RESUMEN
Purpose: Aging is a risk factor for dry eye. We sought to identify changes in the aged mouse corneal epithelial transcriptome and determine how age affects corneal sensitivity, re-epithelialization, and barrier reformation after corneal debridement. Methods: Corneal epithelium of female C57BL/6J (B6) mice of different ages (2, 12, 18, and 24 months) was collected, RNA extracted, and bulk RNA sequencing performed. Cornea sensitivity was measured with an esthesiometer in 2- to 3-month-old, 12- to 13-month-old, 18- to 19-month-old, and 22- to 25-month-old female and male mice. The 2-month-old and 18-month-old female and male mice underwent unilateral corneal debridement using a blunt blade. Wound size and fluorescein staining were visualized and photographed at different time points, and a re-epithelialization rate curve was calculated. Results: There were 157 differentially expressed genes in aged mice compared with young mice. Several pathways downregulated with age control cell migration, proteoglycan synthesis, and collagen trimerization, assembly, biosynthesis, and degradation. Male mice had decreased corneal sensitivity compared with female mice at 12 and 24 months of age. Aged mice, irrespective of sex, had delayed corneal re-epithelialization in the first 48 hours and worse corneal fluorescein staining intensity at day 14 than young mice. Conclusions: Aged corneal epithelium has an altered transcriptome. Aged mice regardless of sex heal more slowly and displayed more signs of corneal epithelial defects after wounding than young mice. These results indicate that aging significantly alters the corneal epithelium and its ability to coordinate healing.
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Envejecimiento , Epitelio Corneal , Ratones Endogámicos C57BL , Transcriptoma , Cicatrización de Heridas , Animales , Epitelio Corneal/metabolismo , Femenino , Ratones , Cicatrización de Heridas/genética , Cicatrización de Heridas/fisiología , Masculino , Envejecimiento/fisiología , Repitelización/fisiología , Repitelización/genética , Lesiones de la Cornea/genética , Lesiones de la Cornea/metabolismo , Desbridamiento , Regulación de la Expresión Génica/fisiología , Modelos Animales de EnfermedadRESUMEN
Keratinocyte migration is vital in the re-epithelialisation of the skin during wound healing. Multiple factors conspire to impair closure of chronic wounds such as diabetic foot ulcers, venous leg ulcers and pressure wounds. Despite deep mechanistic understanding of microRNA (miRNA) biogenesis and function, the translational potential of these small genetic molecules has not been exploited to promote wound repair. In this review, I focus on miRNAs whose importance for wound healing stems from their impact on epidermal keratinocyte behaviour. These include miR-21-5p, miR-31-5p, miR-132-3p, miR-19b, miR-20a, miR-184, miR-129-5p and miR-335-5p which regulate diverse aspect of keratinocyte biology such as migration, proliferation, differentiation, inflammation and wound closure. A combinatorial approach where two or more miRNA mimics targeting distinct but complementary wound healing processes is proposed as this may enhance wound repair more effectively than any single miRNA mimic alone.
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Queratinocitos/metabolismo , MicroARNs/genética , Repitelización/genética , Cicatrización de Heridas/fisiología , Animales , Movimiento Celular/genética , Movimiento Celular/fisiología , Humanos , Queratinocitos/patología , MicroARNs/metabolismo , Repitelización/fisiología , Piel/metabolismo , Piel/patología , Cicatrización de Heridas/genéticaRESUMEN
Chronic infections are considered one of the most severe problems in skin wounds, and bacteria are present in over 90% of chronic wounds. Pseudomonas aeruginosa is frequently isolated from chronic wounds and is thought to be a cause of delayed wound healing. Invariant natural killer T (iNKT) cells, unique lymphocytes with a potent regulatory ability in various inflammatory responses, accelerate the wound healing process. In the present study, we investigated the contribution of iNKT cells in the host defense against P. aeruginosa inoculation at the wound sites. We analyzed the re-epithelialization, bacterial load, accumulation of leukocytes, and production of cytokines and antimicrobial peptides. In iNKT cell-deficient (Jα18KO) mice, re-epithelialization was significantly decreased, and the number of live colonies was significantly increased, when compared with those in wild-type (WT) mice on day 7. IL-17A, and IL-22 production was significantly lower in Jα18KO mice than in WT mice on day 5. Furthermore, the administration of α-galactosylceramide (α-GalCer), a specific activator of iNKT cells, led to enhanced host protection, as shown by reduced bacterial load, and to increased production of IL-22, IL-23, and S100A9 compared that of with WT mice. These results suggest that iNKT cells promote P. aeruginosa clearance during skin wound healing.
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Células T Asesinas Naturales/inmunología , Repitelización/genética , Piel/inmunología , Cicatrización de Heridas/genética , Animales , Calgranulina B/genética , Galactosilceramidas/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Interferón gamma/genética , Interleucina-17/genética , Interleucina-23/genética , Interleucinas/genética , Leucocitos/inmunología , Leucocitos/microbiología , Ratones , Proteínas Citotóxicas Formadoras de Poros/farmacología , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidad , Repitelización/inmunología , Piel/microbiología , Piel/patología , Cicatrización de Heridas/inmunología , Interleucina-22RESUMEN
In self-renewing somatic tissue such as skin epidermis, terminal differentiation genes must be suppressed in progenitors to sustain regenerative capacity. Here we show that hundreds of intronic polyadenylation (IpA) sites are differentially used during keratinocyte differentiation, which is accompanied by downregulation of the Cleavage and Polyadenylation Specificity Factor (CPSF) complex. Sustained CPSF expression in undifferentiated keratinocytes requires the contribution from the transcription factor MYC. In keratinocytes cultured in undifferentiation condition, CSPF knockdown induces premature differentiation and partially affects dynamically used IpA sites. These sites include an IpA site located in the first intron of the differentiation activator GRHL3. CRISPR knockout of GRHL3 IpA increased full-length GRHL3 mRNA expression. Using a targeted genetic screen, we identify that HNRNPA3 interacts with CPSF and enhances GRHL3 IpA. Our data suggest a model where the interaction between CPSF and RNA-binding proteins, such as HNRNPA3, promotes site-specific IpA and suppresses premature differentiation in progenitors.
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Factor de Especificidad de Desdoblamiento y Poliadenilación/metabolismo , Proteínas de Unión al ADN/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/metabolismo , Queratinocitos/fisiología , Repitelización/genética , Células Madre/fisiología , Factores de Transcripción/metabolismo , Sistemas CRISPR-Cas/genética , Diferenciación Celular/genética , Autorrenovación de las Células/genética , Factor de Especificidad de Desdoblamiento y Poliadenilación/genética , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Técnicas de Inactivación de Genes , Células HEK293 , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/genética , Humanos , Intrones/genética , Poliadenilación/genética , Cultivo Primario de Células , Factores de Transcripción/genéticaRESUMEN
Although most acute skin wounds heal rapidly, non-healing skin ulcers represent an increasing and substantial unmet medical need that urgently requires effective therapeutics. Keratinocytes resurface wounds to re-establish the epidermal barrier by transitioning to an activated, migratory state, but this ability is lost in dysfunctional chronic wounds. Small-molecule regulators of keratinocyte plasticity with the potential to reverse keratinocyte malfunction in situ could offer a novel therapeutic approach in skin wound healing. Utilizing high-throughput phenotypic screening of primary keratinocytes, we identify such small molecules, including bromodomain and extra-terminal domain (BET) protein family inhibitors (BETi). BETi induce a sustained activated, migratory state in keratinocytes in vitro, increase activation markers in human epidermis ex vivo and enhance skin wound healing in vivo. Our findings suggest potential clinical utility of BETi in promoting keratinocyte re-epithelialization of skin wounds. Importantly, this novel property of BETi is exclusively observed after transient low-dose exposure, revealing new potential for this compound class.
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Proteínas de Ciclo Celular/genética , Epidermis/efectos de los fármacos , Repitelización/efectos de los fármacos , Úlcera Cutánea/tratamiento farmacológico , Bibliotecas de Moléculas Pequeñas/farmacología , Factores de Transcripción/genética , Heridas no Penetrantes/tratamiento farmacológico , Animales , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/metabolismo , Modelos Animales de Enfermedad , Epidermis/metabolismo , Epidermis/patología , Transferencia Resonante de Energía de Fluorescencia , Regulación de la Expresión Génica , Ensayos Analíticos de Alto Rendimiento , Humanos , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Queratinocitos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Cultivo Primario de Células , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Precursores de Proteínas/antagonistas & inhibidores , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Repitelización/genética , Úlcera Cutánea/genética , Úlcera Cutánea/metabolismo , Úlcera Cutánea/patología , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-Actividad , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/metabolismo , Transcripción Genética , Heridas no Penetrantes/genética , Heridas no Penetrantes/metabolismo , Heridas no Penetrantes/patologíaRESUMEN
In vivo wound healing models are predictive preclinical tests for therapeutics that enhance skin repair or limit scarring. Large animals, such as swine, heal in a manner similar to humans, but testing is impractical and expensive. Experiments in mice are more economic, but may be less translatable as this species heals primarily through contraction, not by the processes of epithelialization and granulation tissue formation as seen in human wounds. Here, we describe a murine model of thermal burn injury that closely mimics human healing, resulting in a large, hypertrophic-like scar. This practical, reproducible model is ideal for testing promising wound-healing therapies, such as virus-derived growth factors and immune-modulatory proteins.
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Quemaduras/patología , Cicatriz/prevención & control , Modelos Animales de Enfermedad , Repitelización/genética , Animales , Quemaduras/genética , Quemaduras/terapia , Cicatriz/genética , Cicatriz/patología , Femenino , Expresión Génica , Calor , Humanos , Factores Inmunológicos/biosíntesis , Factores Inmunológicos/genética , Factores Inmunológicos/farmacología , Péptidos y Proteínas de Señalización Intercelular/biosíntesis , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/farmacología , Ratones , Ratones Endogámicos C57BL , Repitelización/efectos de los fármacos , Piel/efectos de los fármacos , Piel/lesiones , Transgenes , Virus/genéticaRESUMEN
It has been widely recognized that mechanical stretch can regulate the fate of stem cells (SCs). Previous research has shown that short-term mechanical stretch induces SC proliferation by activating the predominant transcription factor YAP, and YAP is a critical modulator in controlling epidermal proliferation. However, our study finds that after this phase, cell growth arrest appears, which is induced by long-term mechanical stretch. In the interfollicular epidermal SCs undergoing long-term mechanical stretch in vivo and in vitro, the level of H3K27me3 and its histone methyltransferase EZH2 are significantly elevated with suppressed expression of the target genes of YAP. EZH2 forms repressive H3K27me3 that suppresses gene transcription. Small-molecule inhibitor of EZH2 rescues significantly the cell growth arrest in interfollicular epidermal SCs induced by long-term mechanical stretch, thus promoting epidermal proliferation in vivo again. These findings reveal that there is an unexpected correlation between SC proliferation and the duration of mechanical stretch regulated by EZH2. This study of long-term mechanical stretch that induces cell growth arrest provides a strategy for clinical translation to promote skin regeneration.
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Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Epidermis/fisiología , Repitelización/genética , Células Madre/fisiología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adolescente , Adulto , Animales , Apoptosis/genética , Proliferación Celular/genética , Niño , Metilación de ADN , Epigénesis Genética , Femenino , Histonas/metabolismo , Humanos , Queratinocitos , Masculino , Ratones , Cultivo Primario de Células , Estrés Mecánico , Factores de Transcripción/metabolismo , Proteínas Señalizadoras YAP , Adulto JovenRESUMEN
BACKGROUND: Drug-eluting stents impair post-angioplasty re-endothelialization thus compromising restenosis prevention while heightening thrombotic risks. We recently found that inhibition of protein kinase RNA-like endoplasmic reticulum kinase (PERK) effectively mitigated both restenosis and thrombosis in rodent models. This motivated us to determine how PERK inhibition impacts re-endothelialization. METHODS: Re-endothelialization was evaluated in endothelial-denuded rat carotid arteries after balloon angioplasty and periadventitial administration of PERK inhibitor in a hydrogel. To study whether PERK in smooth muscle cells (SMCs) regulates re-endothelialization by paracrinally influencing endothelial cells (ECs), denuded arteries exposing SMCs were lentiviral-infected to silence PERK; in vitro, the extracellular vesicles isolated from the medium of PDGF-activated, PERK-upregulating human primary SMCs were transferred to human primary ECs. RESULTS: Treatment with PERK inhibitor versus vehicle control accelerated re-endothelialization in denuded arteries. PERK-specific silencing in the denuded arterial wall (mainly SMCs) also enhanced re-endothelialization compared to scrambled shRNA control. In vitro, while medium transfer from PDGF-activated SMCs impaired EC viability and increased the mRNA levels of dysfunctional EC markers, either PERK inhibition or silencing in donor SMCs mitigated these EC changes. Furthermore, CXCL10, a paracrine cytokine detrimental to ECs, was increased by PDGF activation and decreased after PERK inhibition or silencing in SMCs. CONCLUSIONS: Attenuating PERK activity pharmacologically or genetically provides an approach to accelerating post-angioplasty re-endothelialization in rats. The mechanism may involve paracrine factors regulated by PERK in SMCs that impact neighboring ECs. This study rationalizes future development of PERK-targeted endothelium-friendly vascular interventions.
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Angioplastia de Balón/efectos adversos , Reestenosis Coronaria/prevención & control , Miocitos del Músculo Liso/efectos de los fármacos , Inhibidores de Proteínas Quinasas/administración & dosificación , Repitelización/efectos de los fármacos , eIF-2 Quinasa/antagonistas & inhibidores , Angioplastia de Balón/instrumentación , Animales , Arterias Carótidas/efectos de los fármacos , Arterias Carótidas/patología , Arterias Carótidas/cirugía , Reestenosis Coronaria/etiología , Modelos Animales de Enfermedad , Stents Liberadores de Fármacos/efectos adversos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/patología , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/patología , Humanos , Masculino , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Comunicación Paracrina/efectos de los fármacos , Comunicación Paracrina/genética , ARN Interferente Pequeño/metabolismo , Ratas , Repitelización/genética , eIF-2 Quinasa/genéticaRESUMEN
Wound-induced hair follicle neogenesis (WIHN) has been an important model to study hair follicle regeneration during wound repair. However, the cellular and molecular components of the dermis that make large wounds more regenerative are not fully understood. Here, we compare and contrast recently published scRNA-seq data of small scarring wounds to wounds that regenerate in hope to elucidate the role of fibroblasts lineages in WIHN. Our analysis revealed an over-representation of the newly identified upper wound fibroblasts in regenerative wound conditions, which express the retinoic acid binding protein Crabp1. This regenerative cell type shares a similar gene signature to the murine papillary fibroblast lineage, which are necessary to support hair follicle morphogenesis and homeostasis. RNA velocity analysis comparing scarring and regenerating wounds revealed the divergent trajectories towards upper and lower wound fibroblasts and that the upper populations were closely associated with the specialized dermal papilla. We also provide analyses and explanation reconciling the inconsistency between the histological lineage tracing and the scRNA-seq data from recent reports investigating large wounds. Finally, we performed a computational test to map the spatial location of upper wound fibroblasts in large wounds which revealed that upper peripheral fibroblasts might harbour equivalent regenerative competence as those in the centre. Overall, our scRNA-seq reanalysis combining multiple samples suggests that upper wound fibroblasts are required for hair follicle regeneration and that papillary fibroblasts may migrate from the wound periphery to the centre during wound re-epithelialization. Moreover, data from this publication are made available on our searchable web resource: https://skinregeneration.org/.
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Cicatriz/genética , Fibroblastos/fisiología , Transcriptoma , Cicatrización de Heridas/genética , Heridas y Lesiones/genética , Heridas y Lesiones/patología , Animales , Linaje de la Célula , Bases de Datos Genéticas , Dermis/patología , Fibroblastos/patología , Folículo Piloso/fisiopatología , Factores de Transcripción de Tipo Kruppel/genética , Proteínas Luminiscentes , Ratones , Repitelización/genética , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Piel/lesiones , Proteína Fluorescente RojaRESUMEN
Poor wound closure due to diabetes, aging, stress, obesity, alcoholism, and chronic disease affects millions of people worldwide. Reasons wounds will not close are still unclear, and current therapies are limited. Although stem cell factor (SCF), a cytokine, is known to be important for wound repair, the cellular and molecular mechanisms of SCF in wound closure remain poorly understood. Here, we found that SCF expression in the epidermis is decreased in mouse models of delayed wound closure intended to mimic old age, obesity, and alcoholism. By using SCF conditionally knocked out mice, we demonstrated that keratinocytes' autocrine production of SCF activates a transient c-kit receptor in keratinocytes. Transient activation of the c-kit receptor induces the expression of growth factors and chemokines to promote wound re-epithelialization by increasing migration of skin cells (keratinocytes and fibroblasts) and immune cells (neutrophils) to the wound bed 24-48 h post-wounding. Our results demonstrate that keratinocyte-produced SCF is essential to wound closure due to the increased recruitment of a unique combination of skin cells and immune cells in the early phase after wounding. This discovery is imperative for developing clinical strategies that might improve the body's natural repair mechanisms for treating patients with wound-closure pathologies.
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Queratinocitos/metabolismo , Síndrome Metabólico/fisiopatología , Repitelización/genética , Repitelización/fisiología , Fenómenos Fisiológicos de la Piel , Piel/lesiones , Factor de Células Madre/deficiencia , Factor de Células Madre/fisiología , Cicatrización de Heridas/genética , Cicatrización de Heridas/fisiología , Animales , Modelos Animales de Enfermedad , Expresión Génica , Ratones Endogámicos C57BL , Ratones Noqueados , Neutrófilos , Proteínas Proto-Oncogénicas c-kit/metabolismo , Piel/citología , Factor de Células Madre/genética , Factor de Células Madre/metabolismoRESUMEN
SUMMARY: The treatment of chronic wounds has become a public health issue in recent years mainly due to comorbidities associated with an older population and bacterial resistance. Honey has emerged as an alternative treatment for chronic wounds but lack of knowledge of its mechanism of actionin the treated tissue and low quality of evidence in clinical triads has distanced the medical community from honey as a possible treatment. One of the main processes that is altered in chronic wounds is re-epithelialization mediated by keratinocytes, where proliferation and migration processes are altered. Markers of proliferation, migration and activation of keratinocytes, such as adhesion molecules, growth factors, membrane receptors, signal translating proteins, transcription factors, microRNAs, among others are deregulated in this process. In general, honeys from different floral origins have a positive effect on markers of proliferation and migration in keratinocytes. In conclusion there are still few studies that focus on the molecular action of honey in keratinocytes and fail to report details on the honey used not allowing to achieve the same results.
RESUMEN: El tratamiento de heridas crónicas (HC) se ha vuelto un tema de salud pública en los últimos años, principalmente debido a comorbilidades asociadas a una población de mayor edad y a la resistencia bacteriana. La miel ha surgido como un tratamiento alternativo para HC pero la falta de conocimiento de su mecanismo de acción en el tejido tratado y de la baja calidad de la evidencia en triadas clínicas, ha distanciado a la comunidad médica de la miel como posible tratamiento. Uno de los principales procesos que se ve alterado en las HC es la re-epitelización mediada por queratinocitos, donde se ven alterados los procesos de proliferación y migración. Marcadores de proliferación, migración y activación de queratinocitos, como moléculas de adhesión, factores de crecimiento, receptores de membrana, proteínas traductores de señales, factores de transcripción, microARNs, entre otras, se ven desreguladas en éste proceso. De manera general las mieles de diferentes orígenes florales tienen un efecto positivo en marcadores de proliferación y migración en queratinocitos. En conclusión aún existen pocos estudios que se enfoquen en la acción molecular de la miel en queratinocitos y los pocos que existen fallan en la entrega de información en relación a la miel utilizada que pueda hacer reproducibles los resultados.
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Cicatrización de Heridas/fisiología , Queratinocitos/fisiología , Repitelización/fisiología , Miel , Cicatrización de Heridas/genética , MicroARNs/fisiología , MicroARNs/genética , Repitelización/genéticaRESUMEN
BACKGROUND AND AIMS: Therapeutic targets in ulcerative colitis (UC) have evolved over time from clinical remission to biological and endoscopic remission. Histologic remission remains a debatable outcome due to lack of data regarding its impact on long-term evolution. The development of histologic activity scores has brought standardization. We aimed to identify mucosal markers differentiating histological inflammation from histological remission in UC patients. METHODS: The gene expression levels of 84 genes associated with inflammatory bowel diseases have been analyzed in 43 colonic mucosa samples from 30 patients with UC. The gene expression levels have been correlated with histological inflammation score of Geboes. Patients with endoscopic remission were divided by histological activity into two groups and molecular results were compared in order to identify differences in the mucosal gene expression. RESULTS: We found a significant Pearson correlation (p<0.001 and r>0.5) between the Geboes score and the expression of 29 genes, whereas negative correlation (p<0.001 and r<-0.50) was observed with two genes in the entire UC cohort. In the subgroup of patients with endoscopic remission three transcripts: formyl-peptide receptor 1 (FPR1), matrix metalloproteinases 1 (MMP1) and mucine 1 (MUC1) were significantly up-regulated in patients with histological inflammation compared to patients with histologic remission. CONCLUSION: Our study further emphasizes the importance of histological assessment when endoscopic mucosal healing is present, as FPR1, MMP-1 and MUC1 were all significantly upregulated in patients with histological alterations.
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Colitis Ulcerosa , Colonoscopía/métodos , Mucosa Intestinal , Metaloproteinasa 1 de la Matriz/genética , Mucina-1/genética , Repitelización/genética , Receptores de Formil Péptido/genética , Adulto , Biomarcadores/análisis , Colitis Ulcerosa/genética , Colitis Ulcerosa/inmunología , Colitis Ulcerosa/patología , Colitis Ulcerosa/terapia , Colon/patología , Correlación de Datos , Femenino , Humanos , Inflamación/genética , Mucosa Intestinal/inmunología , Mucosa Intestinal/patología , Masculino , Inducción de Remisión , Transcriptoma , Regulación hacia ArribaRESUMEN
Tunable control of gene expression is an invaluable tool for biological experiments. In this study, we describe a new xylose-inducible promoter system and evaluate it in both Pseudomonas aeruginosa and Pseudomonas fluorescens The Pxut promoter, derived from the P. fluorescensxut operon, was incorporated into a broad-host-range pBBR1-based plasmid and was compared to the Escherichia coli-derived PBAD promoter using gfp as a reporter. Green fluorescent protein (GFP) fluorescence from the Pxut promoter was inducible in both Pseudomonas species, but not in E. coli, which may facilitate the cloning of genes toxic to E. coli to generate plasmids. The Pxut promoter was activated at a lower inducer concentration than PBAD in P. fluorescens, and higher gfp levels were achieved using Pxut Flow cytometry analysis indicated that Pxut was leakier than PBAD in the Pseudomonas species tested but was expressed in a higher proportion of cells when induced. d-Xylose as a sole carbon source did not support the growth of P. aeruginosa or P. fluorescens and is less expensive than many other commonly used inducers, which could facilitate large-scale applications. The efficacy of this system was demonstrated by its use to reveal a role for the P. aeruginosa type II secretion system gene xcpQ in bacterial inhibition of corneal epithelial cell wound closure. This study introduces a new inducible promoter system for gene expression for use in Pseudomonas species.IMPORTANCEPseudomonas species are enormously important in human infections, in biotechnology, and as model systems for investigating basic science questions. In this study, we have developed a xylose-inducible promoter system, evaluated it in P. aeruginosa and P. fluorescens, and found it to be suitable for the strong induction of gene expression. Furthermore, we have demonstrated its efficacy in controlled gene expression to show that a type II secretion system protein from P. aeruginosa, XcpQ, is important for host-pathogen interactions in a corneal wound closure model.
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Proteínas Bacterianas/genética , Proteínas de la Membrana/genética , Regiones Promotoras Genéticas , Pseudomonas aeruginosa/genética , Pseudomonas fluorescens/genética , Repitelización/genética , Sistemas de Secreción Tipo II/genética , Xilosa/metabolismo , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Epitelio Corneal/lesiones , Proteínas de la Membrana/metabolismo , Pseudomonas aeruginosa/metabolismo , Pseudomonas fluorescens/metabolismo , Análisis de Secuencia de ADN , Sistemas de Secreción Tipo II/metabolismoRESUMEN
Signal transducer and activator of transcription 1 (Stat1) is a ubiquitously expressed latent transcription factor that is activated by many cytokines and growth factors. Global Stat1 knockout mice are prone to chemical-induced lung and liver fibrosis, suggesting roles for Stat1 in tissue repair. However, the importance of Stat1 in fibroblast-mediated and vascular smooth muscle cell (VSMC)-mediated injury response has not been directly evaluated in vivo. Here, we focused on two models of tissue repair in conditional Stat1 knockout mice: excisional skin wounding in mice with Stat1 deletion in dermal fibroblasts, and carotid artery ligation in mice with global Stat1 deletion or deletion specific to VSMCs. In the skin model, dermal wounds closed at a similar rate in mice with fibroblast Stat1 deletion and controls, but collagen and α-smooth muscle actin (αSMA) expression were increased in the mutant granulation tissue. Cultured Stat1 -/- and Stat1 +/- dermal fibroblasts exhibited similar αSMA+ stress fiber assembly, collagen gel contraction, proliferation, migration, and growth factor-induced gene expression. In the artery ligation model, there was a significant increase in fibroblast-driven perivascular fibrosis when Stat1 was deleted globally. However, VSMC-driven remodeling and neointima formation were unchanged when Stat1 was deleted specifically in VSMCs. These results suggest an in vivo role for Stat1 as a suppressor of fibroblast mediated, but not VSMC mediated, injury responses, and a suppressor of the myofibroblast phenotype.
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Arterias Carótidas/metabolismo , Fibroblastos/metabolismo , Miocitos del Músculo Liso/metabolismo , Miofibroblastos/metabolismo , Repitelización/genética , Factor de Transcripción STAT1/genética , Piel/metabolismo , Actinas/metabolismo , Animales , Traumatismos de las Arterias Carótidas/metabolismo , Movimiento Celular/genética , Proliferación Celular/genética , Colágeno/metabolismo , Regulación de la Expresión Génica/genética , Tejido de Granulación/metabolismo , Ratones , Ratones Noqueados , Músculo Liso Vascular/metabolismo , Fenotipo , Cicatrización de Heridas/genéticaRESUMEN
Mesenchymal stem cell (MSC) therapies for wound healing are often compromised due to low recruitment and engraftment of transplanted cells, as well as delayed differentiation into cell lineages for skin regeneration. An increased expression of chemokine ligand CXCL16 in wound bed and its cognate receptor, CXCR6, on murine bone-marrow-derived MSCs suggested a putative therapeutic relevance of exogenous MSC transplantation therapy. Induction of the CXCL16-CXCR6 axis led to activation of focal adhesion kinase (FAK), Src, and extracellular signal-regulated kinases 1/2 (ERK1/2)-mediated matrix metalloproteinases (MMP)-2 promoter regulation and expression, the migratory signaling pathways in MSC. CXCL16 induction also increased the transdifferentiation of MSCs into endothelial-like cells and keratinocytes. Intravenous transplantation of allogenic stable MSCs with Cxcr6 gene therapy potentiated skin tissue regeneration by increasing recruitment and engraftment as well as neovascularization and re-epithelialization at the wound site in excisional splinting wounds of type I and II diabetic mice. This study suggests that activation of the CXCL16-CXCR6 axis in bioengineered MSCs with Cxcr6 overexpression provides a promising therapeutic approach for the treatment of diabetic wounds.
Asunto(s)
Diabetes Mellitus Experimental/terapia , Diabetes Mellitus Tipo 1/terapia , Diabetes Mellitus Tipo 2/terapia , Terapia Genética/métodos , Trasplante de Células Madre Mesenquimatosas/métodos , Células Madre Mesenquimatosas/metabolismo , Repitelización/genética , Receptores CXCR6/metabolismo , Piel/metabolismo , Animales , Transdiferenciación Celular/genética , Quimiocina CXCL16/metabolismo , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Neovascularización Fisiológica/genética , Receptores CXCR6/genética , Transfección , Trasplante Homólogo/métodos , Resultado del TratamientoRESUMEN
Chronic wounds-including diabetic foot ulcers, venous leg ulcers, and pressure ulcers-represent a major health problem that demands an urgent solution and new therapies. Despite major burden to patients, health care professionals, and health care systems worldwide, there are no efficacious therapies approved for treatment of chronic wounds. One of the major obstacles in achieving wound closure in patients is the lack of epithelial migration. Here, we used multiple pre-clinical wound models to show that Caveolin-1 (Cav1) impedes healing and that targeting Cav1 accelerates wound closure. We found that Cav1 expression is significantly upregulated in wound edge biopsies of patients with non-healing wounds, confirming its healing-inhibitory role. Conversely, Cav1 was absent from the migrating epithelium and is downregulated in acutely healing wounds. Specifically, Cav1 interacted with membranous glucocorticoid receptor (mbGR) and epidermal growth factor receptor (EGFR) in a glucocorticoid-dependent manner to inhibit cutaneous healing. However, pharmacological disruption of caveolae by MßCD or CRISPR/Cas9-mediated Cav1 knockdown resulted in disruption of Cav1-mbGR and Cav1-EGFR complexes and promoted epithelialization and wound healing. Our data reveal a novel mechanism of inhibition of epithelialization and wound closure, providing a rationale for pharmacological targeting of Cav1 as potential therapy for patients with non-healing chronic wounds.
Asunto(s)
Caveolina 1/genética , Regulación de la Expresión Génica/efectos de los fármacos , Repitelización/genética , Cicatrización de Heridas/efectos de los fármacos , Cicatrización de Heridas/genética , Caveolina 1/metabolismo , Movimiento Celular , Pie Diabético/tratamiento farmacológico , Pie Diabético/etiología , Pie Diabético/metabolismo , Pie Diabético/patología , Receptores ErbB/metabolismo , Expresión Génica , Glucocorticoides/farmacología , Humanos , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Unión Proteica , Receptores de Glucocorticoides/metabolismo , Transducción de Señal/efectos de los fármacos , Piel/efectos de los fármacos , Piel/metabolismo , Piel/patologíaRESUMEN
Cutaneous ulcers are a common cause of morbidity. We have developed a de novo epithelialization method for treating cutaneous ulcers by means of reprogramming wound-resident mesenchymal cells in vivo into cells able to form a stratified epithelium: induced stratified epithelial progenitors (iSEPs). Administration of 4 transcription factors (DNP63A, GRHL2, TFAP2A, and cMYC) expressed via adeno-associated viral vectors enabled generation of epithelial cells and tissues, thereby acheiving de novo epithelialization from the surfaces of cutaneous ulcers in a mouse model. Generated epithelia, having barrier functions equivalent to the original epidermis, were maintained for more than 6 months. Our findings constitute a proof of concept for future development towards innovative therapies for cutaneous ulcers via de novo epithelialization.
Asunto(s)
Terapia Genética/métodos , Repitelización/genética , Úlcera Cutánea/terapia , Factores de Transcripción/genética , Animales , Diferenciación Celular/genética , Dependovirus/genética , Modelos Animales de Enfermedad , Fibroblastos , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Humanos , Células Madre Pluripotentes Inducidas , Queratinocitos , Células Madre Mesenquimatosas/fisiología , Ratones , Ratones Transgénicos , Piel/patología , Úlcera Cutánea/patologíaRESUMEN
Unlike mammals, Xenopus laevis tadpoles have a high regenerative potential. To characterize this regenerative response, we performed single-cell RNA sequencing after tail amputation. By comparing naturally occurring regeneration-competent and -incompetent tadpoles, we identified a previously unrecognized cell type, which we term the regeneration-organizing cell (ROC). ROCs are present in the epidermis during normal tail development and specifically relocalize to the amputation plane of regeneration-competent tadpoles, forming the wound epidermis. Genetic ablation or manual removal of ROCs blocks regeneration, whereas transplantation of ROC-containing grafts induces ectopic outgrowths in early embryos. Transcriptional profiling revealed that ROCs secrete ligands associated with key regenerative pathways, signaling to progenitors to reconstitute lost tissue. These findings reveal the cellular mechanism through which ROCs form the wound epidermis and ensure successful regeneration.
Asunto(s)
Epidermis/fisiología , Repitelización/fisiología , Cola (estructura animal)/fisiología , Xenopus laevis/fisiología , Animales , Células Epidérmicas/fisiología , Repitelización/genética , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Transcriptoma , Proteínas de Xenopus/genética , Xenopus laevis/genéticaRESUMEN
Local transplantation of stem cells has therapeutic effects on skin damage but cannot provide satisfactory wound healing. Studies on the mechanisms underlying the therapeutic effects of stem cells on skin wound healing will be needed. Hence, in the present study, we explored the role of Caveolin-1 in epidermal stem cells (EpiSCs) in the modulation of wound healing. We first isolated EpiSCs from mouse skin tissues and established stable EpiSCs with overexpression of Caveolin-1 using a lentiviral construct. We then evaluated the epidermal growth factor (EGF)-induced cell proliferation ability using cell counting Kit-8 (CCK-8) assay and assessed EpiSC pluripotency by examining Nanog mRNA levels in EpiSCs. Furthermore, we treated mice with skin burn injury using EpiSCs with overexpression of Caveolin-1. Histological examinations were conducted to evaluate re-epithelialization, wound scores, cell proliferation and capillary density in wounds. We found that overexpression of Caveolin-1 in EpiSCs promoted EGF-induced cell proliferation ability and increased wound closure in a mouse model of skin burn injury. Histological evaluation demonstrated that overexpression of Caveolin-1 in EpiSCs promoted re-epithelialization in wounds, enhanced cellularity, and increased vasculature, as well as increased wound scores. Taken together, our results suggested that Caveolin-1 expression in the EpiSCs play a critical role in the regulation of EpiSC proliferation ability and alteration of EpiSC proliferation ability may be an effective approach in promoting EpiSC-based therapy in skin wound healing.
Asunto(s)
Caveolina 1/fisiología , Cicatrización de Heridas/fisiología , Animales , Quemaduras/genética , Quemaduras/patología , Quemaduras/fisiopatología , Caveolina 1/antagonistas & inhibidores , Caveolina 1/genética , Proliferación Celular/genética , Proliferación Celular/fisiología , Células Epidérmicas/patología , Células Epidérmicas/fisiología , Femenino , Expresión Génica , Técnicas de Silenciamiento del Gen , Masculino , Ratones , Ratones Endogámicos BALB C , Neovascularización Fisiológica/genética , Ratas , Repitelización/genética , Repitelización/fisiología , Células Madre/patología , Células Madre/fisiología , Regulación hacia Arriba , Cicatrización de Heridas/genéticaRESUMEN
Wound healing is a complex but a fine-tuned biological process in which human skin has the ability to regenerate itself following damage. However, in particular conditions such as deep burn or diabetes the process of wound healing is compromised. Despite investigations on the potency of a wide variety of stem cells for wound healing, adipose-derived stem cells (ASCs) seem to possess the least limitations for clinical applications, and literature showed that ASCs can improve the process of wound healing very likely by promoting angiogenesis and/or vascularisation, modulating immune response, and inducing epithelialization in the wound. In the present review, advantages and disadvantages of various stem cells which can be used for promoting wound healing are discussed. In addition, potential mechanisms of action by which ASCs may accelerate wound healing are summarised. Finally, clinical studies applying ASCs for wound healing and the associated limitations are reviewed.