Piezo Inhibition Prevents and Rescues Scarring By Targeting the Adipocyte To Fibroblast Transition

Background: While studies have suggested that plasticity exists between dermal fibroblast and adipocytes, it remains unknown whether fat contributes to fibrosis. We hypothesized that mature adipocytes directly participate in wound repair via conversion into fibroblasts, and that adipocyte-derived fibroblasts (ADFs) contribute to skin scarring. Methods: AdipoqCreERT transgenic driver mice were crossed to R26mTmG reporter mice to generate AdipoqCreERT;ROSA26mTmG mice to perform lineage tracing of mature adipocytes. To achieve local adipocyte ablation AdipoqCre;ROSA26mTmG;R26tm1(HBEGF)Awai mice were generated and wounded, and diphtheria toxin (DT) was injected into the wound base. DT- and vehicle control-treated wounds underwent histologic analysis. Lastly, we performed single-cell RNA-sequencing (scRNAseq), Visium gene spatial analysis, and CODEX protein analysis on wounded and unwounded tissue to identify fibrotic ADF subpopulations. Results: Using our AdipoqCre;ROSA26mTmG adipocyte lineage-tracing model, we identified significantly greater number of adiponectin lineage-positive cells (GFP+) within wounds at post-operative day-14 (POD-14) compared to unwounded skin (*P<0.05, n=12). Compared to typical subcutaneous adipocytes, the GFP+ cells exhibited upregulation of fibroblast markers and downregulation of adipocyte markers. FACS further confirmed that the GFP+ cells were fibroblasts and increased to 10% at POD-14. DT-induced ablation of Adipoq lineage-positive cells exhibited reduced scar thickness and collagen deposition at POD-14 compared to control wounds (*P<0.05, n=12). Using an in-vitro collagen gel system we establish that mechanics alone is sufficient to drive the adipocyte-to-fibroblast conversion. Further in vivo analysis using scRNAseq and spatial gene and protein analysis revealed the distinct mechanically sensitive ADF subpopulations expressed high levels of Piezo1 and Piezo2. We further show that Piezo1 or Piezo2 -inhibition yields regenerative healing by preventing adipocytes' activation to fibroblasts, in mouse-wounds using both small molecule Piezo inhibitors (P1i/P2i) and Piezo1/Piezo2 genetic knockouts (P1 KO/P2 KO) (*P < 0.01, n= 12). In addition, P1i overcomes skin scarring in a novel human-xenograft-wound model (n=10). Lastly, we show that P1i was sufficient to induce near-complete wound regeneration of 1- month, 2.5 month and 4 month established mouse scars with return of hair follicles, including full recovery of unwounded-like extracellular architecture, compared to untreated wounds which remained scar-like on histology (n=30). Conclusions: Our findings strongly suggest that mature adipocytes in the skin undergo conversion to pro-fibrotic fibroblasts in response to injury. Importantly, Piezo1 -inhibition induced wound regeneration even in pre-existing established scars, a finding that suggests a role for adipocyte-to-fibroblast transition in wound remodeling, the least-understood phase of wound healing.