Abstract
Purpose :
During wound healing, corneal keratocytes can transform into myofibroblasts. Previous 2D and 3D experiments have indicated that myofibroblast differentiation is influenced by the mechanical microenvironment. However, a comprehensive transcriptional investigation of the mechanotransductive and extracellular matrix (ECM)-related genes associated with this process has been lacking. Here, we analyzed bulk RNA-sequencing data from primary corneal keratocytes cultured in either the presence or absence of TGF-β1 and quantified differences in the expression of genes associated with mechanotransduction or the ECM.
Methods :
Primary rabbit corneal keratocytes were cultured for 5 days on collagen-coated glass coverslips in either the presence or absence of 5 ng/mL TGF-β1 to induce a myofibroblast phenotype. On day 5, RNA was isolated using the BioRad Aurum Total RNA Mini Kit and bulk RNA-sequencing was performed by Novogene. We included RNA from two separate experimental replicates, each with internal technical replicates for each treatment condition. Bioinformatic analysis of differential gene expression was conducted and a significance of padj<0.05 and log2(FoldChange)>1 was considered.
Results :
In the presence of TGF-β1, initial analysis revealed an upregulation of genes known to be associated with elevated levels of myofibroblast differentiation, such as ACTA2, ITGA5, FN1, as well as a down-regulation of KERA and LUM, markers of quiescent corneal keratocytes. Genes associated with focal adhesion formation, PXN and VCL were also upregulated. Surprisingly, we saw an upregulation in several new ECM-related genes such as COL25A1, COL21A1, COL15A1 and COL23A1. There was also an upregulation in the genes involved in cell contractility, such as RASGEF1C and RHOB. Interestingly, we also noticed significant changes in several genes involved in the Hippo signaling pathway, including an increase in the expression of CTGF, a downstream target of YAP activation, a known mechanosensor.
Conclusions :
Taken together, this initial analysis suggests distinct transcriptional differences in the genes associated with mechanotransduction and the ECM in myofibroblasts as compared to quiescent corneal keratocytes. In the future, it would be interesting to investigate the differences in genome expression in TGF-β1-treated corneal keratocytes on substrata of varying stiffness.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.