RESUMEN
BACKGROUND: Recessive dystrophic epidermolysis bullosa (RDEB) is a blistering disease caused by mutations in the gene encoding type VII collagen (C7). RDEB is associated with fibrosis, which is responsible for severe complications. The phenotypic variability observed in siblings with RDEB suggests that epigenetic modifications contribute to disease severity. Identifying epigenetic changes may help to uncover molecular mechanisms underlying RDEB pathogenesis and new therapeutic targets. OBJECTIVES: To investigate histone acetylation in RDEB skin and to explore histone deacetylase inhibitors (HDACi) as therapeutic molecules capable of counteracting fibrosis and disease progression in RDEB mice. METHODS: Acetylated histone levels were detected in human skin by immunofluorescence and in RDEB fibroblasts by enzyme-linked immunosorbent assay (ELISA). The effects of givinostat and valproic acid (VPA) on RDEB fibroblast fibrotic behaviour were assessed by a collagen-gel contraction assay, Western blot and immunocytofluorescence for α-smooth muscle actin, and ELISA for released transforming growth factor (TGF)-ß1. RNA sequencing was performed in HDACi- and vehicle-treated RDEB fibroblasts. VPA was systemically administered to RDEB mice and effects on overt phenotype were monitored. Fibrosis was investigated in the skin using histological and immunofluorescence analyses. Eye and tongue defects were examined microscopically. Mass spectrometry proteomics was performed on skin protein extracts from VPA-treated RDEB and control mice. RESULTS: Histone acetylation decreases in RDEB skin and primary fibroblasts. RDEB fibroblasts treated with HDACi lowered fibrotic traits, including contractility, TGF-ß1 release and proliferation. VPA administration to RDEB mice mitigated severe manifestations affecting the eyes and paws. These effects were associated with fibrosis inhibition. Proteomic analysis of mouse skin revealed that VPA almost normalized protein sets involved in protein synthesis and immune response, processes linked to the increased susceptibility to cancer and bacterial infections seen in people with RDEB. CONCLUSIONS: Dysregulated histone acetylation contributes to RDEB pathogenesis by facilitating the progression of fibrosis. Repurposing of HDACi could be considered for disease-modifying treatments in RDEB.
Recessive dystrophic epidermolysis bullosa (or 'RDEB') is a rare skin disease that affects fewer than 5,000 people in the USA. A similar number of people in Europe are affected. RDEB is caused by mutations in the gene that controls the production of a protein called 'type VII collagen' (or 'C7'). A shortage of C7 causes fragile skin that blisters. In severe forms of RDEB, wounds heal slowly and can even affect a person's life expectancy. Differences in the disease are common in people (even identical twins) with RDEB who have similar levels of C7. This suggests that how severe the disease is could be affected by molecular processes that control other genes. Understanding these processes may help us to find treatments for RDEB. This study was done in Italy, in collaboration with centres in Germany and Switzerland. We wanted to see whether a chemical modification called 'histone acetylation' (which influences gene activity) is different in RDEB and whether it can be targeted by a specific treatment. We found that histone acetylation is reduced in RDEB skin and in skin cells grown in the lab called 'fibroblasts'. When we increased histone acetylation in fibroblasts with two drugs called givinostat and valproic acid, the amount of scar tissue produced decreased. This is important because scar tissue can lead to severe symptoms. We carried out more experiments to study the effects of givinostat and valproic acid in mice with RDEB. We found that valproic acid reduces the severity of RDEB by decreasing the disease's harmful effects and reducing the amount of scar tissue. Our findings suggest that abnormal histone acetylation contributes to the scar tissue seen in RDEB. Our study shows that valproic acid could be useful in treating the scarring seen in RDEB and in reducing the effects of the disease. As this drug is used to treat other diseases, there could be potential for rapid repurposing of it for RDEB.
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Colágeno Tipo VII , Progresión de la Enfermedad , Epidermólisis Ampollosa Distrófica , Fibroblastos , Fibrosis , Inhibidores de Histona Desacetilasas , Piel , Epidermólisis Ampollosa Distrófica/tratamiento farmacológico , Epidermólisis Ampollosa Distrófica/patología , Epidermólisis Ampollosa Distrófica/genética , Animales , Humanos , Inhibidores de Histona Desacetilasas/farmacología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Colágeno Tipo VII/genética , Piel/patología , Piel/efectos de los fármacos , Ratones , Ácido Valproico/farmacología , Histonas/metabolismo , Acetilación/efectos de los fármacos , Masculino , Femenino , Modelos Animales de Enfermedad , Factor de Crecimiento Transformador beta1/metabolismo , Células Cultivadas , Niño , CarbamatosRESUMEN
Analysing the composition and organisation of the fibrous capsule formed as a result of the Foreign Body Response (FBR) to medical devices, is imperative for medical device improvement and biocompatibility. Typically, analysis is performed using histological techniques which often involve random sampling strategies. This method is excellent for acquiring representative values but can miss the unique spatial distribution of features in 3D, especially when analysing devices used in large animal studies. To overcome this limitation, we demonstrate a non-destructive method for high-resolution large sample imaging of the fibrous capsule surrounding human-sized implanted devices using diffusion tensor imaging (DTI). In this study we analyse the fibrous capsule surrounding two unique macroencapsulation devices that have been implanted in a porcine model for 21 days. DTI is used for 3D visualisation of the microstructural organisation and validated using the standard means of fibrous capsule investigation; histological analysis and qualitative micro computed tomography (microCT) and scanning electron microscopy (SEM) imaging. DTI demonstrated the ability to distinguish microstructural differences in the fibrous capsules surrounding two macroencapsulation devices made from different materials and with different surface topographies. DTI-derived metrics yielded insight into the microstructural organisation of both capsules which was corroborated by microCT, SEM and histology. The non-invasive characterisation of the integration of implants in the body has the potential to positively influence analysis methods in pre-clinical studies and accelerate the clinical translation of novel implantable devices.
RESUMEN
Medical devices, such as silicone-based prostheses designed for soft tissue implantation, often induce a suboptimal foreign-body response which results in a hardened avascular fibrotic capsule around the device, often leading to patient discomfort or implant failure. Here, it is proposed that additive manufacturing techniques can be used to deposit durable coatings with multiscale porosity on soft tissue implant surfaces to promote optimal tissue integration. Specifically, the "liquid rope coil effect", is exploited via direct ink writing, to create a controlled macro open-pore architecture, including over highly curved surfaces, while adapting atomizing spray deposition of a silicone ink to create a microporous texture. The potential to tailor the degree of tissue integration and vascularization using these fabrication techniques is demonstrated through subdermal and submuscular implantation studies in rodent and porcine models respectively, illustrating the implant coating's potential applications in both traditional soft tissue prosthetics and active drug-eluting devices.
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Prótesis e Implantes , Siliconas , Animales , Humanos , Ensayo de Materiales , Porosidad , PorcinosRESUMEN
BACKGROUND: Clinical skin manifestations are common in diabetes; however, molecular mechanisms underlying such defects are largely unknown. Several findings indicate a role for microRNAs (miRNAs) in skin homeostasis. OBJECTIVE: To investigate whether miRNA expression is altered in diabetic skin. METHODS: Type 1 and 2 mouse models of diabetes were used. MiRNA profiling was performed on RNA extracted from the skin of type 1 diabetic mice and non-diabetic controls. Expression levels of pri-miRNAs and of miRNA-biogenesis genes were also analyzed. Biogenesis gene expression analysis was performed in human dermal fibroblasts cultured in hyperglycemic, hypoxic or oxidative stress conditions. RESULTS: Several miRNAs were differentially expressed in diabetic skin with a general down-modulation as compared to controls. Bioinformatics analysis of signature-miRNA target genes showed the enrichment in pathways involved in skin homeostasis, such as TGF-ß and Wnt. MiRNA alteration in diabetic skin associated with reduced expression levels of DROSHA, DGCR8, XPO5, DICER1, AGO2, both as mRNA and protein. Reduced biogenesis gene expression did not correlate with accumulation of pri-miRNAs, which displayed differences in expression levels similar to those found for their mature miRNAs. Experiments with cultured fibroblasts showed that hypoxia and oxidative stress induced the down-regulation of miRNA-biogenesis genes in this skin cell type. CONCLUSION: A general down-regulation of differentially expressed miRNAs was found in diabetic skin. This alteration is part of and is dependent from a wider transcriptional defect also affecting the expression of pri-miRNAs and of genes responsible for miRNA biogenesis. Such an alteration is likely contributing to diabetic skin manifestations.
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Diabetes Mellitus Tipo 1/complicaciones , Diabetes Mellitus Tipo 2/complicaciones , Hiperglucemia/complicaciones , MicroARNs/biosíntesis , Enfermedades de la Piel/patología , Animales , Biopsia , Hipoxia de la Célula/genética , Diabetes Mellitus Experimental/sangre , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/inducido químicamente , Regulación hacia Abajo , Fibroblastos , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Hiperglucemia/sangre , Hiperglucemia/inducido químicamente , Hiperglucemia/genética , Masculino , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Estrés Oxidativo/genética , Transducción de Señal/genética , Piel/citología , Piel/patología , Enfermedades de la Piel/sangre , Enfermedades de la Piel/etiologíaRESUMEN
Regenerative medicine approaches, specifically stem cell technologies, have demonstrated significant potential to treat a diverse array of pathologies. However, such approaches have resulted in a modest clinical benefit, which may be attributed to poor cell retention/survival at the disease site. A delivery system that facilitates regional and repeated delivery to target tissues can provide enhanced clinical efficacy of cell therapies when localized delivery of high doses of cells is required. In this study, a new regenerative reservoir platform (Regenervoir) is described for use in large animal models, with relevance to cardiac, abdominal, and soft tissue pathologies. Regenervoir incorporates multiple novel design features essential for clinical translation, with a focus on scalability, mechanism of delivery, fixation to target tissue, and filling/refilling with a therapeutic cargo, and is demonstrated in an array of clinical applications that are easily translated to human studies. Regenervoir consists of a porous reservoir fabricated from a single material, a flexible thermoplastic polymer, capable of delivering cargo via fill lines to target tissues. A radiopaque shear thinning hydrogel can be delivered to the therapy reservoir and multiple fixation methods (laparoscopic tacks and cyanoacrylate bioadhesive) can be used to secure Regenervoir to target tissues through a minimally invasive approach.
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Hidrogeles , Medicina Regenerativa , Animales , Humanos , Modelos Animales , Polímeros , Prótesis e ImplantesRESUMEN
Variably reduced expression of the basement membrane component laminin-332 (α3aß3γ2) causes junctional epidermolysis bullosa generalized intermediate (JEB-GI), a skin fragility disorder with an increased susceptibility to squamous cell carcinoma (SCC) development in adulthood. Laminin-332 is highly expressed in several types of epithelial tumors and is central to signaling pathways that promote SCC tumorigenesis. However, laminin-332 mutations and expression in individuals affected by JEB-GI and suffering from recurrent SCCs have been poorly characterized. We studied a JEB-GI patient who developed over a hundred primary cutaneous SCCs. Molecular analysis combined with gene expression studies in patient skin and primary keratinocytes revealed that the patient is a functional hemizygous for the p.Cys1171* mutant allele which is transcribed in a stable mRNA encoding for a ß3 chain shortened of the last two C-terminal amino acids (Cys1171-Lys1172). The lack of the Cys1171 residue involved in the C-terminal disulphide bond to γ2 chain did not prevent assembly, secretion, and proteolytic processing of the heterotrimeric molecule. Immunohistochemistry of SCC specimens revealed accumulation of mutant laminin-332 at the epithelial-stromal interface of invasive front. We conclude that the C-terminal disulphide bond is a structural element crucial for laminin-332 adhesion function in-vivo. By saving laminin-332 amount, processing, and signaling role the p.Cys1171* mutation may allow intrinsic pro-tumorigenic properties of the protein to be conveyed, thus contributing to invasiveness and recurrence of SCCs in this patient.
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Carcinoma de Células Escamosas , Moléculas de Adhesión Celular , Epidermólisis Ampollosa , Mutación , Proteínas de Neoplasias , Neoplasias Cutáneas , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Epidermólisis Ampollosa/genética , Epidermólisis Ampollosa/metabolismo , Epidermólisis Ampollosa/patología , Humanos , Masculino , Persona de Mediana Edad , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Estabilidad del ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , KalininaRESUMEN
Loss-of-function mutations in the gene encoding type VII collagen underlie recessive dystrophic epidermolysis bullosa (RDEB), a disease characterized by skin and mucosal blistering, impaired wound healing, and diffuse dermal inflammation and fibrosis. Transforming growth factor-ß signaling plays a crucial role in determining RDEB fibrotic microenvironment that leads to the development of disabling secondary disease manifestations, including hand and foot deformities. Experimental findings indicate that expression levels of decorin, a small leucine-rich proteoglycan and an endogenous TGF-ß inhibitor, can modulate RDEB disease phenotype by contrasting dermal fibroblast fibrotic behavior. In this study, the ability of decorin to modify RDEB course was investigated by systemically treating RDEB mice with a lentivirus expressing human decorin. Overexpressed decorin was able to enhance survival, and to limit digit contraction and the development of paw deformities. These effects were associated with decreased TGF-ß1 levels and TGF-ß signaling activation. Fibrotic traits were strongly reduced in paw skin and also attenuated in the non-chronically injured back skin. However, the expression of pro-inflammatory proteins was not decreased in both paw and back skin. Our findings confirm TGF-ß role in promoting fibrosis and disease progression in RDEB, and show that decorin counteracts disease manifestations by inhibiting TGF-ß activation. More generally, our data indicate that modifying extracellular matrix composition is an option to improve RDEB disease course.
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Decorina/genética , Epidermólisis Ampollosa Distrófica/terapia , Vectores Genéticos/administración & dosificación , Factor de Crecimiento Transformador beta1/metabolismo , Animales , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Epidermólisis Ampollosa Distrófica/genética , Epidermólisis Ampollosa Distrófica/metabolismo , Humanos , Lentivirus/genética , Ratones , Transducción de Señal , Análisis de Supervivencia , Factor de Crecimiento Transformador beta/metabolismo , Resultado del TratamientoRESUMEN
Individuals with recessive dystrophic epidermolysis bullosa (RDEB), a rare genetic skin disease, carry mutations in the COL7A1 gene that codes for type VII collagen, an extracellular matrix component of the basement membrane zone forming the anchoring fibrils. As a consequence, RDEB individuals manifest unremitting skin blistering that evolves into chronic wounds, inflammation, and fibrosis. These features play a central role in the development of more severe disease complications, such as mitten deformities of hands and feet and aggressive epithelial cancers. Despite being recognized as a central clinical issue for RDEB, wound healing impairment has been only marginally investigated. Recently, studies with disease mouse models started to shed light on the molecular mechanisms underlying the altered healing response of RDEB. In turn, alterations found in RDEB skin cell behavior fostered the understanding of mechanisms that may be responsible for defective skin repair. This review summarizes findings related to healing impairment in RDEB, and highlights therapeutic strategies for ameliorating healing.
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Colágeno Tipo VII/genética , Epidermólisis Ampollosa Distrófica/genética , Cicatrización de Heridas/genética , Animales , Vesícula , Proliferación Celular , Modelos Animales de Enfermedad , Epidermólisis Ampollosa Distrófica/patología , Epidermólisis Ampollosa Distrófica/terapia , Genes Recesivos/genética , Humanos , Inflamación , Ratones , Mutación , Piel/patologíaRESUMEN
Interleukin-22 (IL-22) belongs to the family of IL-10 cytokines and is involved in a wide number of human diseases, including inflammatory disorders and cancer pathology. The ligand-receptor complex IL-22/IL-22R plays a key role in several pathways especially in the regulation and resolution of immune responses. The identification of novel compounds able to modulate IL-22/IL-22R complex could open the route to new therapeutic strategies in multiple human diseases. In this study, we designed and characterized IL-22 derived peptides at protein interface regions: several sequences revealed able to interfere with the protein complex with IC50 in the micromolar range as evaluated through Surface Plasmon Resonance (SPR) experiments. Their conformational characterization was carried out through Circular Dichroism (CD) and Nuclear Magnetic Resonance (NMR) spectroscopies, shedding new light into the features of IL-22 fragments and on structural determinants of IL-22/IL-22R1 recognition. Finally, several peptides were tested on human keratinocyte cultures for evaluating their ability to mimic the activation of molecular pathways downstream to IL-22R in response to IL-22 binding.
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Interleucinas/química , Modelos Moleculares , Secuencia de Aminoácidos , Humanos , Interleucinas/metabolismo , Queratinocitos/metabolismo , Espectroscopía de Resonancia Magnética , Péptidos , Estructura Terciaria de Proteína , Transducción de Señal , Resonancia por Plasmón de Superficie , Interleucina-22RESUMEN
The HECT-type E3 ubiquitin ligase Itch is absent in the non-agouti-lethal 18H or Itchy mice, which develop a severe immunological disease. Several of the known Itch substrates are relevant for epidermal development and homeostasis, such as p63, Notch, c-Jun and JunB. By analysing Itchy mice before the onset of immunological alterations, we investigated the contribution of Itch in skin development and wound healing. Itchy newborn mice manifested hyperplastic epidermis, which is not present in adulthood. Itch(-/-) cultured keratinocytes showed overexpression of proliferating markers and increased capability to proliferate, migrate and to repair a scratch injury in vitro. These data correlated with improved in vivo wound healing in Itchy mice, at late time points of the repair process when Itch is physiologically upregulated. Despite healing acceleration, epidermal remodelling was delayed in the scars of Itch(-/-) mice, as indicated by enhanced epidermal thickening, keratinocyte proliferation and keratin 6 expression, and retarded keratin 14 polarization to the basal layer. Itch(-/-) keratinocyte prolonged activation was not associated with increased immune cell persistence in the scars. Our in vitro and in vivo results indicate that Itch plays a role in epidermal homeostasis and remodelling and this feature does not seem to depend on immunological alterations.
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Ubiquitina-Proteína Ligasas/deficiencia , Ubiquitina-Proteína Ligasas/metabolismo , Cicatrización de Heridas , Animales , Proliferación Celular , Células Cultivadas , Queratinocitos/citología , Queratinocitos/metabolismo , Ratones , Ratones NoqueadosRESUMEN
Impaired re-epithelialization, imbalanced expression of cytokines and growth factors, and vascular disease contribute to healing impairment in diabetes. IL-22, a pro-inflammatory cytokine mediating a cross-talk between immune system and epithelial cells, has been shown to have a role in repair processes. In this study we aimed to investigate IL-22 regenerative potential in the poor healing context of diabetic wounds. By using streptozotocin-induced diabetic mice, we demonstrated that IL-22 wound treatment significantly accelerated the healing process, by promoting re-epithelialization, granulation tissue formation, and vascularization. Improved re-epithelialization was associated with increased keratinocyte proliferation and signal transducer and activator of transcription 3 (STAT3) activation. We showed that endogenous IL-22 content was reduced at both mRNA and protein level during the inflammatory phase of diabetic wounds, with fewer IL-22-positive cells infiltrating the granulation tissue. We demonstrated that IL-22 treatment promoted proliferation and injury repair of hyperglycemic keratinocytes and induced activation of STAT3 and extracellular signal-regulated kinase transduction pathways in keratinocytes grown in hyperglycemic condition or isolated from diabetic patients. Finally, we demonstrated that IL-22 treatment was able to inhibit diabetic keratinocyte differentiation while promoting vascular endothelial growth factor release. Our data indicate a pro-healing role of IL-22 in diabetic wounds, suggesting a therapeutic potential for this cytokine in diabetic ulcer management.
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Interleucinas/farmacología , Queratinocitos/efectos de los fármacos , Úlcera Cutánea/tratamiento farmacológico , Cicatrización de Heridas/fisiología , Administración Tópica , Animales , Biopsia con Aguja , Western Blotting , Células Cultivadas , Diabetes Mellitus Experimental , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Inmunohistoquímica , Interleucinas/metabolismo , Queratinocitos/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos BALB C , Distribución Aleatoria , Reacción en Cadena en Tiempo Real de la Polimerasa , Factor de Transcripción STAT3/efectos de los fármacos , Factor de Transcripción STAT3/metabolismo , Úlcera Cutánea/metabolismo , Úlcera Cutánea/patología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Interleucina-22RESUMEN
Recessive dystrophic epidermolysis bullosa (RDEB) is a genodermatosis characterized by fragile skin forming blisters that heal invariably with scars. It is due to mutations in the COL7A1 gene encoding type VII collagen, the major component of anchoring fibrils connecting the cutaneous basement membrane to the dermis. Identical COL7A1 mutations often result in inter- and intra-familial disease variability, suggesting that additional modifiers contribute to RDEB course. Here, we studied a monozygotic twin pair with RDEB presenting markedly different phenotypic manifestations, while expressing similar amounts of collagen VII. Genome-wide expression analysis in twins' fibroblasts showed differential expression of genes associated with TGF-ß pathway inhibition. In particular, decorin, a skin matrix component with anti-fibrotic properties, was found to be more expressed in the less affected twin. Accordingly, fibroblasts from the more affected sibling manifested a profibrotic and contractile phenotype characterized by enhanced α-smooth muscle actin and plasminogen activator inhibitor 1 expression, collagen I release and collagen lattice contraction. These cells also produced increased amounts of proinflammatory cytokines interleukin 6 and monocyte chemoattractant protein-1. Both TGF-ß canonical (Smads) and non-canonical (MAPKs) pathways were basally more activated in the fibroblasts of the more affected twin. The profibrotic behaviour of these fibroblasts was suppressed by decorin delivery to cells. Our data show that the amount of type VII collagen is not the only determinant of RDEB clinical severity, and indicate an involvement of TGF-ß pathways in modulating disease variability. Moreover, our findings identify decorin as a possible anti-fibrotic/inflammatory agent for RDEB therapeutic intervention.
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Epidermólisis Ampollosa Distrófica/genética , Fibroblastos/metabolismo , Genotipo , Fenotipo , Piel/metabolismo , Factor de Crecimiento Transformador beta/genética , Gemelos Monocigóticos/genética , Actinas/genética , Actinas/metabolismo , Adulto , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Colágeno Tipo VII/genética , Colágeno Tipo VII/metabolismo , Epidermólisis Ampollosa Distrófica/metabolismo , Epidermólisis Ampollosa Distrófica/patología , Fibroblastos/patología , Regulación de la Expresión Génica , Genes Recesivos , Heterogeneidad Genética , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Masculino , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Inhibidor 1 de Activador Plasminogénico/genética , Inhibidor 1 de Activador Plasminogénico/metabolismo , Índice de Severidad de la Enfermedad , Transducción de Señal , Piel/patología , Proteínas Smad/genética , Proteínas Smad/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Adipose tissue-derived stem cells (ASCs) are gaining increasing consideration in tissue repair therapeutic application. Recent evidence indicates that ASCs enhance skin repair in animal models of impaired wound healing. To assess the therapeutic activity of autologous vs. allogeneic ASCs in the treatment of diabetic ulcers, we functionally characterized diabetic ASCs and investigated their potential to promote wound healing with respect to nondiabetic ones. Adipose tissue-derived cells from streptozotocin-induced type 1 diabetic mice were analyzed either freshly isolated as stromal vascular fraction (SVF), or following a single passage of culture (ASCs). Diabetic ASCs showed decreased proliferative potential and migration. Expression of surface markers was altered in diabetic SVF and cultured ASCs, with a reduction in stem cell marker-positive cells. ASCs from diabetic mice released lower amounts of hepatocyte growth factor, vascular endothelial growth factor (VEGF)-A, and insulin-like growth factor-1, growth factors playing important roles in skin repair. Accordingly, the supernatant of diabetic ASCs manifested reduced capability to promote keratinocyte and fibroblast proliferation and migration. Therapeutic potential of diabetic SVF administered to wounds of diabetic mice was blunted as compared with cells isolated from nondiabetic mice. Our data indicate that diabetes alters ASC intrinsic properties and impairs their function, thus affecting therapeutic potential in the autologous treatment for diabetic ulcers.
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Tejido Adiposo/citología , Diabetes Mellitus Experimental/fisiopatología , Células Madre/fisiología , Cicatrización de Heridas/fisiología , Animales , Movimiento Celular/fisiología , Proliferación Celular , Diabetes Mellitus Experimental/metabolismo , Fibroblastos/fisiología , Factor de Crecimiento de Hepatocito/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Queratinocitos/fisiología , Masculino , Ratones , Células Madre/metabolismo , Células del Estroma , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
BACKGROUND: Vascular endothelial growth factor-C (VEGF-C), a lymphatic vessel growth factor, has been involved in the formation of lymph nodal metastases in different tumor types. Early evidences indicate that VEGF-C expression in human primary melanoma could be predictive of lymph nodal metastases, whereas the role of lymphangiogenesis is still controversial. METHODS: By immunohistochemical analysis, we investigated VEGF-C or CC chemokine receptor 7 expression, together with the lymphatic and blood vessel network, in 36 patients with primary skin melanomas and metastases at the sentinel lymph node biopsy (SLN-positive), and 26 melanoma patients with negative SLN biopsy (SLN-negative). RESULTS: We found that VEGF-C expression in primary melanoma specimens was significantly associated with SLN-positive (p < 0.001), particularly in thin melanomas. An association between augmented peritumoral lymphatic vessel area and SLN-positive (p < 0.02) was also seen. Conversely, no association between either expression of the CC chemokine receptor 7 in the primary tumor, or intratumoral lymphatic vessel or peritumoral and intratumoral blood vessel area, and SLN-positive was found. CONCLUSIONS: Our results, taking into account the expression of either VEGF-C or related histopathological markers, indicated the possibility to use VEGF-C immunohistochemistry as a marker of metastatic progression, especially in thin cutaneous melanomas.
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Biomarcadores de Tumor/biosíntesis , Regulación Neoplásica de la Expresión Génica , Melanoma , Receptores CCR7/biosíntesis , Neoplasias Cutáneas , Factor C de Crecimiento Endotelial Vascular/biosíntesis , Adulto , Anciano , Femenino , Humanos , Metástasis Linfática , Masculino , Melanoma/metabolismo , Melanoma/patología , Persona de Mediana Edad , Estudios Retrospectivos , Biopsia del Ganglio Linfático Centinela , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patologíaRESUMEN
The 14-3-3 protein family controls diverse biochemical processes through interaction with phosphorylated consensus sequences in protein targets. Its epithelial specific member, 14-3-3σ, also known as stratifin, is highly expressed in differentiated keratinocytes, and in vitro evidence indicates that 14-3-3σ downregulation leads to keratinocyte immortalization. To define the role of 14-3-3σ in skin homeostasis in vivo, we generated transgenic mice overexpressing 14-3-3σ in proliferating keratinocytes of the epidermis and hair follicle. Transgenic animals show decreased epidermal thickness and hair follicle density associated with reduced number of proliferating keratinocytes and decreased levels of keratins 14, 5, and 15. Primary keratinocytes isolated from transgenic mice manifest reduced proliferation and migration. Moreover, clonogenicity assessment and label-retaining analysis reveal a reduction in keratinocyte progenitor cell number in transgenic mice. Response to IGF-1 is strongly impaired in cultured transgenic keratinocytes compared with wild-type cells. Consistently, activation of phosphoinositol 3-kinase (PI3K), AKT, and Rac1, all IGF-1 downstream mediators, is reduced. Our results demonstrate that 14-3-3σ controls the in vivo epidermal proliferation-differentiation switch by reducing proliferative potential and forcing keratinocytes to exit the cell cycle, and that this effect associates with inhibition of the IGF-1 pathway.
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Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Epidermis/fisiología , Exonucleasas/genética , Exonucleasas/metabolismo , Folículo Piloso/fisiología , Queratinocitos/fisiología , Animales , División Celular/fisiología , Células Cultivadas , Células Clonales/citología , Células Clonales/fisiología , Células Epidérmicas , Exorribonucleasas , Folículo Piloso/citología , Factor I del Crecimiento Similar a la Insulina/metabolismo , Queratinocitos/citología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Transgénicos , Fenotipo , Regiones Promotoras Genéticas/fisiología , Transducción de Señal/fisiología , Células Madre/citología , Células Madre/fisiologíaRESUMEN
IL-22 has a pathogenetic role in psoriasis, where it is responsible for the altered proliferation and differentiation of keratinocytes and induces inflammatory molecules. The IL-22-induced effects are mediated by STAT3, whose activity is proportional to acetylation in lysine (Lys)685 and phosphorylation in tyrosine (Tyr)705. Lys 685 acetylation of STAT3 is inhibited by sirtuin (SIRT)1, a class III deacetylase promoting keratinocyte differentiation. Due to the opposite effects of IL-22 and SIRT1, we investigated whether IL-22-induced effects in keratinocytes could be regulated by SIRT1 through control of STAT3. We found that SIRT1 opposes the IL-22-induced STAT3 activity by deacetylating STAT3 and reducing STAT3 Tyr705 phosphorylation. By controlling STAT3, SIRT1 also influences the IL-22-induced expression of molecules involved in proliferation and inflammation as well as proliferation and migration processes in cultured keratinocytes. Although SIRT1 levels were similar in keratinocytes of healthy individuals and patients with psoriasis, they were reduced in psoriatic skin lesions, with the lymphokine IFN-γ inhibiting SIRT1 expression. Concomitantly, IFN-γ enhanced basal acetylation of STAT3 and its phosphorylation induced by IL-22. In conclusion, STAT3-dependent IL-22 signaling and effects in keratinocytes are negatively regulated by SIRT1. In skin affected by psoriasis, SIRT1 is down-regulated by IFN-γ, which thus renders psoriatic keratinocytes more prone to respond to IL-22.
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Interleucinas/metabolismo , Queratinocitos/metabolismo , Psoriasis/metabolismo , Factor de Transcripción STAT3/metabolismo , Sirtuina 1/metabolismo , Acetilación/efectos de los fármacos , Adulto , División Celular/fisiología , Movimiento Celular/fisiología , Células Cultivadas , Dermatitis/inmunología , Dermatitis/metabolismo , Histona Desacetilasas/metabolismo , Humanos , Interferón gamma/metabolismo , Interferón gamma/farmacología , Queratinocitos/citología , Queratinocitos/inmunología , Fosforilación/fisiología , Procesamiento Proteico-Postraduccional/fisiología , Psoriasis/inmunología , ARN Interferente Pequeño , Factor de Transcripción STAT3/genética , Transducción de Señal/inmunología , Sirtuina 1/genética , Interleucina-22RESUMEN
Experimental evidence suggests that in autoimmune thyroid diseases (AITDs) the skin is a target of autoantibodies against thyroid-specific antigens; however, the role of these autoantibodies in skin alterations remains unclear. To gain insight into the function of nominally thyroid-specific genes in skin, we analyzed the expression of thyroid-stimulating hormone-receptor (TSH-R), thyroglobulin (Tg), sodium iodide symporter (NIS), and thyroperoxidase (TPO) genes in normal human skin biopsies and cultured primary keratinocytes and dermal fibroblasts. The results revealed the presence of all the transcripts in skin biopsies. However, in keratinocytes and fibroblasts, only TSH-R messenger RNA was always detected. Western blot and immunohistochemical analyses of skin specimens confirmed the presence of TSH-R protein in keratinocytes and fibroblasts. Moreover, TSH treatment induced the proliferation of cultured keratinocytes and fibroblasts and increased keratinocyte intracellular cAMP. Finally, affinity-purified IgGs from serum of patients affected by Graves' disease, but not by chronic lymphocytic thyroiditis, stimulated cAMP accumulation in cultured keratinocytes, as well as their proliferation. In conclusion, the expression of thyroid-specific genes in cultured keratinocytes and fibroblasts and the mitogenic effects of TSH and IgGs on these cells support the concept that autoantibodies against thyroid-specific antigens may contribute to cutaneous symptoms in AITDs.
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Receptores de Tirotropina/genética , Receptores de Tirotropina/metabolismo , Piel/citología , Piel/inmunología , Enfermedades de la Tiroides , Autoanticuerpos/sangre , Autoantígenos/genética , Autoantígenos/inmunología , Autoantígenos/metabolismo , Biopsia , Células Cultivadas , Fibroblastos/citología , Fibroblastos/fisiología , Expresión Génica/fisiología , Humanos , Inmunoglobulina G/sangre , Yoduro Peroxidasa/genética , Yoduro Peroxidasa/inmunología , Yoduro Peroxidasa/metabolismo , Proteínas de Unión a Hierro/genética , Proteínas de Unión a Hierro/inmunología , Proteínas de Unión a Hierro/metabolismo , Queratinocitos/citología , Queratinocitos/fisiología , ARN Mensajero/metabolismo , Receptores de Tirotropina/inmunología , Piel/metabolismo , Simportadores/genética , Simportadores/inmunología , Simportadores/metabolismo , Tiroglobulina/genética , Tiroglobulina/inmunología , Tiroglobulina/metabolismo , Enfermedades de la Tiroides/inmunología , Enfermedades de la Tiroides/metabolismo , Enfermedades de la Tiroides/fisiopatología , Tirotropina/genética , Tirotropina/inmunología , Tirotropina/metabolismoRESUMEN
Th subsets are defined according to their production of lineage-indicating cytokines and functions. In this study, we have identified a subset of human Th cells that infiltrates the epidermis in individuals with inflammatory skin disorders and is characterized by the secretion of IL-22 and TNF-alpha, but not IFN-gamma, IL-4, or IL-17. In analogy to the Th17 subset, cells with this cytokine profile have been named the Th22 subset. Th22 clones derived from patients with psoriasis were stable in culture and exhibited a transcriptome profile clearly separate from those of Th1, Th2, and Th17 cells; it included genes encoding proteins involved in tissue remodeling, such as FGFs, and chemokines involved in angiogenesis and fibrosis. Primary human keratinocytes exposed to Th22 supernatants expressed a transcriptome response profile that included genes involved in innate immune pathways and the induction and modulation of adaptive immunity. These proinflammatory Th22 responses were synergistically dependent on IL-22 and TNF-alpha. Furthermore, Th22 supernatants enhanced wound healing in an in vitro injury model, which was exclusively dependent on IL-22. In conclusion, the human Th22 subset may represent a separate T cell subset with a distinct identity with respect to gene expression and function, present within the epidermal layer in inflammatory skin diseases. Future strategies directed against the Th22 subset may be of value in chronic inflammatory skin disorders.
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Células Epidérmicas , Epidermis/inmunología , Interleucinas/metabolismo , Subgrupos de Linfocitos T/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Adulto , Células Clonales , Dermatitis/genética , Dermatitis/inmunología , Dermatitis/patología , Perfilación de la Expresión Génica , Humanos , Inmunidad Innata , Técnicas In Vitro , Interferón gamma/metabolismo , Interleucina-17/metabolismo , Interleucina-4/metabolismo , Interleucinas/genética , Queratinocitos/inmunología , Psoriasis/genética , Psoriasis/inmunología , Psoriasis/patología , Receptores CCR10/metabolismo , Subgrupos de Linfocitos T/citología , Linfocitos T Colaboradores-Inductores/citología , Factor de Necrosis Tumoral alfa/metabolismo , Cicatrización de Heridas/genética , Cicatrización de Heridas/inmunología , Interleucina-22RESUMEN
Epidermal wound repair is a complex process involving the fine orchestrated regulation of crucial cell functions, such as proliferation, adhesion and migration. Using an in vitro model that recapitulates central aspects of epidermal wound healing, we demonstrate that the transcription factor HIF1 is strongly stimulated in keratinocyte cultures submitted to mechanical injury. Signals generated by scratch wounding stabilise the HIF1alpha protein, which requires activation of the PI3K pathway independently of oxygen availability. We further show that upregulation of HIF1alpha plays an essential role in keratinocyte migration during the in vitro healing process, because HIF1alpha inhibition dramatically delays the wound closure. In this context, we demonstrate that HIF1 controls the expression of laminin-332, one of the major epithelial cell adhesion ligands involved in cell migration and invasion. Indeed, silencing of HIF1alpha abrogates injury-induced laminin-332 expression, and we provide evidence that HIF1 directly regulates the promoter activity of the laminin alpha3 chain. Our results suggest that HIF1 contributes to keratinocyte migration and thus to the re-epithelialisation process by regulating laminin-332.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Movimiento Celular/fisiología , Factor 1 Inducible por Hipoxia/metabolismo , Queratinocitos/fisiología , Animales , Secuencia de Bases , Moléculas de Adhesión Celular/genética , Células Cultivadas , Regulación de la Expresión Génica , Humanos , Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Queratinocitos/citología , Laminina/genética , Laminina/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Fosfatidilinositol 3-Quinasas/metabolismo , Regiones Promotoras Genéticas , Transducción de Señal/fisiología , Cicatrización de Heridas , KalininaRESUMEN
Reduced microcirculation and diminished expression of growth factors contribute to wound healing impairment in diabetes. Placenta growth factor (PlGF), an angiogenic mediator promoting pathophysiological neovascularization, is expressed during cutaneous wound healing and improves wound closure by enhancing angiogenesis. By using streptozotocin-induced diabetic mice, we here demonstrate that PlGF induction is strongly reduced in diabetic wounds. Diabetic transgenic mice overexpressing PlGF in the skin displayed accelerated wound closure compared with diabetic wild-type littermates. Moreover, diabetic wound treatment with an adenovirus vector expressing the human PlGF gene (AdCMV.PlGF) significantly accelerated the healing process compared with wounds treated with a control vector. The analysis of treated wounds showed that PlGF gene transfer improved granulation tissue formation, maturation, and vascularization, as well as monocytes/macrophages local recruitment. Platelet-derived growth factor, fibroblast growth factor-2, and vascular endothelial growth factor mRNA levels were increased in AdCMV.PlGF-treated wounds, possibly enhancing PlGF-mediated effects. Finally, PlGF treatment stimulated cultured dermal fibroblast migration, pointing to a direct role of PlGF in accelerating granulation tissue maturation. In conclusion, our data indicate that reduced PlGF expression contributes to impaired wound healing in diabetes and that PlGF gene transfer to diabetic wounds exerts therapeutic activity by promoting different aspects of the repair process.