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1.
PLoS Biol ; 18(9): e3000849, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32898168

RESUMO

Despite limited genomic diversity, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has shown a wide range of clinical manifestations in different patient populations. The mechanisms behind these host differences are still unclear. Here, we examined host response gene expression across infection status, viral load, age, and sex among shotgun RNA sequencing profiles of nasopharyngeal (NP) swabs from 430 individuals with PCR-confirmed SARS-CoV-2 and 54 negative controls. SARS-CoV-2 induced a strong antiviral response with up-regulation of antiviral factors such as OAS1-3 and IFIT1-3 and T helper type 1 (Th1) chemokines CXCL9/10/11, as well as a reduction in transcription of ribosomal proteins. SARS-CoV-2 culture in human airway epithelial (HAE) cultures replicated the in vivo antiviral host response 7 days post infection, with no induction of interferon-stimulated genes after 3 days. Patient-matched longitudinal specimens (mean elapsed time = 6.3 days) demonstrated reduction in interferon-induced transcription, recovery of transcription of ribosomal proteins, and initiation of wound healing and humoral immune responses. Expression of interferon-responsive genes, including ACE2, increased as a function of viral load, while transcripts for B cell-specific proteins and neutrophil chemokines were elevated in patients with lower viral load. Older individuals had reduced expression of the Th1 chemokines CXCL9/10/11 and their cognate receptor CXCR3, as well as CD8A and granzyme B, suggesting deficiencies in trafficking and/or function of cytotoxic T cells and natural killer (NK) cells. Relative to females, males had reduced B cell-specific and NK cell-specific transcripts and an increase in inhibitors of nuclear factor kappa-B (NF-κB) signaling, possibly inappropriately throttling antiviral responses. Collectively, our data demonstrate that host responses to SARS-CoV-2 are dependent on viral load and infection time course, with observed differences due to age and sex that may contribute to disease severity.


Assuntos
Antivirais/imunologia , Betacoronavirus/fisiologia , Infecções por Coronavirus/imunologia , Pneumonia Viral/imunologia , Adolescente , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/virologia , Feminino , Regulação da Expressão Gênica , Humanos , Imunidade/genética , Cinética , Masculino , Pessoa de Meia-Idade , Nasofaringe/imunologia , Nasofaringe/virologia , Pandemias , Pneumonia Viral/epidemiologia , Pneumonia Viral/virologia , Proteínas Ribossômicas/genética , Fatores Sexuais , Transdução de Sinais/genética , Carga Viral , Cicatrização/genética , Adulto Jovem
2.
Nat Commun ; 11(1): 4678, 2020 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-32938916

RESUMO

Diabetic foot ulcers (DFUs) are a life-threatening disease that often result in lower limb amputations and a shortened lifespan. However, molecular mechanisms contributing to the pathogenesis of DFUs remain poorly understood. We use next-generation sequencing to generate a human dataset of pathogenic DFUs to compare to transcriptional profiles of human skin and oral acute wounds, oral as a model of "ideal" adult tissue repair due to accelerated closure without scarring. Here we identify major transcriptional networks deregulated in DFUs that result in decreased neutrophils and macrophages recruitment and overall poorly controlled inflammatory response. Transcription factors FOXM1 and STAT3, which function to activate and promote survival of immune cells, are inhibited in DFUs. Moreover, inhibition of FOXM1 in diabetic mouse models (STZ-induced and db/db) results in delayed wound healing and decreased neutrophil and macrophage recruitment in diabetic wounds in vivo. Our data underscore the role of a perturbed, ineffective inflammatory response as a major contributor to the pathogenesis of DFUs, which is facilitated by FOXM1-mediated deregulation of recruitment of neutrophils and macrophages, revealing a potential therapeutic strategy.


Assuntos
Pé Diabético/genética , Pé Diabético/imunologia , Proteína Forkhead Box M1/imunologia , Cicatrização/imunologia , Adulto , Idoso , Animais , Proliferação de Células , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/imunologia , Pé Diabético/patologia , Modelos Animais de Doenças , Feminino , Proteína Forkhead Box M1/antagonistas & inibidores , Proteína Forkhead Box M1/metabolismo , Humanos , Inflamação/genética , Inflamação/imunologia , Masculino , Camundongos Endogâmicos , Pessoa de Meia-Idade , Mucosa Bucal/fisiologia , Piridinas/farmacologia , Tiofenos/farmacologia , Transcriptoma/fisiologia , Cicatrização/genética
3.
Nat Commun ; 11(1): 3866, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32737287

RESUMO

Upon severe head injury (HI), blood vessels of the meninges and brain parenchyma are inevitably damaged. While limited vascular regeneration of the injured brain has been studied extensively, our understanding of meningeal vascular regeneration following head injury is quite limited. Here, we identify key pathways governing meningeal vascular regeneration following HI. Rapid and complete vascular regeneration in the meninges is predominantly driven by VEGFR2 signaling. Substantial increase of VEGFR2 is observed in both human patients and mouse models of HI, and endothelial cell-specific deletion of Vegfr2 in the latter inhibits meningeal vascular regeneration. We further identify the facilitating, stabilizing and arresting roles of Tie2, PDGFRß and Dll4 signaling, respectively, in meningeal vascular regeneration. Prolonged inhibition of this angiogenic process following HI compromises immunological and stromal integrity of the injured meninges. These findings establish a molecular framework for meningeal vascular regeneration after HI, and may guide development of wound healing therapeutics.


Assuntos
Traumatismos Craniocerebrais/genética , Células Endoteliais/metabolismo , Neovascularização Fisiológica/genética , Regeneração/genética , Transdução de Sinais/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/patologia , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Circulação Cerebrovascular , Traumatismos Craniocerebrais/metabolismo , Traumatismos Craniocerebrais/patologia , Modelos Animais de Doenças , Células Endoteliais/patologia , Regulação da Expressão Gênica/genética , Humanos , Macrófagos/metabolismo , Macrófagos/patologia , Meninges/lesões , Meninges/metabolismo , Camundongos , Camundongos Knockout , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptor TIE-2/genética , Receptor TIE-2/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Cicatrização/genética
4.
Hum Cell ; 33(4): 990-1005, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32617892

RESUMO

Orchestrated control of multiple overlapping and sequential processes is required for the maintenance of epidermal homeostasis and the response to and recovery from a variety of skin insults. Previous studies indicate that membrane-associated serine protease matriptase and prostasin play essential roles in epidermal development, differentiation, and barrier formation. The control of proteolysis is a highly regulated process, which depends not only on gene expression but also on zymogen activation and the balance between protease and protease inhibitor. Subcellular localization can affect the accessibility of protease inhibitors to proteases and, thus, also represents an integral component of the control of proteolysis. To understand how membrane-associated proteolysis is regulated in human skin, these key aspects of matriptase and prostasin were determined in normal and injured human skin by immunohistochemistry. This staining shows that matriptase is expressed predominantly in the zymogen form at the periphery of basal and spinous keratinocytes, and prostasin appears to be constitutively activated at high levels in polarized organelle-like structures of the granular keratinocytes in the adjacent quiescent skin. The membrane-associated proteolysis appears to be elevated via an increase in matriptase zymogen activation and prostasin protein expression in areas of skin recovering from epidermal insults. There was no noticeable change observed in other regulatory aspects, including the expression and tissue distribution of their cognate inhibitors HAI-1 and HAI-2. This study reveals that the membrane-associated proteolysis may be a critical epidermal mechanism involved in responding to, and recovering from, damage to human skin.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Expressão Gênica , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Fenômenos Fisiológicos da Pele/genética , Pele/lesões , Cicatrização/genética , Cicatrização/fisiologia , Ferimentos e Lesões/genética , Ferimentos e Lesões/metabolismo , Células Cultivadas , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas Secretadas Inibidoras de Proteinases/genética , Proteínas Secretadas Inibidoras de Proteinases/metabolismo , Proteólise , Serina Endopeptidases/fisiologia , Pele/metabolismo
5.
PLoS Pathog ; 16(6): e1008511, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32555671

RESUMO

The clinical importance of microbiomes to the chronicity of wounds is widely appreciated, yet little is understood about patient-specific processes shaping wound microbiome composition. Here, a two-cohort microbiome-genome wide association study is presented through which patient genomic loci associated with chronic wound microbiome diversity were identified. Further investigation revealed that alternative TLN2 and ZNF521 genotypes explained significant inter-patient variation in relative abundance of two key pathogens, Pseudomonas aeruginosa and Staphylococcus epidermidis. Wound diversity was lowest in Pseudomonas aeruginosa infected wounds, and decreasing wound diversity had a significant negative linear relationship with healing rate. In addition to microbiome characteristics, age, diabetic status, and genetic ancestry all significantly influenced healing. Using structural equation modeling to identify common variance among SNPs, six loci were sufficient to explain 53% of variation in wound microbiome diversity, which was a 10% increase over traditional multiple regression. Focusing on TLN2, genotype at rs8031916 explained expression differences of alternative transcripts that differ in inclusion of important focal adhesion binding domains. Such differences are hypothesized to relate to wound microbiomes and healing through effects on bacterial exploitation of focal adhesions and/or cellular migration. Related, other associated loci were functionally enriched, often with roles in cytoskeletal dynamics. This study, being the first to identify patient genetic determinants for wound microbiomes and healing, implicates genetic variation determining cellular adhesion phenotypes as important drivers of infection type. The identification of predictive biomarkers for chronic wound microbiomes may serve as risk factors and guide treatment by informing patient-specific tendencies of infection.


Assuntos
Microbiota , Polimorfismo de Nucleotídeo Único , Infecções por Pseudomonas , Pseudomonas aeruginosa , Infecções Estafilocócicas , Staphylococcus epidermidis , Cicatrização/genética , Infecção dos Ferimentos , Animais , Doença Crônica , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Estudo de Associação Genômica Ampla , Humanos , Masculino , Camundongos , Infecções por Pseudomonas/genética , Infecções por Pseudomonas/metabolismo , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/patologia , Infecções Estafilocócicas/genética , Infecções Estafilocócicas/metabolismo , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/patologia , Talina/genética , Talina/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Infecção dos Ferimentos/genética , Infecção dos Ferimentos/metabolismo , Infecção dos Ferimentos/microbiologia , Infecção dos Ferimentos/patologia
6.
Nat Commun ; 11(1): 2604, 2020 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-32451392

RESUMO

Matrix deposition is essential for wound repair, but when excessive, leads to hypertrophic scars and fibrosis. The factors that control matrix deposition in skin wounds have only partially been identified and the consequences of matrix alterations for the mechanical properties of wounds are largely unknown. Here, we report how a single diffusible factor, activin A, affects the healing process across scales. Bioinformatics analysis of wound fibroblast transcriptome data combined with biochemical and histopathological analyses of wounds and functional in vitro studies identify that activin promotes pro-fibrotic gene expression signatures and processes, including glycoprotein and proteoglycan biosynthesis, collagen deposition, and altered collagen cross-linking. As a consequence, activin strongly reduces the wound and scar deformability, as identified by a non-invasive in vivo method for biomechanical analysis. These results provide mechanistic insight into the roles of activin in wound repair and fibrosis and identify the functional consequences of alterations in the wound matrisome at the biomechanical level.


Assuntos
Subunidades beta de Inibinas/metabolismo , Pele/lesões , Pele/metabolismo , Animais , Fenômenos Biomecânicos , Linhagem Celular , Cicatriz/patologia , Cicatriz/fisiopatologia , Cicatriz Hipertrófica/patologia , Cicatriz Hipertrófica/fisiopatologia , Colágeno/metabolismo , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Feminino , Fibroblastos/metabolismo , Fibrose , Humanos , Subunidades beta de Inibinas/genética , Queratinócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pele/patologia , Transcriptoma , Regulação para Cima , Cicatrização/genética , Cicatrização/fisiologia
7.
J Surg Res ; 254: 102-109, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32422429

RESUMO

BACKGROUND: Wound healing is a complex process aiming at repairing the damaged skin. MiR-23b has been reported to be upregulated during wound healing. In this study, we intended to explore the working mechanism of miR-23b during wound healing. METHODS: Quantitative real-time polymerase chain reaction was performed to detect the enrichment of miR-23b and tissue inhibitor of metalloproteinase-3 (TIMP3) in HaCaT cells. Scratch wound assay was carried out to measure the migration of HaCaT cells. The target of miR-23b was predicted by microT-CDS software, and the combination was confirmed by dual-luciferase reporter assay and RNA immunoprecipitation assay. The abundance of TIMP3 protein was detected by Western blot assay. RESULTS: The abundance of miR-23b was positively related to the concentration and time of transforming growth factor ß1 treatment in HaCaT cells. MiR-23b promoted the migration of keratinocytes. TIMP3 was a direct target of miR-23b and was negatively regulated by miR-23b. TIMP3 inhibited the migration of keratinocytes. MiR-23b accelerated the migration of keratinocytes by downregulating the abundance of TIMP3. CONCLUSIONS: MiR-23b promoted the migration of keratinocytes partly through reducing the enrichment of TIMP3. MiR-23b might be a promising target for the treatment of wound healing-associated diseases.


Assuntos
Movimento Celular/genética , Regulação para Baixo/genética , Queratinócitos/fisiologia , MicroRNAs/fisiologia , Inibidor Tecidual de Metaloproteinase-3/genética , Linhagem Celular , Movimento Celular/fisiologia , Queratinócitos/química , MicroRNAs/análise , Inibidor Tecidual de Metaloproteinase-3/análise , Transfecção , Fator de Crescimento Transformador beta1/farmacologia , Cicatrização/genética
8.
Nat Immunol ; 21(6): 671-683, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32424366

RESUMO

Urinary tract infections (UTIs) typically evoke prompt and vigorous innate bladder immune responses, including extensive exfoliation of the epithelium. To explain the basis for the extraordinarily high recurrence rates of UTIs, we examined adaptive immune responses in mouse bladders. We found that, following each bladder infection, a highly T helper type 2 (TH2)-skewed immune response directed at bladder re-epithelialization is observed, with limited capacity to clear infection. This response is initiated by a distinct subset of CD301b+OX40L+ dendritic cells, which migrate into the bladder epithelium after infection before trafficking to lymph nodes to preferentially activate TH2 cells. The bladder epithelial repair response is cumulative and aberrant as, after multiple infections, the epithelium was markedly thickened and bladder capacity was reduced relative to controls. Thus, recurrence of UTIs and associated bladder dysfunction are the outcome of the preferential focus of the adaptive immune response on epithelial repair at the expense of bacterial clearance.


Assuntos
Cistite/etiologia , Cistite/metabolismo , Ativação Linfocitária/imunologia , Membrana Mucosa/imunologia , Membrana Mucosa/metabolismo , Células Th2/imunologia , Células Th2/metabolismo , Animais , Carga Bacteriana , Biomarcadores , Linhagem Celular , Cistite/patologia , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Camundongos , Camundongos Knockout , Membrana Mucosa/patologia , Células Th1/imunologia , Células Th1/metabolismo , Células Th1/patologia , Infecções Urinárias/etiologia , Infecções Urinárias/metabolismo , Infecções Urinárias/microbiologia , Cicatrização/genética , Cicatrização/imunologia
9.
PLoS One ; 15(4): e0229421, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32320433

RESUMO

Oxidative stress may cause ocular surface damage during the development of dry eye. Mammalian cells have defense systems against oxidative stress. A central regulator of the stress response is nuclear factor-erythroid 2-related factor 2 (NFE2L2). NFE2L2 is activated by the novel triterpenoid RS9 (a biotransformation compound of RTA 402). The purpose of this study was to assess the efficacy of RS9 against dry eye using in vitro and in vivo models. Bioactivity was estimated by the induction of mRNAs for two NFE2L2-targeted genes: NQO1 (prevents radical species) and GCLC (glutathione synthesis), using a corneal epithelial cell line (HCE-T). Protection against oxidation and cell damage was tested in vitro by culturing cells under hyperosmotic stress or by the addition of menadione, a generator of reactive oxygen species (ROS). Dry eye in vivo was induced by the injection of scopolamine into rats. Then, 930 nM of RS9 was applied to both eyes for 2 weeks. Oxidative stress was measured by the accumulation of 8-hydroxy-2'-deoxyguanosine (8-OHdG). Corneal wound healing was measured by scoring for superficial punctate keratitis (SPK). Corneal epithelial cell densities were evaluated histologically. RS9 and RTA 402 induced the expression of NQO1 and GCLC mRNAs in HCE-T cells. And both compounds suppressed hyperosmotic-ROS generation and menadione induced cellular damage. However RS9 had a stronger protective effect than RTA 402. Ocular instillation of RS9 also significantly upregulated the expression of Nqo1 mRNA in the corneal epithelium. Accumulation of 8-OHdG, increase of SPK scores and decrement of basal cell density were observed in corneal epithelium from scopolamine-injected rats. These changes were significantly ameliorated by the topical administration of RS9. RS9 induced Nfe2l2 activation and Nfe2l2-targeted genes, reduced oxidation, and ameliorated symptoms of dry eye using in vitro and in vivo models. Thus, RS9 might be a potent candidate agent against dry eye disease.


Assuntos
Lesões da Córnea/tratamento farmacológico , Síndromes do Olho Seco/tratamento farmacológico , Ceratite/tratamento farmacológico , Fator 2 Relacionado a NF-E2/genética , Triterpenos/farmacologia , 8-Hidroxi-2'-Desoxiguanosina/genética , Animais , Lesões da Córnea/induzido quimicamente , Lesões da Córnea/genética , Lesões da Córnea/patologia , Modelos Animais de Doenças , Síndromes do Olho Seco/induzido quimicamente , Síndromes do Olho Seco/genética , Síndromes do Olho Seco/patologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Epitélio Anterior/efeitos dos fármacos , Epitélio Anterior/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Glutamato-Cisteína Ligase/genética , Humanos , Ceratite/induzido quimicamente , Ceratite/genética , NAD(P)H Desidrogenase (Quinona)/genética , Estresse Oxidativo/efeitos dos fármacos , RNA Mensageiro/efeitos dos fármacos , Ratos , Espécies Reativas de Oxigênio/metabolismo , Escopolamina/toxicidade , Cicatrização/efeitos dos fármacos , Cicatrização/genética
10.
Nature ; 580(7801): 130-135, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32238926

RESUMO

Caspase-dependent apoptosis accounts for approximately 90% of homeostatic cell turnover in the body1, and regulates inflammation, cell proliferation, and tissue regeneration2-4. How apoptotic cells mediate such diverse effects is not fully understood. Here we profiled the apoptotic metabolite secretome and determined its effects on the tissue neighbourhood. We show that apoptotic lymphocytes and macrophages release specific metabolites, while retaining their membrane integrity. A subset of these metabolites is also shared across different primary cells and cell lines after the induction of apoptosis by different stimuli. Mechanistically, the apoptotic metabolite secretome is not simply due to passive emptying of cellular contents and instead is a regulated process. Caspase-mediated opening of pannexin 1 channels at the plasma membrane facilitated the release of a select subset of metabolites. In addition, certain metabolic pathways continued to remain active during apoptosis, with the release of only select metabolites from a given pathway. Functionally, the apoptotic metabolite secretome induced specific gene programs in healthy neighbouring cells, including suppression of inflammation, cell proliferation, and wound healing. Furthermore, a cocktail of apoptotic metabolites reduced disease severity in mouse models of inflammatory arthritis and lung-graft rejection. These data advance the concept that apoptotic cells are not inert cells waiting for removal, but instead release metabolites as 'good-bye' signals to actively modulate outcomes in tissues.


Assuntos
Apoptose/fisiologia , Microambiente Celular , Sistemas do Segundo Mensageiro/fisiologia , Animais , Artrite , Caspases/metabolismo , Linhagem Celular , Proliferação de Células/genética , Sobrevivência Celular/genética , Conexinas/metabolismo , Modelos Animais de Doenças , Rejeição de Enxerto , Humanos , Inflamação/genética , Transplante de Pulmão , Linfócitos/enzimologia , Linfócitos/metabolismo , Macrófagos/enzimologia , Macrófagos/metabolismo , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Fagócitos/metabolismo , Cicatrização/genética
11.
Mem Inst Oswaldo Cruz ; 115: e190361, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32130370

RESUMO

Genes associated with wound healing have been shown to be risk factors for cutaneous leishmaniasis (CL) which is caused by Leishmania braziliensis. In this study, we examined whether the genes previously associated with CL influenced the clinical outcome. Patients were genotyped and retrospectively classified as responders, who were cured with a single course of pentavalent antimony (Sbv), or as refractories, who did not respond to Sbv. Patients characterised as responders showed a stronger response to the leishmanin skin test (LST) when compared to the refractory subjects (p = 0.0003). Furthermore, we observed an association between the FLI1 CC genotype and an increased size of ulcers (p = 0.0170). We suggest that the leishmanin skin test may be a predictive tool for therapeutic outcome and reinforce FLI1 as a potential influencer of susceptibility and lesion size in CL.


Assuntos
Antimônio/uso terapêutico , Antiprotozoários/uso terapêutico , Leishmaniose Cutânea/genética , Cicatrização/genética , Adolescente , Adulto , Estudos de Casos e Controles , Feminino , Predisposição Genética para Doença , Genótipo , Humanos , Leishmaniose Cutânea/tratamento farmacológico , Leishmaniose Cutânea/patologia , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Estudos Retrospectivos , Testes Cutâneos , Adulto Jovem
12.
Clin Orthop Surg ; 12(1): 120-129, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32117548

RESUMO

Background: Platelet-rich plasma (PRP) is a plasma component of autologous blood containing a high concentration of platelets. PRP is used to promote healing of damaged tissues. However, there are not many studies on the composition and expression patterns of active proteins in PRP. The purpose of this study was to identify unknown factors that contribute to tissue healing by proteomic analysis of proteins in PRP. Methods: Three men in their 30s with no basal disease participated in this study. All identified proteins were classified for tissue healing-related functions on the basis of the gene ontology analysis of adhesion molecule with Ig-like domain 2 (AmiGO2). PRP was prepared by using the ACP kit and GPS III kit. Results: We identified a total of 125 proteins related to wound healing, along with three proteins for angiogenesis involved in wound healing, two proteins for fibroblast migration, four proteins for collagen biosynthesis process, two proteins for glycosaminoglycan biosynthesis process, and 13 proteins for glycosaminoglycan binding. So, in addition to the growth factors that have been already known to be involved in tissue healing, 25 new proteins were identified. Conclusions: We identified the unknown proteins associated with tissue healing in PRP. Our findings may serve as a foundation for the establishment of basic medical evidence for PRP applications.


Assuntos
Plasma Rico em Plaquetas/química , Proteômica , Cicatrização/genética , Adulto , Cromatografia Líquida , Voluntários Saudáveis , Humanos , Masculino , Espectrometria de Massas
13.
Artigo em Inglês | MEDLINE | ID: mdl-32159361

RESUMO

More and more evidence advises that circular RNAs (circRNAs) function critically in regulating different disease microenvironments. Our previous study found that autotransplantation of adipose-derived mesenchymal stem cells (ADSCs) promotes diabetes wound healing. Exosomes derived in ADSCs play an important regulatory role. This study aimed to characterize if mmu_circ_0000250 played a role in ADSC-exosome-mediated full-thickness skin wound repair in diabetic rats. Endothelial progenitor cells (EPCs) were selected to study the therapeutic mechanism of exosomes in high-glucose (HG)-induced cell damage and dysfunction. Analysis and luciferase reporter assay were utilized to explore the interaction among mmu_circ_0000250, miRNA (miR)-128-3p, and sirtuin (SIRT)1. The diabetic rats were used to confirm the therapeutic effect of mmu_circ_0000250 against exosome-mediated wound healing. Exosomes containing a high concentration of mmu_circ_0000250 had a greater therapeutic effect on restoration of the function of EPCs by promotion autophagy activation under HG conditions. Expression of mmu_circ_0000250 promoted SIRT1 expression by miR-128-3p adsorption, which was confirmed via luciferase reporter assay and bioinformatics analysis. In vivo, exosomes containing a high concentration of mmu_circ_0000250 had a more therapeutic effect on wound healing when compared with wild-type exosomes from ADSCs. Immunohistochemistry and immunofluorescence detection showed that mmu_circ_0000250 increased angiopoiesis with exosome treatment in wound skin and suppressed apoptosis by autophagy activation. In conclusion, we verified that mmu_circ_0000250 enhanced the therapeutic effect of ADSC-exosomes to promote wound healing in diabetes by absorption of miR-128-3p and upregulation of SIRT1. Therefore, these findings advocate targeting the mmu_circ_0000250/miR-128-3p/SIRT1 axis as a candidate therapeutic option for diabetic ulcers.


Assuntos
Diabetes Mellitus Experimental/terapia , MicroRNAs/genética , RNA Circular/genética , Sirtuína 1/genética , Úlcera/terapia , Animais , Autofagia/genética , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patologia , Modelos Animais de Doenças , Exossomos/genética , Humanos , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Úlcera/complicações , Úlcera/genética , Úlcera/patologia , Cicatrização/genética
14.
Lasers Med Sci ; 35(7): 1577-1588, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32170506

RESUMO

In this study, the effects of miR-27b on angiogenesis in skin repair procedure in rats with deep II degree scald were explored. The rat model of deep II scald was established. miR-27b mimics and inhibitor were injected daily at the wound site for 3 weeks. The healing of scald was observed at 0, 3, 7, 14, and 21 days after the model was established, and the pathological changes of skin were observed by HE and Masson's trichrome stains. Skin tissues were taken 14 days after the operation; CD31 and Ki-67 immunohistochemistry was exerted to evaluate neovascularization and proliferation. Human microvascular endothelial cells (HMEC-1) cells were cultured in vitro. miR-27b mimics or inhibitor was transfected to construct over-expression or inhibition cell lines. MTT assay, scratch test, and angiogenesis test were used to evaluate cell proliferation, migration, and vascular regeneration. Finally, RT-PCR and Western blot were exerted to determine the expression of vascular endothelial growth factor C (VEGF-C), epidermal growth factor (EGF) mRNAs, and protein, respectively. Control, inhibitor, mi-NC, VEGF-C, inhibitor + si-NC, and inhibitor + VEGF-C siRNA groups were used to further analyze the mechanism of miR-27b on VEGF-C; the above experiments were repeated. In contrast to model group, miR-27b inhibitor could significantly promote the healing of scalded skin, alleviate the pathological status of scalded, and promote the angiogenesis and proliferation (p < 0.05). In vitro, miR-27b inhibitor evidently promoted cell proliferation, migration, and angiogenesis and increased the expression of VEGF-C, EGF genes, and protein, while miR-27b mimics significantly reversed the above trends. Further studies shown that downregulation of miR-27b expression can promote the proliferation, migration, and angiogenesis of HMEC-1 cells by promoting the expression of VEGF-C. miR-27b promotes angiogenesis and skin repair in scalded rats through regulating VEGF-C expression.


Assuntos
Queimaduras/genética , Queimaduras/patologia , Regulação da Expressão Gênica , MicroRNAs/metabolismo , Neovascularização Fisiológica/genética , Pele/patologia , Fator C de Crescimento do Endotélio Vascular/genética , Cicatrização , Animais , Sequência de Bases , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Colágeno/metabolismo , Modelos Animais de Doenças , Regulação para Baixo , Células Endoteliais/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Humanos , Antígeno Ki-67/metabolismo , Masculino , MicroRNAs/genética , Microvasos/patologia , Ratos Sprague-Dawley , Fator C de Crescimento do Endotélio Vascular/metabolismo , Cicatrização/genética
15.
Adv Wound Care (New Rochelle) ; 9(4): 145-160, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32117579

RESUMO

Objective: Insufficient knowledge about the molecular pathology of diabetic foot ulcer (DFU) impedes the development of effective wound treatment. Circular RNAs (circRNAs) are a novel class of RNA recently discovered to be widely expressed and have important biological functions; however, their role in skin wound healing remains largely unexplored. In this study, we investigated the role of circRNAs in DFU. Approach: CircRNA expression was profiled in normal wounds (NWs) and DFUs by microarray analysis, and hsa_circ_0084443 was identified as differentially expressed. The circularity and subcellular localization of hsa_circ_0084443 were characterized by northern blotting, real-time PCR, and fluorescence in situ hybridization. Cell migration, cell growth, and the transcriptome of human primary keratinocytes were analyzed after overexpression or RNA interference of hsa_circ_0084443. Results: hsa_circ_0084443 is downregulated in NWs compared with intact skin, and its level is higher in DFUs than NWs. We confirmed its circularity and presence in the cytoplasm of human epidermal keratinocytes. We showed that hsa_circ_0084443 reduced motility while enhancing the growth of keratinocytes. Furthermore, we identified a gene network with the potential to mediate the biological effect of hsa_circ_0084443. Innovation: CircRNAs have a functional role and a potential clinical significance in skin wound healing. Conclusions: We identified hsa_circ_0084443, a circRNA downregulated during NW healing, as a negative regulator of keratinocyte migration. Higher levels of hsa_circ_0084443 were detected in DFU samples, suggesting that it plays a role in pathology. These findings pave the way to understanding the functional role of circRNAs in human skin wound healing.


Assuntos
Movimento Celular/genética , Proliferação de Células/genética , Pé Diabético/genética , Queratinócitos/metabolismo , RNA Circular/genética , Regulação para Cima/genética , Cicatrização/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Northern Blotting , Estudos de Coortes , Feminino , Humanos , Hibridização in Situ Fluorescente , Masculino , Pessoa de Meia-Idade , RNA Circular/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transcriptoma
16.
Adv Wound Care (New Rochelle) ; 9(4): 161-173, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32117580

RESUMO

Objective: Activation of epidermal stem cells (EpSCs) from their quiescent niche is an integral component of wound reepithelialization and involves Wnt/ß-catenin (ß-Cat) signaling and remodeling of the actin cytoskeleton. The aim of this study was to investigate the effect of Flightless I (Flii), a cytoskeletal protein and inhibitor of wound healing, on EpSC activation during wound repair. Approach: Genetically modified Flii mice (Flii knockdown: Flii+/- , wild type: WT, Flii overexpressing: FliiTg/Tg ) received two incisional wounds along the lateral axis of the dorsal skin. Indicators of EpSC activation (epidermal growth factor receptor 1 [EGFR1], leucine-rich repeats and immunoglobulin-like domains-1 [Lrig1], K14), Wnt/ß-Cat signaling (Lgr6, Flap2, ß-Cat, and axis inhibition protein 2 [Axin2]), and cell proliferation (proliferating cell nuclear antigen [PCNA]) were assessed using immunohistochemistry. ß-Cat stabilization was examined using western blotting with cell cycling and differentiation of isolated CD34+ITGA6high EpSCs examined using real time-quantitative polymerase chain reaction after treatment with wound-conditioned media. Results: Flii+/- led to increased numbers of activated EpSCs expressing PCNA, elevated EGFR1, and decreased Lrig1. EpSCs in Flii+/- hair follicle niches adjacent to the wounds also showed expression of Wnt-activation markers including increased ß-Cat and Lgr6, and decreased Axin2. EpSCs (CD34+ITGA6high) isolated from Flii+/- unwounded skin showed elevated expression of cell-cycling genes including ΔNp63, filaggrin (Fila), involucrin (Invo), cyclin D1 (Ccnd1), and cell-division cycle protein-20 (Cdc20); and elevated ΔNp63 and Invo after treatment with wound-conditioned media compared with WT and FliiTg/Tg counterparts. Innovation: Flii was identified as an inhibitor of EpSC activation that may explain its negative effects on wound reepithelialization. Conclusion: Flii may inhibit EpSC activation by interrupting Wnt/ß-Cat signaling. Strategies that reduce Flii may increase activation of EpSCs and promote reepithelialization of wounds.


Assuntos
Células Epidérmicas/metabolismo , Proteínas dos Microfilamentos/metabolismo , Nicho de Células-Tronco/genética , Células-Tronco/metabolismo , Transativadores/metabolismo , Cicatrização/genética , Animais , Proliferação de Células/genética , Modelos Animais de Doenças , Feminino , Técnicas de Silenciamento de Genes , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Proteínas dos Microfilamentos/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Pele/lesões , Pele/metabolismo , Transativadores/genética , Via de Sinalização Wnt/genética , beta Catenina/metabolismo
17.
Proc Natl Acad Sci U S A ; 117(10): 5339-5350, 2020 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-32094197

RESUMO

Aging manifests with architectural alteration and functional decline of multiple organs throughout an organism. In mammals, aged skin is accompanied by a marked reduction in hair cycling and appearance of bald patches, leading researchers to propose that hair follicle stem cells (HFSCs) are either lost, differentiate, or change to an epidermal fate during aging. Here, we employed single-cell RNA-sequencing to interrogate aging-related changes in the HFSCs. Surprisingly, although numbers declined, aging HFSCs were present, maintained their identity, and showed no overt signs of shifting to an epidermal fate. However, they did exhibit prevalent transcriptional changes particularly in extracellular matrix genes, and this was accompanied by profound structural perturbations in the aging SC niche. Moreover, marked age-related changes occurred in many nonepithelial cell types, including resident immune cells, sensory neurons, and arrector pili muscles. Each of these SC niche components has been shown to influence HF regeneration. When we performed skin injuries that are known to mobilize young HFSCs to exit their niche and regenerate HFs, we discovered that aged skin is defective at doing so. Interestingly, however, in transplantation assays in vivo, aged HFSCs regenerated HFs when supported with young dermis, while young HFSCs failed to regenerate HFs when combined with aged dermis. Together, our findings highlight the importance of SC:niche interactions and favor a model where youthfulness of the niche microenvironment plays a dominant role in dictating the properties of its SCs and tissue health and fitness.


Assuntos
Folículo Piloso/fisiologia , Regeneração/fisiologia , Envelhecimento da Pele/fisiologia , Nicho de Células-Tronco/fisiologia , Células-Tronco/fisiologia , Animais , Derme/fisiologia , Células Epidérmicas/fisiologia , Epiderme/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Músculos/fisiologia , Reepitelização , Regeneração/genética , Células Receptoras Sensoriais/fisiologia , Envelhecimento da Pele/genética , Nicho de Células-Tronco/genética , Transplante de Células-Tronco , Transcriptoma , Cicatrização/genética , Cicatrização/fisiologia
18.
Artigo em Inglês | MEDLINE | ID: mdl-32100325

RESUMO

This study was conducted to determine the effects of a co-infection with Moritella viscosa at different exposure levels of sea lice Lepeophtheirus salmonis in Atlantic salmon (Salmo salar). M. viscosa (1.14 × 106  cfu/ml) was introduced to all experimental tanks at 10 days post-lice infection (dpLs). Mean lice counts decreased over time in both the medium lice co-infection (31.5 ± 19.0 at 7 dpLs; 16.9 ± 9.3 at 46 dpLs) and high lice co-infection (62.0 ± 10.8 at 7 dpLs; 37.6 ± 11.3 at 46 dpLs). There were significantly higher mortalities and more severe skin lesions in the high lice co-infected group compared to medium lice co-infected group or M. viscosa-only infection. Quantitative gene expression analysis detected a significant upregulation of genes in skin from the high lice co-infection group consistent with severe inflammation (il-8, mmp-9, hep, saa). Skin lesions retrieved throughout the study were positive for M. viscosa growth, but these were rarely located in regions associated with lice. These results suggest that while M. viscosa infection itself may induce skin lesion development in salmon, co-infection with high numbers of lice can enhance this impact and significantly reduce the ability of these lesions to resolve, resulting in increased mortality.


Assuntos
Coinfecção/veterinária , Copépodes/fisiologia , Doenças dos Peixes/mortalidade , Infecções por Bactérias Gram-Negativas/veterinária , Moritella/fisiologia , Salmo salar , Dermatopatias Bacterianas/veterinária , Animais , Aquicultura , Coinfecção/imunologia , Coinfecção/microbiologia , Coinfecção/parasitologia , Feminino , Doenças dos Peixes/imunologia , Doenças dos Peixes/microbiologia , Doenças dos Peixes/parasitologia , Infecções por Bactérias Gram-Negativas/mortalidade , Imunidade Inata , Inflamação/imunologia , Inflamação/microbiologia , Inflamação/parasitologia , Inflamação/veterinária , Masculino , Dermatopatias Bacterianas/epidemiologia , Dermatopatias Bacterianas/microbiologia , Dermatopatias Bacterianas/parasitologia , Cicatrização/genética
19.
Biochem Biophys Res Commun ; 524(3): 533-541, 2020 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-32014254

RESUMO

The early-phase wound repair response of the intestinal epithelium is characterized by rapid and organized cell migration. This response is regulated by several humoral factors, including TGF-ß. However, due to a lack of appropriate models, the precise response of untransformed intestinal epithelial cells (IECs) to those factors is unclear. In this study, we established an in vitro wound repair model of untransformed IECs, based on native type-I collagen. In our system, IECs formed a uniform monolayer in a two-chamber culture insert and displayed a stable wound repair response. Gene expression analysis revealed significant induction of Apoa1, Apoa4, and Wnt4 during the collagen-guided wound repair response. The wound repair response was enhanced significantly by the addition of TGF-ß. Surprisingly, addition of TGF-ß induced a set of genes, including Slc28a2, Tubb2a, and Cpe, that were expressed preferentially in fetal IECs. Moreover, TGF-ß significantly increased the peak velocity of migrating IECs and, conversely, reduced the time required to reach the peak velocity, as confirmed by the motion vector prediction (MVP) method. Our current in vitro system could be employed to assess other humoral factors involved in IEC migration and could contribute to a deeper understanding of the wound repair potentials of untransformed IECs.


Assuntos
Movimento Celular/genética , Células Epiteliais/patologia , Feto/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Intestinos/patologia , Modelos Biológicos , Fator de Crescimento Transformador beta/farmacologia , Cicatrização/genética , Animais , Movimento Celular/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feto/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Organoides/efeitos dos fármacos , Organoides/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética , Cicatrização/efeitos dos fármacos
20.
Mar Biotechnol (NY) ; 22(2): 285-307, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32016679

RESUMO

Regeneration of a limb or tissue can be achieved through multiple different pathways and mechanisms. The sea anemone Exaiptasia pallida has been observed to have excellent regenerative proficiency, but this has not yet been described transcriptionally. In this study, we examined the genetic expression changes during a regenerative timecourse and reported key genes involved in regeneration and wound healing. We found that the major response was an early (within the first 8 h) upregulation of genes involved in cellular movement and cell communication, which likely contribute to a high level of tissue plasticity resulting in the rapid regeneration response observed in this species. We find the immune system was only transcriptionally active in the first 8 h post-amputation and conclude, in accordance with previous literature, that the immune system and regeneration have an inverse relationship. Fifty-nine genes (3.8% of total) differentially expressed during regeneration were identified as having no orthologues in other species, indicating that regeneration in E. pallida may rely on the activation of species-specific novel genes. Additionally, taxonomically restricted novel genes, including species-specific novels, and highly conserved genes were identified throughout the regenerative timecourse, showing that both may work in concert to achieve complete regeneration.


Assuntos
Regeneração/genética , Anêmonas-do-Mar/genética , Animais , Comunicação Celular/genética , Movimento Celular/genética , Perfilação da Expressão Gênica , Regeneração/fisiologia , Anêmonas-do-Mar/imunologia , Anêmonas-do-Mar/metabolismo , Cicatrização/genética
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