Genomic and functional responses of lens epithelial cells to acute and fractionated ionizing radiation

Abstract

The lens of the eye, a crucial component in focusing light onto the retina, plays an integral role in our ability to see clearly. Cataracts, defined as the clouding of the lens, can significantly impair vision and, in severe cases, lead to vision loss. This condition, affecting millions worldwide, has been studied extensively to understand its causes and develop preventive measures. One of the known causes of cataract formation is ionizing radiation (IR). Notably, recent recommendations from international regulatory bodies suggest that the threshold radiation dose for cataract formation is lower than previously thought, at an absorbed dose of just 0.5 Gy from low linear energy transfer (LET) radiation. Despite this, the exact mechanisms behind radiation-induced cataracts remain unclear, necessitating further research in this field. This thesis explores the impact of acute and fractionated ionizing radiation on the lens of the eye, concentrating on genomic and functional alterations, to gain a deeper understanding of the causes of radiation-induced cataractogenesis. First, we focused on uncovering acute radiation-induced changes in a lens epithelial cell (LEC) cell line. X-ray irradiation at 0.25 Gy significantly affected cell function, reducing adhesion, initially decreasing proliferation followed by an increase, and stimulating migration at both 12 hours and 7 days post-irradiation. Gene expression analysis indicated the involvement of FGF2, MAPK1, TGFB2, PDGFD, IGF1, MMP9, ITGA5, ICAM1, and CDH2. A non-linear dose response suggested a threshold around 0.25 Gy. Next, we examined functional changes in cultured LEC following fractionated radiation exposure. Exposure to 0.25 Gy significantly affected proliferation and migration over 14 days, peaking at 7 days. High-dose fractionated irradiation contrarily reduced proliferation and migration rates. Transcriptomic analysis revealed dose-dependent gene expression patterns and highlighted biological processes like cell migration and differentiation. Upstream regulator analysis identified TWIST1, BMP2, and NR2F2 as key regulators, elucidating radiation's impact on cellular signaling pathways. Finally, we explored the effects of acute radiation (0.25 Gy and 2 Gy) on embryonic stem cell- derived lentoid bodies over 48 hours using transcriptomic analysis, which identified dose- and time-dependent gene expression changes. At 0.25 Gy, the most significant changes occurred 24 hours post-exposure, while at 2 Gy, they peaked at 48 hours. Gene ontology analysis emphasized impacts on cell fate, developmental processes, migration, and adhesion. Overall, this thesis further elucidates the complex biological response of LEC to acute and fractionated ionizing radiation using both cell line and organoid models. This research helps to further our understanding of the mechanisms behind radiation induced cataracts, which can be used in determining biologically relevant radiation protection standards and assisting in developing mitigation strategies.