Plastic Surgery Research Council

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Inhibition of Mechanotransduction Yields Regenerative Wound Healing by Engrailed1-negative Fibroblasts
Shamik Mascharak, Alessandra L. Moore, Heather E. desJardins-Park, Maria R. Borrelli, Bryan Duoto, Malini Chinta, Deshka S. Foster, Hermann P. Lorenz, Michael T. Longaker.
Stanford University, Stanford, CA, USA.

PURPOSE: Skin scarring poses a significant medical burden for tens of millions of patients every year. Recently, Engrailed1-positive fibroblasts (EPFs) were shown to be responsible for the majority of scarring on the dorsal skin after embryonic day (e)18.5 in mice. However, comparatively little is known about the postnatal function of Engrailed1-negative fibroblasts (ENFs), which are present in all layers of the dermis and are putatively non-scarring. We sought to characterize the ENF lineage and assess if ENF-mediated wound healing leads to more ordered repair of skin with regeneration of dermal appendages.
METHODS: Experiments were performed in En1Cre;R26mTmG (En1mTmG) and En1Cre;Ai6 (En1Ai6) mice, with Engrailed1-positive cells expressing GFP and Engrailed1-negative cells expressing RFP or no reporter, respectively. Postnatal (p)30 mice underwent dorsal excisional wounding, and healed wound histology was assessed at 2 or 4 weeks. For mechanotransduction inhibition, the wound bed was injected with 30 uL of 50 uM Verteporfin in PBS. Engrailed1-negative fibroblasts were isolated from whole dorsal p1 skin using fluorescence-activated cell sorting (FACS). In vivo behavior of ENFs was assessed by injecting FACS-sorted ENFs into p30 mouse dorsal skin 2 days prior to wounding. Transcriptomic analysis of ENFs and EPFs was performed using RNA-sequencing of plated cells.
RESULTS: FACS-isolated ENFs from p1 mice activate Engrailed1 after 7 days of culture on plastic (Figure 1A top row), as indicated by transition from red to green fluorescence. However, ENFs do not activate Engrailed1 in vitro after inhibition of stiffness sensing (ROCK inhibitor Y-27632) or culture in soft three-dimensional collagen hydrogels, suggesting a mechanotransduction-mediated mechanism for postnatal Engrailed1 expression (Figure 1A bottom row). Postnatal ENFs transplanted into dorsal skin also show activation of Engrailed1 after wounding (Figure 1B). Transcriptomic analysis by RNA-sequencing reveals that postnatal ENF to EPF transition is accompanied by expression of genes related to fibrosis (e.g., WNT/TGFb) and mechanotransduction signaling, including several target genes of Yes-associated protein (YAP). Accordingly, wounds treated with a single administration of YAP inhibitor Verteporfin yield scars with markedly fewer EPFs, reduced fibrosis, and greater ENF presence at 2 weeks. After 4 weeks, Verteporfin-treated wounds show sustained presence of ENFs, as well as regeneration of dermal appendages (Figure 1C top vs. bottom row).
CONCLUSIONS: Postnatal ENFs activate Engrailed1 in vitro by canonical mechanotransduction signaling and take on a fibrotic phenotype; a similar process occurs in the in vivo wound environment. We demonstrate that inhibition of YAP signaling promotes ENF-mediated wound healing with reduced fibrosis and regeneration of secondary elements. Our findings suggest that ENFs may play a critical role in scarring by activating Engrailed1 in response to mechanical cues within the wound bed. Considering that the Engrailed1-negative lineage represents several distinct subpopulations of cells, we aim to assess postnatal Engrailed1 activation in the papillary, reticular, and hypodermal layers to identify a specific ENF population that contributes to scarring.


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