Short-Term UVB Irradiation Causes Persistent DNA Damage in Corneal Limbal Epithelial Stem Cells

Abstract

Purpose : The characterisation of UVB damage in corneal epithelial cells and the effectiveness of two DNA damage treatment methods: CPD photolyase and T4 Endonuclease V.

Methods : The effect of these CPD photolyase (CPDPL) and T4 endonuclease V (T4N5) was evaluated through proteomics and immunofluorescence measurements of UVB-induced CPDs before and after treatment. This was tested on extracted human limbal epithelial cells and murine eyes. Cells were exposed to 0.03J/cm2 UVB and murine eyes were exposed to 1J/cm2. Photoreactivation to activated CPDPL was 30min. Treatment with T4N5 went up to 48h.

Results : The mice used were between the ages of 12 weeks and 36 weeks. Limbal stem cell marker p63a was used to locate stem cells in the murine eyes. Full CPD repair normally takes approximately 48h in cell cultures, the addiction of T4N5 shortened this repair to less than 12h. In mouse eyes, CPD was mostly eliminated through CPDPL photoreactivation, although some persistent CPD would be observed in p63a-positive cells. Proteomics data showed an increase in angiogenic and inflammatory activity following T4N5 treatment of cultured primary cells.

Conclusions : CPD repair was achieved in human cell cultures and murine eyes. Further analysis in vitro revealed increased inflammation and angiogenesis. Further work in the mouse eyes revealed that the total clearance of CPDs in the limbus was not achieved.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.