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Redirecting valvular myofibroblasts into dormant fibroblasts through light-mediated reduction in substrate modulus.

ABSTRACT: Fibroblasts residing in connective tissues throughout the body are responsible for extracellular matrix (ECM) homeostasis and repair. In response to tissue damage, they activate to become myofibroblasts, which have organized contractile cytoskeletons and produce a myriad of proteins for ECM remodeling. However, persistence of myofibroblasts can lead to fibrosis with excessive collagen deposition and tissue stiffening. Thus, understanding which signals regulate de-activation of myofibroblasts during normal tissue repair is critical. Substrate modulus has recently been shown to regulate fibrogenic properties, proliferation and apoptosis of fibroblasts isolated from different organs. However, few studies track the cellular responses of fibroblasts to dynamic changes in the microenvironmental modulus. Here, we utilized a light-responsive hydrogel system to probe the fate of valvular myofibroblasts when the Young's modulus of the substrate was reduced from ~32 kPa, mimicking pre-calcified diseased tissue, to ~7 kPa, mimicking healthy cardiac valve fibrosa. After softening the substrata, valvular myofibroblasts de-activated with decreases in ?-smooth muscle actin (?-SMA) stress fibers and proliferation, indicating a dormant fibroblast state. Gene signatures of myofibroblasts (including ?-SMA and connective tissue growth factor (CTGF)) were significantly down-regulated to fibroblast levels within 6 hours of in situ substrate elasticity reduction while a general fibroblast gene vimentin was not changed. Additionally, the de-activated fibroblasts were in a reversible state and could be re-activated to enter cell cycle by growth stimulation and to express fibrogenic genes, such as CTGF, collagen 1A1 and fibronectin 1, in response to TGF-?1. Our data suggest that lowering substrate modulus can serve as a cue to down-regulate the valvular myofibroblast phenotype resulting in a predominantly quiescent fibroblast population. These results provide insight in designing hydrogel substrates with physiologically relevant stiffness to dynamically redirect cell fate in vitro.

SUBMITTER: Wang H

PROVIDER: S-EPMC3396623 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Redirecting valvular myofibroblasts into dormant fibroblasts through light-mediated reduction in substrate modulus.

Wang Huan H Wang Huan H Haeger Sarah M SM Kloxin April M AM Leinwand Leslie A LA Anseth Kristi S KS

PloS one 20120713 7

Fibroblasts residing in connective tissues throughout the body are responsible for extracellular matrix (ECM) homeostasis and repair. In response to tissue damage, they activate to become myofibroblasts, which have organized contractile cytoskeletons and produce a myriad of proteins for ECM remodeling. However, persistence of myofibroblasts can lead to fibrosis with excessive collagen deposition and tissue stiffening. Thus, understanding which signals regulate de-activation of myofibroblasts dur ...[more]

PMID: 22808079

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Project description: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.

Dataset Information

Redirecting valvular myofibroblasts into dormant fibroblasts through light-mediated reduction in substrate modulus.

Publications

Redirecting valvular myofibroblasts into dormant fibroblasts through light-mediated reduction in substrate modulus.

Similar Datasets

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