RESUMEN
A major challenge for clinicians treating (arterio) venous leg ulcers is to decide between standard therapy and advanced interventions. Here, we developed a simple method to collect human material representative of the ulcer wound bed, which can be used to identify biomarkers for prognostic test development. Superficial surgical debridement was performed using a small vidal curette during the weekly visit to the outpatient clinic. Moist, easily removable debridement material essentially blood free (including necrotic and nonviable slough) was collected from the surface of the ulcer. The amount ranged from 5.5 mg to 78 mg material per ulcer. Seventeen cytokines, chemokines, and growth factors were extracted and analyzed by enzyme-linked immunosorbent assay (concentration range: 0.0005-78 ng/mg total protein). Notably, CXCL8 was by far the most abundant protein present. Inflammatory mediators were more abundant than anti-inflammatory mediators (e.g., interleukin (IL)-10 and transforming growth factor-ß1). Bioactivity assays showed chronic wound extracts to be capable of stimulating fibroblast migration in a chemokine-dependent manner and also capable of stimulating healthy cells within skin substitutes to secrete wound healing mediators (CCL2, CXCL1, CXCL8, IL-6) in an IL-1α dependent manner. Collection of debridement tissue enables investigation of the ulcer environment in an easy noninvasive manner that may be suitable for prognostic test development.
Asunto(s)
Quimiocina CCL20/metabolismo , Quimiocina CXCL1/metabolismo , Exudados y Transudados/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Úlcera Varicosa/inmunología , Cicatrización de Heridas/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores/metabolismo , Células Cultivadas , Enfermedad Crónica , Desbridamiento/métodos , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Masculino , Persona de Mediana Edad , Valor Predictivo de las PruebasRESUMEN
Wounds in the mouth heal faster and with less scarification and inflammation than those in the skin. Saliva is thought to be essential for the superior oral wound healing, but the involved mechanism is still unclear. We have previously discovered that a human-specific peptide, histatin, might be implicated in the wound-healing properties of saliva. Here we report that histatin enhances reepithelialization in a human full-skin wound model closely resembling normal skin. The peptide does not stimulate proliferation but induces cell spreading and migration, two key initiating steps in reepithelialization. Activation of cells by histatin requires a G-protein-coupled receptor that activates the ERK1/2 pathway. Using a stepwise-truncation method, we determined the minimal domain (SHREFPFYGDYGS) of the 38-mer-parent peptide that is required for activity. Strikingly, N- to C-terminal cyclization of histatin-1 potentiates the molar activity approximately 1000-fold, indicating that the recognition of histatin by its cognate receptor requires a specific spatial conformation of the peptide. Our results emphasize the importance of histatin in human saliva for tissue protection and recovery and establish the experimental basis for the development of synthetic histatins as novel skin wound-healing agents.
Asunto(s)
Histatinas/fisiología , Cicatrización de Heridas/fisiología , Secuencia de Aminoácidos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Ciclización , Sinergismo Farmacológico , Humanos , Recién Nacido , Queratinocitos/efectos de los fármacos , Masculino , Modelos Biológicos , Saliva/química , Relación Estructura-Actividad , Cicatrización de Heridas/efectos de los fármacosRESUMEN
Many cell-based regenerative medicine strategies toward tissue-engineered constructs are currently being explored. Cell-cell interactions and interactions with different biomaterials are extensively investigated, whereas very few studies address how cultured cells will interact with soluble wound-healing mediators that are present within the wound bed after transplantation. The aim of this study was to determine how adipose tissue-derived mesenchymal stem cells (ASC), dermal fibroblasts, and keratinocytes will react when they come in contact with the deep cutaneous burn wound bed. Burn wound exudates isolated from deep burn wounds were found to contain many cytokines, including chemokines and growth factors related to inflammation and wound healing. Seventeen mediators were identified by ELISA (concentration range 0.0006-9 ng/mg total protein), including the skin-specific chemokine CCL27. Burn wound exudates activated both ASC and dermal fibroblasts, but not keratinocytes, to increase secretion of CXCL1, CXCL8, CCL2, and CCL20. Notably, ASC but not fibroblasts or keratinocytes showed significant increased secretion of vascular endothelial growth factor (5-fold) and interleukin-6 (253-fold), although when the cells were incorporated in bi-layered skin substitute (SS) these differences were less pronounced. A similar discrepancy between ASC and dermal fibroblast mono-cultures was observed when recombinant human-CCL27 was used instead of burn wound exudates. Although CCL27 did not stimulate the secretion of any of the wound-healing mediators by keratinocytes, these cells, in contrast to ASC or dermal fibroblasts, showed increased proliferation and migration. Taken together, these results indicate that on transplantation, keratinocytes are primarily activated to promote wound closure. In contrast, dermal fibroblasts and, in particular, ASC respond vigorously to factors present in the wound bed, leading to increased secretion of angiogenesis/granulation tissue formation factors. Our findings have implications for the choice of cell type (ASC or dermal fibroblast) to be used in regenerative medicine strategies and indicate the importance of taking into account interactions with the wound bed when developing advanced therapies for difficult-to-close cutaneous wounds.
Asunto(s)
Tejido Adiposo/citología , Quemaduras/patología , Quimiocina CCL27/metabolismo , Exudados y Transudados/metabolismo , Fibroblastos/metabolismo , Queratinocitos/metabolismo , Células Madre Mesenquimatosas/metabolismo , Adulto , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Dermis/metabolismo , Dermis/patología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Fibroblastos/efectos de los fármacos , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Queratinocitos/efectos de los fármacos , Masculino , Células Madre Mesenquimatosas/efectos de los fármacos , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Especificidad de Órganos/efectos de los fármacos , Proteínas Recombinantes/farmacología , Solubilidad , Cicatrización de Heridas/efectos de los fármacosRESUMEN
This study identifies chemokine receptors involved in an autocrine regulation of re-epithelialization after skin tissue damage. We determined which receptors, from a panel of 13, are expressed in healthy human epidermis and which monospecific chemokine ligands, secreted by keratinocytes, were able to stimulate migration and proliferation. A reconstructed epidermis cryo(freeze)-wound model was used to assess chemokine secretion after wounding and the effect of pertussis toxin (chemokine receptor blocker) on re-epithelialization and differentiation. Chemokine receptors CCR1, CCR3, CCR4, CCR6, CCR10, CXCR1, CXCR2, CXCR3, and CXCR4 were expressed in epidermis. No expression of CCR2, CCR5, CCR7, and CCR8 was observed by either immunostaining or flow cytometry. Five chemokine receptors (CCR1, CCR10, CXCR1, CXCR2, and CXCR3) were identified, the corresponding monospecific ligands (CCL14, CCL27, CXCL8, CXCL1, CXCL10, respectively) of which were not only able to stimulate keratinocyte migration and/or proliferation but were also secreted by keratinocytes after introducing cryo-wounds into epidermal equivalents. Blocking of receptor-ligand interactions with pertussis toxin delayed re-epithelialization, but did not influence differentiation (as assessed by formation of basal layer, spinous layer, granular layer, and stratum corneum) after cryo-wounding. Taken together, these results confirm that an autocrine positive-feedback loop of epithelialization exists in order to stimulate wound closure after skin injury.
Asunto(s)
Comunicación Autocrina/inmunología , Epidermis/inmunología , Epidermis/lesiones , Queratinocitos/inmunología , Receptores de Quimiocina/inmunología , Cicatrización de Heridas/inmunología , Adulto , División Celular/inmunología , Movimiento Celular/inmunología , Células Cultivadas , Células Epidérmicas , Células Epiteliales/citología , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Humanos , Queratinocitos/citología , Queratinocitos/metabolismo , Receptores CCR1/inmunología , Receptores CCR1/metabolismo , Receptores CCR10/inmunología , Receptores CCR10/metabolismo , Receptores CCR4/inmunología , Receptores CCR4/metabolismo , Receptores CCR6/inmunología , Receptores CCR6/metabolismo , Receptores CXCR3/inmunología , Receptores CXCR3/metabolismo , Receptores CXCR4/inmunología , Receptores CXCR4/metabolismo , Receptores de Quimiocina/metabolismo , Receptores de Interleucina-8A/inmunología , Receptores de Interleucina-8A/metabolismo , Receptores de Interleucina-8B/inmunología , Receptores de Interleucina-8B/metabolismoRESUMEN
The ability of stem cells to self-renew as well as their multilineage differentiation potential makes them ideal candidates for skin regeneration strategies. Mesenchymal stem cells residing in human adult dermis, in contrast to adipose tissue, have not yet been described. The objective of this study was to determine the stemness and chemokine-mediated homing potential of dermal stromal cells (DSC) and to compare this with adipose stem cells (ASC). DSC have a less stellate form than ASC, confirming that DSC and ASC are two different types of mesenchymal cell populations. However, DSC display a mesenchymal stem cell phenotype (CD31(-), CD34(+), CD45(-), CD54(+), CD90(+), CD105(+), and CD166(+) similar to ASC and are also multipotent in their ability to differentiate into adipocytes, chondrocytes, and osteoblasts. Both ASC and DSC display a similar set of chemokine receptors (CCR3, CCR4, CCR6, CCR10, CXCR1, and CXCR2). Several ligands for these receptors, with CCL5/RANTES being the most potent, can induce migration of ASC and DSC in an in vitro wound-healing assay. Taken together, these results show that a population of mesenchymal stem cells resides in the dermis of human adult skin and these dermal-derived stem cells have a phenotypic and chemokine-mediated homing potential similar to adipose stem cells, which to our knowledge is previously unreported.