Inhibition of Yes-Associated Protein Promotes Skin Wound Regeneration in Large Animals

INTRODUCTION: Inhibiting mechanotransduction signaling has been shown in preclinical rodent studies to reduce scar formation; however, rodents are loose-skinned mammals and may not fully replicate human healing. METHODS: Full-thickness excisional wounds (2 cm × 5 cm) were produced on the dorsal skin of adult red Duroc pigs. PBS or verteporfin (a YAP inhibitor) were injected into wounds, followed by primary repair with 3-0 Vicryl deep dermal and 3-0 Monocryl running subcuticular sutures. Cutometer measurements were taken to measure stiffness of normal skin and scars every 2 weeks. Immunohistochemistry was performed for collagen proteins, scarring fibroblast markers, cytokeratins, and YAP. Images were evaluated using a machine learning algorithm to stratify tissue architecture. To measure wound breaking strength, tissue strips spanning each region were excised and evaluated using a MTS Bionix force transducer. scRNA-seq was performed using the 10X Chromium system. Postprocessing dimensional reduction was achieved using parametrically optimized UMAP analysis. Pseudotime (Monocle 3) receptor-ligand (CellChat) and RNA velocity (scVelo) analyses were performed in R. RESULTS: Verteporfin treatment was found to significantly reduce scarring and promote skin regeneration, including recovery of hair follicles and glands. scRNA-seq analysis identified 2 novel fibroblast subpopulations, one unique to verteporfin-treated and unwounded skin and the other present in control wounds only. Pseudotime trajectory constructs demonstrated putative state transitions between these two populations, which were supported by prospective FACS isolation and force manipulation in a 3D culture system. CONCLUSION: One-time local administration of verteporfin after injury significantly reduces scarring and induces regenerative healing in a large animal model.