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1.
Int J Mol Sci ; 22(10)2021 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-34065474

RESUMO

Obesity-induced adipose tissue dysfunction and disorders of glycolipid metabolism have become a worldwide research priority. Zfp217 plays a crucial role in adipogenesis of 3T3-L1 preadipocytes, but about its functions in animal models are not yet clear. To explore the role of Zfp217 in high-fat diet (HFD)-induced obese mice, global Zfp217 heterozygous knockout (Zfp217+/-) mice were constructed. Zfp217+/- mice and Zfp217+/+ mice fed a normal chow diet (NC) did not differ significantly in weight gain, percent body fat mass, glucose tolerance, or insulin sensitivity. When challenged with HFD, Zfp217+/- mice had less weight gain than Zfp217+/+ mice. Histological observations revealed that Zfp217+/- mice fed a high-fat diet had much smaller white adipocytes in inguinal white adipose tissue (iWAT). Zfp217+/- mice had improved metabolic profiles, including improved glucose tolerance, enhanced insulin sensitivity, and increased energy expenditure compared to the Zfp217+/+ mice under HFD. We found that adipogenesis-related genes were increased and metabolic thermogenesis-related genes were decreased in the iWAT of HFD-fed Zfp217+/+ mice compared to Zfp217+/- mice. In addition, adipogenesis was markedly reduced in mouse embryonic fibroblasts (MEFs) from Zfp217-deleted mice. Together, these data indicate that Zfp217 is a regulator of energy metabolism and it is likely to provide novel insight into treatment for obesity.


Assuntos
Metabolismo Energético/fisiologia , Obesidade/metabolismo , Obesidade/fisiopatologia , Transativadores/metabolismo , Adipócitos Brancos/metabolismo , Adipócitos Brancos/fisiologia , Adipogenia/fisiologia , Tecido Adiposo Branco/metabolismo , Tecido Adiposo Branco/fisiopatologia , Animais , Dieta Hiperlipídica , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Resistência à Insulina/fisiologia , Metabolismo dos Lipídeos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Obesos , Termogênese/fisiologia , Ganho de Peso/fisiologia
2.
Int J Mol Sci ; 22(10)2021 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-34069872

RESUMO

Loss-of-function mutations in the synaptosomal-associated protein 29 (SNAP29) lead to the rare autosomal recessive neurocutaneous cerebral dysgenesis, neuropathy, ichthyosis, and keratoderma (CEDNIK) syndrome. SNAP29 is a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein. So far, it has been shown to be involved in membrane fusion, epidermal differentiation, formation of primary cilia, and autophagy. Recently, we reported the successful generation of two mouse models for the human CEDNIK syndrome. The aim of this investigation was the generation of a CRISPR/Cas9-mediated SNAP29 knockout (KO) in an immortalized human cell line to further investigate the role of SNAP29 in cellular homeostasis and signaling in humans independently of animal models. Comparison of different methods of delivery for CRISPR/Cas9 plasmids into the cell revealed that lentiviral transduction is more efficient than transfection methods. Here, we reported to the best of our knowledge the first successful generation of a CRISPR/Cas9-mediated SNAP29 KO in immortalized human MRC5Vi fibroblasts (c.169_196delinsTTCGT) via lentiviral transduction.


Assuntos
Fibroblastos/metabolismo , Técnicas de Inativação de Genes/métodos , Proteínas Qb-SNARE/genética , Proteínas Qc-SNARE/genética , Animais , Autofagia/genética , Sistemas CRISPR-Cas/genética , Diferenciação Celular/genética , Linhagem Celular , Fibroblastos/fisiologia , Humanos , Ceratodermia Palmar e Plantar/genética , Fusão de Membrana/genética , Mutação/genética , Síndromes Neurocutâneas/genética , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/metabolismo , Proteínas SNARE/genética , Proteínas SNARE/metabolismo
3.
Int J Mol Sci ; 22(10)2021 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-34067929

RESUMO

Cutaneous melanoma (CM) tissue represents a network constituted by cancer cells and tumor microenvironment (TME). A key feature of CM is the high structural and cellular plasticity of TME, allowing its evolution with disease and adaptation to cancer cell and environmental alterations. In particular, during melanoma development and progression each component of TME by interacting with each other and with cancer cells is subjected to dramatic structural and cellular modifications. These alterations affect extracellular matrix (ECM) remodelling, phenotypic profile of stromal cells, cancer growth and therapeutic response. The stromal fibroblast populations of the TME include normal fibroblasts and melanoma-associated fibroblasts (MAFs) that are highly abundant and flexible cell types interacting with melanoma and stromal cells and differently influencing CM outcomes. The shift from the normal microenvironment to TME and from normal fibroblasts to MAFs deeply sustains CM growth. Hence, in this article we review the features of the normal microenvironment and TME and describe the phenotypic plasticity of normal dermal fibroblasts and MAFs, highlighting their roles in normal skin homeostasis and TME regulation. Moreover, we discuss the influence of MAFs and their secretory profiles on TME remodelling, melanoma progression, targeted therapy resistance and immunosurveillance, highlighting the cellular interactions, the signalling pathways and molecules involved in these processes.


Assuntos
Fibroblastos/fisiologia , Melanoma/metabolismo , Microambiente Tumoral/fisiologia , Fibroblastos Associados a Câncer/metabolismo , Comunicação Celular , Plasticidade Celular/fisiologia , Matriz Extracelular/metabolismo , Humanos , Melanoma/patologia , Melanoma/fisiopatologia , Transdução de Sinais , Neoplasias Cutâneas/patologia , Células Estromais/metabolismo
4.
Food Chem ; 358: 129910, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-33957602

RESUMO

Sodium metabisulfite (SMB), an antioxidant agent, is extensively used as a preservative in food industry. The current study was aimed to clarify its potential toxic effects on human fetal foreskin fibroblasts (HFFF2) cells, in vitro. Subsequently, MTT results illustrated that exposure to SMB significantly (p < 0.0001) decreased HFFF2 cell viability in a dose-dependent manner, and the concentration of 25 µM reduced cell survival rates to 50% as the half-maximal inhibitory concentration of SMB. It was further shown that SMB exerted this cytotoxic effect on HFFF2 cells through apoptosis induction. qRT-PCR and western blotting results showed that treatment of HFFF2 cells with this food additive led to significant upregulation of Bax, caspase 8, and caspase 9 pro-apoptotic genes and downregulation of Bcl-2 expression as a pro-survival agent. Furthermore, SMB remarkably increased caspase 3 levels and promoted its activation through cleavage in treated cells. Besides, exposure to SMB increased ROS levels and activated autophagy in treated cells, which are considered as the other indicators for cell damage. Taken together, our findings suggested that SMB could exert remarkable toxic effects on human normal cells through multiple mechanisms, including apoptosis activation, and its widespread usage in food safety should be reconsidered.


Assuntos
Apoptose/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Aditivos Alimentares/toxicidade , Sulfitos/toxicidade , Apoptose/genética , Autofagia/efeitos dos fármacos , Autofagia/genética , Caspase 3/genética , Caspase 8/genética , Caspase 9/genética , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Fibroblastos/fisiologia , Aditivos Alimentares/administração & dosagem , Prepúcio do Pênis/citologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Espécies Reativas de Oxigênio/metabolismo , Sulfitos/administração & dosagem , Proteína X Associada a bcl-2/genética
5.
Nat Commun ; 12(1): 2989, 2021 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-34017000

RESUMO

The allogeneic transplantation of primordial germ cells (PGCs) derived from somatic cells overcomes the limitation of avian cloning. Here, we transdifferentiate chicken embryo fibroblasts (CEFs) from black feathered Langshan chickens to PGCs and transplant them into White Plymouth Rock chicken embryos to produce viable offspring with characteristics inherited from the donor. We express Oct4/Sox2/Nanog/Lin28A (OSNL) to reprogram CEFs to induced pluripotent stem cells (iPSCs), which are further induced to differentiate into PGCs by BMP4/BMP8b/EGF. DNA demethylation, histone acetylation and glycolytic activation elevate the iPSC induction efficiency, while histone acetylation and glycolytic inhibition facilitate PGCs formation. The induced PGCs (iPGCs) are transplanted into the recipients, which are self-crossed to produce 189/509 somatic cells derived chicken with the donor's characteristics. Microsatellite analysis and genome sequencing confirm the inheritance of genetic information from the donor. Thus, we demonstrate the feasibility of avian cloning from somatic cells.


Assuntos
Transdiferenciação Celular/genética , Clonagem de Organismos/métodos , Células Germinativas/transplante , Células-Tronco Pluripotentes Induzidas/fisiologia , Criação de Animais Domésticos/métodos , Animais , Proteína Morfogenética Óssea 4/genética , Células Cultivadas , Embrião de Galinha/citologia , Galinhas , Fator de Crescimento Epidérmico/genética , Estudos de Viabilidade , Fibroblastos/fisiologia , Células Germinativas/fisiologia , Proteína Homeobox Nanog/genética , Fator 3 de Transcrição de Octâmero/genética , Proteínas de Ligação a RNA/genética , Fatores de Transcrição SOXB1/genética , Transplante Homólogo/métodos
6.
Int J Mol Sci ; 22(9)2021 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-33922891

RESUMO

(1) Background: A simplistic understanding of the central dogma falls short in correlating the number of genes in the genome to the number of proteins in the proteome. Post-transcriptional alternative splicing contributes to the complexity of the proteome and is critical in understanding gene expression. mRNA-sequencing (RNA-seq) has been widely used to study the transcriptome and provides opportunity to detect alternative splicing events among different biological conditions. Despite the popularity of studying transcriptome variants with RNA-seq, few efficient and user-friendly bioinformatics tools have been developed for the genome-wide detection and visualization of alternative splicing events. (2) Results: We propose AS-Quant, (Alternative Splicing Quantitation), a robust program to identify alternative splicing events from RNA-seq data. We then extended AS-Quant to visualize the splicing events with short-read coverage plots along with complete gene annotation. The tool works in three major steps: (i) calculate the read coverage of the potential spliced exons and the corresponding gene; (ii) categorize the events into five different categories according to the annotation, and assess the significance of the events between two biological conditions; (iii) generate the short reads coverage plot for user specified splicing events. Our extensive experiments on simulated and real datasets demonstrate that AS-Quant outperforms the other three widely used baselines, SUPPA2, rMATS, and diffSplice for detecting alternative splicing events. Moreover, the significant alternative splicing events identified by AS-Quant between two biological contexts were validated by RT-PCR experiment. (3) Availability: AS-Quant is implemented in Python 3.0. Source code and a comprehensive user's manual are freely available online.


Assuntos
Processamento Alternativo , Análise de Sequência de RNA/métodos , Software , Animais , Biologia Computacional/métodos , Visualização de Dados , Éxons , Fibroblastos/citologia , Fibroblastos/fisiologia , Camundongos , Anotação de Sequência Molecular
7.
Mar Drugs ; 19(3)2021 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-33809867

RESUMO

While electrospun chitosan membranes modified to retain nanofibrous morphology have shown promise for use in guided bone regeneration applications in in vitro and in vivo studies, their mechanical tear strengths are lower than commercial collagen membranes. Elastin, a natural component of the extracellular matrix, is a protein with extensive elastic property. This work examined the incorporation of elastin into electrospun chitosan membranes to improve their mechanical tear strengths and to further mimic the native extracellular composition for guided bone regeneration (GBR) applications. In this work, hydrolyzed elastin (ES12, Elastin Products Company, USA) was added to a chitosan spinning solution from 0 to 4 wt% of chitosan. The chitosan-elastin (CE) membranes were examined for fiber morphology using SEM, hydrophobicity using water contact angle measurements, the mechanical tear strength under simulated surgical tacking, and compositions using Fourier-transform infrared spectroscopy (FTIR) and post-spinning protein extraction. In vitro experiments were conducted to evaluate the degradation in a lysozyme solution based on the mass loss and growth of fibroblastic cells. Chitosan membranes with elastin showed significantly thicker fiber diameters, lower water contact angles, up to 33% faster degradation rates, and up to seven times higher mechanical strengths than the chitosan membrane. The FTIR spectra showed stronger amide peaks at 1535 cm-1 and 1655 cm-1 in membranes with higher concentrated elastin, indicating the incorporation of elastin into electrospun fibers. The bicinchoninic acid (BCA) assay demonstrated an increase in protein concentration in proportion to the amount of elastin added to the CE membranes. In addition, all the CE membranes showed in vitro biocompatibility with the fibroblasts.


Assuntos
Materiais Biocompatíveis , Quitosana/química , Elastina/química , Membranas Artificiais , Animais , Proliferação de Células , Elasticidade , Fibroblastos/fisiologia , Camundongos , Estrutura Molecular , Células NIH 3T3 , Relação Estrutura-Atividade , Propriedades de Superfície , Resistência à Tração
8.
Science ; 372(6540)2021 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-33888614

RESUMO

Skin scarring, the end result of adult wound healing, is detrimental to tissue form and function. Engrailed-1 lineage-positive fibroblasts (EPFs) are known to function in scarring, but Engrailed-1 lineage-negative fibroblasts (ENFs) remain poorly characterized. Using cell transplantation and transgenic mouse models, we identified a dermal ENF subpopulation that gives rise to postnatally derived EPFs by activating Engrailed-1 expression during adult wound healing. By studying ENF responses to substrate mechanics, we found that mechanical tension drives Engrailed-1 activation via canonical mechanotransduction signaling. Finally, we showed that blocking mechanotransduction signaling with either verteporfin, an inhibitor of Yes-associated protein (YAP), or fibroblast-specific transgenic YAP knockout prevents Engrailed-1 activation and promotes wound regeneration by ENFs, with recovery of skin appendages, ultrastructure, and mechanical strength. This finding suggests that there are two possible outcomes to postnatal wound healing: a fibrotic response (EPF-mediated) and a regenerative response (ENF-mediated).


Assuntos
Cicatriz/patologia , Fibroblastos/fisiologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Regeneração , Pele/lesões , Cicatrização , Animais , Cicatriz/prevenção & controle , Fibroblastos/transplante , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Mecanotransdução Celular , Camundongos , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-yes/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-yes/genética , Proteínas Proto-Oncogênicas c-yes/metabolismo , Transdução de Sinais , Estresse Mecânico , Ativação Transcricional , Transcriptoma , Verteporfina/farmacologia
9.
Nat Metab ; 3(4): 469-484, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33846639

RESUMO

Brown adipose tissue can expend large amounts of energy, and therefore increasing its size or activity is a promising therapeutic approach to combat metabolic disease. In humans, major deposits of brown fat cells are found intimately associated with large blood vessels, corresponding to perivascular adipose tissue (PVAT). However, the cellular origins of PVAT are poorly understood. Here, we determine the identity of perivascular adipocyte progenitors in mice and humans. In mice, thoracic PVAT develops from a fibroblastic lineage, consisting of progenitor cells (Pdgfra+, Ly6a+ and Pparg-) and preadipocytes (Pdgfra+, Ly6a+ and Pparg+), which share transcriptional similarity with analogous cell types in white adipose tissue. Interestingly, the aortic adventitia of adult animals contains a population of adipogenic smooth muscle cells (Myh11+, Pdgfra- and Pparg+) that contribute to perivascular adipocyte formation. Similarly, human PVAT contains presumptive fibroblastic and smooth muscle-like adipocyte progenitor cells, as revealed by single-nucleus RNA sequencing. Together, these studies define distinct populations of progenitor cells for thermogenic PVAT, providing a foundation for developing strategies to augment brown fat activity.


Assuntos
Adipócitos Marrons/fisiologia , Tecido Adiposo Marrom/fisiologia , Linhagem da Célula/fisiologia , Termogênese/fisiologia , Adipócitos Brancos/fisiologia , Adipogenia/fisiologia , Tecido Adiposo Marrom/crescimento & desenvolvimento , Animais , Animais Recém-Nascidos , Aorta/citologia , Aorta/fisiologia , Vasos Sanguíneos/fisiologia , Linhagem da Célula/genética , Fibroblastos/fisiologia , Regulação da Expressão Gênica/fisiologia , Humanos , Recém-Nascido , Camundongos , Camundongos Endogâmicos C57BL , Miócitos de Músculo Liso/fisiologia , Células-Tronco/fisiologia , Termogênese/genética
10.
Mol Cell ; 81(8): 1715-1731.e6, 2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33784494

RESUMO

Heat shock instantly reprograms transcription. Whether gene and enhancer transcription fully recover from stress and whether stress establishes a memory by provoking transcription regulation that persists through mitosis remained unknown. Here, we measured nascent transcription and chromatin accessibility in unconditioned cells and in the daughters of stress-exposed cells. Tracking transcription genome-wide at nucleotide-resolution revealed that cells precisely restored RNA polymerase II (Pol II) distribution at gene bodies and enhancers upon recovery from stress. However, a single heat exposure in embryonic fibroblasts primed a faster gene induction in their daughter cells by increasing promoter-proximal Pol II pausing and by accelerating the pause release. In K562 erythroleukemia cells, repeated stress refined basal and heat-induced transcription over mitotic division and decelerated termination-coupled pre-mRNA processing. The slower termination retained transcripts on the chromatin and reduced recycling of Pol II. These results demonstrate that heat-induced transcriptional memory acts through promoter-proximal pause release and pre-mRNA processing at transcription termination.


Assuntos
Mitose/genética , Regiões Promotoras Genéticas/genética , Estresse Fisiológico/genética , Transcrição Genética/genética , Linhagem Celular Tumoral , Cromatina/genética , Fibroblastos/fisiologia , Regulação da Expressão Gênica/genética , Genoma/genética , Resposta ao Choque Térmico/genética , Humanos , Células K562 , RNA Polimerase II/genética , RNA Mensageiro/genética
11.
Carbohydr Polym ; 261: 117810, 2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-33766329

RESUMO

Chitosan-based hydrogels have been widely used for various biomedical applications due to their versatile properties such as biocompatibility, biodegradability, muco-adhesiveness, hemostatic effect and so on. However, the inherent rigidity and brittleness of pure chitosan hydrogels are still unmanageable, which has limited their potential use in biomaterial research. In this study, we developed in situ forming chitosan/PEG hydrogels with improved mechanical properties, using the enzymatic crosslinking reaction of horseradish peroxidase (HRP). The effect of PEG on physico-chemical properties of hybrid hydrogels was thoroughly elucidated by varying the content (0-100 %), molecular weight (4, 10 and 20 kDa) and geometry (linear, 4-arm) of the PEG derivatives. The resulting hydrogels demonstrated excellent hemostatic ability and are highly biocompatible in vivo, comparable to commercially available fibrin glue. We suggest these chitosan/PEG hybrid hydrogels with tunable physicochemical and tissue adhesive properties have great potential for a wide range of biomedical applications in the near future.


Assuntos
Quitosana/química , Hidrogéis/síntese química , Adesivos Teciduais , Adesividade , Animais , Células Cultivadas , Derme/citologia , Derme/efeitos dos fármacos , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Hemostasia/efeitos dos fármacos , Humanos , Hidrogéis/química , Hidrogéis/farmacologia , Injeções , Masculino , Teste de Materiais , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Polimerização , Polímeros/administração & dosagem , Polímeros/síntese química , Polímeros/química , Polímeros/farmacologia , Ratos , Ratos Sprague-Dawley , Estresse Mecânico , Adesivos Teciduais/administração & dosagem , Adesivos Teciduais/síntese química , Adesivos Teciduais/química , Adesivos Teciduais/farmacologia , Engenharia Tecidual/métodos
12.
Methods Mol Biol ; 2273: 151-158, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33604851

RESUMO

The first differentiation event in mammalian embryos is the formation of the trophectoderm, which is the progenitor of the outer epithelial component of the placenta and supports the fetus during intrauterine life. Our understanding of these events is limited, particularly in human, because of ethical and legal restrictions and availability of adequate in vitro models would be very advantageous. Here we describe a method that converts human fibroblasts into trophoblast-like cells, combining the use of 5-azacytidine-CR (5-aza-CR) to erase the original cell phenotype and a cocktail containing bone morphogenetic protein 4 (BMP4) with inhibitors of the Activin/Nodal/ERK signaling pathways, to drive erased fibroblasts into the trophoblastic differentiation. This innovative method uses very easily accessible cells to derive trophoblast-like cells and it can be useful to study embryo implantation disorders related to aging.


Assuntos
Técnicas de Cultura de Células/métodos , Fibroblastos/citologia , Trofoblastos/citologia , Ativinas/antagonistas & inibidores , Animais , Azacitidina/farmacologia , Proteína Morfogenética Óssea 4/metabolismo , Proteína Morfogenética Óssea 4/farmacologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Implantação do Embrião , Embrião de Mamíferos/metabolismo , Células-Tronco Embrionárias/citologia , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Proteína Nodal/antagonistas & inibidores , Placenta/citologia , Gravidez , Transdução de Sinais , Pele/citologia , Pele/crescimento & desenvolvimento
13.
Rev. bras. oftalmol ; 80(1): 8-11, jan.-fev. 2021. graf
Artigo em Português | LILACS | ID: biblio-1251324

RESUMO

RESUMO Objetivo: Avaliar a inibição da proliferação de fibroblastos in vitro das conjuntivas obtidas através de exérese de pterígios de pacientes utilizando mitomicina C (MMC) e ciclofosfamida (CF). Métodos: Os pterígios foram retirados de 7 pacientes e submetidos a cultivo celular. Após o cultivo, 3 fragmentos de dimensões iguais deste material foram colhidos de áreas adjacentes do pterígio removido de cada paciente. Eles foram randomicamente selecionados de tal forma que: um fragmento de cada paciente foi exposto: ao meio de cultura (grupo controle), a MMC e a CF por igual período de tempo nas concentrações de 0,4 mg/ml e 10 mg/ml respectivamente. Após este período realizou-se a contagem celular de fibroblastos destes 3 grupos. Cada grupo continha 7 fragmentos. Resultados: Com a utilização da MMC tivemos uma taxa de 95% da inibição da proliferação dos fibroblastos, enquanto com a CF 100%. Conclusões: Ambas as drogas apresentaram elevada taxa da inibição da proliferação de fibroblastos, porém a CF apresentou inibição maior que a MMC.


Abstract Objective: To evaluate the inhibition of fibroblast proliferation in vitro of conjunctiva obtained by excision of pterygium from patients using mitomycin (MMC) and cyclophosphamide (CF). Methods: Pterygiums were removed from 7 patients and subjected to cell culture. After cell cultivation, 3 fragments of equal dimensions of these tissues were collected from adjacent areas of each patient removed pterygium. They were randomly selected in such a way that one fragment of each patient was exposed to: the culture medium (group control), to MMC and to CF for an equal period of time at concentrations of 0,4 mg/dl and 10 mg/dl respectively. After this period, the fibroblast cell count of these groups were performed. Each group had seven fragments. Results: With the use of MMC we had a 95% rate of inhibition of fibroblast proliferation, while with CF 100%. Conclusion: Both drugs showed a high rate of inhibition of fibroblast proliferation, but CF showed greater inhibition than MMC.


Assuntos
Humanos , Masculino , Feminino , Adulto , Pessoa de Meia-Idade , Cicatrização , Pterígio/cirurgia , Mitomicina/efeitos adversos , Ciclofosfamida/efeitos adversos , Proliferação de Células/fisiologia , Antimitóticos/efeitos adversos , Fibroblastos/fisiologia , Técnicas In Vitro
14.
Aging (Albany NY) ; 13(3): 3239-3253, 2021 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-33510044

RESUMO

The naked mole rat (NMR), Heterocephalus glaber, is the longest-living rodent species, and is extraordinarily resistant to cancer and aging-related diseases. The molecular basis for these unique phenotypic traits of the NMR is under extensive research. However, the role of regulated cell death (RCD) in the longevity and the protection from cancer in the NMR is still largely unknown. RCD is a mechanism restricting the proliferation of damaged or premalignant cells, which counteracts aging and oncotransformation. In this study, DNA damage-induced cell death in NMR fibroblasts was investigated in comparison to RCD in fibroblasts from Mus musculus. The effects of methyl methanesulfonate, 5-fluorouracil, and etoposide in both cell types were examined using contemporary cell death analyses. Skin fibroblasts from Heterocephalus glaber were found to be more resistant to the action of DNA damaging agents compared to fibroblasts from Mus musculus. Strikingly, our results revealed that NMR cells also exhibit a limited apoptotic response and seem to undergo regulated necrosis. Taken together, this study provides new insights into the mechanisms of cell death in NMR expanding our understanding of longevity, and it paves the way towards the development of innovative therapeutic approaches.


Assuntos
Longevidade/fisiologia , Ratos-Toupeira/fisiologia , Morte Celular Regulada/fisiologia , Animais , Células Cultivadas , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/fisiologia , Fibroblastos/citologia , Fibroblastos/fisiologia , Metanossulfonato de Metila/toxicidade , Camundongos , Morte Celular Regulada/efeitos dos fármacos
15.
J Orthop Surg Res ; 16(1): 1, 2021 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-33397415

RESUMO

BACKGROUND: The over-proliferation of fibroblasts is considered to be the main cause of scar adhesion after joint surgery. Hydroxycamptothecin (HCPT), though as a potent antineoplastic drug, shows preventive effects on scar adhesion. This study aimed to investigate the role of activating transcription factor 6 (ATF-6) in the HCPT-induced inhibition of fibroblast viability. METHODS: The cell counting kit-8 (CCK-8) assay, western blot analysis, lentivirus-mediated gene silencing, transmission electron microscopy (TEM) analysis, immunofluorescent staining for autophagy-related protein light chain 3 (LC3) were used to explore the effect of HCPT on triggering fibroblast apoptosis and inhibiting fibroblast proliferation, and the involvement of possible signaling pathways. RESULTS: It was found that HCPT exacerbated fibroblast apoptosis and repressed its proliferation. Subsequently, endoplasmic reticulum stress (ERS)-related proteins were determined by western blot prior to ATF6 p50 was screened out and reexamined after it was silenced. As a result, ATF6-mediated ERS played a role in HCPT-induced fibroblast apoptosis. Autophagy-related proteins and autophagosomes were detected after the HCPT administration using western blot and TEM analyses, respectively. Autophagy was activated after the HCPT treatment. With the co-treatment of autophagy inhibitor 3-methyladenine (3-MA), both the western blot analysis and the CCK-8 assay showed inhibited autophagy, which indicated that the effect of HCPT on fibroblast proliferation was partially reversed. Besides, the LC3 immunofluorescence staining revealed suppressed autophagy after silencing ATF6 p50. CONCLUSION: Our results demonstrate that HCPT acts as a facilitator of fibroblast apoptosis and inhibitor of fibroblast proliferation for curbing the postoperative scar adhesion, in which the ATF6-mediated ERS pathway and autophagy are involved.


Assuntos
Fator 6 Ativador da Transcrição/fisiologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Autofagia/efeitos dos fármacos , Autofagia/genética , Camptotecina/análogos & derivados , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Fibroblastos/fisiologia , Camptotecina/farmacologia , Linhagem Celular , Cicatriz/prevenção & controle , Estresse do Retículo Endoplasmático/genética , Estresse do Retículo Endoplasmático/fisiologia , Humanos , Transdução de Sinais/genética , Transdução de Sinais/fisiologia
16.
J Orthop Surg Res ; 16(1): 9, 2021 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-33407698

RESUMO

BACKGROUND: The prevention of surgery-induced intraarticular fibrosis remains a challenge following orthopedic surgery. Homoharringtonine (HHT) has been reported to have positive effects in preventing various kinds of fibrosis. However, little is known regarding its effect as well as the potential mechanism of HHT in preventing surgery-induced intraarticular fibrosis. METHODS: Various concentrations of HHTs were locally applied in vivo to reduce knee intraarticular fibrosis in rabbits. Histological macroscopic assessments such as hematoxylin and eosin (HE) staining, Masson's trichrome staining, and Picric-sirius red polarized light were used to evaluate the effect of HHT in reducing intraarticular fibrosis. CCK-8, cell cycle assay, and EdU incorporation assay were used in vitro to detect HHT's effect on inhibiting fibroblast viability and proliferation. The effect of HHT on fibroblast differentiation, extracellular matrix production, and apoptosis were evaluated by western blot, flow cytometry, immunofluorescent staining, and TUNEL analysis. Moreover, the expressions of PI3K/AKT/mTOR signaling pathway were detected. RESULTS: The results demonstrated that HHT could reduce the formation of intraarticular fibrosis. HHT was also found to induce fibroblast apoptotic cell death in a dose- and time-dependent manner in vitro. Moreover, HHT could effectively inhibit the production of the extracellular matrix secreted by fibroblasts and inhibited the expression of p-PI3K, p-AKT, and p-mTOR in a dose-dependent manner. After treating with insulin-like growth factor-1 (IGF-1), an activator of the PI3K/AKT axis, the expressions of pro-apoptosis-related proteins were decreased, and the fibroblast apoptosis rate was also inhibited. CONCLUSIONS: In conclusion, this study demonstrated that HHT could reduce the formation of intraarticular fibrosis through the inhibition of fibroblast proliferation, extracellular matrix production, and the induction of fibroblast apoptotic cell death. Furthermore, its potential mechanism may be through the suppression of the PI3K/AKT/mTOR signaling pathway.


Assuntos
Apoptose/efeitos dos fármacos , Apoptose/genética , Proliferação de Células/efeitos dos fármacos , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Mepesuccinato de Omacetaxina/farmacologia , Articulação do Joelho/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Complicações Pós-Operatórias/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Serina-Treonina Quinases TOR/metabolismo , Linhagem Celular , Depressão Química , Fibrose/prevenção & controle , Humanos , Complicações Pós-Operatórias/prevenção & controle
17.
Exp Cell Res ; 399(2): 112464, 2021 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-33385416

RESUMO

AIMS/HYPOTHESIS: MicroRNA-21 has been implicated in diabetic complication, including diabetic cardiomyopathy. However, there is limited information regarding the biological role of the miR-21 passenger strand (miR-21-3p) in diabetic cardiac fibrosis. The aim of this study was to investigate the role of miR-21-3p and its target androgen receptor in STZ-induced diabetic cardiac fibrosis. METHODS: The pathological changes and collagen depositions was analyzed by HE, Sirius Red staining and Masson's Trichrome Staining. MiR-21-3p, AR, NLRP3, caspase1 and collagen I expression were analyzed by western blotting, immunohistochemistry, immunofluorescence, qRT-PCR, miR one step qRT-PCR, respectively. A luciferase reporter assay was used to verify the interaction between miR-21 and the 3' untranslated region (3'UTR) of AR. RESULTS: Our results indicated that miR-21-3p level was up-regulated, while AR was decreased in STZ-induced diabetic cardiac fibrosis tissues and cardiac fibroblast. High glucose triggers cardiac fibroblasts pyroptosis and collagen deposition. Gain-of-function and loss-of-function assays demonstrated that miR-21-3p mediated the crucial role in diabetic cardiac fibrosis. Our results show that miR-21-3p bound to the 3'UTR of AR post-transcriptionally repressed its expression. We also found AR, which regulates cardiac fibroblasts pyroptosis and collagen deposition through caspase1 signaling. CONCLUSIONS: /interpretation: Taken together, our study showed that miR-21-3p aggravates STZ-induced diabetic cardiac fibrosis through the caspase1 pathways by suppressing AR expression.


Assuntos
Cardiomiopatias Diabéticas/genética , Fibroblastos/fisiologia , MicroRNAs/fisiologia , Miocárdio/patologia , Piroptose/genética , Animais , Animais Recém-Nascidos , Células Cultivadas , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patologia , Cardiomiopatias Diabéticas/metabolismo , Cardiomiopatias Diabéticas/patologia , Fibroblastos/patologia , Fibrose/genética , Masculino , MicroRNAs/genética , Miocárdio/metabolismo , Interferência de RNA/fisiologia , Ratos , Ratos Sprague-Dawley , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Transdução de Sinais/genética , Estreptozocina
18.
Invest Ophthalmol Vis Sci ; 62(1): 27, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33502460

RESUMO

Purpose: The purpose of this study was to describe the cellular architecture of normal human peripapillary sclera (PPS) and evaluate surface topography's role in fibroblast behavior. Methods: PPS cryosections from nonglaucomatous eyes were labelled for nuclei, fibrillar actin (FA), and alpha smooth muscle actin (αSMA) and imaged. Collagen fibrils were imaged using second harmonic generation. Nuclear density and aspect ratio of the internal PPS (iPPS), outer PPS (oPPS), and peripheral sclera were determined. FA and αSMA fibril alignment with collagen extracellular matrix (ECM) was determined. PPS fibroblasts were cultured on smooth or patterned membranes under mechanical strain and in the presence of TGFß1 and 2. Results: The iPPS (7.1 ± 2.0 × 10-4, P < 0.0001) and oPPS (5.3 ± 1.4 × 10-4, P = 0.0013) had greater nuclei density (nuclei/µm2) than peripheral sclera (2.5 ± 0.8 × 10-4). The iPPS (2.0 ± 0.3, P = 0.002) but not oPPS (2.4 ± 0.4, P = 0.45) nuclei had smaller aspect ratios than peripheral (2.7 ± 0.5) nuclei. FA was present throughout the scleral stroma and was more aligned with oPPS collagen (9.6 ± 1.9 degrees) than in the peripheral sclera (15.9 ± 3.9 degrees, P =0.002). The αSMA fibers in the peripheral sclera were less aligned with collagen fibrils (26.4 ± 4.8 degrees) than were FA (15.9 ± 3.9 degrees, P = 0.0002). PPS fibroblasts cultured on smooth membranes shifted to an orientation perpendicular to the direction of cyclic uniaxial strain (1 Hz, 5% strain, 42.2 ± 7.1 degrees versus 62.0 ± 8.5 degrees, P < 0.0001), whereas aligned fibroblasts on patterned membranes were resistant to strain-induced reorientation (5.9 ± 1.4 degrees versus 10 ± 3.3 degrees, P = 0.21). Resistance to re-orientation was reduced by TGFß treatment (10 ± 3.3 degrees without TGFß1 compared to 23.1 ± 4.5 degrees with TGFß1, P < 0.0001). Conclusions: Regions of the posterior sclera differ in cellular density and nuclear morphology. Topography alters the cellular response to mechanical strain.


Assuntos
Fibroblastos/citologia , Esclera/citologia , Actinas/metabolismo , Idoso , Idoso de 80 Anos ou mais , Anatomia Regional , Biomarcadores/metabolismo , Contagem de Células , Células Cultivadas , Colágeno/metabolismo , Feminino , Fibroblastos/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Disco Óptico/anatomia & histologia , Doadores de Tecidos
19.
Commun Biol ; 4(1): 81, 2021 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-33469145

RESUMO

Ageing in humans is associated with the decreased capacity to regulate cell physiology. Cellular properties, such as cell morphology and mechanics, encode ageing information, and can therefore be used as robust biomarkers of ageing. Using a panel of dermal fibroblasts derived from healthy donors spanning a wide age range, we observe an age-associated decrease in cell motility. By taking advantage of the single-cell nature of our motility data, we classified cells based on spatial and activity patterns to define age-dependent motility states. We show that the age-dependent decrease in cell motility is not due to the reduced motility of all cells, but results from the fractional re-distribution among motility states. These findings highlight an important feature of ageing cells characterized by a reduction of cellular heterogeneity in older adults relative to post-adolescent/adults. Furthermore, these results point to a mechanistic framework of ageing, with potential applications in deciphering emergent ageing phenotypes and biomarker development.


Assuntos
Envelhecimento/fisiologia , Movimento Celular/fisiologia , Adolescente , Adulto , Fatores Etários , Idoso , Envelhecimento/metabolismo , Criança , Pré-Escolar , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Humanos , Pessoa de Meia-Idade , Modelos Teóricos , Fenótipo , Pele/metabolismo
20.
EMBO J ; 40(1): e102236, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33034061

RESUMO

The generation of induced pluripotent stem cells (iPSCs) from somatic cells provides an excellent model to study mechanisms of transcription factor-induced global alterations of the epigenome and genome function. Here, we have investigated the early transcriptional events of cellular reprogramming triggered by the co-expression of Oct4, Sox2, Klf4, and c-Myc (OSKM) in mouse embryonic fibroblasts (MEFs) and mouse hepatocytes (mHeps). In this analysis, we identified a gene regulatory network composed of nine transcriptional regulators (9TR; Cbfa2t3, Gli2, Irf6, Nanog, Ovol1, Rcan1, Taf1c, Tead4, and Tfap4), which are directly targeted by OSKM, in vivo. Functional studies using single and double shRNA knockdowns of any of these factors caused disruption of the network and dramatic reductions in reprogramming efficiency, indicating that this network is essential for the induction and establishment of pluripotency. We demonstrate that the stochastic co-expression of 9TR network components occurs in a remarkably small number of cells, approximating the percentage of terminally reprogrammed cells as a result of dynamic molecular events. Thus, the early DNA-binding patterns of OSKM and the subsequent probabilistic co-expression of essential 9TR components in subpopulations of cells undergoing reprogramming steer the reconstruction of a gene regulatory network marking the transition to pluripotency.


Assuntos
Reprogramação Celular/genética , Fibroblastos/fisiologia , Redes Reguladoras de Genes/genética , Hepatócitos/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Animais , Células-Tronco Embrionárias/fisiologia , Feminino , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Gravidez , Fatores de Transcrição/genética , Transcrição Genética/genética
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