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2.
Nature ; 623(7988): 792-802, 2023 Nov.
Article in English | MEDLINE | ID: mdl-37968392

ABSTRACT

Optimal tissue recovery and organismal survival are achieved by spatiotemporal tuning of tissue inflammation, contraction and scar formation1. Here we identify a multipotent fibroblast progenitor marked by CD201 expression in the fascia, the deepest connective tissue layer of the skin. Using skin injury models in mice, single-cell transcriptomics and genetic lineage tracing, ablation and gene deletion models, we demonstrate that CD201+ progenitors control the pace of wound healing by generating multiple specialized cell types, from proinflammatory fibroblasts to myofibroblasts, in a spatiotemporally tuned sequence. We identified retinoic acid and hypoxia signalling as the entry checkpoints into proinflammatory and myofibroblast states. Modulating CD201+ progenitor differentiation impaired the spatiotemporal appearances of fibroblasts and chronically delayed wound healing. The discovery of proinflammatory and myofibroblast progenitors and their differentiation pathways provide a new roadmap to understand and clinically treat impaired wound healing.


Subject(s)
Endothelial Protein C Receptor , Fascia , Wound Healing , Animals , Mice , Cell Differentiation , Cell Hypoxia , Cell Lineage , Disease Models, Animal , Endothelial Protein C Receptor/metabolism , Fascia/cytology , Fascia/injuries , Fascia/metabolism , Fibroblasts/cytology , Fibroblasts/metabolism , Gene Expression Profiling , Inflammation/metabolism , Inflammation/pathology , Myofibroblasts/cytology , Myofibroblasts/metabolism , Signal Transduction , Single-Cell Gene Expression Analysis , Skin/cytology , Skin/injuries , Skin/metabolism , Tretinoin/metabolism
3.
Nat Protoc ; 18(10): 2876-2890, 2023 10.
Article in English | MEDLINE | ID: mdl-37558896

ABSTRACT

Connective tissues are essential building blocks for organ development, repair and regeneration. However, we are at the early stages of understanding connective tissue dynamics. Here, we detail a method that enables in vivo fate mapping of organ extracellular matrix (ECM) by taking advantage of a crosslinking chemical reaction between amine groups and N-hydroxysuccinimide esters. This methodology enables robust labeling of ECM proteins, which complement previous affinity-based single-protein methods. This protocol is intended for entry-level scientists and the labeling step takes between 5 and 10 min. ECM 'tagging' with fluorophores using N-hydroxysuccinimide esters enables visualization of ECM spatial modifications and is particularly useful to study connective tissue dynamics in organ fibrosis, tumor stroma formation, wound healing and regeneration. This in vivo chemical fate mapping methodology is highly versatile, regardless of the tissue/organ system, and complements cellular fate-mapping techniques. Furthermore, as the basic chemistry of proteins is highly conserved between species, this method is also suitable for cross-species comparative studies of ECM dynamics.


Subject(s)
Extracellular Matrix , Succinimides , Extracellular Matrix/metabolism , Connective Tissue , Extracellular Matrix Proteins/metabolism
4.
Nat Immunol ; 23(4): 518-531, 2022 04.
Article in English | MEDLINE | ID: mdl-35354953

ABSTRACT

Internal organs heal injuries with new connective tissue, but the cellular and molecular events of this process remain obscure. By tagging extracellular matrix around the mesothelium lining in mouse peritoneum, liver and cecum, here we show that preexisting matrix was transferred across organs into wounds in various injury models. Using proteomics, genetic lineage-tracing and selective injury in juxtaposed organs, we found that the tissue of origin for the transferred matrix likely dictated the scarring or regeneration of the healing tissue. Single-cell RNA sequencing and genetic and chemical screens indicated that the preexisting matrix was transferred by neutrophils dependent on the HSF-integrin AM/B2-kindlin3 cascade. Pharmacologic inhibition of this axis prevented matrix transfer and the formation of peritoneal adhesions. Matrix transfer was thus an early event of wound repair and provides a therapeutic window to dampen scaring across a range of conditions.


Subject(s)
Neutrophils , Peritoneum , Animals , Epithelium , Extracellular Matrix , Mice , Peritoneum/injuries , Wound Healing
5.
Matrix Biol ; 97: 58-71, 2021 03.
Article in English | MEDLINE | ID: mdl-33508427

ABSTRACT

Deep and voluminous skin wounds are repaired with scars, by mobilization of fibroblasts and extracellular matrix from fascia, deep below the skin. The molecular trigger of this novel repair mechanism is incompletely understood. Here we reveal that the gap junction alpha-1 protein (Connexin43, Cx43) is the key to patch repair of deep wounds. By combining full-thickness wound models with fibroblast lineage specific transgenic lines, we show Cx43 expression is substantially upregulated in specialized fibroblasts of the fascia deep beneath the skin that are responsible for scar formation. Using live imaging of fascia fibroblasts and fate tracing of the fascia extracellular matrix we show that Cx43 inhibition disrupts calcium oscillations in cultured fibroblasts and that this inhibits collective migration of fascia EPFs necessary to mobilize fascia matrix into open wounds. Cell-cell communication through Cx43 thus mediates matrix movement and scar formation, and is necessary for patch repair of voluminous wounds. These mechanistic findings have broad clinical implications toward treating fibrosis, aggravated scarring and impaired wound healing.


Subject(s)
Connexin 43/genetics , Connexin 43/metabolism , Wound Healing , Animals , Calcium/metabolism , Cell Communication , Disease Models, Animal , Extracellular Matrix/metabolism , Fascia/metabolism , Humans , Mice , Mice, Transgenic , NIH 3T3 Cells
6.
Nat Commun ; 11(1): 5653, 2020 11 06.
Article in English | MEDLINE | ID: mdl-33159076

ABSTRACT

Scars are more severe when the subcutaneous fascia beneath the dermis is injured upon surgical or traumatic wounding. Here, we present a detailed analysis of fascia cell mobilisation by using deep tissue intravital live imaging of acute surgical wounds, fibroblast lineage-specific transgenic mice, and skin-fascia explants (scar-like tissue in a dish - SCAD). We observe that injury triggers a swarming-like collective cell migration of fascia fibroblasts that progressively contracts the skin and form scars. Swarming is exclusive to fascia fibroblasts, and requires the upregulation of N-cadherin. Both swarming and N-cadherin expression are absent from fibroblasts in the upper skin layers and the oral mucosa, tissues that repair wounds with minimal scar. Impeding N-cadherin binding inhibits swarming and skin contraction, and leads to reduced scarring in SCADs and in animals. Fibroblast swarming and N-cadherin thus provide therapeutic avenues to curtail fascia mobilisation and pathological fibrotic responses across a range of medical settings.


Subject(s)
Cicatrix/metabolism , Fascia/injuries , Fibroblasts/metabolism , Wounds and Injuries/metabolism , Adult , Aged , Animals , Cadherins/metabolism , Cell Movement , Cicatrix/physiopathology , Fascia/cytology , Fascia/metabolism , Female , Fibroblasts/cytology , Humans , Male , Mice , Mice, Inbred C57BL , Middle Aged , Skin/cytology , Skin/injuries , Skin/metabolism , Skin/physiopathology , Wound Healing , Wounds and Injuries/physiopathology , Young Adult
7.
Nat Commun ; 11(1): 3068, 2020 06 17.
Article in English | MEDLINE | ID: mdl-32555155

ABSTRACT

Surgical adhesions are bands of scar tissues that abnormally conjoin organ surfaces. Adhesions are a major cause of post-operative and dialysis-related complications, yet their patho-mechanism remains elusive, and prevention agents in clinical trials have thus far failed to achieve efficacy. Here, we uncover the adhesion initiation mechanism by coating beads with human mesothelial cells that normally line organ surfaces, and viewing them under adhesion stimuli. We document expansive membrane protrusions from mesothelia that tether beads with massive accompanying adherence forces. Membrane protrusions precede matrix deposition, and can transmit adhesion stimuli to healthy surfaces. We identify cytoskeletal effectors and calcium signaling as molecular triggers that initiate surgical adhesions. A single, localized dose targeting these early germinal events completely prevented adhesions in a preclinical mouse model, and in human assays. Our findings classifies the adhesion pathology as originating from mesothelial membrane bridges and offer a radically new therapeutic approach to treat adhesions.


Subject(s)
Calcium/chemistry , Epithelium/metabolism , Tissue Adhesions/metabolism , Animals , Calcium Signaling , Cell Adhesion , Cell Line , Cell Membrane/metabolism , Computational Biology , Cytoskeleton/metabolism , Cytosol/metabolism , Disease Models, Animal , Female , Humans , Imaging, Three-Dimensional , Male , Mice , Mice, Inbred C57BL , Postoperative Complications , Principal Component Analysis , RNA, Small Interfering/metabolism , Single-Cell Analysis
8.
Nature ; 576(7786): 287-292, 2019 12.
Article in English | MEDLINE | ID: mdl-31776510

ABSTRACT

Mammals form scars to quickly seal wounds and ensure survival by an incompletely understood mechanism1-5. Here we show that skin scars originate from prefabricated matrix in the subcutaneous fascia. Fate mapping and live imaging revealed that fascia fibroblasts rise to the skin surface after wounding, dragging their surrounding extracellular jelly-like matrix, including embedded blood vessels, macrophages and peripheral nerves, to form the provisional matrix. Genetic ablation of fascia fibroblasts prevented matrix from homing into wounds and resulted in defective scars, whereas placing an impermeable film beneath the skin-preventing fascia fibroblasts from migrating upwards-led to chronic open wounds. Thus, fascia contains a specialized prefabricated kit of sentry fibroblasts, embedded within a movable sealant, that preassemble together diverse cell types and matrix components needed to heal wounds. Our findings suggest that chronic and excessive skin wounds may be attributed to the mobility of the fascia matrix.


Subject(s)
Fascia/pathology , Wound Healing , Animals , Biomarkers/analysis , Cell Movement , Fascia/transplantation , Fibroblasts , Keloid , Mice, Inbred C57BL
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