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1.
Nat Commun ; 12(1): 2951, 2021 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-34012031

RESUMO

The muscular dystrophies encompass a broad range of pathologies with varied clinical outcomes. In the case of patients carrying defects in fukutin-related protein (FKRP), these diverse pathologies arise from mutations within the same gene. This is surprising as FKRP is a glycosyltransferase, whose only identified function is to transfer ribitol-5-phosphate to α-dystroglycan (α-DG). Although this modification is critical for extracellular matrix attachment, α-DG's glycosylation status relates poorly to disease severity, suggesting the existence of unidentified FKRP targets. Here we reveal that FKRP directs sialylation of fibronectin, a process essential for collagen recruitment to the muscle basement membrane. Thus, our results reveal that FKRP simultaneously regulates the two major muscle-ECM linkages essential for fibre survival, and establishes a new disease axis for the muscular dystrophies.


Assuntos
Fibronectinas/metabolismo , Glicosiltransferases/metabolismo , Distrofias Musculares/metabolismo , Distrofias Musculares/patologia , Distrofia Muscular Animal/metabolismo , Distrofia Muscular Animal/patologia , Pentosiltransferases/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Membrana Basal/metabolismo , Membrana Basal/patologia , Linhagem Celular , Modelos Animais de Doenças , Técnicas de Inativação de Genes , Glicosilação , Glicosiltransferases/deficiência , Glicosiltransferases/genética , Humanos , Masculino , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Distrofias Musculares/genética , Distrofia Muscular do Cíngulo dos Membros/genética , Distrofia Muscular do Cíngulo dos Membros/metabolismo , Distrofia Muscular do Cíngulo dos Membros/patologia , Distrofia Muscular Animal/genética , Mutação , Mioblastos Esqueléticos/metabolismo , Mioblastos Esqueléticos/patologia , Pentosiltransferases/deficiência , Pentosiltransferases/genética , Fenótipo , Peixe-Zebra , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genética
2.
Int J Mol Sci ; 22(9)2021 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-33946948

RESUMO

Neurodegenerative diseases are characterized by increased permeability of the blood-brain barrier (BBB) due to alterations in cellular and structural components of the neurovascular unit, particularly in association with neuroinflammation. A previous screening study of peptide ligands to identify molecular alterations of the BBB in neuroinflammation by phage-display, revealed that phage clone 88 presented specific binding affinity to endothelial cells under inflammatory conditions in vivo and in vitro. Here, we aimed to identify the possible target receptor of the peptide ligand 88 expressed under inflammatory conditions. A cross-link test between phage-peptide-88 with IL-1ß-stimulated human hCMEC cells, followed by mass spectrometry analysis, was used to identify the target of peptide-88. We modeled the epitope-receptor molecular interaction between peptide-88 and its target by using docking simulations. Three proteins were selected as potential target candidates and tested in enzyme-linked immunosorbent assays with peptide-88: fibronectin, laminin subunit α5 and laminin subunit ß-1. Among them, only laminin subunit ß-1 presented measurable interaction with peptide-88. Peptide-88 showed specific interaction with laminin subunit ß-1, highlighting its importance as a potential biomarker of the laminin changes that may occur at the BBB endothelial cells under pathological inflammation conditions.


Assuntos
Barreira Hematoencefálica , Células Endoteliais/metabolismo , Inflamação/metabolismo , Laminina/metabolismo , Animais , Bacteriófago M13 , Biomarcadores , Células Cultivadas , Reagentes para Ligações Cruzadas , Fibronectinas/metabolismo , Ontologia Genética , Humanos , Interleucina-1beta/farmacologia , Modelos Moleculares , Simulação de Acoplamento Molecular , Biblioteca de Peptídeos , Ligação Proteica , Conformação Proteica , Mapeamento de Interação de Proteínas , Ratos
3.
Int J Mol Sci ; 22(5)2021 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-33800150

RESUMO

Celiac disease (CD) is a frequent intestinal inflammatory disease occurring in genetically susceptible individuals upon gluten ingestion. Recent studies point to a role in CD for genes involved in cell shape, adhesion and actin rearrangements, including a Rho family regulator, Rho GTPase-activating protein 31 (ARHGAP31). In this study, we investigated the morphology and actin cytoskeletons of peripheral monocyte-derived dendritic cells (DCs) from children with CD and controls when in contact with a physiological substrate, fibronectin. DCs were generated from peripheral blood monocytes of pediatric CD patients and controls. After adhesion on fibronectin, DCs showed a higher number of protrusions and a more elongated shape in CD patients compared with controls, as assessed by immunofluorescence actin staining, transmitted light staining and video time-lapse microscopy. These alterations did not depend on active intestinal inflammation associated with gluten consumption and were specific to CD, since they were not found in subjects affected by other intestinal inflammatory conditions. The elongated morphology was not a result of differences in DC activation or maturation status, and did not depend on the human leukocyte antigen (HLA)-DQ2 haplotype. Notably, we found that ARH-GAP31 mRNA levels were decreased while RhoA-GTP activity was increased in CD DCs, pointing to an impairment of the Rho pathway in CD cells. Accordingly, Rho inhibition was able to prevent the cytoskeleton rearrangements leading to the elongated morphology of celiac DCs upon adhesion on fibronectin, confirming the role of this pathway in the observed phenotype. In conclusion, adhesion on fibronectin discriminated CD from the controls' DCs, revealing a gluten-independent CD-specific cellular phenotype related to DC shape and regulated by RhoA activity.


Assuntos
Actinas/metabolismo , Doença Celíaca/metabolismo , Forma Celular , Células Dendríticas/imunologia , Monócitos/metabolismo , Doença Celíaca/patologia , Adesão Celular , Criança , Pré-Escolar , Células Dendríticas/patologia , Feminino , Fibronectinas/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Antígenos HLA-DQ/metabolismo , Humanos , Masculino , Monócitos/patologia , Fosfoproteínas/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo
4.
Int J Mol Sci ; 22(6)2021 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-33805743

RESUMO

Idiopathic pulmonary fibrosis (IPF) is a lethal age-related lung disease whose pathogenesis involves an aberrant response of alveolar epithelial cells (AEC). Activated epithelial cells secrete mediators that participate in the activation of fibroblasts and the excessive deposition of extracellular matrix proteins. Previous studies indicate that matrix metalloproteinase 14 (MMP14) is increased in the lung epithelium in patients with IPF, however, the role of this membrane-type matrix metalloproteinase has not been elucidated. In this study, the role of Mmp14 was explored in experimental lung fibrosis induced with bleomycin in a conditional mouse model of lung epithelial MMP14-specific genetic deletion. Our results show that epithelial Mmp14 deficiency in mice increases the severity and extension of fibrotic injury and affects the resolution of the lesions. Gain-and loss-of-function experiments with human epithelial cell line A549 demonstrated that cells with a deficiency of MMP14 exhibited increased senescence-associated markers. Moreover, conditioned medium from these cells increased fibroblast expression of fibrotic molecules. These findings suggest a new anti-fibrotic mechanism of MMP14 associated with anti-senescent activity, and consequently, its absence results in impaired lung repair. Increased MMP14 in IPF may represent an anti-fibrotic mechanism that is overwhelmed by the strong profibrotic microenvironment that characterizes this disease.


Assuntos
Células Epiteliais/patologia , Fibrose Pulmonar Idiopática/genética , Metaloproteinase 14 da Matriz/genética , Alvéolos Pulmonares/metabolismo , Células A549 , Actinas/genética , Actinas/metabolismo , Animais , Bleomicina/administração & dosagem , Senescência Celular/genética , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Modelos Animais de Doenças , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Fibronectinas/genética , Fibronectinas/metabolismo , Regulação da Expressão Gênica , Humanos , Hipoxantina Fosforribosiltransferase/genética , Hipoxantina Fosforribosiltransferase/metabolismo , Fibrose Pulmonar Idiopática/induzido quimicamente , Fibrose Pulmonar Idiopática/metabolismo , Fibrose Pulmonar Idiopática/patologia , Metaloproteinase 14 da Matriz/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Cultura Primária de Células , Alvéolos Pulmonares/efeitos dos fármacos , Alvéolos Pulmonares/patologia , Succinato Desidrogenase/genética , Succinato Desidrogenase/metabolismo , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo
5.
DNA Cell Biol ; 40(5): 694-705, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33751897

RESUMO

Several studies have reported that miR-885-5p was dysregulated in a variety of cancers. However, there are few studies on the biological function of miR-885-5p in gastric cancer (GC). In this study, we investigated the biological function and underlying mechanism of miR-885-5p in GC. Quantitative real-time PCR was used to examine the expression of miR-885-5p in GC. Bioinformatics analysis was used to predict the target of miR-885-5p and confirmed using the luciferase reporter assay. Wound-healing and Transwell assay were conducted to evaluate the biological function of miR-885-5p and malic enzyme 1 (ME1). Western blotting was used to assess molecular changes. Hepatic and lung metastasis models were constructed and used to verify the role of miR-885-5p. We found that the expression of miR-885-5p was significantly downregulated in GC. Overexpression of miR-885-5p inhibited invasion and metastasis of GC in vivo and in vitro, while inhibition of miR-885-5p has the opposite result in vitro. ME1 is a direct target of miR-885-5p, overexpressed in GC, associated with poor prognosis. Overexpression of miR-885-5p negatively regulates ME1 and causes changes in downstream molecules Vimentin and Fibronectin. Our research found that miR-885-5p plays a tumor suppressor gene and could potentially serve as a biomarker and therapeutic target in GC.


Assuntos
Malato Desidrogenase/metabolismo , MicroRNAs/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Animais , Sequência de Bases , Linhagem Celular Tumoral , Movimento Celular/genética , Regulação para Baixo/genética , Feminino , Fibronectinas/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Metástase Linfática/patologia , Masculino , Camundongos Nus , MicroRNAs/genética , Pessoa de Meia-Idade , Invasividade Neoplásica , Prognóstico , Regulação para Cima/genética , Vimentina/metabolismo
6.
J Psychiatr Res ; 136: 173-183, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33607579

RESUMO

Various exercise-training types are known to prevent depression, but mechanisms underlying their beneficial effects remain unknown. In the present study, the preconditioning effect of continuous and interval exercise on stress-induced depression was evaluated. Adult male Wistar rats in the exercise groups were made to run on a motorized treadmill, five sessions per week for six weeks. After that, to induce the depression model, the rats were exposed to chronic unpredictable stress for three weeks. Behavioral tests were assessed by open field, elevated plus maze, and forced swim tests. Hippocampal PGC-1α, FNDC5, and BDNF protein expression by Western blot and serum corticosterone by ELISA were detected. In the present results, after continuous and interval exercise periods, locomotor activity, the number of entries and time spent in the open arms were increased, and immobility time was significantly reduced. PGC-1α, FNDC5, and BDNF protein levels had a significant increase, and serum corticosterone did not change. Also, interval exercise training increased PGC-1α and FNDC5 more than continuous. Chronic unpredictable stress reduced the positive changes caused by exercise training, although, except FNDC5, exercise preconditioned groups experienced less significant adverse changes in most variables. These findings showed that both continuous and interval exercise preconditioning with increasing hippocampal PGC-1α, FNDC5, and BDNF proteins and improve the anxiety- and depression-like behaviors have a protective effect against chronic unpredictable stress.


Assuntos
Fator Neurotrófico Derivado do Encéfalo , Fibronectinas , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Fibronectinas/metabolismo , Hipocampo/metabolismo , Masculino , Ratos , Ratos Wistar , Fatores de Transcrição/metabolismo
7.
Nat Commun ; 12(1): 1140, 2021 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-33602902

RESUMO

Clostridioides difficile spores produced during infection are important for the recurrence of the disease. Here, we show that C. difficile spores gain entry into the intestinal mucosa via pathways dependent on host fibronectin-α5ß1 and vitronectin-αvß1. The exosporium protein BclA3, on the spore surface, is required for both entry pathways. Deletion of the bclA3 gene in C. difficile, or pharmacological inhibition of endocytosis using nystatin, leads to reduced entry into the intestinal mucosa and reduced recurrence of the disease in a mouse model. Our findings indicate that C. difficile spore entry into the intestinal barrier can contribute to spore persistence and infection recurrence, and suggest potential avenues for new therapies.


Assuntos
/fisiologia , Infecções por Clostridium/microbiologia , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Intestinos/microbiologia , Intestinos/patologia , Esporos Bacterianos/fisiologia , Animais , Aderência Bacteriana/efeitos dos fármacos , Proteínas de Bactérias/metabolismo , Linhagem Celular , /ultraestrutura , Colágeno/metabolismo , Endocitose , Células Epiteliais/ultraestrutura , Feminino , Fibronectinas/metabolismo , Humanos , Integrinas/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Masculino , Camundongos Endogâmicos C57BL , Nistatina/farmacologia , Ligação Proteica/efeitos dos fármacos , Recidiva , Esporos Bacterianos/efeitos dos fármacos , Esporos Bacterianos/ultraestrutura , Ácido Taurocólico/farmacologia , Vitronectina/metabolismo
8.
Medicine (Baltimore) ; 100(5): e24061, 2021 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-33592858

RESUMO

ABSTRACT: Irisin, a novel myokine, is believed to be the crucial factor in converting white adipose tissue to beige adipose tissue. For this paper, we studied the relationship among irisin and components of metabolic syndrome (MetS), and insulin secretion and resistance in schoolchildren of Taiwan.Subjects receiving routine annual health examination at elementary school were enrolled. Demographic data, anthropometry, MetS components, irisin, and insulin secretion and resistance were collected. Subjects were divided into normal, overweight, and obese groups for evaluation of irisin in obesity. Finally, the relationship between irisin and MetS was analyzed.There were 376 children (179 boys and 197 girls), aged 10.3 ±â€Š1.5 years, were enrolled. In boys, irisin levels were not associated with body mass index percentile, body fat, blood pressure, lipid profiles, insulin secretion or resistance. After adjusting for age, the irisin level in boys was negatively related to fasting plasma glucose (FPG) (r = -0.21, P = .006). In girls, after adjusting for age, the irisin levels were positively related only to FPG (r = 1.49, P = .038). In both genders, irisin levels were similar among normal, overweight, and obese groups, and between subjects with and without MetS.The irisin levels were not associated with MetS in either boys or girls. In girls, circulating irisin levels have a nonsignificant declining trend in overweight and obese girls. However, irisin levels were negatively related to FPG in boys and positively related to FPG in girls. The contrary relationship between irisin and FPG in boys and girls needs further exploration.


Assuntos
Tecido Adiposo/metabolismo , Fibronectinas , Secreção de Insulina/fisiologia , Insulina , Síndrome Metabólica , Sobrepeso , Antropometria/métodos , Determinação da Pressão Arterial/métodos , Índice de Massa Corporal , Criança , Estudos Transversais , Feminino , Fibronectinas/sangue , Fibronectinas/metabolismo , Humanos , Insulina/sangue , Insulina/metabolismo , Resistência à Insulina/fisiologia , Masculino , Síndrome Metabólica/sangue , Síndrome Metabólica/diagnóstico , Síndrome Metabólica/epidemiologia , Obesidade/diagnóstico , Obesidade/epidemiologia , Obesidade/metabolismo , Sobrepeso/diagnóstico , Sobrepeso/epidemiologia , Sobrepeso/metabolismo , Serviços de Saúde Escolar/estatística & dados numéricos , Taiwan/epidemiologia
9.
BMC Cancer ; 21(1): 156, 2021 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-33579227

RESUMO

BACKGROUND: Matricellular glycoprotein, SPARC is a secreted molecule, that mediates the interaction between cells and extracellular matrix. SPARC functions as a regulator of matrix organization and modulates cell behavior. In various kinds of cancer, strong SPARC expression was observed in stromal tissues as well as in cancer epithelial cells. The function of SPARC in cancer cells is somewhat controversial and its impact on peritumoral stromal cells remains to be resolved. METHODS: We investigated the effects of SPARC expression in endometrial cancer cells on the surrounding stromal fibroblasts using in vitro co-culture system. Changes in characteristics of fibroblasts were examined by analysis of fibroblast-specific markers and in vitro contraction assay. RESULTS: SPARC induced AKT phosphorylation and epithelial-to-mesenchymal transition, consistent with previous reports. Cancer-associated fibroblasts of endometrial cancer expressed higher levels of mesenchymal- and fibroblast-associated factors and had a stronger contraction ability. Unexpectedly, cancer-associated fibroblasts expressed comparable levels of SPARC compared with fibroblasts from normal endometrium. However, co-culture of normal fibroblasts with SPARC-expressing Ishikawa cells resulted in activation of the fibroblasts. Immunodepletion of SPARC did not affect the activation of fibroblasts. CONCLUSIONS: Our data indicated that SPARC activated fibroblasts only in the presence of fibronectin, which was abundantly secreted from SPARC-expressing endometrial cancer cells. These results suggested that a SPARC-fibronectin-mediated activation of fibroblasts might be involved in enhanced mobility and invasion of cancer cells.


Assuntos
Movimento Celular , Neoplasias do Endométrio/patologia , Transição Epitelial-Mesenquimal , Fibroblastos/metabolismo , Fibronectinas/metabolismo , Osteonectina/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Neoplasias do Endométrio/metabolismo , Matriz Extracelular/metabolismo , Feminino , Humanos , Osteonectina/genética , Fosforilação , Proteínas Proto-Oncogênicas c-akt/genética
10.
Int J Mol Sci ; 22(4)2021 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-33557232

RESUMO

Fibrosis is characterized by excessive production of disorganized collagen- and fibronectin-rich extracellular matrices (ECMs) and is driven by the persistence of myofibroblasts within tissues. A key protein contributing to myofibroblast differentiation is extra domain A fibronectin (EDA-FN). We sought to target and interfere with interactions between EDA-FN and its integrin receptors to effectively inhibit profibrotic activity and myofibroblast formation. Molecular docking was used to assist in the design of a blocking polypeptide (antifibrotic 38-amino-acid polypeptide, AF38Pep) for specific inhibition of EDA-FN associations with the fibroblast-expressed integrins α4ß1 and α4ß7. Blocking peptides were designed and evaluated in silico before synthesis, confirmation of binding specificity, and evaluation in vitro. We identified the high-affinity EDA-FN C-C' loop binding cleft within integrins α4ß1 and α4ß7. The polypeptide with the highest predicted binding affinity, AF38Pep, was synthesized and could achieve specific binding to myofibroblast fibronectin-rich ECM and EDA-FN C-C' loop peptides. AF38Pep demonstrated potent myofibroblast inhibitory activity at 10 µg/mL and was not cytotoxic. Treatment with AF38Pep prevented integrin α4ß1-mediated focal adhesion kinase (FAK) activation and early signaling through extracellular-signal-regulated kinases 1 and 2 (ERK1/2), attenuated the expression of pro-matrix metalloproteinase 9 (MMP9) and pro-MMP2, and inhibited collagen synthesis and deposition. Immunocytochemistry staining revealed an inhibition of α-smooth muscle actin (α-SMA) incorporation into actin stress fibers and attenuated cell contraction. Increases in the expression of mRNA associated with fibrosis and downstream from integrin signaling were inhibited by treatment with AF38Pep. Our study suggested that AF38Pep could successfully interfere with EDA-FN C-C' loop-specific integrin interactions and could act as an effective inhibitor of fibroblast of myofibroblast differentiation.


Assuntos
Desenho de Fármacos , Fibroblastos/efeitos dos fármacos , Fibronectinas/metabolismo , Fibrose/tratamento farmacológico , Integrinas/metabolismo , Miofibroblastos/efeitos dos fármacos , Peptídeos/farmacologia , Sítios de Ligação , Diferenciação Celular , Matriz Extracelular/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibronectinas/química , Fibrose/metabolismo , Fibrose/patologia , Humanos , Integrinas/química , Pulmão/citologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Simulação de Acoplamento Molecular , Miofibroblastos/citologia , Miofibroblastos/metabolismo , Ligação Proteica , Domínios Proteicos , Transdução de Sinais
12.
Mol Biol Rep ; 48(1): 763-772, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33389537

RESUMO

Physical exercise is an effective strategy for improving human health. Various organs, including the heart, lung and kidney, can benefit from exercise. However, the underlying molecular mechanisms by which exercise protects organs remain unknown. Irisin, a myokine secreted from muscle in response to exercise, has attracted increased attention from researchers. The role of irisin in multiorgan protection has been gradually revealed, and this muscle-derived circulating factor is regarded as an essential bridge linking exercise and organ health. The mechanisms by which irisin protects diverse organs are different. Here, we review the research progress on the multiorgan protective effects of irisin and discuss the underlying molecular mechanisms.


Assuntos
Fibronectinas/genética , Regulação da Expressão Gênica , Coração/fisiologia , Rim/fisiologia , Pulmão/fisiologia , Músculo Esquelético/metabolismo , Caspase 3/genética , Caspase 3/metabolismo , Comunicação Celular/genética , Exercício Físico/fisiologia , Fibronectinas/metabolismo , Humanos , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Músculo Esquelético/citologia , Transdução de Sinais , Proteína Desacopladora 2/genética , Proteína Desacopladora 2/metabolismo
13.
Exp Cell Res ; 399(2): 112489, 2021 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-33453237

RESUMO

Cardiac fibroblasts and myofibroblasts assemble and maintain extracellular matrix during normal development and following injury. Culture expansion of these cells yield a bioengineered matrix that could lead to intriguing therapeutic opportunities. For example, we reported that cultured rat cardiac fibroblasts form a matrix that can be used to delivery therapeutic stem cells. Furthermore, we reported that matrix derived from cultured human cardiac fibroblasts/myofibroblasts converted monocytes into macrophages that express interesting anti-inflammatory and pro-angiogenic properties. Expanding these matrix investigations require characterization of the source cells for quality control. In these efforts, we observed and herein report that Sushi Containing Domain 2 (SUSD2) is a novel and consistent marker for cultured human cardiac fibroblast and myofibroblasts.


Assuntos
Matriz Extracelular/metabolismo , Glicoproteínas de Membrana/metabolismo , Miocárdio/metabolismo , Biomarcadores/metabolismo , Células Cultivadas , Matriz Extracelular/fisiologia , Feminino , Fibroblastos/metabolismo , Fibronectinas/metabolismo , Humanos , Masculino , Glicoproteínas de Membrana/genética , Miocárdio/citologia , Miofibroblastos/metabolismo
14.
Int J Mol Sci ; 22(3)2021 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-33498156

RESUMO

Excessive cross-linking is a major factor in the resistance to the remodelling of the extracellular matrix (ECM) during fibrotic progression. The role of TGFß signalling in impairing ECM remodelling has been demonstrated in various fibrotic models. We hypothesised that increased ECM cross-linking by TGFß contributes to skin fibrosis in Systemic Sclerosis (SSc). Proteomics was used to identify cross-linking enzymes in the ECM of primary human dermal fibroblasts, and to compare their levels following treatment with TGFß-1. A significant upregulation and enrichment of lysyl-oxidase-like 1, 2 and 4 and transglutaminase 2 were found. Western blotting confirmed the upregulation of lysyl hydroxylase 2 in the ECM. Increased transglutaminase activity in TGFß-1 treated ECM was revealed from a cell-based assay. We employed a mass spectrometry-based method to identify alterations in the ECM cross-linking pattern caused by TGFß-1. Cross-linking sites were identified in collagens I and V, fibrinogen and fibronectin. One cross-linking site in fibrinogen alpha was found only in TGFß-treated samples. In conclusion, we have mapped novel cross-links between ECM proteins and demonstrated that activation of TGFß signalling in cultured dermal fibroblasts upregulates multiple cross-linking enzymes in the ECM.


Assuntos
Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Aminoácido Oxirredutases/metabolismo , Células Cultivadas , Colágeno/química , Colágeno/metabolismo , Reagentes para Ligações Cruzadas/química , Derme/citologia , Matriz Extracelular/química , Matriz Extracelular/efeitos dos fármacos , Feminino , Fibrinogênio/química , Fibrinogênio/metabolismo , Fibronectinas/química , Fibronectinas/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Humanos , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Transglutaminases/metabolismo
15.
Life Sci ; 267: 118954, 2021 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-33359670

RESUMO

The scientific interest in irisin, a myokine discovered in 2012, has grown exponentially in recent years. Irisin, which is mainly produced in skeletal muscle, influences the browning process of adipose tissue and lipid and energy metabolism. Recent discoveries highlight that the potential of this hormone may have been underestimated. In the first part of this review, reports on irisin structure and molecules involved in its metabolic pathway are shown. Furthermore, data related to unclear aspects are also reported: distribution, different gene expression of its precursors in different tissues, physiological levels of circulating irisin, and pharmacokinetic and pharmacodynamic profile. The second part of this work focuses on exogenous stimuli and pharmacological agents which regulate the metabolic pathway of irisin and its serum concentration. In addition to physical exercise and exposure to low temperatures, which were early recognized as exogenous stimuli able to promote the production of this myokine, preclinical and clinical evidence demonstrates the ability of natural and synthetic molecules to interfere with this metabolic pathway. Current experimental data on irisin cannot dissolve all doubts related to this interesting molecule, but they certainly underline its potential for therapeutic purposes. Thus, identification of new pharmacological tools able to act on the irisin pathway is a challenging issue for biomedical research.


Assuntos
Fibronectinas/metabolismo , Fibronectinas/farmacologia , Fibronectinas/fisiologia , Tecido Adiposo/metabolismo , Tecido Adiposo Marrom/metabolismo , Animais , Metabolismo Energético/fisiologia , Exercício Físico/fisiologia , Humanos , Músculo Esquelético/metabolismo , Fatores de Transcrição/metabolismo
16.
Microvasc Res ; 133: 104061, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32827495

RESUMO

BACKGROUND: The pathological character of cerebral small vessel disease (CSVD) is the dysfunction of cerebral small arteries caused by risk factors. A switch from the contractile phenotype to the synthetic phenotype of vascular smooth muscle cells (SMCs) can decrease the contractility of arteries. The alteration of the vascular wall extracellular matrix (ECM) is found to regulate the process. We speculated that SMCs phenotype changes may also occur in CSVD induced by hypertension and the alteration of ECM especially fibronectin and laminin may regulate the process. METHOD: Male spontaneously hypertensive rats (SHR) were used as a CSVD animal model. SMCs phenotypic markers and the ECM expression of the cerebral small arteries of SHR at different ages were evaluated by immunofluorescence. The phenotype changes of primary brain microvascular SMCs cultured on laminin-coating dish or fibronectin-coating dish were evaluated by western blot. RESULT: A switch from the contractile phenotype to synthetic phenotype in SHR at 10 and 22 weeks of age was observed. Meanwhile, increased expression of fibronectin and a temporary decline of laminin was found in small arteries of SHR at 22 weeks. In vitro experiments also convinced that SMCs cultured on a fibronectin-coating dish failed to maintain contractile phenotype. While at 50 weeks, significant drops of both synthetic and contractile phenotypic markers were witnessed in SHR, with high expressions of four kinds of ECM. CONCLUSION: SMCs in cerebral small arteries exhibited a switch from the contractile phenotype to synthetic phenotype during the chronic process of hypertension and aging. Moreover, the change of fibronectin and laminin may regulate the process.


Assuntos
Doenças de Pequenos Vasos Cerebrais/etiologia , Matriz Extracelular/metabolismo , Hipertensão/complicações , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Fatores Etários , Animais , Biomarcadores/metabolismo , Células Cultivadas , Artérias Cerebrais/metabolismo , Artérias Cerebrais/patologia , Artérias Cerebrais/fisiopatologia , Doenças de Pequenos Vasos Cerebrais/metabolismo , Doenças de Pequenos Vasos Cerebrais/patologia , Doenças de Pequenos Vasos Cerebrais/fisiopatologia , Modelos Animais de Doenças , Matriz Extracelular/patologia , Fibronectinas/metabolismo , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Laminina/metabolismo , Masculino , Músculo Liso Vascular/patologia , Músculo Liso Vascular/fisiopatologia , Miócitos de Músculo Liso/patologia , Fenótipo , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Remodelação Vascular , Vasoconstrição
17.
Methods Mol Biol ; 2217: 183-195, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33215382

RESUMO

Surface nanopatterning allows for the creation of spatially controlled binding sites for extracellular matrix ligands and the modulation of receptor binding sites. Here we describe the preparation of gold nanopatterned substrates using diblock micellar nanolithography to immobilize integrin ligands at defined spacing and combined with molecular tension sensors to measure molecular forces as function of integrin lateral clustering.


Assuntos
Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Adesões Focais/metabolismo , Integrinas/química , Oligodesoxirribonucleotídeos/química , Estereolitografia , Animais , Sítios de Ligação , Adesão Celular , Movimento Celular , Cloretos/química , Matriz Extracelular/ultraestrutura , Fibroblastos/ultraestrutura , Fibronectinas/química , Fibronectinas/metabolismo , Adesões Focais/ultraestrutura , Compostos de Ouro/química , Integrinas/metabolismo , Ligantes , Camundongos , Micelas , Microscopia de Fluorescência/métodos , Nanoestruturas/química , Polietilenoglicóis/química , Polimerização , Ligação Proteica , Piridinas/química , Propriedades de Superfície
18.
Methods Mol Biol ; 2217: 301-311, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33215388

RESUMO

In endothelial cells (ECs), the onset of apicobasal polarity is primarily regulated by the interaction of integrins with the surrounding extracellular matrix (ECM). ECs secrete and polymerize fibronectin (FN), a unique, permissive substrate that allows for vascular morphogenesis and lumen formation. We previously identified a signaling pathway that, under the control of the adhesion site adaptor protein PPFIA1, integrates the polarized secretion of freshly synthesized FN with the recycling of conformationally active α5ß1 integrin, the main FN receptor in ECs. To characterize the functional role of PPFIA1-dependent signaling in ECs, we set up a Transwell-based assay to quantify the polarized secretion of ECM proteins. To this aim, we allowed ECs to form a confluent monolayer on the Transwell membrane and checked its integrity by measuring transendothelial electric resistance and controlling the stability of tight junctions over time by fluorescent confocal microscope analysis. Finally, we quantified apical and basolateral FN secretion in control and PPFIA1-silenced EC culture medium by western blot analysis coupled to spike-in normalization.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Células Endoteliais/metabolismo , Matriz Extracelular/metabolismo , Fibronectinas/genética , Integrina alfa5beta1/genética , Junções Íntimas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Transporte Biológico , Polaridade Celular , Cultura em Câmaras de Difusão , Células Endoteliais/ultraestrutura , Matriz Extracelular/ultraestrutura , Fibronectinas/metabolismo , Regulação da Expressão Gênica , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Humanos , Integrina alfa5beta1/metabolismo , Microscopia de Fluorescência/métodos , Cultura Primária de Células , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Junções Íntimas/ultraestrutura
19.
Exp Eye Res ; 203: 108421, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33359326

RESUMO

Glaucoma, the second leading cause of blindness worldwide, is characterized by aberrant elevations of intraocular pressure (IOP), which can damage the optic nerve. IOP reduction is the only effective therapy for prevention of visual impairment and blindness in both hypertensive and normotensive individuals, and in some cases, trabeculectomy is a major surgical procedure that can lower IOP in patients with glaucoma. No matter how surgical technique and postoperative care advances, excessive scarring and tissue fibrosis could result from increased human conjunctival fibroblast (HCF) proliferation and extracellular matrix (ECM) deposition of the subconjunctival tissue and scleral flaps would persist after trabeculectomy. And these issues are major impediments to IOP reduction and filtering of bleb formations, so the modulation of the factors which can induce fibrosis could used as a novel strategy to control scarring after trabeculectomy. In this study, we examined the effects of mammalian target of rapamycin (mTOR) inhibitors (rapamycin or Torin1) on the fibrotic response induced by transforming growth factor-beta 1 (TGF-ß1) in cultured human conjunctival fibroblast (HCF) cells. The study also examined the effects of mTOR inhibitor on fibrosis after trabeculectomy in rabbit eyes. In in vitro studies, we stimulated HCFs with TGF-ß1, and confirmed that the expression levels of fibronectin, collagen type I alpha 1 chain (COL1A1), and α-smooth muscle actin (SMA) were significantly upregulated in HCFs with TGF-ß1, by means of quantitative real-time polymerase chain reaction and immunocytochemistry. And those TGF-ß1-induced changes were significantly attenuated with mTOR inhibitors, rapamycin or Torin1. Additionally the migration rate of HCFs was examined under conditions of TGF-ß1 induction, TGF-ß1-induced changes were significantly attenuated with mTOR inhibitors. A rabbit model of trabeculectomy was examined in vivo, and the effects of topical mTOR inhibitor were also examined, and found that topical treatment with mTOR inhibitor significantly suppressed collagen deposition in rabbit eyes after trabeculectomy. These results have demonstrated that mTOR inhibitors may provide a novel treatment modality for reducing the fibrotic response in HCFs and improving bleb scarring after filtration surgery.


Assuntos
Túnica Conjuntiva/patologia , Naftiridinas/farmacologia , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Trabeculectomia , Actinas/genética , Actinas/metabolismo , Animais , Western Blotting , Células Cultivadas , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Túnica Conjuntiva/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibronectinas/genética , Fibronectinas/metabolismo , Fibrose , Humanos , Imuno-Histoquímica , Masculino , RNA Mensageiro/genética , Coelhos , Reação em Cadeia da Polimerase em Tempo Real , Fator de Crescimento Transformador beta1/farmacologia
20.
Mol Med Rep ; 23(1)2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33215221

RESUMO

Scaffold­based bone tissue engineering has therapeutic potential in the regeneration of osseous defects. The present study aimed to explore the adhesion and cell viability of a co­culture system composed of vascular endothelial cells PI­/Annexin V+ represents early apoptotic cells, and PI+/Annexin V+ represents late apoptotic cells (VECs) and adipose­derived stem cells (ADSCs) on partially deproteinized biologic bone (PDPBB) in vitro, and determine the optimum time period for maximum cell viability that could possibly be used for standardizing the scaffold transplant into the in vivo system. VECs and ADSCs were isolated from pregnant Sprague­Dawley rats and confirmed by immunostaining with von Willebrand factor and CD90, respectively. PDPBB was prepared using standardized protocols involving coating partially deproteinized bone with fibronectin. PDPBB was incubated in a mono­culture with VECs or ADSCs, or in a co­culture with both of these cells at a ratio of 1:1. An MTT assay was used to assess the adhesion and cell viability of VECs and ADSCs on PDPBB in the three different cultures. Scanning electron microscopy was used to observe the adhesion, cell viability and morphology of the different types of cells on PDPBB. It was observed that the absorbance of each group increased gradually and peaked on the 10th day; the highest absorbance was found for the co­cultured cells group. The difference of cell viability between each cell group was statistically significant. On the 10th day, in the co­cultured cells group, several cells adhered on the PDPBB material and a nest­like distribution morphology was observed. Therefore, the adhesion and cell viability of the co­cultured cells was higher compared with the mono­cultures of VECs or ADSCs. As cell viability was highest on the 10th day, this could be the optimal length of time for incubation and therefore could be used for in vivo experiments.


Assuntos
Tecido Adiposo/crescimento & desenvolvimento , Osso e Ossos/metabolismo , Técnicas de Cocultura/métodos , Células Endoteliais/metabolismo , Células-Tronco/metabolismo , Engenharia Tecidual/métodos , Tecido Adiposo/citologia , Tecido Adiposo/metabolismo , Animais , Osso e Ossos/citologia , Adesão Celular , Diferenciação Celular , Sobrevivência Celular , Células Cultivadas , Células Endoteliais/citologia , Feminino , Sangue Fetal , Fibronectinas/metabolismo , Imunofluorescência , Microscopia Eletrônica de Varredura , Ratos Sprague-Dawley , Células-Tronco/citologia , Fatores de Tempo , Tecidos Suporte
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