RNA-sequencing demonstrates transcriptional differences between human vocal fold fibroblasts and myofibroblasts.

Abstract

Background: Differentiation of fibroblasts into myofibroblasts is necessary for wound healing, but excessive myofibroblast presence and persistence can result in scarring. Treatment for scarring is limited largely due to a lack of comprehensive understanding of how fibroblasts and myofibroblasts differ at the transcript level. The purpose of this study was to characterize transcriptional profiles of injured fibroblasts relative to normal fibroblasts, utilizing fibroblasts from the vocal fold as a model.

Results: Utilizing bulk RNA sequencing technology, we identified differentially expressed genes between four cell lines of normal fibroblasts (cVFF), one line of scarred fibroblasts (sVFF), and four lines of fibroblasts treated with transforming growth factor-beta 1 (TGF-β1), representing an induced-scar phenotype (tVFF). Principal component analysis revealed clustering of normal fibroblasts separate from the clustering of fibroblasts treated with TGF-β1; scarred fibroblasts were more similar to normal fibroblasts than fibroblasts treated with TGF-β1. Enrichment analyses revealed pathways related to cell signaling, receptor-ligand activity, and regulation of cell functions in scarred fibroblasts, pathways related to cell adhesion in normal fibroblasts, and pathways related to ECM binding in fibroblasts treated with TGF-β1. Although transcriptomic profiles between scarred fibroblasts and fibroblasts treated with TGF-β1 were relatively dissimilar, the most highly co-expressed genes were enriched in pathways related to actin cytoskeleton binding, which supports the use of fibroblasts treated with TGF-β1 to represent a scarred cell phenotype.

Conclusions: Transcriptomics of normal fibroblasts differ from myofibroblasts, including from those retrieved from scar and those treated with TGF-β1. Despite large differences in transcriptomics between tVFF and sVFF, tVFF serve as a useful in vitro model of myofibroblasts and highlight key similarities to myofibroblasts extracted from scar pathology, as well as expected differences related to normal fibroblasts from healthy vocal folds.