Abstract
Purpose :
The purpose of this study was to test whether retinal pigment epithelium (RPE) cells express vascular endothelial growth factor (VEGF) in a circadian manner. We tested the hypothesis that 12 hour alternating intervals of dopamine then an experimental drug would induce RPE cells into a circadian rhythm that controlled VEGF expression.
Methods :
Induced pluripotent stem cells were used to create RPE monolayers on snap-well filters, which were maintained in static culture conditions for at least six months, during which time they became pigmented and well differentiated. We used a continuous perfusion system thereafter, and collected separate apical and basal effluents every 2 hours. A baseline of VEGF secretion was established over a 3-week period. After the baseline was established, the cells were perfused with media containing 1uM dopamine for 12 hours, then media containing 1uM of the experimental drug for the next 12 hours. In this perfusion system, the drug concentrations began at zero, and gradually increased to the full concentration over 12 hours, when the next drug was begun. Initiation of the next drug dilutes the previous over 12 hours while simultaneously ramping up the new drug. Cells were maintained in this circadian rhythm for at least three cycles. Samples were collected at 2-hour intervals and VEGF was quantified via ELISA. The amount of VEGF was plotted against the time of perfusion and fitted using the Cosinor method to find a length of circadian cycle and corresponding p-value for statistical analysis.
Results :
The baseline expression of VEGF in the perfusion system was below the limit of detection prior to addition of the circadian drugs. When the cells were perfused with the 12-hour intervals of dopamine then the experimental drug, the amount of VEGF expressed showed a circadian cycle of 23.5 hours (p < .05) for the basal secretion, but only 16.1 hours (p < .05) for the apical side of the cells. The difference between the apical and basal periodicity is attributable to the volume and exchange rate differences between the apical and basal chambers. Apical volume was 4.4ml while the basal volume was 1.76ml, but we collected 575ml/hr from each side.
Conclusions :
Our results show that circadian agents can alter RPE VEGF expression in a rhythmic manner, and suggest that dopamine receptors increase RPE VEGF secretion, while the experimental drug may antagonize dopamine effects.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.