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Tomas White, Neha Deshpande, Ula V Jurkunas; UV-A light induces G2/M phase arrest and subsequent endothelial-mesenchymal transition in Fuchs Endothelial Corneal Dystrophy. Invest. Ophthalmol. Vis. Sci. 2019;60(9):2179.
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Fuchs endothelial corneal dystrophy (FECD) is a genetic and oxidative stress disorder of post-mitotic human corneal endothelial cells, with the hallmark being aberrant ECM deposition in the form of guttae. Given that the human cornea is exposed daily to UV radiation, which is known to initiate oxidative stress, the aim of this study was to investigate the effects of UV-A irradiation on human and mouse corneal endothelial cells (CEnCs), specifically focusing on endothelial-mesenchymal transition (EMT) and senescence.
Immortalized HCEnCs were subjected to UV-A light irradiation at a fluence of 25J/cm2, followed by a recovery period of 24 hours. Additionally, seven to nine-week-old mice were irradiated with 1000J/cm2 UV-A in one eye, with the other eye used as a control. Gene and protein expression analysis was carried out using RT-PCR and western blot, respectively. Cell cycle data was collected using flow cytometry. Cells in the G0/G1 phase were sorted from cells in G2/M phase of the cell cycle with FACS to compare gene expression profiles. RO-3306 was used to arrest cells in G2/M phase.
UV-A light induced the formation of FECD- associated rosettes and resulted in significant upregulation of EMT-related genes SNAI1 (4-fold, P<0.001) and ACTA2 (8-fold, P<0.001). Furthermore, UV-A irradiation also induced cellular senescence, as observed with SA-β-GAL staining and a 6-fold increase in p21 mRNA expression (P<0.001). Cell cycle analysis revealed G2/M phase arrest in UV-A treated normal HCEnCs due to activation of ATR signaling pathway. Arresting cells in G2/M with cell cycle inhibitor produced a gene expression profile that mirrored UV-A treated cells. FACS analysis revealed a significant increase SNAI2, ACTA2 and CDKN1A (P<0.05) in G2/M phase cells compared to G0/G1 cells (P<0.05). Untreated FECD cells displayed a cell cycle profile similar to that of UV-A treated normal HCEnCs. p21 and cyclin B1 protein levels were significantly upregulated in UV-A irradiated mouse endothelial cells, with similar findings observed in human FECD specimens.
UV-A irradiation induced EMT and senescence-like changes in corneal endothelium in vitro and in vivo. These changes appear to stem from G2/M phase cell cycle arrest, indicating a temporal relationship between these two processes in FECD pathogenesis.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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