A tension offloading patch mitigates dermal fibrosis induced by pro-fibrotic skin injections.

Skin fibrosis is a clinical problem with devastating impacts but limited treatment options. In the setting of diabetes, insulin administration often causes local dermal fibrosis, leading to a range of clinical sequelae including impeded insulin absorption. Mechanical forces are important drivers of fibrosis and, clinically, physical tension offloading at the skin level using an elastomeric patch significantly reduces wound scarring. However, it is not known whether tension offloading could similarly prevent skin fibrosis in the setting of pro-fibrotic injections. Here, we develop a porcine model using repeated local injections of bleomycin to recapitulate key features of insulin-induced skin fibrosis. Using histologic, tissue ultrastructural, and biomechanical analyses, we show that application of a tension-offloading patch both prevents and rescues existing skin fibrosis from bleomycin injections. By applying single-cell transcriptomic analysis, we find that the fibrotic response to bleomycin involves shifts in myeloid cell dynamics from favoring putatively pro-regenerative to pro-fibrotic myeloid subtypes; in a mechanomodulatory in vitro platform, we show that these shifts are mechanically driven and reversed by exogenous IL4. Finally, using a human foreskin xenograft model, we show that IL4 treatment mitigates bleomycin-induced dermal fibrosis. Overall, this study highlights that skin tension offloading, using an FDA cleared, commercially available patch, could have significant potential clinical benefit for the millions of patients dependent on insulin.

Allele-specific expression reveals genetic drivers of tissue regeneration in mice.

In adult mammals, skin wounds typically heal by scarring rather than through regeneration. In contrast, "super-healer" Murphy Roths Large (MRL) mice have the unusual ability to regenerate ear punch wounds; however, the molecular basis for this regeneration remains elusive. Here, in hybrid crosses between MRL and non-regenerating mice, we used allele-specific gene expression to identify cis-regulatory variation associated with ear regeneration. Analyzing three major cell populations (immune, fibroblast, and endothelial), we found that genes with cis-regulatory differences specifically in fibroblasts were associated with wound-healing pathways and also co-localized with quantitative trait loci for ear wound-healing. Ectopic treatment with one of these proteins, complement factor H (CFH), accelerated wound repair and induced regeneration in typically fibrotic wounds. Through single-cell RNA sequencing (RNA-seq), we observed that CFH treatment dramatically reduced immune cell recruitment to wounds, suggesting a potential mechanism for CFH's effect. Overall, our results provide insights into the molecular drivers of regeneration with potential clinical implications.

Piezo inhibition prevents and rescues scarring by targeting the adipocyte to fibroblast transition.

While past studies have suggested that plasticity exists between dermal fibroblasts and adipocytes, it remains unknown whether fat actively contributes to fibrosis in scarring. We show that adipocytes convert to scar-forming fibroblasts in response to Piezo -mediated mechanosensing to drive wound fibrosis. We establish that mechanics alone are sufficient to drive adipocyte-to- fibroblast conversion. By leveraging clonal-lineage-tracing in combination with scRNA-seq, Visium, and CODEX, we define a "mechanically naïve" fibroblast-subpopulation that represents a transcriptionally intermediate state between adipocytes and scar-fibroblasts. Finally, we show that Piezo1 or Piezo2 -inhibition yields regenerative healing by preventing adipocytes' activation to fibroblasts, in both mouse-wounds and a novel human-xenograft-wound model. Importantly, Piezo1 -inhibition induced wound regeneration even in pre-existing established scars, a finding that suggests a role for adipocyte-to-fibroblast transition in wound remodeling, the least-understood phase of wound healing. Adipocyte-to-fibroblast transition may thus represent a therapeutic target for minimizing fibrosis via Piezo -inhibition in organs where fat contributes to fibrosis.

Multiplexed evaluation of mouse wound tissue using oligonucleotide barcoding with single-cell RNA sequencing.

Despite its rapidly increased availability for the study of complex tissue, single-cell RNA sequencing remains prohibitively expensive for large studies. Here, we present a protocol using oligonucleotide barcoding for the tagging and pooling of multiple samples from healing wounds, which are among the most challenging tissue types for this application. We describe steps to generate skin wounds in mice, followed by tissue harvest and oligonucleotide barcoding. This protocol is also applicable to other species including rats, pigs, and humans. For complete details on the use and execution of this protocol, please refer to Stoeckius et al. (2018),1 Galiano et al. (2004),2 and Mascharak et al. (2022).3.

Multiomic analysis reveals conservation of cancer-associated fibroblast phenotypes across species and tissue of origin.

Cancer-associated fibroblasts (CAFs) are integral to the solid tumor microenvironment. CAFs were once thought to be a relatively uniform population of matrix-producing cells, but single-cell RNA sequencing has revealed diverse CAF phenotypes. Here, we further probed CAF heterogeneity with a comprehensive multiomics approach. Using paired, same-cell chromatin accessibility and transcriptome analysis, we provided an integrated analysis of CAF subpopulations over a complex spatial transcriptomic and proteomic landscape to identify three superclusters: steady state-like (SSL), mechanoresponsive (MR), and immunomodulatory (IM) CAFs. These superclusters are recapitulated across multiple tissue types and species. Selective disruption of underlying mechanical force or immune checkpoint inhibition therapy results in shifts in CAF subpopulation distributions and affected tumor growth. As such, the balance among CAF superclusters may have considerable translational implications. Collectively, this research expands our understanding of CAF biology, identifying regulatory pathways in CAF differentiation and elucidating therapeutic targets in a species- and tumor-agnostic manner.

Wound healing, fibroblast heterogeneity, and fibrosis.

Fibroblasts are highly dynamic cells that play a central role in tissue repair and fibrosis. However, the mechanisms by which they contribute to both physiologic and pathologic states of extracellular matrix deposition and remodeling are just starting to be understood. In this review article, we discuss the current state of knowledge in fibroblast biology and heterogeneity, with a primary focus on the role of fibroblasts in skin wound repair. We also consider emerging techniques in the field, which enable an increasingly nuanced and contextualized understanding of these complex systems, and evaluate limitations of existing methodologies and knowledge. Collectively, this review spotlights a diverse body of research examining an often-overlooked cell type-the fibroblast-and its critical functions in wound repair and beyond.

Multi-omic analysis reveals divergent molecular events in scarring and regenerative wound healing.

Regeneration is the holy grail of tissue repair, but skin injury typically yields fibrotic, non-functional scars. Developing pro-regenerative therapies requires rigorous understanding of the molecular progression from injury to fibrosis or regeneration. Here, we report the divergent molecular events driving skin wound cells toward scarring or regenerative fates. We profile scarring versus YAP-inhibition-induced wound regeneration at the transcriptional (single-cell RNA sequencing), protein (timsTOF proteomics), and tissue (extracellular matrix ultrastructural analysis) levels. Using cell-surface barcoding, we integrate these data to reveal fibrotic and regenerative "molecular trajectories" of healing. We show that disrupting YAP mechanotransduction yields regenerative repair by fibroblasts with activated Trps1 and Wnt signaling. Finally, via in vivo gene knockdown and overexpression in wounds, we identify Trps1 as a key regulatory gene that is necessary and partially sufficient for wound regeneration. Our findings serve as a multi-omic map of wound regeneration and could have therapeutic implications for pathologic fibroses.