Corneal keratocytes, fibroblasts, and myofibroblasts exhibit distinct transcriptional profiles in vitro
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ABSTRACT:
PROVIDER: PRJNA1215274 | ENA |
REPOSITORIES: ENA
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Project description:After stromal injury to the cornea, the release of growth factors and pro-inflammatory cytokines promotes the activation of quiescent keratocytes into a migratory fibroblast and/or fibrotic myofibroblast phenotype. Persistence of the myofibroblast phenotype can lead to corneal fibrosis and scarring, which are leading causes of blindness worldwide. This study aims to establish comprehensive transcriptional profiles for cultured corneal keratocytes, fibroblasts, and myofibroblasts to gain insights into the mechanisms through which these phenotypic changes occur. Primary rabbit corneal keratocytes were cultured in either defined serum-free media (SF), fetal bovine serum (FBS) containing media, or in the presence of TGF-β1 to induce keratocyte, fibroblast, or myofibroblast phenotypes, respectively. Bulk RNA sequencing followed by bioinformatic analyses was performed to identify significant differentially expressed genes (DEGs) and enriched biological pathways for each phenotype. Genes commonly associated with keratocytes, fibroblasts, or myofibroblasts showed high relative expression in SF, FBS, or TGF-β1 culture conditions, respectively. Differential expression and functional analyses revealed novel DEGs for each cell type, as well as enriched pathways indicative of differences in proliferation, apoptosis, extracellular matrix (ECM) synthesis, cell-ECM interactions, cytokine signaling, and cell mechanics. Overall, these data demonstrate distinct transcriptional differences among cultured corneal keratocytes, fibroblasts, and myofibroblasts. We have identified genes and signaling pathways that may play important roles in keratocyte differentiation, including many related to mechanotransduction and ECM biology. Our findings have revealed novel molecular markers for each cell type, as well as possible targets for modulating cell behavior and promoting physiological corneal wound healing.
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Project description:Background: Asthma is the most common chronic lung disease in children and young adults worldwide. Airway remodelling (including increased fibroblasts and myofibroblasts in airway walls due to chronic inflammation) differentiates asthmatic from non-asthmatic airways. The increase in airway fibroblasts and myofibroblasts occurs via epithelial to mesenchymal transition (EMT) where epithelial cells lose their tight junctions and are transdifferentiated to mesenchymal cells, with further increases in myofibroblasts occurring via fibroblast-myofibroblast transition (FMT). Transforming growth factor (TGF)-β is the central EMT- and FMT-inducing cytokine. In this study, we have used next generation sequencing to delineate the changes in the fibroblast transcriptome induced by TGF-β treatment in both the short term and after differentiation into myofibroblasts, to gain an understanding of the contribution of TGF-b induced transdifferentiation to the asthmatic phenotype. The data obtained from RNAseq analysis was confirmed by quantitative PCR (qPCR). Results: As expected, we found that genes coding for intermediates in the TGF-β signalling pathways (SMADs) were differentially expressed after treatment, and genes involved in cytoskeletal pathways (FN1, LAMA, ITGB1) were differentially expressed in myofibroblasts compared to fibroblasts. Importantly, genes that were previously shown to be changed in asthmatic lungs (ADAMTS1, DSP, TIMPs, MMPs) were differentially expressed in myofibroblasts, strongly suggesting that TGF-β mediated differentiation of fibroblasts to myofibroblasts may underlie important changes in the asthmatic airway. We also identified new signalling pathways (AKT, PTEN) that are changed in myofibroblasts compared to fibroblasts. Conclusion: We have found a significant number of genes that are altered after differentiation of fibroblasts into myofibroblasts by TGF-β treatment, many of which were expected or predicted. However, we also identified novel genes and pathways that were affected after treatment of fibroblasts with TGF-β, which suggests additional pathways that that are activated during the transition between fibroblasts and myofibroblasts, and may contribute to the asthma phenotype.
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