Abstract
Purpose :
Degenerative retinal diseases including Age Related Degeneration have many complex risk factors, including environmental and genetic factors.
In the retina the outer Blood retinal barrier (oBRB) allows uptake of cholesterol via a lipoprotein based/receptor-mediated mechanism. Sterols can readily pass from blood-borne lipoproteins into the Retinal Pigment epithelial cells (RPE) and all layers of the neural retina. Cholesterol may be oxidized, leading to the production of oxysterols. The aims of this study are to:
Determine the effects of Sulforaphane, a naturally occurring anti-oxidant and epigenetic modulator, on ARPE-19 Tight Junction (TJ) gene expression.
Determine the effects of 7-ketocholesterol (7KC), 22R- and 22S-hydroxycholesterol 22(R)OHC, and 22(S)OHC), and 25-hydroxycholesterol (25OHC), on RPE TJ expression and permeability.
Methods :
ARPE-19 cells were cultured for 3-4 months and treated for 24h with oxysterols (5– 50uM), or Sulforaphane (0.1uM – 50uM) and cell viability analysed by the Alamar Blue dye binding assay. TEER values were obtained and Fluorescein isocyanate (FITC) dextran permeability studies carried out. Gene expression was determined by quantitative real-time PCR and protein expression was determined by Western blotting. Statistical analysis was performed using the T test. P<0.05 was considered significant.
Results :
ARPE-19 cells treated with a 5uM concentration of oxysterols showed 90% cell viability. A reduction in the mRNA expression of Claudins and Occludin was found for RPE cells treated with 5uM 7KC, 22ROHC, 22(S)OHC or 25OHC. In addition, TEER values and FITC dextran permeability studies showed outer Blood retinal barrier permeability was affected.
Cells treated with 5uM Sulforphane showed 70% cell viability. Sulforaphane treatment led to a reduction in Claudin 1 expression levels and a modulation in expression of NRF2, superoxide dismutase 1 and thioredoxin-1.
Conclusions :
In conclusion our results show oxysterols and Sulforaphane may affect oBRB function and the mechanisms are the subject of ongoing investigations.
This is a 2020 ARVO Annual Meeting abstract.