Investigating the role of TGF β signaling pathways in improving human corneal endothelial cell culture

Abstract

Purpose : Corneal endothelial dystrophy is the first indication for corneal transplantation. Due to a significant shortage of donated corneas worldwide, cell therapy and/or bioengineering of endothelial grafts to replace corneal transplants is the paramount topic in this field. The key to success lies in the optimization of the culture method of human corneal endothelial cells (hCECs). It is well known that TGF β plays a detrimental role on hCECs, the purpose of this study was to identify the signaling pathways responsible for the detrimental effect of TGF β in order to optimize the culture of hCECs through their inhibition.

Methods : Amplified hCECs from primary culture were used. After confirming the detrimental effect of TGF β1, 2, 3 on hCECs, the different TGF β signaling pathways were evaluated using the specific inhibitors. Their effect on hCECs was assessed by 2 criteria: 1) endothelial cell density (ECD), which was assessed using DAPI nuclear staining; and 2) CEC differentiation and endothelial-mesenchymal transition (EnMT) status, which were assessed by immunofluorescence (IF) using a preselected biomarker from a set of CEC and EnMT biomarkers. N≥8 for each statistical test.

Results : TGF β1 and β3 had a greater effect on EnMT induction than TGF β2. NCAM (CD56) was chosen as a reliable biomarker to assess both differentiation and EnMT of hCECs. Inhibition of the p38-MAPK pathway, the ROCK pathway, and TGF β receptor (total TGF β signaling) all showed a beneficial effect on hCEC culture with decreasing degree; inhibition of the MEK-ERK pathway had a detrimental effect.

Conclusions : Inhibitors of p38-MAPK, such as SB202190, TA-02, or SB203580, are the most effective molecules for enhancing hCEC culture; a combination of P38-MAPK, ROCK, and TGF β receptor inhibitors could be considered for a beneficial and cumulative effect; activation of the MEK-ERK signaling pathway appears to be important for hCEC growth and should be maintained.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.