Epigenetic Modulation of TGF-beta Signaling Underlies the Scarring Fibroblast Phenotype
Heather E. desJardins-Park, AB1, Alessandra L. Moore, MD1, Ulrike Litzenburger, PhD2, Clement D. Marshall, MD1, Leandra A. Barnes, AB1, Shamik Mascharak, BS1, Bryan Duoto, MS1, Ryan C. Ransom, BS1, Deshka S. Foster, MD1, Michael S. Hu, MD1, H. Peter Lorenz, MD1, Howard Y. Chang, MD2,3, Michael T. Longaker, MD1,4.
1Department of Surgery, Division of Plastic and Reconstructive Surgery, Stanford University School of Medicine, Stanford, CA, USA, 2Center for Personal Dynamic Regulomes, Stanford University School of Medicine, Stanford, CA, USA, 3Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA, USA, 4Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, USA.
PURPOSE: Scars represent a massive clinical and financial burden on healthcare systems. Interestingly, mammalian fetuses are capable of scarless wound healing. However, little progress has been made toward scarless healing in adult patients, as underlying molecular pathways remain poorly elucidated. The murine dorsal "scarring fibroblast" lineage can be identified by embryonic expression of Engrailed1. Intriguingly, these Engrailed1-positive fibroblasts (EPFs) are present in the dermis prior to the developmental transition to scarring healing between embryonic day (e)16.5-e18.5. We sought to determine the molecular basis for the development of the EPF scarring phenotype.
METHODS: Experiments were performed in En1Cre;R26mTmG (En1mTmG) mice, in which Engrailed1 lineage-positive cells express GFP and Engrailed1 lineage-negative cells express RFP. En1mTmG fetal mice underwent dorsal excisional wounding at e16.5 and e18.5, and wounds and controls (unwounded dorsal skin) were harvested at 48h post-wounding and processed for histology. EPFs and Engrailed1-negative fibroblasts (ENFs; non-scarring resident dermal fibroblasts) were isolated from skin at e16.5, e18.5, postnatal day (p)1, and p60 using fluorescence-activated cell sorting (FACS). In vivo EPF phenotype was analyzed by injecting EPFs from e16.5 or p1 into the dorsal dermis of C57BL/6J mice at p1 or e16.5, respectively. Epigenetic analysis was performed using the Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq).
RESULTS: Healed e16.5 wounds are indistinguishable from unwounded skin, whereas e18.5 wounds form distinct scars; however, EPFs contribute to healing at both timepoints (Figure 1A,B). EPFs from a non-scarring timepoint transplanted into a scarring niche exhibit linear morphology and 2.13% colocalization with type I collagen, whereas EPFs from a scarring timepoint transplanted into a scarless niche exhibit scar-like morphology and 24.18% collagen colocalization (Figure 1C,D). EPFs and ENFs are epigenetically similar at e16.5 but diverge at e18.5 (~100 vs >3000 significantly different peaks, *P<0.05). Principal component analysis of ATAC-seq data revealed that while ENFs undergo gradual epigenetic "drift" over time, EPFs exhibit a dramatic shift between scarless (e16.5) and scarring (e18.5, p1, and p60) timepoints. At e18.5, 2436 chromatin regions close and 995 open in EPFs, >10 times more significant changes (*P<1x10-15) than at any other developmental timepoint. Chromatin regions opening in EPFs between e16.5-e18.5 are enriched in promoter regions, abnormal fibroblast proliferation gene ontology (GO) terms, and transcription factor motifs relating to chromatin remodeling and TGF-β signaling. However, no significant change is observed in accessibility of TGF-β itself.
CONCLUSIONS: EPFs contribute to scarless healing during early fetal development then transition to a scarring phenotype later in gestation. We reveal that EPF phenotype is cell-intrinsic rather than microenvironment-dependent. EPFs undergo a dramatic epigenetic shift between e16.5-e18.5, corresponding precisely to the time of scarless-to-scarring transition. Analysis of changing genomic regions reveals that epigenetic modulation of downstream TGF-β signaling pathway components may underlie the shift from scarless to scarring EPF phenotype. We plan to perform targeted perturbation of these putative "scarring genes" with the ultimate goal of identifying novel anti-scarring therapeutic targets.
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