Abstract
Purpose :
Fuchs endothelial corneal dystrophy (FECD) is characterized by corneal endothelial (CE) cell loss along with the development of extracellular matrix deposits known as guttae, resulting in loss of corneal function and edema. The gradual loss of CE cells that are post-mitotic state and exhibit limited proliferative capacity in vivo, is accompanied by neighboring cell enlargement to maintain the intact monolayer. However, the mechanism of cell loss in FECD has not been studied. Here, we investigate whether replacement of lost cells occurs via whole genome duplication (i.e. polyploidy), where adjacent cells by enlarging their cell size and nuclear content cause abnormal tissue repair response and lead to pathologic findings seen in FECD.
Methods :
8 weeks old female C57BL/6 mice were irradiated on the right eye with 500 Joules/cm2 of ultraviolet-A (UVA) light while the unexposed left eye served as control. Nuclear ploidy of CE cells was analyzed by measuring DAPI intensity within each nuclear boundary normalized to an internal control, corneal epithelial cells, which are known to be diploid. Changes in cell size and development of multinucleated cells was evaluated using ZO-1 immunostaining. The development of senescence and fibrosis was analyzed by immunostaining against H3K9me3 and FN-1, respectively. One-Way ANOVA with Tukey’s HSD was used for statistics.
Results :
After day-2 post UVA, CE cells underwent whole genome duplication, engaging in one round of DNA synthesis without nuclear division thus generating giant polyploid nuclei. This resulted in increased nuclear ploidy at day-3 (10.9±0.4C) compared to control (5.4±0.0C) (p<0.0001). Subsequently, post day-7, the large polyploid nuclei divided into multiple nuclei, giving rise to multinucleated polyploid cells. Junctional staining with ZO-1 revealed a progressive increase in the percentage of multinucleated cells, reaching its maximum at day-14 (21±3%vs 0±0% in control) (p<0.001). Additionally, the percentage of senescent cells (35±11% vs 0±0% in control) (p<0.0001) and FN-1 immunointensity (254±41at day-14 vs 65±7 in control) (p<0.01) increased at day-14, parallel to the evolution of the multinucleated, polyploid cells.
Conclusions :
We demonstrate that UVA-induced oxidative stress triggers polyploidy in CE cells resulting in the formation of multinucleated polyploid cells which corelates with development of cellular senescence and fibrosis in FECD.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.