Abstract
Purpose :
Typical Fuchs endothelial corneal dystrophy (FECD), induced by exposing ultraviolet A (UVA), affects corneal endothelial barrier and pumping functions since the UV exposed cells release reactive oxygen species (ROS). However, the correlations between UV exposure level and TRPV activation has not yet been explored. In this study, TRPV 1 and 4 activation levels are evaluated by measuring cornea endothelial responses to chronic UV exposure, in addition to junction protein and cell size, to determine how UVA exposure can activate these ROS sensitive ion channels which contribute to the development of the disease of corneal endothelium.
Methods :
HCEC-B4G12 was cultured for 7 days from the initial seeding density at 1x104 cells/cm2 for creating monolayered cornea endothelium before applying UVA. Using an UVA light source (365 nm, 2.4mW/cm2), we irradiated UVA daily for 4 weeks to the cells with 2 J/cm2 (4 min), 4 J/cm2 (8 min), and 8 J/cm2 (16 min) energy levels based on the damage threshold of cornea endothelium to simulate chronic FECD. During the experiments, the cell morphology, size, junction protein, and TRPV 1 and 4 were observed. For quantification of these images, MATLAB and image J were utilized for accurate data analysis.
Results :
Before exposing UVA, we confirmed the expression of ZO-1 and hexagonal morphology of the corneal endothelium. As increasing UVA intensity, cell enlargement and shape distortion from initial hexagonal shape have been observed without significant alteration of ZO-1 expression. Compared to each group, the most substantial size alterations were observed from the case of the 8 J/cm2. For TRPV 1 and 4 expression studies, we were able to confirm that UVA showed agonistic effect for both TRPV channels. There was no TRPV1 expression for intact corneal endothelial cells, whereas UVA treatment induced TRPV1 expression with the UVA exposure level. For TRPV4 expression, we observed the expression on intact corneal endothelial cells and the level of TRPV4 expression increased as UVA exposure level increased.
Conclusions :
This study supports the hypothesis that UV exposure activates TRPV channels and contributes to the endothelial pumping dysfunction related cornea diseases. This TRPV activation along with morphological changes show similar trend with FECD progression. With further studies on the interrelation, we will be able to develop a therapeutic strategy for FECD by modulating TRPV channels.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.