Defining the role of mechanoresponsive adipocyte-to-fibroblast transition in wound fibrosis.

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7. Project Summary/Abstract Adult human skin heals by developing fibrotic scar tissue, which can result in devastating disfigurement, growth restriction, and permanent functional loss. Despite a plethora of clinical options, no current treatment strategies successfully prevent or reverse this fibrotic process, and scars and their sequelae cost the United States over $20 billion every year. Progress towards the development of new therapies has been significantly hindered by a lack of understanding of the cell populations responsible for scarring and their molecular dynamics. Studies in recent years have reported that adipocytes in wounds are capable of transitioning into fibroblasts (and vice versa); however, the extent to which adipocyte-to-fibroblast transition contributes to wound fibrosis (scarring), and whether this process can be targeted to prevent scarring, remain unknown. In this proposal, we explore for the first time the role of tissue mechanics in conversion of dermal adipocytes to scarring fibroblasts within the wound environment. First, employing genetic lineage tracing, we will use histology, immunohistochemistry, and flow cytometry to study adipocyte-to-fibroblast transition and to interrogate the molecular phenotype of adipocyte lineage-derived fibroblasts within wounds. Second, we will use a Rainbow mouse model to interrogate clonal dynamics of adipocyte-to-fibroblast transition in wounds, and will apply an integrated multi-omic analysis, with single-cell transcriptomic (scRNA-seq) and epigenomic (scATAC-seq), spatial transcriptomic (Visium) and proteomic (CODEX), and quantitative extracellular matrix (ECM) ultrastructural analyses, in order to robustly define the molecular drivers and pathways involved in adipocyte-to-fibroblast conversion during scarring. Third, as our preliminary data strongly support a mechanotransduction mechanism underlying adipocyte-to-fibroblast transition during wound healing, we will inhibit mechanical signaling in adipocytes using both small molecule and transgenic approaches in order to block adipocyte-to-fibroblast transition. We will apply a similar multi-omic analysis to elucidate the molecular dynamics that differentiate wound adipocyte dynamics in the context of intact versus blocked mechanical signaling and determine how inhibiting mechanically driven adipocyte-to-fibroblast conversion may reduce fibrosis and yield wound regeneration. Our ultimate translational goal is to develop therapeutics that target fibrogenic wound cell dynamics to promote regenerative healing. Collectively, the proposed work will significantly enhance our understanding of the key molecular and cellular determinants of cutaneous scarring, inform the development of novel anti-scarring therapies, and shed light on the contributions of adipose tissue to wound fibrosis.