Elucidating the role of tissue mechanics in wound repair and regeneration

Wound healing problems affect hundreds of millions of individuals worldwide. Scarring is one of the most common issues in wound healing: any postnatal injury involving the dermis will result in some degree of fibrotic scar formation. Scar tissue differs from unwounded skin in multiple critical ways, including lacking skin's appendages (such as hair follicles and glands) and possessing decreased strength and elasticity compared to skin, due to scars' abnormal tissue ultrastructure. The ideal healing outcome would be wound repair via regeneration of normal tissue; however, no targeted therapies currently exist that can prevent or reverse scarring to yield regenerative wound healing. Therapeutic advancements have been limited, in part, by a lack of understanding of the precise molecular mechanisms that drive scarring versus those that could promote regeneration. In recent years, both basic and translational studies have illuminated a potential role for tissue mechanics in driving fibrosis. Mechanical forces at the tissue level are communicated into molecular changes at the cellular level via cell mechanotransduction signaling. Fibroblasts, the end cellular mediators of fibrosis, are particularly mechanosensitive cells, and it has been shown that mechanical stimuli and activated mechanotransduction signaling drive pro-fibrotic changes in fibroblasts. This dissertation explores, through the lenses of multiple distinct cellular and molecular pathways, how mechanically-targeted approaches may be able to block scarring and instead drive regenerative healing of mammalian wounds. The Introduction surveys existing knowledge and literature in key background topics, including wound repair, fibroblast biology and heterogeneity, and the roles of tissue mechanics and mechanotransduction in fibrosis. Part I of the Introduction reviews clinical issues in wound healing, including the clinical burden of scarring and fibrosis, to contextualize the need for novel anti-scarring therapies. Part II discusses basic science research into fibroblasts and fibrosis. Fibroblasts are a highly heterogeneous and plastic cell type; this section provides an overview of fibroblast subpopulations identified in the skin and other organs and their distinct roles in fibrosis and other physiologic and pathologic processes. Finally, Part III examines current knowledge of the role(s) of tissue mechanics in driving fibrosis and the cell populations and molecular signaling pathways involved in the mechanically-driven scarring response. Chapter I presents a study in which, through targeted mechanomodulation in postnatal dermal fibroblasts, complete, drug-induced wound regeneration was achieved for the first time in adult mouse wounds. Engrailed-1 lineage-positive fibroblasts (EPFs) are known to mediate dorsal scarring in mice; however, it was not previously known whether or how Engrailed-1 lineage-negative fibroblasts (ENFs) might contribute to postnatal wound repair. Using multiple in vivo and in vitro approaches, we show that ENFs activate Engrailed-1 expression and thereby convert into pro-fibrotic, postnatally-derived EPFs in response to mechanical forces in the wound environment, via a process of canonical mechanotransduction requiring Yes-associated protein (YAP). Next, we demonstrate that YAP inhibition, via either verteporfin (small molecule YAP inhibitor) treatment or fibroblast-targeted transgenic YAP knockout, blocks postnatal Engrailed-1 activation and ENF-to-EPF conversion and yields ENF-mediated, regenerative wound repair without scarring. These findings suggest that postnatal mammalian skin is capable of either a fibrotic or a regenerative wound response and the balance between these outcomes can be shifted by modulating YAP signaling. Chapter II presents a study wherein an integrated, multi-omic analytic approach is used to reveal divergent mechanisms of regenerative versus fibrotic wound repair. We use single-cell RNA-sequencing, timsTOF high-throughput shotgun proteomic sequencing, and a quantitative extracellular matrix ultrastructural analysis to profile both scarring (control) and regenerating (verteporfin-treated) wounds over the course of healing. By integrating these datasets at the single-mouse level, we reveal dynamic molecular trajectories characteristic of scarring and regenerative wound repair; the former is dominated by mechanical and fibrotic signaling, while the latter exhibits activated developmental, including Wnt pathway, signaling. Finally, using in vivo gene knockdown and overexpression, we show that fibroblast Trps1 expression is both necessary (in the context of YAP inhibition) and partially sufficient for wound regeneration. The results of this study represent an in-depth, multimodal analysis of a novel example of mammalian wound regeneration and may serve as a resource for future studies of regenerative versus fibrotic tissue repair. Chapter III presents a study in which our initial findings of YAP inhibition-induced wound regeneration in mice are translated to a rigorous, preclinical, large-animal model (excisional wound healing in red Duroc pigs). As rodent skin and wound healing differ from those of humans in several key ways, including having dramatically different mechanical properties, a critical component of translation is validating mouse findings in more human-like large animal models. Wound healing in red Duroc pigs is considered the gold standard preclinical model for human scarring. Here, we show that a single treatment with verteporfin at the time of wounding is sufficient to significantly reduce scarring and induce regeneration in high-tension red Duroc pig excisional wounds. We perform single-cell RNA-sequencing for the first time on porcine wounds and show that verteporfin-induced wound regeneration involves shifts in fibroblast subpopulation profiles. Further, using transcriptomic analyses of cell differentiation dynamics and in vitro fibroblast mechanomodulation studies, we find that modulation of mechanical stimuli and YAP signaling can shift the balance between putative pro-regenerative versus pro-fibrotic porcine dermal fibroblast subtypes. Finally, we validate our findings of verteporfin-induced wound regeneration in a human skin xenograft model. This study's findings are highly promising for the future translation of verteporfin treatment for clinical applications in preventing scarring. Lastly, Chapter IV presents a study in which wound mechanical forces are shown to activate dermal fibroblasts to a pro-fibrotic scar fibroblast fate. Using multiple in vitro and in vivo models, we show that adipocytes transition into scar-forming fibroblasts in response to wound mechanical forces by activating Piezo mechanosignaling. We elucidate the molecular dynamics of this process by using single-cell RNA-sequencing to show that adipocyte-to-fibroblast conversion proceeds via a "mechanically naïve" fibroblast transcriptional intermediate. Finally, we show that adipocyte-targeted Piezo inhibition prevents adipocytes' transition into fibroblasts and can both prevent (in new wounds) and reverse (in existing scars) wound fibrosis to yield skin regeneration. Piezo inhibition was also found to prevent human fibroblast-mediated scarring in a human skin xenograft model. These results demonstrate the critical role of adipocyte-to-fibroblast conversion in scarring and highlight a novel mechanism by which tissue mechanics drive adipocytes' contributions to fibrosis, which may represent a new therapeutic target for not only preventing but also reversing existing scarring

Advisor: Czechowicz Agnieszka
Wernig Gerlinde