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1.
PLoS One ; 15(8): e0237040, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32764823

RESUMO

As type-I-allergies show an increasing prevalence in the general populace, orthodontic patients may also be affected by histamine release during treatment. Human periodontal ligament fibroblasts (PDLF) are regulators of orthodontic tooth movement. However, the impact of histamine on PDLF in this regard is unknown. Therefore PDLF were incubated without or with an orthodontic compressive force of 2g/cm2 with and without additional histamine. To assess the role of histamine-1-receptor (H1R) H1R-antagonist cetirizine was used. Expression of histamine receptors and important mediators of orthodontic tooth movement were investigated. PDLF expressed histamine receptors H1R, H2R and H4R, but not H3R. Histamine increased the expression of H1R, H2R and H4R as well as of interleukin-6, cyclooxygenase-2, and prostaglandin-E2 secretion even without pressure application and induced receptor activator of NF-kB ligand (RANKL) protein expression with unchanged osteoprotegerin secretion. These effects were not observed in presence of H1R antagonist cetirizine. By expressing histamine receptors, PDLF seem to be able to respond to fluctuating histamine levels in the periodontal tissue. Increased histamine concentration was associated with enhanced expression of proinflammatory mediators and RANKL, suggesting an inductive effect of histamine on PDLF-mediated osteoclastogenesis and orthodontic tooth movement. Since cetirizine inhibited these effects, they seem to be mainly mediated via histamine receptor H1R.


Assuntos
Histamina/farmacologia , Ligamento Periodontal/efeitos dos fármacos , Ligamento Periodontal/fisiologia , Técnicas de Movimentação Dentária , Células Cultivadas , Cetirizina/farmacologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Expressão Gênica/efeitos dos fármacos , Histamina/fisiologia , Antagonistas dos Receptores Histamínicos H1/farmacologia , Humanos , Mediadores da Inflamação/metabolismo , Modelos Biológicos , Osteogênese/efeitos dos fármacos , Osteogênese/fisiologia , Osteoprotegerina/genética , Osteoprotegerina/metabolismo , Ligamento Periodontal/citologia , Ligante RANK/genética , Ligante RANK/metabolismo , Receptores Histamínicos H1/fisiologia , Estresse Mecânico
2.
Mol Cell ; 79(4): 660-676.e8, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32755593

RESUMO

Specific combinations of two transcription factors (Hnf4α plus Foxa1, Foxa2, or Foxa3) can induce direct conversion of mouse fibroblasts into hepatocyte-like cells. However, the molecular mechanisms underlying hepatic reprogramming are largely unknown. Here, we show that the Foxa protein family members and Hnf4α sequentially and cooperatively bind to chromatin to activate liver-specific gene expression. Although all Foxa proteins bind to and open regions of closed chromatin as pioneer factors, Foxa3 has the unique potential of transferring from the distal to proximal regions of the transcription start site of target genes, binding RNA polymerase II, and co-traversing target genes. These distinctive characteristics of Foxa3 are essential for inducing the hepatic fate in fibroblasts. Similar functional coupling of transcription factors to RNA polymerase II may occur in other contexts whereby transcriptional activation can induce cell differentiation.


Assuntos
Fator 3-gama Nuclear de Hepatócito/metabolismo , Fator 4 Nuclear de Hepatócito/metabolismo , Fígado/citologia , Fígado/fisiologia , Ativação Transcricional , Animais , Sítios de Ligação , Células Cultivadas , Reprogramação Celular/fisiologia , Cromatina/metabolismo , DNA Polimerase II/genética , DNA Polimerase II/metabolismo , Fibroblastos/citologia , Fibroblastos/fisiologia , Regulação da Expressão Gênica , Fator 3-gama Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/genética , Camundongos Endogâmicos C57BL , Domínios Proteicos , Sítio de Iniciação de Transcrição
3.
Nat Commun ; 11(1): 3953, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32769974

RESUMO

Many important cell types in adult vertebrates have a mesenchymal origin, including fibroblasts and vascular mural cells. Although their biological importance is undisputed, the level of mesenchymal cell heterogeneity within and between organs, while appreciated, has not been analyzed in detail. Here, we compare single-cell transcriptional profiles of fibroblasts and vascular mural cells across four murine muscular organs: heart, skeletal muscle, intestine and bladder. We reveal gene expression signatures that demarcate fibroblasts from mural cells and provide molecular signatures for cell subtype identification. We observe striking inter- and intra-organ heterogeneity amongst the fibroblasts, primarily reflecting differences in the expression of extracellular matrix components. Fibroblast subtypes localize to discrete anatomical positions offering novel predictions about physiological function(s) and regulatory signaling circuits. Our data shed new light on the diversity of poorly defined classes of cells and provide a foundation for improved understanding of their roles in physiological and pathological processes.


Assuntos
Diferenciação Celular , Fibroblastos/fisiologia , Células-Tronco Mesenquimais/fisiologia , Miócitos de Músculo Liso/fisiologia , Pericitos/fisiologia , Animais , Separação Celular , Vasos Coronários/citologia , Matriz Extracelular/metabolismo , Fibroblastos/citologia , Citometria de Fluxo , Intestinos/irrigação sanguínea , Intestinos/citologia , Masculino , Camundongos , Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/citologia , Músculo Liso Vascular/citologia , Miocárdio/citologia , Miócitos de Músculo Liso/citologia , Pericitos/citologia , RNA-Seq , Análise de Célula Única , Bexiga Urinária/irrigação sanguínea , Bexiga Urinária/citologia
4.
Ecotoxicol Environ Saf ; 202: 110896, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32622306

RESUMO

Exposure to fine particulate matter (PM) comprising toxic compounds arising from air pollution is a major human health concern. It is linked to increased mortality and incidence of various lung diseases. However, the mechanisms underlying the toxic effects of PM on lung fibroblasts have not been fully explored. We used targeted quantitative metabolomics and lipidomics analysis along with cytotoxicity studies to comprehensively characterize the alterations in the metabolite profiles of human lung fibroblasts (HEL 299) upon exposure to PM2.5 and PM10. This exposure at 50 µg/mL for 72 h induced an abnormally high apoptotic response via triggering intracellular reactive oxygen species (ROS) production and mitochondrial dysfunction through an imbalance between pro- and anti-apoptotic signaling pathways. The cytotoxic effects of PM2.5 were more severe than those of PM10. Metabolomics and lipidomics analyses revealed that PM exposure triggered substantial changes in the cellular metabolite profile, which involved reduced mitochondria-related metabolites such as tricarboxylic acid (TCA) cycle intermediates, amino acids, and free fatty acids as well as increased lysoglycerophospholipids (LPLs) containing polyunsaturated fatty acids. The decrease in mitochondria-related metabolites suggested that PM exposure led to reduced TCA cycle capacity and energy production. Apoptotic and inflammatory responses as well as mitochondrial dysfunction were likely to be accelerated because of excessive accumulation of LPLs, contributing to the disruption of membrane rafts and Ca2+ homeostasis and causing increased mitochondrial ROS formation. These results provide valuable insights regarding the toxic effects of PM exposure. Our study also provides a new direction for research on PM exposure-related health disorders using different cell lines.


Assuntos
Poluentes Atmosféricos/toxicidade , Fibroblastos/fisiologia , Material Particulado/toxicidade , Fosfolipídeos/metabolismo , Poluentes Atmosféricos/análise , Poluição do Ar/análise , Apoptose , Linhagem Celular , Fibroblastos/efeitos dos fármacos , Homeostase , Humanos , Lipidômica , Pulmão/efeitos dos fármacos , Pneumopatias , Metabolômica , Mitocôndrias/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
5.
PLoS One ; 15(6): e0234180, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32511278

RESUMO

The autophagy-endolysosomal pathway is an evolutionally conserved degradation system that is tightly linked to a wide variety of physiological processes. Dysfunction of this system is associated with many pathological conditions such as cancer, inflammation and neurodegenerative diseases. Therefore, monitoring the cellular autophagy-endolysosomal activity is crucial for studies on the pathogenesis as well as therapeutics of such disorders. To this end, we here sought to create a novel means exploiting Keima, an acid-stable fluorescent protein possessing pH-dependent fluorescence excitation spectra, for precisely monitoring the autophagy-endolysosomal system. First, we generated three lines of transgenic (tg) mouse expressing monomeric Keima-fused MAP1LC3B (mKeima-LC3B). Then, these tg mice were subjected to starvation by food-restriction, and also challenged to neurodegeneration by genetically crossing with a mouse model of amyotrophic lateral sclerosis; i.e., SOD1H46R transgenic mouse. Unexpectedly, despite that a lipidated-form of endogenous LC3 (LC3-II) was significantly increased, those of mKeima-LC3B (mKeima-LC3B-II) were not changed under both stressed conditions. It was also noted that mKeima-LC3B-positive aggregates were progressively accumulated in the spinal cord of SOD1H46R;mKeima-LC3B double-tg mice, suggestive of acid-resistance and aggregate-prone natures of long-term overexpressed mKeima-LC3B in vivo. Next, we characterized mouse embryonic fibroblasts (MEFs) derived from mKeima-LC3B-tg mice. In contrast with in vivo, levels of mKeima-LC3B-I were decreased under starved conditions. Furthermore, when starved MEFs were treated with chloroquine (CQ), the abundance of mKeima-LC3B-II was significantly increased. Remarkably, when cultured medium was repeatedly changed between DMEM (nutrient-rich) and EBSS (starvation), acidic/neutral signal ratios of mKeima-LC3B-positive compartments were rapidly and reversibly shifted, which were suppressed by the CQ treatment, indicating that intraluminal pH of mKeima-LC3B-positive vesicles was changeable upon nutritional conditions of culture media. Taken together, although mKeima-LC3B-tg mice may not be an appropriate tool to monitor the autophagy-endolysosomal system in vivo, mKeima-LC3B must be one of the most sensitive reporter molecules for monitoring this system under in vitro cultured conditions.


Assuntos
Autofagia/fisiologia , Endossomos/metabolismo , Proteínas Luminescentes/genética , Lisossomos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Animais , Células Cultivadas , Meios de Cultura/farmacologia , Endossomos/genética , Feminino , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Humanos , Concentração de Íons de Hidrogênio , Proteínas Luminescentes/metabolismo , Lisossomos/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Inanição , Superóxido Dismutase-1/genética , Imagem com Lapso de Tempo
6.
Ann Rheum Dis ; 79(9): 1227-1233, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32482644

RESUMO

OBJECTIVES: Coactivators are a heterogeneous family of transcriptional regulators that are essential for modulation of transcriptional outcomes and fine-tune numerous cellular processes. The aim of the present study was to evaluate the role of the coactivator peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) in the pathogenesis of systemic sclerosis (SSc). METHODS: Expression of PGC-1α was analysed by real-time PCR, western blot and immunofluorescence. Modulation of autophagy was analysed by reporter studies by expression of autophagy-related genes. The effects of PGC-1α knockdown on collagen production and myofibroblast differentiation were analysed in cultured human fibroblasts and in two mouse models with fibroblast-specific knockout of PGC-1α. RESULTS: The expression of PGC-1α was induced in dermal fibroblasts of patients with SSc and experimental murine fibrosis. Transforming growth factor beta (TGFß), hypoxia and epigenetic mechanisms regulate the expression of PGC-1α in fibroblasts. Knockdown of PGC-1α prevented the activation of autophagy by TGFß and this translated into reduced fibroblast-to-myofibroblast differentiation and collagen release. Knockout of PGC-1α in fibroblasts prevented skin fibrosis induced by bleomycin and by overexpression of a constitutively active TGFß receptor type I. Moreover, pharmacological inhibition of PGC-1α by SR18292 induced regression of pre-established, bleomycin-induced skin fibrosis. CONCLUSION: PGC-1α is upregulated in SSc and promotes autophagy to foster TGFß-induced fibroblast activation. Targeting of PGC-1α prevents aberrant autophagy, inhibits fibroblast activation and tissue fibrosis and may over therapeutic potential.


Assuntos
Autofagia/genética , Fibroblastos/fisiologia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/fisiologia , Escleroderma Sistêmico/genética , Escleroderma Sistêmico/patologia , Animais , Bleomicina/farmacologia , Western Blotting , Colágeno/biossíntese , Modelos Animais de Doenças , Fibrose , Imunofluorescência , Humanos , Camundongos , Reação em Cadeia da Polimerase , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta/metabolismo , Regulação para Cima
7.
Adv Gerontol ; 33(1): 46-54, 2020.
Artigo em Russo | MEDLINE | ID: mdl-32362083

RESUMO

Short peptides are applied for supporting skin function during ageing, because they can permeate the intact stratum corneum of the epidermis and affect the cells of the dermis. Short peptides are part of natural metabolism of cells and many of them have geroprotective properties. In the review we are considering the base sorts of peptides that are used for normalized skin fibroblasts function: matrikines, carnosine, collagen peptides, cytokine and growth factor analogs, defensins, immunoprotective peptides and polyfunctional peptides. Polyfunctional peptides (AcSDKP, KED, AEDG, AED) have geroprotective properties, slow apoptosis and stimulate skin cell proliferation, also increase functional activity of skin fibroblasts, normalize intracellular matrix hemostasis. Polyfunctional peptides are the antioxidants and immunoprotectors and can activate microcirculation in dermis. Peptide regulation of skin function during ageing are the fast-developing and prospective area in molecular gerontology.


Assuntos
Derme/fisiologia , Peptídeos/fisiologia , Envelhecimento da Pele , Apoptose , Fibroblastos/fisiologia , Humanos
8.
Nat Commun ; 11(1): 2585, 2020 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-32444791

RESUMO

Cardiac maturation lays the foundation for postnatal heart development and disease, yet little is known about the contributions of the microenvironment to cardiomyocyte maturation. By integrating single-cell RNA-sequencing data of mouse hearts at multiple postnatal stages, we construct cellular interactomes and regulatory signaling networks. Here we report switching of fibroblast subtypes from a neonatal to adult state and this drives cardiomyocyte maturation. Molecular and functional maturation of neonatal mouse cardiomyocytes and human embryonic stem cell-derived cardiomyocytes are considerably enhanced upon co-culture with corresponding adult cardiac fibroblasts. Further, single-cell analysis of in vivo and in vitro cardiomyocyte maturation trajectories identify highly conserved signaling pathways, pharmacological targeting of which substantially delays cardiomyocyte maturation in postnatal hearts, and markedly enhances cardiomyocyte proliferation and improves cardiac function in infarcted hearts. Together, we identify cardiac fibroblasts as a key constituent in the microenvironment promoting cardiomyocyte maturation, providing insights into how the manipulation of cardiomyocyte maturity may impact on disease development and regeneration.


Assuntos
Fibroblastos/fisiologia , Infarto do Miocárdio/patologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/fisiologia , Fatores Etários , Animais , Animais Recém-Nascidos , Meios de Cultivo Condicionados/farmacologia , Feminino , Fibroblastos/citologia , Coração/crescimento & desenvolvimento , Humanos , Masculino , Camundongos Endogâmicos C57BL , Transdução de Sinais , Análise de Célula Única
9.
PLoS One ; 15(5): e0232899, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32392240

RESUMO

Various nanopatterning techniques have been developed to improve cell proliferation and differentiation efficiency. As we previously reported, nanopillars and pores are able to sustain human pluripotent stem cells and differentiate pancreatic cells. From this, the nanoscale patterns would be effective environment for the co-culturing of epithelial and mesenchymal cell types. Interestingly, the nanopatterning selectively reduced the proliferative rate of mesenchymal cells while increasing the expression of adhesion protein in epithelial type cells. Additionally, co-cultured cells on the nanopatterning were not negatively affected in terms of cell function metabolic ability or cell survival. This is in contrast to conventional co-culturing methods such as ultraviolet or chemical treatments. The nanopatterning appears to be an effective environment for mesenchymal co-cultures with typically low proliferative rates cells such as astrocytes, neurons, melanocytes, and fibroblasts without using potentially damaging treatments.


Assuntos
Técnicas de Cocultura/instrumentação , Células Epiteliais , Células-Tronco Mesenquimais , Nanoestruturas , Animais , Adesão Celular , Proliferação de Células , Sobrevivência Celular , Células Epiteliais/citologia , Células Epiteliais/fisiologia , Fibroblastos/citologia , Fibroblastos/fisiologia , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/fisiologia , Camundongos , Propriedades de Superfície
10.
Res Vet Sci ; 131: 222-231, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32413795

RESUMO

Skin lesions are normal to all species, regardless of gender or age. The skin, the largest organ of the body, has function as a primary barrier to the chemical, physical and biological aggressions of the environment. In animals, these lesions may be due to fights and/or predations, also as in humans, there is a very common cause of dermal lesions that are caused by burns and carcinomas. Looking for new techniques of tissue bioengineering, studies have been shown promising results for formulations of acellular biological scaffolds from tissue decellularization for the reconstitution of these lesions. The decellularization has its proof by a varied range of tests such as scanning electron microscopy and residual genomic DNA tests. Subsequently the tissue can go through the process of recellularization using cells of interest, even the animal that will receive this tissue, reducing the risks of rejection and improving the response to tissue transplantation. Thus, this manuscript aimed at the decellularization of the tissue with the use of chemical and physical means followed by sterilization and the establishment of a protocol for the recellularization of a decellularized scaffold from the Wistar rat dermis using murine fibroblasts and mesenchymal stem cells from canine adipose tissue for 7 days. After efficacy tests, the tissue recellularization were confirmed by immunofluorescence assays and scanning electron microscopy where the adherence of the cells in the biological scaffold was observed.


Assuntos
Derme , Fibroblastos/fisiologia , Células-Tronco Mesenquimais/fisiologia , Engenharia Tecidual/métodos , Tecidos Suporte , Animais , Cães , Camundongos , Ratos , Ratos Wistar
11.
Gene ; 743: 144592, 2020 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-32198125

RESUMO

FOXOs transcription factors not only play key roles in glucose metabolism, muscle atrophy and energy homeostasis but also play crucial transcriptional regulatory roles in the cell's metabolism, orchestrating programs of gene expression that regulate cell apoptosis, cell-cycle progression and oxidative stress resistance. However, the specific function of FOXOs promoting fibroblasts proliferation and apoptosis are still unknown. Thus, we used the High-Resolution Melting (HRM) and RNA interference methods to detect SNPs and function. We found one SNP in the exon of FOXO1, three SNPs were identified in the exon of FOXO3, and three SNPs and production traits were significantly different. The siRNA sequence of yak FOXO1 and FOXO3 were transfected into the yak fibroblasts, and effects were detected by a series of assays to reveal the function in yak fibroblasts. The results demonstrated that down-regulated expression of FOXO1 and FOXO3 resulted in up-regulated the expression of BAX, Caspase9 and Caspase3, and down-regulated the expression level of anti-apoptotic gene of BCL2. The apoptotic situation was consistent with results of the flow cytometry and Tunel test cell cycle and cell vitality results revealed that knockdown FOXO1 and FOXO3 resulted in increased P27 expression level and decreased CyclinD1. Meanwhile, cell vitality was also decreased. These results demonstrated that FOXO1 and FOXO3 are two novel regulatory factors to suppress cells proliferation and promote cells apoptosis. Furthermore, these results provide evidence that FOXO1 and FOXO3 play a functional role in cell apoptosis.


Assuntos
Proteínas Reguladoras de Apoptose/genética , Fibroblastos/fisiologia , Proteína Forkhead Box O1/metabolismo , Proteína Forkhead Box O3/metabolismo , Regulação da Expressão Gênica , Animais , Apoptose/genética , Bovinos , Proliferação de Células/genética , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O3/genética , Técnicas de Silenciamento de Genes , Masculino , Polimorfismo de Nucleotídeo Único , Interferência de RNA , Transdução de Sinais/genética
12.
J Med Chem ; 63(8): 3896-3907, 2020 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-32191456

RESUMO

The critical consequences of human cytomegalovirus (HCMV) infection in the transplant population and in congenitally infected infants, the limited treatment options for HCMV, and the rise of resistant mutants toward existing therapies has fueled the search for new anti-HCMV agents. A pp28-luciferase recombinant HCMV was used as a reporter system for high-throughput screening of HCMV inhibitors. Approximately 400 000 compounds from existing libraries were screened. Subsequent validation assays using resynthesized compounds, several virus strains, and detailed virology assays resulted in the identification of five structurally unique and selective HCMV inhibitors, active at sub to low micromolar concentrations. Further characterization revealed that each compound inhibited a specific stage of HCMV replication. One compound was also active against herpes simplex virus (HSV1 and HSV2), and another compound was active against Epstein-Barr virus (EBV). Drug combination studies revealed that all five compounds were additive with ganciclovir or letermovir. Future studies will focus on optimization of these new anti-HCMV compounds along with mechanistic studies.


Assuntos
Antivirais/química , Antivirais/farmacologia , Citomegalovirus/efeitos dos fármacos , Descoberta de Drogas/métodos , Animais , Antivirais/uso terapêutico , Células Cultivadas , Citomegalovirus/fisiologia , Infecções por Citomegalovirus/tratamento farmacológico , Infecções por Citomegalovirus/fisiopatologia , Relação Dose-Resposta a Droga , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Fibroblastos/virologia , Humanos , Masculino , Camundongos
13.
Nat Protoc ; 15(3): 750-772, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32051617

RESUMO

Single-cell technologies are offering unparalleled insight into complex biology, revealing the behavior of rare cell populations that are masked in bulk population analyses. One current limitation of single-cell approaches is that lineage relationships are typically lost as a result of cell processing. We recently established a method, CellTagging, permitting the parallel capture of lineage information and cell identity via a combinatorial cell indexing approach. CellTagging integrates with high-throughput single-cell RNA sequencing, where sequential rounds of cell labeling enable the construction of multi-level lineage trees. Here, we provide a detailed protocol to (i) generate complex plasmid and lentivirus CellTag libraries for labeling of cells; (ii) sequentially CellTag cells over the course of a biological process; (iii) profile single-cell transcriptomes via high-throughput droplet-based platforms; and (iv) generate a CellTag expression matrix, followed by clone calling and lineage reconstruction. This lentiviral-labeling approach can be deployed in any organism or in vitro culture system that is amenable to viral transduction to simultaneously profile lineage and identity at single-cell resolution.


Assuntos
Linhagem da Célula , Rastreamento de Células/métodos , Fibroblastos/fisiologia , Animais , Linhagem Celular , Escherichia coli , Regulação da Expressão Gênica , Humanos , Camundongos
14.
Science ; 367(6479): 800-806, 2020 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-32054765

RESUMO

Circadian (~24 hour) clocks have a fundamental role in regulating daily physiology. The transcription factor BMAL1 is a principal driver of a molecular clock in mammals. Bmal1 deletion abolishes 24-hour activity patterning, one measure of clock output. We determined whether Bmal1 function is necessary for daily molecular oscillations in skin fibroblasts and liver slices. Unexpectedly, in Bmal1 knockout mice, both tissues exhibited 24-hour oscillations of the transcriptome, proteome, and phosphoproteome over 2 to 3 days in the absence of any exogenous drivers such as daily light or temperature cycles. This demonstrates a competent 24-hour molecular pacemaker in Bmal1 knockouts. We suggest that such oscillations might be underpinned by transcriptional regulation by the recruitment of ETS family transcription factors, and nontranscriptionally by co-opting redox oscillations.


Assuntos
Fatores de Transcrição ARNTL/genética , Fatores de Transcrição ARNTL/fisiologia , Relógios Circadianos/genética , Ritmo Circadiano/genética , Fígado/fisiologia , Fenômenos Fisiológicos da Pele , Animais , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Deleção de Genes , Regulação da Expressão Gênica , Fígado/metabolismo , Camundongos , Camundongos Knockout , Fosfoproteínas/metabolismo , Proteoma/metabolismo , Proteoma/fisiologia , Transcrição Genética , Transcriptoma/fisiologia
15.
Invest Ophthalmol Vis Sci ; 61(2): 28, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-32084275

RESUMO

Purpose: This review highlights the roles of fibrocytes-their origin, markers, regulation and functions-including contributions to corneal wound healing and fibrosis. Methods: Literature review. Results: Peripheral blood fibroblast-like cells, called fibrocytes, are primarily generated as mature collagen-producing cells in the bone marrow. They are likely derived from the myeloid lineage, although the exact precursor remains unknown. Fibrocytes are identified by a combination of expressed markers, such as simultaneous expression of CD34 or CD45 or CD11b and collagen type I or collagen type III. Fibrocytes migrate into the wound from the blood where they participate in pathogen clearance, tissue regeneration, wound closure and angiogenesis. Transforming growth factor beta 1 (TGF-ß1) and adiponectin induce expression of α-smooth muscle actin and extracellular matrix proteins through activation of Smad3 and adenosine monophosphate-activated protein kinase pathways, respectively. Fibrocytes are important contributors to the cornea wound healing response and there are several mechanisms through which fibrocytes contribute to fibrosis in the cornea and other organs, such as their differentiation into myofibroblasts, production of matrix metalloproteinase, secretion of tissue inhibitor of metalloproteinase, and release of TGF-ß1. In some tissues, fibrocytes may also contribute to the basement membrane regeneration and to the resolution of fibrosis. Conclusions: New methods that block fibrocyte generation, fibrocyte migration, and their differentiation into myofibroblasts, as well as their production of matrix metalloproteinases, tissue inhibitor of metalloproteinase, and TGF-ß1, have therapeutic potential to reduce the accumulation of collagens, maintain tissue integrity and retard or prevent the development of fibrosis.


Assuntos
Lesões da Córnea , Fibroblastos/fisiologia , Miofibroblastos/fisiologia , Cicatrização/fisiologia , Diferenciação Celular/fisiologia , Movimento Celular/fisiologia , Lesões da Córnea/metabolismo , Lesões da Córnea/patologia , Proteínas da Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Fibrose/metabolismo , Humanos , Fator de Crescimento Transformador beta1/metabolismo
16.
Development ; 147(4)2020 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-31988189

RESUMO

Cellular proliferation is a basic process during organ development, tissue homeostasis and disease progression. Likewise, after injury typically multiple cell lineages respond to various cues and proliferate to initiate repair and/or remodeling of the injured tissue. Unravelling the specific role of proliferation of one cell type and its lineage in the context of the whole organism during tissue regeneration and/or disease progression would provide valuable information on these processes. Here, we report a new genetic system that allows cell proliferation to be inhibited in a tissue-specific manner. We generated Cre- or Dre-inducible p21-GFP (ip21-GFP) transgenic mice that enable experimentally induced permanent cell cycle arrest of specific cell lineages of interest, while genetically marking these cells. This system allows for the inhibition of pathogenic cell proliferation. We found that cardiac fibroblast proliferation inhibition significantly reduced scar formation, and promoted neovascularization and cardiomyocyte survival. Additionally, we found that inhibition of one type of cell proliferation (namely, hepatocytes) induces the lineage conversion of another type cells (i.e. ductal cells) during tissue regeneration. These results validate the use of ip21-GFP mice as a new genetic tool for cell lineage-specific inhibition of cell proliferation in vivo.


Assuntos
Proliferação de Células , Regulação da Expressão Gênica , Técnicas Genéticas , Alelos , Animais , Linhagem da Célula , Inibidor de Quinase Dependente de Ciclina p21/fisiologia , Feminino , Fibroblastos/fisiologia , Proteínas de Fluorescência Verde , Coração/crescimento & desenvolvimento , Coração/fisiologia , Hepatócitos/citologia , Hepatócitos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Infarto do Miocárdio/patologia , Miócitos Cardíacos/citologia
17.
Neurology ; 94(5): e474-e480, 2020 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-31892634

RESUMO

OBJECTIVE: Varicella zoster virus (VZV) can spread anterogradely and infect cerebral arteries causing VZV vasculopathy and arterial ischemic stroke. In this study, we tested the hypothesis that virus-infected cerebrovascular fibroblasts undergo phenotypic changes that promote vascular remodeling and facilitate virus transmission in an in vitro model of VZV vasculopathy. The aims of this project were therefore to examine the changes that virus-infected human brain adventitial vascular fibroblasts (HBVAFs) undergo in an in vitro model of VZV vasculopathy and to identify disease biomarkers relating to VZV-related vasculopathy. METHODS: HBVAFs were infected with VZV, and their ability to migrate, proliferate, transdifferentiate, and interact with endothelial cells was studied with flow cytometry. Microparticles (MPs) released from these cells were isolated and imaged with transmission electron microscopy, and their protein content was analyzed with mass spectrometry. Circulating MP profiles were also studied in children with VZV and non-VZV vasculopathy and compared with controls. RESULTS: VZV-infected HBVAFs transdifferentiated into myofibroblasts with enhanced proliferative and migratory capacity. Interaction of VZV-infected HBVAFs with endothelial cells resulted in endothelial dysfunction. These effects were, in part, mediated by the release of MPs from VZV-infected HBVAFs. These MPs contained VZV virions that could transmit VZV to neighboring cells, highlighting a novel model of VZV cell-to-cell viral dissemination. MPs positive for VZV were significantly higher in children with VZV-related vasculopathy compared to children with non-VZV vasculopathy (p = 0.01) and controls (p = 0.007). CONCLUSIONS: VZV-infected HBVAFs promote vascular remodeling and facilitate virus transmission. These effects were mediated by the release of apoptotic MPs that could transmit VZV infection to neighboring cells through a Trojan horse means of productive viral infection. VZV+ MPs may represent a disease biomarker worthy of further study.


Assuntos
Movimento Celular , Proliferação de Células , Transdiferenciação Celular , Micropartículas Derivadas de Células/virologia , Transtornos Cerebrovasculares/virologia , Fibroblastos/virologia , Miofibroblastos/virologia , Remodelação Vascular , Adolescente , Túnica Adventícia , Micropartículas Derivadas de Células/ultraestrutura , Artérias Cerebrais , Transtornos Cerebrovasculares/fisiopatologia , Criança , Pré-Escolar , Células Endoteliais , Feminino , Fibroblastos/fisiologia , Fibroblastos/ultraestrutura , Herpesvirus Humano 3 , Humanos , Técnicas In Vitro , Masculino , Espectrometria de Massas , Microscopia Eletrônica de Transmissão , Miofibroblastos/fisiologia , Miofibroblastos/ultraestrutura , Acidente Vascular Cerebral/fisiopatologia , Acidente Vascular Cerebral/virologia , Cultura de Vírus
18.
Am J Physiol Renal Physiol ; 318(3): F689-F701, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31928224

RESUMO

Acute kidney injury (AKI) is a highly prevalent medical syndrome associated with high mortality and morbidity. Several types of cells, including epithelial cells, vascular endothelial cells, pericytes, and macrophages, participate in the development of AKI. Recently, renal fibroblasts were found to play an important role in the regulation of tubular injury, repair, and recovery after AKI. However, the mechanisms underlying fibroblast activation and proliferation during the progression of AKI remain unclear. In the present study, we found many activated myofibroblasts located in the renal interstitium with an abundance of extracellular matrix deposition following folic acid-induced AKI. The proliferative pattern of tubular epithelial cells and interstitial cells following acute injury was different, indicating that the proliferation of fibroblasts followed the proliferation of tubular epithelial cells. Furthermore, we observed that proliferative tubular epithelial cells preferred aerobic glycolysis as the dominating metabolic pathway in the progression of AKI. Lactate generated from injured tubules was taken up by interstitial fibroblasts in the later stages of AKI, which induced fibroblast activation and proliferation in vitro. Early inhibition of lactate production in tubules by glycolytic inhibitors suppressed fibroblast activation after folic acid-induced injury. Collectively, these results support the important role of fibroblasts in the development of AKI and suggest that lactate produced by glycolysis in tubular epithelial cells is a novel regulator of fibroblast activation and proliferation.


Assuntos
Lesão Renal Aguda/metabolismo , Fibroblastos/fisiologia , Túbulos Renais/metabolismo , Lactatos/metabolismo , Animais , Apoptose , Nitrogênio da Ureia Sanguínea , Linhagem Celular , Creatinina/sangue , Hepatócitos , Lactatos/sangue , Lipocalina-2/urina , Masculino , Camundongos
19.
FASEB J ; 34(1): 386-398, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31914653

RESUMO

To date, there is no direct evidence of telomerase activity in adult lung epithelial cells, but typical culture conditions only support cell proliferation for 30-40 population doublings (PD), a point at which telomeres remain relatively long. Here we report that in in vitro low stress culture conditions consisting of a fibroblast feeder layer, rho-associated coiled coil protein kinase inhibitor (ROCKi), and low oxygen (2%), normal human bronchial epithelial basal progenitor cells (HBECs) divide for over 200 PD without engaging a telomere maintenance mechanism (almost four times the "Hayflick limit"). HBECs exhibit critically short telomeres at 200 PD and the population of cells start to undergo replicative senescence. Subcloning these late passage cells to clonal density, to mimic lung injury in vivo, selects for rare subsets of HBECs that activate low levels of telomerase activity to maintain short telomeres. CRISPR/Cas9 knockout of human telomerase reverse transcriptase or treatment with the telomerase-mediated telomere targeting agent 6-thio-2'deoxyguanosine abrogates colony growth in these late passage cultures (>200 PD) but not in early passage cultures (<200 PD). To our knowledge, this is the first study to report such long-term growth of HBECs without a telomere maintenance mechanism. This report also provides direct evidence of telomerase activation in HBECs near senescence when telomeres are critically short. This novel cell culture system provides an experimental model to understand how telomerase is regulated in normal adult tissues.


Assuntos
Brônquios/citologia , Técnicas de Cultura de Células/métodos , Proliferação de Células , Senescência Celular , Células Epiteliais/citologia , Fibroblastos/citologia , Telômero/fisiologia , Adulto , Brônquios/fisiologia , Divisão Celular , Células Cultivadas , Células Epiteliais/fisiologia , Fibroblastos/fisiologia , Humanos , Telomerase/metabolismo , Encurtamento do Telômero
20.
Ann Neurol ; 87(2): 217-232, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31794073

RESUMO

OBJECTIVE: Recently, the ASC-1 complex has been identified as a mechanistic link between amyotrophic lateral sclerosis and spinal muscular atrophy (SMA), and 3 mutations of the ASC-1 gene TRIP4 have been associated with SMA or congenital myopathy. Our goal was to define ASC-1 neuromuscular function and the phenotypical spectrum associated with TRIP4 mutations. METHODS: Clinical, molecular, histological, and magnetic resonance imaging studies were made in 5 families with 7 novel TRIP4 mutations. Fluorescence activated cell sorting and Western blot were performed in patient-derived fibroblasts and muscles and in Trip4 knocked-down C2C12 cells. RESULTS: All mutations caused ASC-1 protein depletion. The clinical phenotype was purely myopathic, ranging from lethal neonatal to mild ambulatory adult patients. It included early onset axial and proximal weakness, scoliosis, rigid spine, dysmorphic facies, cutaneous involvement, respiratory failure, and in the older cases, dilated cardiomyopathy. Muscle biopsies showed multiminicores, nemaline rods, cytoplasmic bodies, caps, central nuclei, rimmed fibers, and/or mild endomysial fibrosis. ASC-1 depletion in C2C12 and in patient-derived fibroblasts and muscles caused accelerated proliferation, altered expression of cell cycle proteins, and/or shortening of the G0/G1 cell cycle phase leading to cell size reduction. INTERPRETATION: Our results expand the phenotypical and molecular spectrum of TRIP4-associated disease to include mild adult forms with or without cardiomyopathy, associate ASC-1 depletion with isolated primary muscle involvement, and establish TRIP4 as a causative gene for several congenital muscle diseases, including nemaline, core, centronuclear, and cytoplasmic-body myopathies. They also identify ASC-1 as a novel cell cycle regulator with a key role in cell proliferation, and underline transcriptional coregulation defects as a novel pathophysiological mechanism. ANN NEUROL 2020;87:217-232.


Assuntos
Sistema y+ de Transporte de Aminoácidos/fisiologia , Ciclo Celular/fisiologia , Doenças Musculares/fisiopatologia , Fatores de Transcrição/genética , Adulto , Sistema y+ de Transporte de Aminoácidos/metabolismo , Células Cultivadas , Criança , Pré-Escolar , Feminino , Fibroblastos/fisiologia , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Proteínas Musculares/genética , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Doenças Musculares/genética , Mutação , Linhagem , Fenótipo
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