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Inhibiting Mechanotransduction Signaling Changes Fibroblast Heterogeneity And Promotes Tissue Regeneration In Healing Wounds
Kellen Chen, PhD, Sun Hyung Kwon, PhD, Dominic Henn, MD, Britta A. Kuehlmann, MD, Clark A. Bonham, BS, Jagannath Padmanabhan, PhD, Chikage Noishiki, MD PhD, Janos Barrera, MD, Michael T. Longaker, MD, Michael Januszyk, MD PhD, Geoffrey C. Gurtner, MD.
Stanford University, Stanford, CA, USA.

BACKGROUND: The ability of cells to respond to injury through either a fibrotic or regenerative phenotype plays an important role in wound healing processes throughout the body. Currently, clinically effective treatment strategies for scars and fibrotic conditions are very limited. In the past decade, we have demonstrated that mechanical stress is a critical component of hypertrophic scar formation, acting via mechanisms that promote fibrotic cellular activities.
METHODS: We created large deep partial-thickness wounds on the dorsum of red duroc pigs. Immediately upon injury, wounds were treated with either focal adhesion kinase inhibitor (FAKI) or placebo-releasing hydrogels or standard bandages as controls. Wound healing and scar quality was assessed over time by serial quantification of scar area in photographic images as well as cutometer measurements of the scars over time. We then isolated and cultured dermal fibroblasts from both porcine skin as well as human skin collected from various surgical procedures. Fibroblasts were incorporated into 3D collagen lattices. The cell-populated collagen lattices were either stretched, stretched and treated with FAKI, or left unstretched for 2 days before being processed for droplet-based microfluidic single cell RNA sequencing (scRNAseq) using the 10X Genomics platform. Data were log-normalized and partitioned using UMAP based density mappings.
RESULTS: Disruption of mechanotransduction using FAKI resulted in significantly accelerated wound closure, lower Visual Analog Scale (VAS) score, decreased firmness and increased elasticity in the cutometer measurements, and regrowth of hair follicles as well as other dermal appendages. FAKI treated wounds also demonstrated significantly lower expression of alpha smooth muscle actin (αSMA). ScRNAseq analysis indicated that mechanical stretch in the 3D collagen lattice system shifted fibroblast gene expression profiles to distinctly different subpopulations in UMAP based density mappings which showed a higher expression of ACTA2 (αSMA), as well as classical pro-fibrotic markers (PDGFR, COL1A1). Treatment of stretched fibroblasts with FAKI shifted fibroblast heterogeneity into a subpopulation that had decreased expression of pro-fibrotic markers. These findings were observed in porcine fibroblasts as well as fibroblasts originating from various human patients and procedures, demonstrating the robustness of our findings.
CONCLUSIONS: Small molecule disruption of FAK-mediated mechanotransduction promotes wound healing and improves the quality of resultant scar likely due to an enhanced regeneration of dermal appendages. Moreover, our data indicate that FAKI treatment is able to mitigate the transcriptional pro-fibrotic signatures which we induced in our 3D collagen lattice system by shifting fibroblast heterogeneity from a pro-fibrotic into a more regenerative subpopulation.


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