Abstract
Despite recent advances in understanding skin scarring, mechanisms triggering hypertrophic scar formation are still poorly understood. In the present study, we investigate mature human hypertrophic scars and developing scars in mice at single cell resolution. Compared to normal skin, we find significant differences in gene expression in most cell types present in scar tissue. Fibroblasts show the most prominent alterations in gene expression, displaying a distinct fibrotic signature. By comparing genes upregulated in murine fibroblasts during scar development with genes highly expressed in mature human hypertrophic scars, we identify a group of serine proteases, tentatively involved in scar formation. Two of them, dipeptidyl-peptidase 4 (DPP4) and urokinase (PLAU), are further analyzed in functional assays, revealing a role in TGFβ1-mediated myofibroblast differentiation and over-production of components of the extracellular matrix in vitro. Topical treatment with inhibitors of DPP4 and PLAU during scar formation in vivo shows anti-fibrotic activity and improvement of scar quality, most prominently after application of the PLAU inhibitor BC-11. In this study, we delineate the genetic landscape of hypertrophic scars and present insights into mechanisms involved in hypertrophic scar formation. Our data suggest the use of serine protease inhibitors for the treatment of skin fibrosis.
Highlights
Despite recent advances in understanding skin scarring, mechanisms triggering hypertrophic scar formation are still poorly understood
To elucidate the complex biological processes of scar formation, we performed droplet-based single-cell transcriptome analysis of human hypertrophic scar tissue and healthy skin[34] (Fig. 1A). In both samples, Unsupervised Uniform Manifold Approximation and Projection (UMAP)-clustering revealed 21 cell clusters, which were further classified as specific cell types by well-established marker genes (Figure S2A), expression patterns of all clusters (Figure S2B), and transcriptional cluster proximity via a phylogenetic clustertree (Fig. 1B)
We identified seven FB clusters, smooth muscle cells and pericytes (SMC/Peri), three clusters of endothelial cells (EC), and lymphatic endothelial cells (LECs), two clusters of T cells and of dendritic cells (DC), macrophages (Mac), three keratinocyte (KC) clusters, and melanocytes (Mel)
Summary
Despite recent advances in understanding skin scarring, mechanisms triggering hypertrophic scar formation are still poorly understood. By comparing genes upregulated in murine fibroblasts during scar development with genes highly expressed in mature human hypertrophic scars, we identify a group of serine proteases, tentatively involved in scar formation. Compression or massage therapy, intralesional injection of triamcinolone (TAC), corticosteroids, or 5-Fluorouracil (5-FU), laser ablation, and surgery are the most commonly used options for prevention or treatment of hypertrophic scars[6,9,10,11] Many of these therapies lack evidence of efficacy and safety, show high recurrence rates, and mechanisms of actions are still unclear[10,12]. The group of trypsins comprises proteases contributing to vital processes such as blood coagulation, fibrinolysis, apoptosis, and immunity[16] Members of this family include urokinase, HTRA1/3 (high-temperature requirement A1/3 peptidase), several coagulation factors and complement components, PRSS-like serine proteases, granzymes, and cathepsin G16,17. Even though scRNAseq was previously performed to identify factors important for embryonic[30] and postnatal[31] skin development as well as for tissue regeneration[32] by investigating murine wound healing[33], scar tissue on single-cell level has not been investigated yet
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