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Inhibiting Yes-associated Protein Prevents Scarring And Promotes Regeneration In A Large Animal Model Of Wound Repair
Michelle Griffin, MSc MRes MD PhD MRCS, Mascharak Shamik, MD PhD, Heather Talbot, PhD, Jason Guo, PhD, Jennifer Parker, BSc, Dayan Li, MD PhD, Max Kuhnertm, BSc, Annah Morgan, BSc, Caleb Valencia, BSc, Michael Januszyk, MD PhD, H. Peter Lorenz, MD, Derrick Wan, MD, Michael T. Longaker, MD MBA.
Stanford University, Palo Alto, CA, USA.
Introduction: We recently showed that inhibiting mechanotransduction (Yes-associated protein [YAP]) in mouse wounds yields regeneration without scarring. However, rodents are loose-skinned and fail to recapitulate key aspects of human wound repair. We sought to elucidate the effects of YAP inhibition in red Duroc pig wounds, the most human-like model of scarring.
Methods: Full-thickness excisional wounds (2x5cm hexagons) were produced on the dorsum of adult pigs (n=6). Wounds received intradermal verteporfin (YAP inhibitor; 2mg/mL) or vehicle control (PBS) followed by primary repair with 3-0 Vicryl deep dermal and 3-0 Monocryl running subcuticular sutures. Cutometer measurements were obtained to assess tissue stiffness every two weeks. Wounds and unwounded skin were harvested after 2, 4, 8, and 16 weeks for histologic (hematoxylin and eosin staining), mechanical (Instron strength testing), scRNA-seq (10X Chromium) analyses and CODEX protein spatial analysis.
Results: Grossly and histologically, verteporfin treatment significantly reduced scarring and promoted skin regeneration, including recovery of hair follicles/glands (
Figure 1A-B). Verteporfin-treated wounds were both significantly less stiff (
Figure 1C) and stronger (
Figure 1D) than PBS-treated wounds, with mechanical properties similar to those of unwounded skin. scRNA-seq identified two novel fibroblast subpopulations, one enriched in regenerating (verteporfin) wounds and unwounded skin, the other enriched in scarring (PBS) wounds (
Figure 1E-F; clusters 1 and 4, respectively). 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. CODEX protein spatial analysis further supported the fibroblast dynamics among treatment groups as that observed by scRNAseq. CODEX spatial protein expression analysis confirmed that fibrotic and regenerative fibroblast subtype markers identified by scRNA-seq at the protein level, with differential distribution and cell-cell interactions in PBS-versus YAPi-treated wounds.
Conclusions: One-time local administration of verteporfin following injury significantly reduces scarring and induces regenerative healing in a large animal model. Collectively, our findings provide critical support for the future translation of local mechanotransduction inhibitors to prevent human skin scarring.
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