TGF-β–Mediated Modulation of Cell–Cell Interactions in Postconfluent Maturing Corneal Endothelial Cells

Abstract

Purpose Transforming growth factor-beta (TGF-β) is known to influence many cell functions. In the corneal endothelium, TGF-β1 exerts contextual effects, promoting endothelial–mesenchymal transition in proliferating cells and enhancing barrier integrity in early confluent maturing cells. Herein, we studied how TGF-β isoforms participate in the formation of corneal endothelial intercellular junctions. Methods Corneal endothelial cells (CECs) were cultured using a two-phase media approach. When CECs reached confluence, the proliferation medium was replaced with maturation medium, which was supplemented or not with TGF-β isoforms. The cell morphology (circularity index), intercellular junction protein expression, trans-endothelial electrical resistance (TEER), and permeability of 7-day postconfluent CECs were assessed. Gene transcription and signaling pathways that were activated following maturation in the presence of TGF-β2 were also studied. The beneficial effect of TGF-β2 on CEC maturation was evaluated using ex vivo corneas mounted on a corneal bioreactor. Results The results showed increases in circularity index, membrane localization of junction-related proteins, and TEER when TGF-β isoforms were individually added during the maturation phase, and TGF-β2 was the most effective isoform. Gene profiling revealed an increase in extracellular matrix-related gene expression. In ex vivo cell adhesion experiments, CECs that were matured in the presence of TGF-β2 had a higher circularity index and cell density and exhibited cell membrane-localized junction-related protein expression at earlier time points. Conclusions These results suggest that TGF-β2 can strengthen cell–cell and cell–substrate adhesion, which accelerates barrier integrity establishment and thus enhances CEC functionality.

Effects of Autologous Serum and Platelet-Rich Plasma on Human Corneal Endothelial Cell Regeneration: A Comparative Study

“…The interaction and balance between these two factors play a key role in regulating corneal endothelial cell number in vivo. [29][30][31][32][33] This study also evaluated the TGF-b1 levels among the study groups and demonstrated that AS and PRP reduced the TGF-b1 levels, whereas Y-27632 showed no significant effect on TGF-b1. As mentioned above, multifactorial nature of AS and PRP may have had a balancing effect on the TGF-b1 concentration, while providing growth factors for HCEC proliferation.…”

“…The interaction and balance between these two factors play a key role in regulating corneal endothelial cell number in vivo. 29–33…”

Investigating the Role of TGF-β Signaling Pathways in Human Corneal Endothelial Cell Primary Culture

“…When added to confluent cells cultured in a maturation medium, TGF-β1 can enhance the endothelial phenotype, in contrast to its induction of EndMT when added to cells grown in a proliferation medium [ 50 ]. Similarly, the improvement in the endothelial phenotype exerted by TGF-β2 in the same maturation medium was even greater than that of TGF-β1 [ 51 ]. We found some differences between the maturation medium and our medium without FBS.…”

“…Referring to the trabecular meshwork cells, which are neighboring cells of hCECs, we speculate that low concentrations of TGF-βs could protect hCECs from EndMT through the antioxidant effect via the PI3K/AKT/mTOR pathway. Additionally, hCECs treated in maturation medium containing TGF-β at 2 ng/mL also showed stronger activation of the AKT pathway [ 51 ].…”

Squishy matters – Corneal mechanobiology in health and disease

“…Importantly, blockade of TGFβ1 preserves corneal endothelial cell hexagonal shape and downregulate EnMT markers ( Wang et al, 2022b ). By contrast, the TGFβ2 isoform strengthens cell-cell and cell-substrate adhesion, accelerate monolayer barrier integrity establishment, and thus enhance corneal endothelial functionality ( Santerre et al, 2022 ). Continued investigations into the contextual effects of TGFβ isoforms will likely be of growing importance to efforts to develop engineered corneal endothelial cell grafts.…”