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Transcriptomic changes during TGF-?-mediated differentiation of airway fibroblasts to myofibroblasts.

ABSTRACT: 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)-? is the central EMT- and FMT-inducing cytokine. In this study, we have used next generation sequencing to delineate the changes in the transcriptome induced by TGF-? treatment of WI-38 airway fibroblasts in both the short term and after differentiation into myofibroblasts, to gain an understanding of the contribution of TGF-? induced transdifferentiation to the asthmatic phenotype. The data obtained from RNAseq analysis was confirmed by quantitative PCR (qPCR) and protein expression investigated by western blotting. As expected, we found that genes coding for intermediates in the TGF-? signalling pathways (SMADs) were differentially expressed after TGF-? treatment, SMAD2 being upregulated and SMAD3 being downregulated as expected. Further, genes involved in cytoskeletal pathways (FN1, LAMA, ITGB1) were upregulated in myofibroblasts compared to fibroblasts. Importantly, genes that were previously shown to be changed in asthmatic lungs (ADAMTS1, DSP, TIMPs, MMPs) were similarly differentially expressed in myofibroblasts, strongly suggesting that TGF-? mediated differentiation of fibroblasts to myofibroblasts may underlie important changes in the asthmatic airway. We also identified new intermediates of signalling pathways (PKB, PTEN) that are changed in myofibroblasts compared to fibroblasts. We have found a significant number of genes that are altered after TGF-? induced transdifferentiation of WI-38 fibroblasts into myofibroblasts, many of which were expected or predicted. We also identified novel genes and pathways that were affected after TGF-? treatment, suggesting additional pathways are activated during the transition between fibroblasts and myofibroblasts and may contribute to the asthma phenotype.

SUBMITTER: Walker EJ

PROVIDER: S-EPMC6937312 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Transcriptomic changes during TGF-β-mediated differentiation of airway fibroblasts to myofibroblasts.

Walker Erin Joanne EJ Heydet Deborah D Veldre Timothy T Ghildyal Reena R

Scientific reports 20191230 1

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 myofibrobl ...[more]

PMID: 31889146

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Project description:Cardiac fibroblasts and their activated derivatives, myofibroblasts, play a critical role in wound healing after myocardial injury and often contribute to long-term pathological outcomes, such as excessive fibrosis. Thus, defining the microenvironmental factors that regulate the phenotype of cardiac fibroblasts and myofibroblasts could lead to new therapeutic strategies. Both chemical and biomechanical cues have previously been shown to induce myofibroblast differentiation in many organs and species. For example, transforming growth factor beta 1, a cytokine secreted by neutrophils, and rigid extracellular matrix environments have both been shown to promote differentiation. However, the relative contributions of transforming growth factor beta 1 and extracellular matrix rigidity, two hallmark cues in many pathological myocardial microenvironments, to the phenotype of human cardiac fibroblasts are unclear. We hypothesized that transforming growth factor beta 1 and rigid extracellular matrix environments would potentially have a synergistic effect on the differentiation of human cardiac fibroblasts to myofibroblasts. To test this, we seeded primary human adult cardiac fibroblasts onto coverslips coated with polydimethylsiloxane of various elastic moduli, introduced transforming growth factor beta 1, and longitudinally quantified cell phenotype by measuring expression of α-smooth muscle actin, the most robust indicator of myofibroblasts. Our data indicate that, although extracellular matrix rigidity influenced differentiation after one day of transforming growth factor beta 1 treatment, ultimately transforming growth factor beta 1 superseded extracellular matrix rigidity as the primary regulator of myofibroblast differentiation. We also measured expression of POSTN, FAP, and FSP1, proposed secondary indicators of fibroblast/myofibroblast phenotypes. Although these genes partially trended with α-smooth muscle actin expression, they were relatively inconsistent. Finally, we demonstrated that activated myofibroblasts incompletely revert to a fibroblast phenotype after they are re-plated onto new surfaces without transforming growth factor beta 1, suggesting differentiation is partially reversible. Our results provide new insights into how microenvironmental cues affect human cardiac fibroblast differentiation in the context of myocardial pathology, which is important for identifying effective therapeutic targets and dictating supporting cell phenotypes for engineered human cardiac disease models. Impact statement Heart disease is the leading cause of death worldwide. Many forms of heart disease are associated with fibrosis, which increases extracellular matrix (ECM) rigidity and compromises cardiac output. Fibrotic tissue is synthesized primarily by myofibroblasts differentiated from fibroblasts. Thus, defining the cues that regulate myofibroblast differentiation is important for understanding the mechanisms of fibrosis. However, previous studies have focused on non-human cardiac fibroblasts and have not tested combinations of chemical and mechanical cues. We tested the effects of TGF-β1, a cytokine secreted by immune cells after injury, and ECM rigidity on the differentiation of human cardiac fibroblasts to myofibroblasts. Our results indicate that differentiation is initially influenced by ECM rigidity, but is ultimately superseded by TGF-β1. This suggests that targeting TGF-β signaling pathways in cardiac fibroblasts may have therapeutic potential for attenuating fibrosis, even in rigid microenvironments. Additionally, our approach can be leveraged to engineer more precise multi-cellular human cardiac tissue models.

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Transcriptomic changes during TGF-?-mediated differentiation of airway fibroblasts to myofibroblasts.

Publications

Transcriptomic changes during TGF-β-mediated differentiation of airway fibroblasts to myofibroblasts.

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