Abstract
Purpose :
The cornea, the outer layer of the vertebrate eye, maintains its transparency through the action of the corneal endothelium, which regulates hydration levels. In Fuchs Corneal Endothelial Dystrophy (FECD), corneal endothelial cells show abnormal composition of Descemet’s membrane and decreased stiffness. This project investigates how alterations in Descemet’s membrane stiffness and composition affect corneal endothelial cell health and viability.
Methods :
To assess the impact of ECM stiffness on bovine corneal endothelial cells (BCEC), we used CytoSoft® plates with stiffness levels mimicking healthy and FECD-like conditions - 8kPa (rigid) and 2kPa (soft) plates, consistent with reported changes. We used immunofluorescence to identify changes in the oxidative stress and morphology of BCEC using antibodies, Nitrotyrosine and ZO-1, respectively. To detect changes in Epithelial-mesenchymal transition (EMT) markers, we tested the expression of Snail, Zeb-1, and N-cadherin using Jess immunoassay. To evaluate the effects on the integrin pathway, we measured the expression of Integrins α5/β3, Focal Adhesion Kinase (FAK), and phosphorylated FAK. We also measured the levels of antioxidant proteins, SOD2, and catalase.
Results :
BCEC cultured on 2 kPa substrates exhibited heightened nitrotyrosine expression and diminished ZO-1 expression compared to those on 8 kPa surfaces, suggesting a departure from typical endothelial cell characteristics and a potential increase in reactive oxygen species (ROS) production. BCEC displayed an upregulation of the proteins Snail (34%) and Zeb-1 (18%) and a downregulation of N-cadherin (34%) expression at 2kPa stiffness level indicative of increased EMT. Additionally, we observed reduced expression of the antioxidant enzymes SOD2 (27%) and Catalase (82%) in BCEC plated at 2 kPa, further supporting the notion that stiffness alterations may influence ROS production in these cells. We observed decreased Integrins α5/β3 (28%/38%) expression and activities in BCEC cells cultured on 2 kPa substrates.
Conclusions :
Our study highlights the complex interplay between ECM stiffness and cell health in the corneal endothelium. These findings contribute to understanding FECD mechanisms and may lead to potential therapeutic insights.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.