J.B. Parker1,2, M. Griffin1, A. Tomasso1, J.L. Guo1, C. Valencia1, A. Morgan1, M. Kuhnert1, M. Januszyk1, M.T. Longaker1,2, D. Wan1 1Stanford University, Division Of Plastic And Reconstructive Surgery, Stanford, CA, USA 2Stanford University, Institute Of Stem Cell Biology And Regenerative Medicine, Palo Alto, CA, USA
Introduction: Although early stages of wound healing have been explored in depth, molecular investigations into later-stage remodeling have not yet been conducted. Our group has previously shown the role of mechanotransduction pathways during scar formation, and has demonstrated that inhibition of Piezo1 (P1i) signaling at time of wounding leads to scar prevention. Here, we conducted molecular investigations into later-stage scar remodeling, and explored how P1i can lead to reduced scarring on existing scars.
Methods: Adult C57Bl/6 mice received 8-mm stented dorsal excisional wounds, which were harvested at days 2, 7 and 14 (early-stage wound healing), and days 60, 105, and 150 following wounding (late-stage wound healing) for histologic comparison. Half of the late-stage scars were also treated with P1i via intra-dermal injection 30 days prior to tissue collection (days 30, 75, and 120 following wounding). Whole tissues were analyzed for histology and confocal microscopy. Wounds from the same timepoints were also prepared for Chromium Single Cell RNA sequencing (scRNAseq), spatial transcriptomics, and CODEX spatial proteomics (Fig. 1A).
Results: Grossly, scars from these initial wounds remained visible 150 days following wounding (Fig 1B). Remodelled wounds exhibited characteristic features of early-stage scars, including an absence of hair follicles and other skin appendages (Fig. 1B). Interestingly, ultrastructure analysis of Picrosirius red staining illustrated distinct separation of early and late-stage scars, with both groups spatially separating from unwounded skin. P1i treatment of existing scars led to a rescued phenotype, with the reappearance of skin appendage such as hair follicles normally absent in scars. Notably, similar to what has been overserved in the earlier stages of wound healing, spatial proteomic (Fig. 1D) and transcriptomic (Fig. 1E) analyses confirmed the continued prominence of mechanical signaling pathways in remodelled control wounds, with subpopulations of fibroblasts and adipocytes expressing different degrees of mechanical markers PTK2, YAP1, PIEZO1, and PIEZO2 (Fig. 1D). Strikingly, mechanically sensitive fibroblast and adipocyte subpopulations were reduced in P1i-treating scars, suggesting that P1i can modulate the dermal spatial environment up until 120 days following injury (Fig. 1D-E).
Conclusion: These results demonstrate that remodeling remains a dynamic process with cellular and signaling components within the tissue driving continued maintenance of dermal fibrosis months following injury. Our findings also suggest that P1i can modulate the dermal spatial environment up until 120 days following injury, and may hold promise as a potential therapeutic target for established pathologic scarring.