Abstract
Purpose :
Macular edema is a consequence of breakdown of the blood retinal barrier (BRB) and is an early event in the development of diabetic retinopathy (DR), a leading cause of preventable blindness in developed countries. The BRB is comprised of an inner and outer barrier. The outer barrier is formed by tight junctions between the retinal pigment epithelial (RPE) cells, and the inner barrier is formed by tight junctions between the retinal vascular endothelial cells. While the inner BRB has been well studied, studies on the development and regulation of the RPE barrier in the context of DR are relatively sparse. Previous studies suggest that human RPE is a claudin-19 dominant epithelium. We propose to test the hypothesis that alterations in claudin-19 in diabetes leads to breakdown of the outer BRB. Using in vivo and in vitro approaches we will evaluate the molecular mechanisms by which hyperglycemia regulates claudin-19 and tight junction/barrier function in RPE cells. Understanding the molecular mechanisms involved in breakdown of the outer blood retinal barrier will help identify therapeutic targets for macular edema.
Methods :
Real time quantitative PCR (RT-qPCR) was used to evaluate claudin 19 expression in primary porcine RPE cells. siRNA knock-down approaches was used to determine the role of claudin 19 on the barrier properties of the RPE. Knock down efficiency was evaluated by RT-qPCR, western blotting and immunofluorescence. Impedance spectroscopy was used to evaluate the transepithelial electrical resistance (TEER) as well as capacitance of primary porcine RPE. Effect of hyperglycemia on claudin-19 and tight junctions was also evaluated.
Results :
Claudin-19 is the predominant claudin in primary pig RPE cells and remains so following culture for at least 4 weeks. The integrity of the RPE barrier was compromised in cells treated with the claudin 19 siRNA, while the control and cells treated with the scramble siRNA maintained their barrier properties.
Conclusions :
These results suggests that claudin 19 plays a critical role in maintaining the integrity of the outer blood retinal barrier.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.