Abstract
Purpose :
To evaluate the expression of endothelial-mesenchymal transition (EMT) proteins in Fuchs endothelial corneal dystrophy (FECD) cells ex vivo and in vitro.
Methods :
End-stage FECD and healthy Eye bank ex vivo corneal endothelium and Descemet membrane were immunostained against the following EMT-related proteins: β-catenin, Smad 2 Phosphorylated (Smad2P), Smad 3 Phosphorylated (Smad3P), LOX, Slug, c-Jun, S100A4 Snail, ZEB1, Active β-catenin (ABC). In parallel, post-confluent (passage 3 to 6) FECD and healthy CECs were irradiated daily (or not) with UVA (2kJ/m2) for 14 days. Their conditioned media were used to detect the expression of secreted EMT-related proteins (TGF-β2, LOX and Wnt4) using ELISA assays or Western blots. Cells were then fixed and immunostained as above.
Results :
Cells on ex vivo FECD specimens expressed β-catenin in their cytoplasm (near the nucleus), while healthy cells’ expression was near the cell membrane. Smad2P and Smad3P were present in ex vivo FECD cells’ cytoplasm, but were expressed in the nuclei in healthy cells. LOX was expressed in FECD specimens, both in the fibrillar layer of Descemet membrane and in the cell cytoplasm, and was absent in healthy specimens. Slug protein expression was absent in ex vivo FECD cells, and was present in the nuclei of healthy cells. C-Jun was absent in FECD cells, but was expressed in 1/4 healthy specimens (nuclear expression). S100A4, Snail, ZEB-1 and ABC’s expression was similar in FECD and healthy specimens.
In vitro, non-irradiated mature CECs expressed β-catenin, LOX and Snail in the nuclei of FECD cells, while in healthy cells β-catenin was present near the membrane, LOX in the cytoplasm, and Snail was not expressed. Irradiated FECD CECs expressed Snail in their cytoplasm and nuclei, while being only expressed in their nuclei when non-irradiated. LOX and Wnt4 were not found in the conditioned media. TGF-β2 was secreted only by FECD cells.
Conclusions :
Ex vivo, the expression of LOX, Slug, β-catenin, Smad2P, and Smad3P differed between FECD and healthy specimens, while in vitro, LOX, Snail, β-catenin and TGF-β2 were expressed differently in FECD and healthy CECs. Of the 12 EMT-related proteins studied, irradiation only influenced Snail expression in culture. Overall, the results show that CECs present a unique EMT-like profile in end-stage FECD and in cultured cells.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.