RESUMO
The hallmark of epidermolysis bullosa (EB) is fragile attachment of epithelia due to genetic variants in cell adhesion genes. We describe 16 EB patients treated in the ear, nose, and throat department of a tertiary pediatric hospital linked to the United Kingdom's national EB unit between 1992 and 2023. Patients suffered a high degree of morbidity and mortality from laryngotracheal stenosis. Variants in laminin subunit alpha-3 (LAMA3) were found in 10/15 patients where genotype was available. LAMA3 encodes a subunit of the laminin-332 heterotrimeric extracellular matrix protein complex and is expressed by airway epithelial basal stem cells. We investigated the benefit of restoring wild-type LAMA3 expression in primary EB patient-derived basal cell cultures. EB basal cells demonstrated weak adhesion to cell culture substrates, but could otherwise be expanded similarly to non-EB basal cells. In vitro lentiviral overexpression of LAMA3A in EB basal cells enabled them to differentiate in air-liquid interface cultures, producing cilia with normal ciliary beat frequency. Moreover, transduction restored cell adhesion to levels comparable to a non-EB donor culture. These data provide proof of concept for a combined cell and gene therapy approach to treat airway disease in LAMA3-affected EB.
Assuntos
Adesão Celular , Epidermólise Bolhosa , Laminina , Lentivirus , Humanos , Laminina/metabolismo , Laminina/genética , Epidermólise Bolhosa/genética , Epidermólise Bolhosa/metabolismo , Epidermólise Bolhosa/terapia , Epidermólise Bolhosa/patologia , Criança , Lentivirus/genética , Masculino , Feminino , Pré-Escolar , Terapia Genética/métodos , Vetores Genéticos/genética , Células Epiteliais/metabolismo , Células Cultivadas , Expressão Gênica , Adolescente , LactenteRESUMO
Epidermolysis bullosa (EB) is a heritable skin blistering disease caused by variants in genes coding for proteins that secure cell-cell adhesion and attachment of the epidermis to the dermis. Interestingly, several proteins involved in inherited EB are also associated with autoimmune blistering diseases (AIBD). In this study, we present a long-term follow-up of 15 patients suffering from recessive dystrophic or junctional EB. From these patients, 62 sera were analysed for the presence of autoantibodies associated with AIBD. We show that patients suffering from recessive dystrophic EB (RDEB) are more susceptible to developing autoantibodies against skin proteins than patients suffering from junctional EB (70% vs. 20%, respectively). Interestingly, no correlation with age was observed. Most patients showed reactivity to Type XVII collagen/linear IgA bullous dermatosis autoantigen (n = 5; 33%), followed by BP230 (n = 4; 27%), Type VII collagen (n = 4; 27%) and laminin-332 (n = 1; 7%). The pathogenicity of these autoantibodies remains a subject for future experiments.
Assuntos
Doenças Autoimunes , Epidermólise Bolhosa Distrófica , Epidermólise Bolhosa Juncional , Epidermólise Bolhosa , Humanos , Epidermólise Bolhosa Distrófica/genética , Autoanticorpos , Pele/metabolismo , Epidermólise Bolhosa/metabolismo , Epidermólise Bolhosa Juncional/genéticaRESUMO
Epidermolysis bullosa (EB), a phenotypically and genetically heterogeneous disorder, has been linked to mutations in the genes encoding structural proteins that reinforce skin integrity via dermal-epidermal adhesion. Breakdowns in these adhesion mechanisms result in four different subtypes of EB classified on the basis of the level of tissue separation within the cutaneous basement membrane zone (BMZ). Mutations in as many as 17 distinct genes that encode structural proteins in the BMZ have been linked to EB. Despite the clinical and histopathological confirmation of EB, many cases remain genetically unsolved. Technical advancements in next-generation sequencing have paved the way for the identification of genes involved in the pathophysiology of EB. Structural proteins have long been identified as the candidate molecules altered in EB, however, recently non-structural proteins, encoded for example by PLOD3, USB1, EXPH5, and KLHL24, involved in enzymatic modification or migration of structural proteins have been implicated. In this overview, we discuss recent work regarding these proteins vis-à-vis their function, associated clinical manifestations, and involvement in the pathogenesis of EB.
Assuntos
Epidermólise Bolhosa , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Membrana Basal/metabolismo , Epiderme/patologia , Epidermólise Bolhosa/metabolismo , Humanos , Mutação , Diester Fosfórico Hidrolases/genética , Pele/metabolismoRESUMO
Junctional epidermolysis bullosa (JEB) is a debilitating hereditary skin disorder caused by mutations in genes encoding laminin-332, type XVII collagen (C17), and integrin-α6ß4, which maintain stability between the dermis and epidermis. We designed patient-specific Cas9-nuclease- and -nickase-based targeting strategies for reframing a common homozygous deletion in exon 52 of COL17A1 associated with a lack of full-length C17 expression. Subsequent characterization of protein restoration, indel composition, and divergence of DNA and mRNA outcomes after treatment revealed auspicious efficiency, safety, and precision profiles for paired nicking-based COL17A1 editing. Almost 46% of treated primary JEB keratinocytes expressed reframed C17. Reframed COL17A1 transcripts predominantly featured 25- and 37-nt deletions, accounting for >42% of all edits and encoding C17 protein variants that localized accurately to the cell membrane. Furthermore, corrected cells showed accurate shedding of the extracellular 120-kDa C17 domain and improved adhesion capabilities to laminin-332 compared with untreated JEB cells. Three-dimensional (3D) skin equivalents demonstrated accurate and continuous deposition of C17 within the basal membrane zone between epidermis and dermis. Our findings constitute, for the first time, gene-editing-based correction of a COL17A1 mutation and demonstrate the superiority of proximal paired nicking strategies based on Cas9 D10A nickase over wild-type Cas9-based strategies for gene reframing in a clinical context.
Assuntos
Autoantígenos , Epidermólise Bolhosa Juncional , Epidermólise Bolhosa , Colágenos não Fibrilares , Autoantígenos/genética , Desoxirribonuclease I/genética , Epidermólise Bolhosa/metabolismo , Epidermólise Bolhosa Juncional/genética , Epidermólise Bolhosa Juncional/terapia , Homozigoto , Humanos , Laminina/genética , Mutação , Colágenos não Fibrilares/genética , Deleção de Sequência , Colágeno Tipo XVIIRESUMO
KLHL24 is an E3 ubiquitin ligase. Variants in the start codon of KLHL24 result in truncated KLHL24 protein lacking the initial 28 amino acids (KLHL24-ΔN28). KLHL24-ΔN28 is more stable than wild-type KLHL24 and causes excessive degradation of keratin 14, leading to epidermolysis bullosa. Patients with KLHL24-related epidermolysis bullosa usually develop alopecia, which is uncommon in patients with epidermolysis bullosa. The mechanisms by which KLHL24 variants cause alopecia is currently unclear. In this study, we show that KLHL24 regulates hair maintenance by mediating the stability of keratin 15. Using a Klhl24c.3G>T knock-in mouse model, we identify that KLHL24-ΔN28 disrupts the structure of hair follicle stem cells (HFSCs). Destructed HFSCs cannot anchor hairs and cause premature hair loss. Long-term destruction of HFSCs causes their exhaustion and hair follicle degeneration. Mechanically, KLHL24 mediates the ubiquitination and proteasomal degradation of keratin 15, an intermediate filament composing the HFSC cytoskeleton network. Keratin 15 is dramatically decreased in the skin of Klhl24c.3G>T mice and in patients with KLHL24-related epidermolysis bullosa. These findings show that KLHL24 plays a role in hair maintenance by regulating the cytoskeleton structure of HFSCs and highlight the importance of the ubiquitinâproteasome system in the stability of HFSCs.
Assuntos
Alopecia , Epidermólise Bolhosa , Folículo Piloso , Proteínas Repressoras , Células-Tronco , Animais , Camundongos , Alopecia/genética , Alopecia/metabolismo , Epidermólise Bolhosa/metabolismo , Folículo Piloso/metabolismo , Queratina-15 , Proteínas Repressoras/genéticaRESUMO
Impaired wound healing complicates a wide range of diseases and represents a major cost to healthcare systems. Here we describe the use of discarded wound dressings as a novel, cost effective, accessible, and non-invasive method of isolating viable human cells present at the site of skin wounds. By analyzing 133 discarded wound dressings from 51 patients with the inherited skin-blistering disease epidermolysis bullosa (EB), we show that large numbers of cells, often in excess of 100 million per day, continually infiltrate wound dressings. We show, that the method is able to differentiate chronic from acute wounds, identifying significant increases in granulocytes in chronic wounds, and we show that patients with the junctional form of EB have significantly more cells infiltrating their wounds compared with patients with recessive dystrophic EB. Finally, we identify subsets of granulocytes and T lymphocytes present in all wounds paving the way for single cell profiling of innate and adaptive immune cells with relevance to wound pathologies. In summary, our study delineates findings in EB that have potential relevance for all chronic wounds, and presents a method of cellular isolation that has wide reaching clinical application.
Assuntos
Bandagens , Separação Celular , Epidermólise Bolhosa , Granulócitos , Linfócitos T , Cicatrização , Doença Aguda , Adulto , Doença Crônica , Epidermólise Bolhosa/metabolismo , Epidermólise Bolhosa/patologia , Epidermólise Bolhosa/terapia , Granulócitos/metabolismo , Granulócitos/patologia , Humanos , Masculino , Linfócitos T/metabolismo , Linfócitos T/patologiaRESUMO
Recessive dystrophic epidermolysis bullosa (RDEB) is a severe skin disease caused by mutation of the COL7A1 gene. RDEB is associated with high levels of TGF-ß1, which is likely to be involved in the fibrosis that develops in this disease. Endoglin (CD105) is a type III coreceptor for TGF-ß1 and its overexpression in fibroblasts deregulates physiological Smad/Alk1/Alk5 signalling, repressing the synthesis of TGF-ß1 and extracellular matrix (ECM) proteins. Raloxifene is a specific estrogen receptor modulator designated as an orphan drug for hereditary hemorrhagic telangiectasia, a rare vascular disease. Raloxifene stimulates endoglin synthesis, which could attenuate fibrosis. By contrast, the antioxidant N-acetylcysteine may have therapeutic value to rectify inflammation, fibrosis and endothelial dysfunction. Thus, we present here a repurposing strategy based on the molecular and functional screening of fibroblasts from RDEB patients with these drugs, leading us to propose the repositioning of these two well-known drugs currently in clinical use, raloxifene and N-acetylcysteine, to counteract fibrosis and inflammation in RDEB. Both compounds modulate the profibrotic events that may ultimately be responsible for the clinical manifestations in RDEB, suggesting that these findings may also be relevant for other diseases in which fibrosis is an important pathophysiological event.
Assuntos
Acetilcisteína/farmacologia , Reposicionamento de Medicamentos , Epidermólise Bolhosa/genética , Fibroblastos/efeitos dos fármacos , Cloridrato de Raloxifeno/farmacologia , Fator de Crescimento Transformador beta1/genética , Receptores de Activinas Tipo II/genética , Receptores de Activinas Tipo II/metabolismo , Antioxidantes/farmacologia , Estudos de Casos e Controles , Colágeno Tipo VII/genética , Colágeno Tipo VII/metabolismo , Endoglina/genética , Endoglina/metabolismo , Epidermólise Bolhosa/metabolismo , Epidermólise Bolhosa/patologia , Antagonistas de Estrogênios/farmacologia , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose , Regulação da Expressão Gênica , Humanos , Padrões de Herança , Cultura Primária de Células , Receptor do Fator de Crescimento Transformador beta Tipo I/genética , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Índice de Gravidade de Doença , Transdução de Sinais , Pele/efeitos dos fármacos , Pele/metabolismo , Pele/patologia , Proteínas Smad/genética , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Systemic skin-selective therapeutics would be a major advancement in the treatment of diseases affecting the entire skin, such as recessive dystrophic epidermolysis bullosa (RDEB), which is caused by mutations in the COL7A1 gene and manifests in transforming growth factor-ß (TGF-ß)-driven fibrosis and malignant transformation. Homing peptides containing a C-terminal R/KXXR/K motif (C-end rule [CendR] sequence) activate an extravasation and tissue penetration pathway for tumor-specific drug delivery. We have previously described a homing peptide CRKDKC (CRK) that contains a cryptic CendR motif and homes to angiogenic blood vessels in wounds and tumors, but it cannot penetrate cells or tissues. In this study, we demonstrate that removal of the cysteine from CRK to expose the CendR sequence confers the peptide novel ability to home to normal skin. Fusion of the truncated CRK (tCRK) peptide to the C terminus of an extracellular matrix protein decorin (DCN), a natural TGF-ß inhibitor, resulted in a skin-homing therapeutic molecule (DCN-tCRK). Systemic DCN-tCRK administration in RDEB mice led to inhibition of TGF-ß signaling in the skin and significant improvement in the survival of RDEB mice. These results suggest that DCN-tCRK has the potential to be utilized as a novel therapeutic compound for the treatment of dermatological diseases such as RDEB.
Assuntos
Epidermólise Bolhosa/etiologia , Epidermólise Bolhosa/metabolismo , Peptídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Animais , Biomarcadores , Modelos Animais de Doenças , Epidermólise Bolhosa/patologia , Fibrose , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Neuropilina-1/metabolismo , Peptídeos/química , Peptídeos/farmacologia , Ligação Proteica , Proteínas Recombinantes de Fusão/farmacologia , Pele/efeitos dos fármacos , Pele/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Cicatrização/efeitos dos fármacosRESUMO
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.
Assuntos
Carcinoma de Células Escamosas , Moléculas de Adesão Celular , Epidermólise Bolhosa , Mutação , Proteínas de Neoplasias , Neoplasias Cutâneas , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Epidermólise Bolhosa/genética , Epidermólise Bolhosa/metabolismo , Epidermólise Bolhosa/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Estabilidade de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , CalininaAssuntos
Vesícula/metabolismo , Vesícula/patologia , Epidermólise Bolhosa/metabolismo , Epidermólise Bolhosa/patologia , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas de Neoplasias/metabolismo , Doenças Periodontais/metabolismo , Doenças Periodontais/patologia , Transtornos de Fotossensibilidade/metabolismo , Transtornos de Fotossensibilidade/patologia , Transativadores/metabolismo , Adulto , Feminino , Humanos , MasculinoRESUMO
Epidermolysis bullosa (EB) is a heterogeneous group of inherited skin disorders determined by mutations in genes encoding for structural components of the cutaneous basement membrane zone. Disease hallmarks are skin fragility and unremitting blistering. The most disabling EB (sub)types show defective wound healing, fibrosis and inflammation at lesional skin. These features expose patients to serious disease complications, including the development of cutaneous squamous cell carcinomas (SCCs). Almost all subjects affected with the severe recessive dystrophic EB (RDEB) subtype suffer from early and extremely aggressive SCCs (RDEB-SCC), which represent the first cause of death in these patients. The genetic determinants of RDEB-SCC do not exhaustively explain its unique behavior as compared to low-risk, ultraviolet-induced SCCs in the general population. On the other hand, a growing body of evidence points to the key role of tumor microenvironment in initiation, progression and spreading of RDEB-SCC, as well as of other, less-investigated, EB-related SCCs (EB-SCCs). Here, we discuss the recent advances in understanding the complex series of molecular events (i.e., fibrotic, inflammatory, and immune processes) contributing to SCC development in EB patients, cross-compare tumor features in the different EB subtypes and report the most promising therapeutic approaches to counteract or delay EB-SCCs.
Assuntos
Carcinoma de Células Escamosas/etiologia , Suscetibilidade a Doenças , Epidermólise Bolhosa/complicações , Animais , Carcinoma de Células Escamosas/diagnóstico , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/terapia , Gerenciamento Clínico , Epidermólise Bolhosa/genética , Epidermólise Bolhosa/metabolismo , Humanos , Vigilância da População , Transdução de Sinais , Microambiente Tumoral , CicatrizaçãoRESUMO
Human keratinocyte cultures contain keratinocyte stem cells, and have been involved in significant progress regarding stem cell biology as well as keratinocyte biology. Such cultures have also been applied in cell therapy for extensive severe burns for more than three decades, and in genetic disorders of the skin recently. Human keratinocyte stem cells were firstly characterized as holoclones by ex post clonal analysis, but in situ identification of keratinocyte stem cells is required for clinical applications. Recently, it was demonstrated that human keratinocyte stem cells display a unique rotational motion at early stages of culture, with subsequent dynamic collective motion at later stages. This finding enables image-based identification of keratinocyte stem cells, and noninvasive evaluation of their proliferative capacity, which can be applied for the quality assurance of human keratinocyte cultures. This review summarizes the historical development of human keratinocyte cultures and its applications for cell biology and cell therapy. This article also introduces recent advances in keratinocyte stem cell research with medical relevance and discusses the next-generation of regenerative medicine using human keratinocyte stem cells.
Assuntos
Terapia Baseada em Transplante de Células e Tecidos/métodos , Queratinócitos/citologia , Regeneração , Células-Tronco/citologia , Animais , Autoenxertos , Queimaduras/metabolismo , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Células Epidérmicas , Epidermólise Bolhosa/metabolismo , Humanos , Camundongos , Proteínas Recombinantes/metabolismo , Medicina Regenerativa/métodos , Pele/metabolismo , Fenômenos Fisiológicos da PeleRESUMO
Recessive dystrophic epidermolysis bullosa (RDEB) is a severe congenital skin fragility disease caused by COL7A1 mutations that result in type VII collagen (C7) deficiency. Herein, we report a synergistic polyplex system that can efficiently restore C7 expression in RDEB keratinocytes. A highly branched multifunctional poly(ß-amino ester) (HPAE), termed as HC32-122, was optimized systematically as the high-performance gene delivery vector for keratinocytes, achieving much higher transfection capability than polyethylenimine, SuperFect, and Lipofectamine 2000 without inducing obvious cytotoxicity. Concurrently, a 12 kb length minicircle DNA encoding â¼9 kb full-length COL7A1 (MCC7) devoid of bacterial sequence was biosynthesized as the therapeutic gene. Combining the highly potent polymer and the miniaturized gene structure, HC32-122/MCC7 polyplexes achieve 96.4% cellular uptake efficiency, 4019-fold COL7A1 mRNA enhancement, and robust recombinant C7 expression. Structure-property investigations reveal that HC32-122 can effectively condense MCC7 to form small, uniform, compact, and positively charged spherical nanoparticles with high DNA release flexibility. Moreover, formulation study shows that sucrose is conductive to lyophilized HC32-122/DNA polyplexes for maintaining the transfection capability. Direct frozen polyplexes can maintain full gene transfection capability after one-year storage. High efficiency, biocompatibility, facile manipulation, and long-term stability make the HC32-122/MCC7 system a promising bench-to-bed candidate for treating the debilitating RDEB.
Assuntos
Colágeno Tipo VII , Epidermólise Bolhosa , Técnicas de Transferência de Genes , Terapia Genética , Queratinócitos , Nanopartículas/química , Animais , Linhagem Celular , Colágeno Tipo VII/biossíntese , Colágeno Tipo VII/genética , Epidermólise Bolhosa/genética , Epidermólise Bolhosa/metabolismo , Epidermólise Bolhosa/patologia , Epidermólise Bolhosa/terapia , Humanos , Queratinócitos/metabolismo , Queratinócitos/patologia , Polímeros/química , Polímeros/farmacologiaRESUMO
Epidermolysis bullosa (EB) is a group of conditions characterized by severe fragility of the skin that causes recurring blistering. The recessive dystrophic subtype of EB (RDEB) has a strong impact on the nutritional status. We evaluated the resting energy expenditure (REE) and presence of protein catabolism in patients with RDEB. REE was assessed in 10 subjects (7 females; age range 4-33 years) by indirect calorimetry and using a predictive equation. Nitrogen balance was calculated by protein intake and 24 h urinary urea excretion estimations. An assessment of body surface area (BSA) with infected and non-infected skin lesions was applied to the nitrogen balance burn equation that was adapted to EB. The REE values predicted by the equation were consistently lower than the ones measured, except for two subjects. All subjects recorded high protein and energy intake, with protein intake being higher than 4 g protein/kg/day for five subjects. Even so, protein catabolism was observed in six subjects, three of whom had infected wounds. This study raises the hypothesis that the clinical and nutritional risks of people with RDEB are associated with an increased REE and negative nitrogen balance, which reinforces the importance of nutritional support.
Assuntos
Metabolismo Basal , Epidermólise Bolhosa/metabolismo , Proteínas/metabolismo , Adolescente , Adulto , Calorimetria Indireta , Criança , Pré-Escolar , Estudos Transversais , Feminino , Humanos , Masculino , Nitrogênio/metabolismo , Adulto JovemRESUMO
Laminin 332-deficient junctional epidermolysis bullosa (JEB) is a severe genetic skin disease. JEB is marked by epidermal stem cell depletion, the origin of which is unknown. We show that dysregulation of the YAP and TAZ pathway underpins such stem cell depletion. Laminin 332-mediated YAP activity sustains human epidermal stem cells, detected as holoclones. Ablation of YAP selectively depletes holoclones, while enforced YAP blocks conversion of stem cells into progenitors and indefinitely extends the keratinocyte lifespan. YAP is dramatically decreased in JEB keratinocytes, which contain only phosphorylated, inactive YAP. In normal keratinocytes, laminin 332 and α6ß4 ablation abolish YAP activity and recapitulate the JEB phenotype. In JEB keratinocytes, laminin 332-gene therapy rescues YAP activity and epidermal stem cells in vitro and in vivo. In JEB cells, enforced YAP recapitulates laminin 332-gene therapy, thus uncoupling adhesion from proliferation in epidermal stem cells. This work has important clinical implication for ex vivo gene therapy of JEB.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Moléculas de Adesão Celular , Epiderme/metabolismo , Epidermólise Bolhosa , Terapia Genética , Células-Tronco/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Adesão Celular , Moléculas de Adesão Celular/biossíntese , Moléculas de Adesão Celular/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Epiderme/patologia , Epidermólise Bolhosa/genética , Epidermólise Bolhosa/metabolismo , Epidermólise Bolhosa/patologia , Epidermólise Bolhosa/terapia , Humanos , Queratinócitos/metabolismo , Queratinócitos/patologia , Camundongos , Células-Tronco/patologia , Fatores de Transcrição/genética , Proteínas de Sinalização YAP , CalininaRESUMO
BACKGROUND: Missense mutations in keratin 5 and 14 genes cause the severe skin fragility disorder epidermolysis bullosa simplex (EBS) by collapsing of the keratin cytoskeleton into cytoplasmic protein aggregates. Despite intense efforts, no molecular therapies are available, mostly due to the complex phenotype of EBS, comprising cell fragility, diminished adhesion, skin inflammation and itch. METHODS: We extensively characterized KRT5 and KRT14 mutant keratinocytes from patients with severe generalized EBS following exposure to the chemical chaperone 4-phenylbutyrate (4-PBA). FINDINGS: 4-PBA diminished keratin aggregates within EBS cells and ameliorated their inflammatory phenotype. Chemoproteomics of 4-PBA-treated and untreated EBS cells revealed reduced IL1ß expression- but also showed activation of Wnt/ß-catenin and NF-kB pathways. The abundance of extracellular matrix and cytoskeletal proteins was significantly altered, coinciding with diminished keratinocyte adhesion and migration in a 4-PBA dose-dependent manner. INTERPRETATION: Together, our study reveals a complex interplay of benefits and disadvantages that challenge the use of 4-PBA in skin fragility disorders.
Assuntos
Epidermólise Bolhosa/metabolismo , Epidermólise Bolhosa/patologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Queratinas/metabolismo , Fenilbutiratos/farmacologia , Animais , Apoptose/genética , Biomarcadores , Biópsia , Adesão Celular , Comunicação Celular , Linhagem Celular , Citoesqueleto/metabolismo , Modelos Animais de Doenças , Epidermólise Bolhosa/etiologia , Matriz Extracelular/metabolismo , Humanos , Imuno-Histoquímica , Queratinócitos/patologia , Camundongos , Fenótipo , Fenilbutiratos/uso terapêutico , Transporte Proteico , Proteoma , Proteômica/métodos , Transdução de Sinais , Pele/efeitos dos fármacos , Pele/metabolismo , Pele/patologiaRESUMO
BACKGROUND: Osteopenia or osteoporosis is one of the many comorbidities in patients with Epidermolysis Bullosa (EB). Current literature on the prevalence of osteoporosis in EB is scarce. OBJECTIVE: This review will analyse the current literature in the field of patients with compromised bone health in EB and any articles on the prevalence of such diseases in EB groups. METHODS: A systematic search for articles related to bone health and epidermolysis bullosa (EB) (1946-2017) was performed on seven databases: MEDLINE, EMBASE and EBM, PubMed, ProQuest, Scopus and Web of Science. Search terms: epidermolysis bullosa, osteop*, bone mass, bone mineral*, fracture, dual X-ray absorptiometry, vitamin D, calcium, nutrition, exercise and physical activity. Abstracts from all search results were screened, and reference scanning of the search results was performed. Eighty-three articles met the selection criteria and were considered for review. Letters to the editor and abstract-only articles were excluded. Articles were favoured based on citation count, impact factor of their journal and study sample size. The search included all languages. RESULTS: The searches yielded a total of 1309 articles including 717 duplicates. The remaining 592 articles were screened by title and abstracts. Eighty-three full-text articles were analysed. Twenty-one articles directly relating to bone health in EB were included. Three descriptive studies and one case-control study were found, indicating a need for research of larger scale. CONCLUSION: Further investigations into osteoporosis in EB, especially the milder forms of EB, are valuable in providing evidence to support guideline developments for EB bone health management.
Assuntos
Doenças Ósseas Metabólicas/etiologia , Epidermólise Bolhosa/complicações , Adulto , Doenças Ósseas Metabólicas/epidemiologia , Criança , Epidermólise Bolhosa/metabolismo , Medicina Baseada em Evidências , Tolerância ao Exercício , Fraturas Espontâneas/epidemiologia , Fraturas Espontâneas/etiologia , Trato Gastrointestinal/fisiopatologia , Transtornos do Crescimento/etiologia , Transtornos do Crescimento/fisiopatologia , Hormônios/fisiologia , Humanos , Inflamação , Absorção Intestinal , Mucosa Intestinal/patologia , Desnutrição/etiologia , Desnutrição/fisiopatologia , Osteoporose/epidemiologia , Osteoporose/etiologia , Prevalência , Puberdade Tardia/etiologia , Puberdade Tardia/fisiopatologiaRESUMO
Integrin α3ß1, a major epidermal adhesion receptor is critical for organization of the basement membrane during development and wound healing. Integrin α3 deficiency leads to interstitial lung disease, nephrotic syndrome and epidermolysis bullosa (ILNEB), an autosomal recessive multiorgan disease characterized by basement membrane abnormalities in skin, lung and kidney. The pathogenetic chains from ITGA3 mutation to tissue abnormalities are still unclear. Although integrin α3 was reported to regulate multiple extracellular proteins, the composition of the extracellular compartment of integrin α3-negative keratinocytes has not been resolved so far. In a comprehensive approach, quantitative proteomics of deposited extracellular matrix, conditioned cultured media as well as of the intracellular compartment of keratinocytes isolated from an ILNEB patient and from normal skin were performed. By mass spectrometry-based proteomics, 167 proteins corresponding to the GO terms "extracellular" and "cell adhesion", or included in the "human matrisome" were identified in the deposited extracellular matrix, and 217 in the conditioned media of normal human keratinocytes. In the absence of integrin α3, 33% and 26% respectively were dysregulated. Dysregulated proteins were functionally related to integrin α3 or were known interaction partners. The results show that in the absence of integrin α3 ILNEB keratinocytes produce a fibronectin-rich microenvironment and make use of fibronectin-binding integrin subunits αv and α5. The most important results were validated in monolayer and organotypic coculture models. Finally, the in vivo relevance of the most dysregulated components was demonstrated by immunostainings of skin, kidney and lung samples of three ILNEB patients.
Assuntos
Epidermólise Bolhosa/metabolismo , Integrina alfa3/genética , Integrina alfa3/metabolismo , Queratinócitos/citologia , Doenças Pulmonares Intersticiais/metabolismo , Síndrome Nefrótica/metabolismo , Membrana Basal/metabolismo , Adesão Celular , Técnicas de Cultura de Células , Linhagem Celular , Microambiente Celular , Meios de Cultivo Condicionados/química , Epidermólise Bolhosa/genética , Matriz Extracelular/metabolismo , Fibronectinas , Regulação da Expressão Gênica , Humanos , Queratinócitos/metabolismo , Doenças Pulmonares Intersticiais/genética , Síndrome Nefrótica/genética , Mapeamento de Interação de Proteínas , ProteômicaRESUMO
Recessive dystrophic epidermolysis bullosa (RDEB) patients suffer from chronic and repeatedly infected wounds predisposing them to the development of aggressive and life-threatening skin cancer in these areas. Vitamin D3 is an often neglected but critical factor for wound healing. Intact skin possesses the entire enzymatic machinery required to produce active 1-alpha,25-dihydroxyvitamin D3 (calcitriol), underscoring its significance to proper skin function. Injury enhances calcitriol production, inducing the expression of calcitriol target genes including the antimicrobial peptide cathelicidin (hCAP18), an essential component of the innate immune system and an important wound healing factor. We found significantly reduced hCAP18 expression in a subset of RDEB keratinocytes which could be restored by calcipotriol treatment. Reduced scratch closure in RDEB cell monolayers was enhanced up to 2-fold by calcipotriol treatment, and the secretome of calcipotriol-treated cells additionally showed increased antimicrobial activity. Calcipotriol exhibited anti-neoplastic effects, suppressing the clonogenicity and proliferation of RDEB tumor cells. The combined wound healing, anti-microbial, and anti-neoplastic effects indicate that calcipotriol may represent a vital therapeutic option for RDEB patients which we could demonstrate in a single-patient observation study.
Assuntos
Antibacterianos/farmacologia , Antineoplásicos/farmacologia , Calcitriol/análogos & derivados , Fármacos Dermatológicos/farmacologia , Epidermólise Bolhosa/metabolismo , Queratinócitos/efeitos dos fármacos , Cicatrização , Idoso , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/metabolismo , Calcitriol/farmacologia , Linhagem Celular , Células Cultivadas , Epidermólise Bolhosa/patologia , Humanos , Queratinócitos/metabolismo , Masculino , CatelicidinasRESUMO
Epidermolysis bullosa (EB), a group of heritable skin fragility disorders, is characterized by blistering, erosions and chronic ulcers in the skin and mucous membranes. In some forms, the blistering phenotype is associated with extensive mutilating scarring and development of aggressive squamous cell carcinomas. The skin findings can be associated with extracutaneous manifestations in the ocular as well as gastrointestinal and vesico-urinary tracts. The phenotypic heterogeneity reflects the presence of mutations in as many as 20 different genes expressed in the cutaneous basement membrane zone, and the types and combinations of the mutations and their consequences at the mRNA and protein levels contribute to the spectrum of severity encountered in different subtypes of EB. This overview highlights the molecular genetics of EB based on mutations in the genes encoding type VII and XVII collagens as well as laminin-332. The mutations identified in these protein components of the extracellular matrix attest to their critical importance in providing stability to the cutaneous basement membrane zone, with implications for heritable and acquired diseases.