Abstract
Purpose :
Our previous work investigating corneal endothelial cell response to an engineered FGF1 (TTHX1114) following wounding demonstrated a 2.5-fold increase in regeneration of the endothelial layer with TTHX1114 treatment. However, these results have not yet been connected to specific changes in gene or pathway expression. We performed RNA-seq analysis to identify changes in gene expression and pathway enrichment resulting from treatment with TTHX1114 using an organ culture model with human donor corneas.
Methods :
Five pairs of healthy human donor corneas were scratch wounded before incubating in the presence or absence of TTHX1114 continuously for 3 days to capture early shifts in wound healing pathway expression. Healing progress was evaluated using Trypan staining and was quantified using ImageJ. After 3 days, corneal endothelial cells were harvested for RNA extraction and sequencing. RNA-seq data processing and analysis were performed using ROSALIND software.
Results :
Within the 3-day treatment period, TTHX1114-treated corneas experienced 44.23% ± 9.45% (mean ± SD) healing of the wound area as measured by Trypan staining compared to 34.66% ± 15.44% healing in untreated corneas. RNA sequencing data revealed 321 genes to be differentially expressed (padj < 0.05, fold-change > 1.5 or <-1.5). The most highly differentially expressed genes assessed by fold-change were DUSP6, PIK3R6, and FAM20C which were upregulated in TTHX1114-treated corneas, and PLA2G2A, CNTD2, and CAV3 which were downregulated. Pathway analysis of differentially expressed genes performed on ROSALIND revealed enrichment of terms for fibroblast growth factor response, endothelial cell migration, cell proliferation, and wound healing.
Conclusions :
RNA sequencing results confirm that treatment of wounded corneal endothelium with TTHX1114 does generate a statistically significant shift in gene expression, and specifically produces a response in FGF signaling. Furthermore, pathway analysis results indicate that TTHX1114 treatment leads to shifts in expression of proliferative and wound healing pathways, which may explain the 2.5x greater wound healing in treated corneas that has been reported in our previous work.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.