RPE tight junction and visual gene expression.
Methods :
Cell viability following cytokine or oxysterol (0-10000ng/ml) treatment was determined by the Alamar Blue dye binding assay.
ARPE-19 cells were treated with various cytokines (TNF-α (0-50ng/ml), IL1-β( 0-100ng/ml), PLGF (0-100ng/ml), VEGF (0-100ng/ml), and oxysterols (7ketocholesterol and 25hydrox cholesterol) for 18-24h under serum-free conditions.
Gene expression was determined by quantitative real-time PCR for claudins 1,2,4 and 5, occludin, RPE65, CYP27A1 and GLUT12.
Results :
ARPE-19 cells treated with oxysterols showed a decrease in gene expression for claudin 1 and occludin whilst cells treated with 7ketocholesterol showed an increase in RPE65 gene expression. 25hydroxycholesterol had no impact on RPE65 expression. An increased expression of CYP27A1 was found with cells treated with both oxysterols tested.
Cells treated with IL1-β showed a decrease in expression for claudin 1 and occludin genes and cells treated with PLGF showed an increase in the same two genes.
Low levels of expression of claudins 2, 4 and 5 were detected on control and cytokine-treated ARPE-19 cells.
Cells treated with TNF-α or VEGF showed a decrease in expression of claudin 1 and occludin gene expression and no change in RPE65 expression.
Conclusions :
We report 7ketocholesterol modulates RPE65 gene expression. RPE65 is a key gene involved in the visual cycle and a change in expression may lead to pathological changes in the retina.
We show the expression of several tight junction genes, important for the function of the oBRB, are decreased in RPE cells exposed to VEGF, IL1-β or TNF-α. We conclude VEGF and proinflammatory cytokines may affect the oBRB, and in diseases including AMD, this could contribute to oBRB dysfunction and breakdown.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.