6. Multi-omic Approach to the Remodeling Phase of Wound Healing: Attenuating Existing Scars Via Piezo 1 Inhibition

INTRODUCTION: Wound healing occurs in a series stages: coagulation, inflammation, cell proliferation, and remodeling. Although early stages of wound healing have been explored in depth, molecular investigations into later-stage remodeling have not yet been conducted. Notably, mechanotransduction is known to play a crucial role during scar formation; however, whether these signals participate in scar homeostasis during late-stage scar remodeling is incompletely understood. Further, our group has previously demonstrated that inhibition of Piezo1 (P1i) signaling at time of wounding prevents scar formation in mice. Applying a multi-omic approach, we investigated the molecular dynamics of later-stage scar remodeling, and how P1i leads to attenuation of existing, long-term scars. METHODS: Adult C57Bl/6 mice received 8-mm stented dorsal excisional wounds, which were harvested at post-injury days 2, 7 and 14 (early-stage wound healing), and days 60, 105, and 150 (late-stage wound healing) for histologic analysis. Half of the late-stage scars were also treated with P1i via local intra-dermal injection within scars 30 days prior to tissue collection (days 30, 75, and 120 following wounding). Tissue sections were analyzed for histology and confocal microscopy. Additional wounds from the same timepoints were processed for single cell RNA sequencing (scRNAseq), spatial transcriptomics, and PhenoCycler spatial proteomics. RESULTS: Grossly, scars remained visible 150 days following wounding. Remodeled wounds exhibited characteristic features of early-stage scars, including an absence of skin appendages such as hair follicles. Interestingly, Picrosirius Red ultrastructure analysis illustrated distinct separation of early and late-stage scars, while both early and late-stage scars remained distinct from unwounded skin ultrastructure. P1i application alleviated existing scars, with the reappearance of skin appendages such as hair follicles, as well as recovery of unwounded-like extracellular matrix architecture. Interestingly, similar to what has been observed in early-staged wound healing, spatial proteomics and transcriptomics demonstrated the continued relevance of mechanical signaling pathways in late-stage control wounds; fibroblast subpopulations and adipocytes expressed different degrees of mechanical markers including PTK2, YAP1, PIEZO1, and PIEZO2. Notably, presence of mechanically sensitive adipocyte and fibroblast subpopulations was reduced among P1i scars, indicated that inhibition of PIEZO1 can modulate dermal spatial environment up until 120 days following injury. CONCLUSIONS: We provide in-depth characterization of the remodeling phase of wound healing for which, to our knowledge, few analyses have been performed to date. Histologic, transcriptomic, and proteomic analyses reveal that the remodeling phase remains a dynamic process. The continued expression of mechanotransduction signaling at both transcriptional and proteomic levels in fibroblasts and adipocytes within scar beds suggest a potential mechanism by which dermal fibrosis is maintained months following injury. These data also provide encouraging results that P1i can modulate the dermal spatial environment, even 120 days following injury, and holds promise as a potential therapeutic target for established pathologic scarring.