Decreased substrate stiffness leads to mitochondrial dysfunctions and Endothelial to Mesenchymal transition through Focal Adhesion Kinase activity in corneal endothelial cells.

Purpose: Fuchs' Endothelial Corneal Dystrophy (FECD), a degenerative corneal disorder, is marked by the thickening of Descemet's membrane and a progressive loss of corneal endothelial cells, ultimately leading to vision loss. A feature associated with the disease is the reduced stiffness of Descemet's membrane. However, the effects of this change in Descemet's membrane, on corneal endothelial cell health are not well understood. To explore this, we used Methods: For in-vitro studies, we cultured bovine corneal endothelial cells for 96 hours on stiff (32 kPa) and soft (8 kPa) substrate CytoSoft plates. By using Jess immunoassay and traditional western blotting, we evaluated changes in integrin signaling components, endothelial-to-mesenchymal transition, apoptosis, autophagy, and ubiquitin-proteasome pathway markers. Mitochondrial health and mitochondrial superoxide levels were assessed using commercial kits. We assessed the protein levels of the above-mentioned markers in the Results: We observed increased levels of phosphorylated FAK, integrins α4 and α5 in bovine corneal endothelial cells cultured on soft substrate. We also found upregulated endothelial-to-mesenchymal transition (EndMT) markers, mitochondrial dysfunction, and apoptosis in cells grown on soft substrate. In the Conclusion: In this study, we explored how changes in the physical characteristics of the Descemet's membrane impact corneal endothelial cell health. While we discovered activation of Focal adhesion kinase as a result of stiffness changes, its inhibition alone was insufficient to improve cell health in an FECD mouse model.