Transcription factor 4 promotes increased corneal endothelial cellular migration by altering microtubules in Fuchs endothelial corneal dystrophy.

Fuchs endothelial corneal dystrophy (FECD) is a complex corneal disease characterized by the progressive decline and morphological changes of corneal endothelial cells (CECs) that leads to corneal edema and vision loss. The most common mutation in FECD is an intronic CTG repeat expansion in transcription factor 4 (TCF4) that leads to its altered expression. Corneal endothelial wound healing occurs primarily through cell enlargement and migration, and FECD CECs have been shown to display increased migration speeds. In this study, we aim to determine whether TCF4 can promote cellular migration in FECD CECs. We generated stable CEC lines derived from FECD patients that overexpressed different TCF4 isoforms and investigated epithelial-to-mesenchymal (EMT) expression, morphological analysis and cellular migration speeds. We found that full length TCF4-B isoform overexpression promotes cellular migration in FECD CECs in an EMT-independent manner. RNA-sequencing identified several pathways including the negative regulation of microtubules, with TUBB4A (tubulin beta 4A class IVa) as the top upregulated gene. TUBB4A expression was increased in FECD ex vivo specimens, and there was altered expression of cytoskeleton proteins, tubulin and actin, compared to normal healthy donor ex vivo specimens. Additionally, there was increased acetylation and detyrosination of microtubules in FECD supporting that microtubule stability is altered in FECD and could promote cellular migration. Future studies could be aimed at investigating if targeting the cytoskeleton and microtubules would have therapeutic potential for FECD by promoting cellular migration and regeneration.

Validation of preloaded DMEK donor tissues: a laboratory-based study on endothelial cell viability and comparison of two F-mark inks.

OBJECTIVE: To investigate endothelial cell loss (ECL) associated with Descemet membrane endothelial keratoplasty (DMEK) donor tissues preloaded in the DMEK RAPID transport system after 1 and 5 days and to compare prestamping with 2 different F-mark inks. METHODS: DMEK donor tissues were stripped, marked with gentian violet dye applied as an F-mark, trephined, stained with trypan blue, and then preloaded into the DMEK RAPID transport system by an eye bank technician. Preloaded DMEK tissues were then unfolded and stained with calcein AM after 1 or 5 days of storage. Tissues were imaged, analyzed for total tissue ECL, and immunostained for corneal endothelium markers zonular occludens-1 and xCD166. Additionally, ECL and the intensity of an F-mark caused by 2 different inks were quantified. RESULTS: Preloaded DMEK tissues displayed an average ECL of 11.9% ± 4.5% (n = 8) at 1 day and 9.9% ± 4.2% (n = 9) at 5 days. No difference was found between the 2 groups. Zonular occludens-1 and activated leukocyte cell adhesion molecule (ALCAM; also know as CD166) staining showed that the corneal endothelial monolayer remained intact on preloaded tissues. On 5-day preloaded DMEK tissues, the average ECL and mean grayscale caused by the Keir Surgical ink F-mark and the Cardinal Health ink F-mark were 4.3% ± 0.8% and 158.5 ± 13.9% and 5.0% ± 1.1% and 142.9% ± 20.0%, respectively. No difference was found between the F-mark inks. CONCLUSION: Preloaded DMEK donor tissues resulted in an acceptable ECL range after 1 and 5 days of storage and were deemed suitable for transplantation. Both F-mark inks are acceptable for prestamping preloaded DMEK tissues prior to surgical transplantation with comparable ECL and intensities.