Investigating transcriptional differences in mechanotransductive and ECM related genes in cultured primary corneal keratocytes, fibroblasts and myofibroblasts.

After a stromal injury in the cornea, the release of growth factors and pro-inflammatory cytokines typically results in the activation of quiescent keratocytes toward migratory fibroblast and/or fibrotic myofibroblast phenotypes. The persistence of the myofibroblast phenotype can lead to corneal fibrosis and scarring, which are leading causes of blindness worldwide. The primary goal of this study was to establish comprehensive transcriptional profiles for cultured corneal keratocytes, fibroblasts, and myofibroblasts to gain insights into the mechanisms through which changes in phenotype may occur. Here, we cultured primary rabbit corneal keratocytes on collagen-coated glass coverslips in serum free media (SF), serum containing media (FBS), or in the presence of TGF-β1 to induce keratocyte, fibroblast, and myofibroblast phenotypes, respectively. Total RNA was collected and sent to Novogene for bulk RNA sequencing. Subsequent bioinformatic analysis included gene expression quantification, differential expression, and functional analysis. When comparing FBS and TGF-β1 conditions to SF, genes characteristic of a quiescent keratocyte phenotype were downregulated (e.g. KERA, LUM, ALDH1A1), while genes commonly associated with fibroblasts or myofibroblasts were upregulated (e.g. VIM, TNC, FN1, ITGA5, ACTA2). Functional analysis of genes differentially expressed between fibroblasts and keratocytes highlighted pathways related to proliferation (e.g. DNA replication, PI3K-Akt signaling) and cell migration (e.g. Rap1 signaling, ECM-receptor interactions). Enriched pathways for the comparison of myofibroblasts to keratocytes included focal adhesion, regulation of actin cytoskeleton, hippo signaling, and ECM-receptor interaction pathways. Together, these pathways support changes in cytoskeletal organization, cell contractility, mechanotransduction, and cell-ECM interactions in myofibroblasts compared to keratocytes. Overall, these data demonstrate that there are distinct transcriptional differences between cultured corneal keratocytes, fibroblasts, and myofibroblasts. In our initial analysis, we have identified genes and signaling pathways that may play important roles in keratocyte differentiation, including many related to proliferation, cell mechanical activity, and ECM interactions. Furthermore, our findings reveal novel markers for each cell type as well as possible targets for modulating cell behavior and differentiation to promote physiological corneal wound healing.