RESUMEN
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.
Asunto(s)
Fascia/patología , Cicatrización de Heridas , Animales , Biomarcadores/análisis , Movimiento Celular , Fascia/trasplante , Fibroblastos , Queloide , Ratones Endogámicos C57BLRESUMEN
Deep skin wounds rapidly heal by mobilizing extracellular matrix and cells from the fascia, deep beneath the dermal layer of the skin, to form scars. Despite wounds being an extensively studied area and an unmet clinical need, the biochemistry driving this patch-like repair remains obscure. Lacking also are efficacious therapeutic means to modulate scar formation in vivo. In this study, we identify a central role for p120 in mediating fascia mobilization and wound repair. Injury triggers p120 expression, largely within engrailed-1 lineage-positive fibroblasts of the fascia that exhibit a supracellular organization. Using adeno-associated virusâmediated gene silencing, we show that p120 establishes the supracellular organization of fascia engrailed-1 lineage-positive fibroblasts, without which fascia mobilization is impaired. Gene silencing of p120 in fascia fibroblasts disentangles their supracellular organization, reducing the transfer of fascial cells and extracellular matrix into wounds and augmenting wound healing. Our findings place p120 as essential for fascia mobilization, opening, to our knowledge, a previously unreported therapeutic avenue for targeted intervention in the treatment of a variety of skin scar conditions.
Asunto(s)
Cicatriz , Cicatrización de Heridas , Humanos , Cicatriz/genética , Cicatriz/terapia , Cicatriz/metabolismo , Cicatrización de Heridas/genética , Piel/patología , Fascia/patología , Fibroblastos/metabolismoRESUMEN
The origins of wound myofibroblasts and scar tissue remains unclear, but it is assumed to involve conversion of adipocytes into myofibroblasts. Here, we directly explore the potential plasticity of adipocytes and fibroblasts after skin injury. Using genetic lineage tracing and live imaging in explants and in wounded animals, we observe that injury induces a transient migratory state in adipocytes with vastly distinct cell migration patterns and behaviours from fibroblasts. Furthermore, migratory adipocytes, do not contribute to scar formation and remain non-fibrogenic in vitro, in vivo and upon transplantation into wounds in animals. Using single-cell and bulk transcriptomics we confirm that wound adipocytes do not convert into fibrogenic myofibroblasts. In summary, the injury-induced migratory adipocytes remain lineage-restricted and do not converge or reprogram into a fibrosing phenotype. These findings broadly impact basic and translational strategies in the regenerative medicine field, including clinical interventions for wound repair, diabetes, and fibrotic pathologies.
Asunto(s)
Cicatriz , Piel , Animales , Cicatriz/patología , Piel/patología , Miofibroblastos/patología , Adipocitos/patología , Cicatrización de Heridas , Fibroblastos/patología , FibrosisRESUMEN
The mammalian global response to sealing deep tissue wounds is through scar formation and tissue contraction, mediated by specialized fascia fibroblasts. Despite the clinical significance of scar formation and impaired wound healing, our understanding of fascia fibroblast dynamics in wound healing is cursory due to the lack of relevant assays that enable direct visualization of fibroblast choreography and dynamics in complex environments such as in skin wounds. This paper presents a protocol to generate ex- situ skin scars using SCAD or "SCar-like tissue in A Dish" that emulate the complex environment of skin wounds. In this assay, 2 mm full-thickness skin is excised and cultured upside down in media for 5 days, during which scars and skin contractures develop uniformly. This methodology, coupled with fibroblast-lineage specific transgenic mouse models, enables visualization of individual fibroblast lineages across the entire wound repair process. Overall, this protocol aids researchers in understanding fundamental processes and mechanisms of wound repair, directly exploring the effects of modulators on wound healing outcomes.
Asunto(s)
Cicatriz , Contractura , Animales , Cicatriz/patología , Contractura/patología , Fibroblastos/fisiología , Mamíferos , Ratones , Piel/patología , Cicatrización de Heridas/fisiologíaRESUMEN
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.
Asunto(s)
Conexina 43/genética , Conexina 43/metabolismo , Cicatrización de Heridas , Animales , Calcio/metabolismo , Comunicación Celular , Modelos Animales de Enfermedad , Matriz Extracelular/metabolismo , Fascia/metabolismo , Humanos , Ratones , Ratones Transgénicos , Células 3T3 NIHRESUMEN
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.
Asunto(s)
Cicatriz/metabolismo , Fascia/lesiones , Fibroblastos/metabolismo , Heridas y Lesiones/metabolismo , Adulto , Anciano , Animales , Cadherinas/metabolismo , Movimiento Celular , Cicatriz/fisiopatología , Fascia/citología , Fascia/metabolismo , Femenino , Fibroblastos/citología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Piel/citología , Piel/lesiones , Piel/metabolismo , Piel/fisiopatología , Cicatrización de Heridas , Heridas y Lesiones/fisiopatología , Adulto JovenRESUMEN
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.
Asunto(s)
Calcio/química , Epitelio/metabolismo , Adherencias Tisulares/metabolismo , Animales , Señalización del Calcio , Adhesión Celular , Línea Celular , Membrana Celular/metabolismo , Biología Computacional , Citoesqueleto/metabolismo , Citosol/metabolismo , Modelos Animales de Enfermedad , Femenino , Humanos , Imagenología Tridimensional , Masculino , Ratones , Ratones Endogámicos C57BL , Complicaciones Posoperatorias , Análisis de Componente Principal , ARN Interferente Pequeño/metabolismo , Análisis de la Célula IndividualRESUMEN
During fetal development, mammalian back-skin undergoes a natural transition in response to injury, from scarless regeneration to skin scarring. Here, we characterize dermal morphogenesis and follow two distinct embryonic fibroblast lineages, based on their history of expression of the engrailed 1 gene. We use single-cell fate-mapping, live three dimensional confocal imaging and in silico analysis coupled with immunolabelling to reveal unanticipated structural and regional complexity and dynamics within the dermis. We show that dermal development and regeneration are driven by engrailed 1-history-naive fibroblasts, whose numbers subsequently decline. Conversely, engrailed 1-history-positive fibroblasts possess scarring abilities at this early stage and their expansion later on drives scar emergence. The transition can be reversed, locally, by transplanting engrailed 1-naive cells. Thus, fibroblastic lineage replacement couples the decline of regeneration with the emergence of scarring and creates potential clinical avenues to reduce scarring.