Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 304
Filtrar
1.
Int J Mol Sci ; 22(19)2021 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-34638669

RESUMO

Extracellular vesicles (EVs) are found in all biological fluids, providing potential for the identification of disease biomarkers such as colorectal cancer (CRC). EVs are heavily glycosylated with specific glycoconjugates such as tetraspanins, integrins, and mucins, reflecting the characteristics of the original cell offering valuable targets for detection of CRC. We report here on europium-nanoparticle (EuNP)-based assay to detect and characterize different surface glycoconjugates of EVs without extensive purification steps from five different CRC and the HEK 293 cell lines. The promising EVs candidates from cell culture were clinically evaluated on small panel of serum samples including early-stage (n = 11) and late-stage (n = 11) CRC patients, benign condition (n = 11), and healthy control (n = 10). The majority of CRC cell lines expressed tetraspanin sub-population and glycovariants of integrins and conventional tumor markers. The subpopulation of CD151 having CD63 expression (CD151CD63) was significantly (p = 0.001) elevated in early-stage CRC (8 out of 11) without detecting any benign and late-stage samples, while conventional CEA detected mostly late-stage CRC (p = 0.045) and with only four early-stage cases. The other glycovariant assays such as CEACon-A, CA125WGA, CA 19.9Ma696, and CA 19.9Con-A further provided some complementation to the CD151CD63 assay. These results indicate the potential application of CD151CD63 assay for early detection of CRC patients in human serum.


Assuntos
Neoplasias Colorretais/sangue , Neoplasias Colorretais/metabolismo , Vesículas Extracelulares/metabolismo , Glicoconjugados/sangue , Glicoconjugados/metabolismo , Nanopartículas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/metabolismo , Antígeno Ca-125/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Feminino , Células HEK293 , Humanos , Masculino , Pessoa de Meia-Idade , Tetraspanina 30/metabolismo , Tetraspaninas/metabolismo , Adulto Jovem
2.
Int J Mol Sci ; 22(17)2021 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-34502227

RESUMO

Tissue Inhibitor of Metalloproteases 1, also known as TIMP-1, is named for its well-established function of inhibiting the proteolytic activity of matrix metalloproteases. Given this function, many studies were carried out to verify if TIMP-1 was able to interrupt processes such as tumor cell invasion and metastasis. In contrast, many studies have shown that TIMP-1 expression is increased in several types of tumors, and this increase was correlated with a poor prognosis and lower survival in cancer patients. Later, it was shown that TIMP-1 is also able to modulate cell behavior through the induction of signaling pathways involved in cell growth, proliferation, and survival. The mechanisms involved in the regulation of the pleiotropic functions of TIMP-1 are still poorly understood. Thus, this review aimed to present literature data that show its ability to form a membrane complex with CD63 and ß1-integrin, and point to N-glycosylation as a potential regulatory mechanism of the functions exerted by TIMP-1. This article reviewed the characteristics and functions performed individually by TIMP1, CD63, and ß1-integrin, the roles of the TIMP-1/CD63/ß1-integrin complex, both in a physiological context and in cancer, and the regulatory mechanisms involved in its assembly.


Assuntos
Integrina beta1/metabolismo , Neoplasias/patologia , Domínios e Motivos de Interação entre Proteínas , Tetraspanina 30/metabolismo , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Humanos , Neoplasias/metabolismo , Transdução de Sinais
3.
Nat Commun ; 12(1): 4389, 2021 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-34282141

RESUMO

Despite their roles in intercellular communications, the different populations of extracellular vesicles (EVs) and their secretion mechanisms are not fully characterized: how and to what extent EVs form as intraluminal vesicles of endocytic compartments (exosomes), or at the plasma membrane (PM) (ectosomes) remains unclear. Here we follow intracellular trafficking of the EV markers CD9 and CD63 from the endoplasmic reticulum to their residency compartment, respectively PM and late endosomes. We observe transient co-localization at both places, before they finally segregate. CD9 and a mutant CD63 stabilized at the PM are more abundantly released in EVs than CD63. Thus, in HeLa cells, ectosomes are more prominent than exosomes. By comparative proteomic analysis and differential response to neutralization of endosomal pH, we identify a few surface proteins likely specific of either exosomes (LAMP1) or ectosomes (BSG, SLC3A2). Our work sets the path for molecular and functional discrimination of exosomes and small ectosomes in any cell type.


Assuntos
Exossomos/metabolismo , Tetraspanina 29/metabolismo , Tetraspanina 30/metabolismo , Comunicação Celular , Membrana Celular/metabolismo , Endossomos/metabolismo , Vesículas Extracelulares/metabolismo , Cadeia Pesada da Proteína-1 Reguladora de Fusão , Técnicas de Inativação de Genes , Células HeLa , Humanos , Proteínas de Membrana/metabolismo , Transporte Proteico , Proteômica
4.
Nat Commun ; 12(1): 4548, 2021 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-34315885

RESUMO

Autosomal dominant polycystic kidney disease (ADPKD) is caused by germline mutations of PKD1 or PKD2 on one allele and a somatic mutation inactivating the remaining normal allele. However, if and how null ADPKD gene renal epithelial cells affect the biology and function of neighboring cells, including heterozygous renal epithelial cells, fibroblasts and macrophages during cyst initiation and expansion remains unknown. Here we address this question with a "cystic extracellular vesicles/exosomes theory". We show that cystic cell derived extracellular vesicles and urinary exosomes derived from ADPKD patients promote cyst growth in Pkd1 mutant kidneys and in 3D cultures. This is achieved by: 1) downregulation of Pkd1 gene expression and upregulation of specific miRNAs, resulting in the activation of PKD associated signaling pathways in recipient renal epithelial cells and tissues; 2) the activation of fibroblasts; and 3) the induction of cytokine expression and the recruitment of macrophages to increase renal inflammation in cystic kidneys. Inhibition of exosome biogenesis/release with GW4869 significantly delays cyst growth in aggressive and milder ADPKD mouse models, suggesting that targeting exosome secretion has therapeutic potential for ADPKD.


Assuntos
Células Epiteliais/patologia , Exossomos/metabolismo , Rim/patologia , Rim Policístico Autossômico Dominante/patologia , Compostos de Anilina/farmacologia , Animais , Compostos de Benzilideno/farmacologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Colágeno , Cistos/patologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Exossomos/efeitos dos fármacos , Fibrose , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Modelos Biológicos , Mutação/genética , Rim Policístico Autossômico Dominante/genética , Rim Policístico Autossômico Dominante/urina , Ratos , Transdução de Sinais/efeitos dos fármacos , Canais de Cátion TRPP/metabolismo , Tetraspanina 30/metabolismo
5.
PLoS Biol ; 19(7): e3001271, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34232954

RESUMO

Leukotriene B4 (LTB4) is secreted by chemotactic neutrophils, forming a secondary gradient that amplifies the reach of primary chemoattractants. This strategy increases the recruitment range for neutrophils and is important during inflammation. Here, we show that LTB4 and its synthesizing enzymes localize to intracellular multivesicular bodies, which, upon stimulation, release their content as exosomes. Purified exosomes can activate resting neutrophils and elicit chemotactic activity in an LTB4 receptor-dependent manner. Inhibition of exosome release leads to loss of directional motility with concomitant loss of LTB4 release. Our findings establish that the exosomal pool of LTB4 acts in an autocrine fashion to sensitize neutrophils towards the primary chemoattractant, and in a paracrine fashion to mediate the recruitment of neighboring neutrophils in trans. We envision that this mechanism is used by other signals to foster communication between cells in harsh extracellular environments.


Assuntos
Quimiotaxia de Leucócito/fisiologia , Exossomos/metabolismo , Leucotrieno B4/metabolismo , Neutrófilos/metabolismo , Araquidonato 5-Lipoxigenase/metabolismo , Transporte Biológico , Humanos , N-Formilmetionina Leucil-Fenilalanina/administração & dosagem , Ativação de Neutrófilo , Receptores de Laminina/metabolismo , Proteínas Ribossômicas/metabolismo , Tetraspanina 30/metabolismo
6.
ACS Appl Mater Interfaces ; 13(28): 32837-32844, 2021 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-34236165

RESUMO

Exosomes, which can transfer and deliver information about the original cell, are considered to be ideal candidates for early cancer diagnosis and evaluation of therapeutic efficacy due to their high abundance and stability. However, the highly expressed proteins on the surface of exosomes are usually associated with a variety of cancers; it is difficult to distinguish them by a single marker. Herein, a controlled self-assembly of gold nanorod (AuNR) arrays was prepared to construct a surface-enhanced Raman spectroscopy (SERS) sensor for the specific detection of exosomes secreted by SK-Br-3 cells based on a designed colocalization-dependent system (Co-DNA-Locker) and ratiometric strategy. After the exosomes are captured in the sensing array by the EpCAM aptamer modified on the surface of AuNRs, the DNA logic process occurs because the other two proteins, CD63 and HER2, are expressed simultaneously on the surface of exosomes secreted by SK-Br-3 cells, and the SERS signal intensity of the Rhodamine 6G (R6G) tagged on the terminal of DNA TE increased with an increase in the concentration of the exosomes, while the SERS signal intensity of Cy5 linked on the terminal of the EpCAM aptamer, which acts as an internal standard, remains stable. The AuNRs are uniformly arranged in a hexagonal shape, and the dense "hot spots" produce "hot surfaces," which greatly improve the sensitivity and uniformity of detection. In the presence of target exosomes, the DNA colocalization three-signal input switch and the ratiometric strategy realize the specific and accurate detection of exosomes. This sensing strategy achieves a wide detection range (1.0 × 104-5.0 × 106 particles/mL) and a lower detection limit (5.3 × 103 particles/mL), without using any signal amplification mechanism, demonstrating promising applications in health care monitoring and clinical diagnostics.


Assuntos
Aptâmeros de Nucleotídeos/química , DNA/química , Exossomos , Nanotubos/química , Aptâmeros de Nucleotídeos/genética , Aptâmeros de Nucleotídeos/metabolismo , Técnicas Biossensoriais/métodos , Carbocianinas/química , Linhagem Celular Tumoral , DNA/genética , DNA/metabolismo , Exossomos/química , Exossomos/metabolismo , Corantes Fluorescentes/química , Ouro/química , Células HEK293 , Humanos , Limite de Detecção , Hibridização de Ácido Nucleico , Receptor ErbB-2/metabolismo , Rodaminas/química , Análise Espectral Raman/métodos , Tetraspanina 30/metabolismo
7.
Biochem Biophys Res Commun ; 562: 50-54, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34034093

RESUMO

Mitochondria are eukaryotic organelles that consist of outer and inner bilayer membranes with a positive potential (H+) in the intermembrane space. This organelle plays an important role in ATP production and apoptosis. To observe the mitochondria in living cells, several fluorescent dyes (such as MitoTracker® [a standard mitochondrial imager] or rhodamine 123) have been developed. However, these reagents are unstable and exhibit a wide range of emission spectra, thereby hampering double staining results. Using recombinant DNA techniques, green or red fluorescent protein (GFP or RFP)-tagged proteins are now available for multi-color labeling of mitochondria. Here, we have discussed the development of the novel mitochondrial live imagers MitoMM1/2, derivatives of ATTO565; furthermore, MitoMM1/2 are sensitive to the membrane potential, resistant to detergents, and the fluorescence of MitoMM1/2 does not overlap with green fluorescence.


Assuntos
Corantes Fluorescentes/metabolismo , Mitocôndrias/metabolismo , Imagem Molecular , Corantes Fluorescentes/química , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Potencial da Membrana Mitocondrial , Mitofagia , Tetraspanina 30/metabolismo
8.
Biochem Pharmacol ; 190: 114624, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34052187

RESUMO

Non-alcoholic steatohepatitis (NASH) has evolved as the most common and devastating chronic liver disease. This study aimed to explore the underlined mechanism for the therapeutic potentials of bone marrow mesenchymal stem cells (BM-MSCs) and their derived exosomes (BM-MSCs-Exo) in an experimental model of high fat diet (HFD) induced NASH. Rats were fed with HFD for 12 weeks. At the seventh week, BM-MSCs were given at a dose of 1x106 cell i.v., per rat. A total of three doses of BM-MSCs were given per each rat in six weeks. BM-MSCs-Exo were given at a dose of 15, 30 and 120 µg/kg i.v., twice per week for six weeks. Perfect homing to the liver was detected. Beneficial effects were reported to BM-MSCs or BM-MSCs-Exo cotreatment; where the highest anti-steatotic effects were attributed to BM-MSCs-Exo (120 µg/kg) showing significant downregulation of fatty acid synthesis (SREB1, 2, ACC), downregulation in lipid uptake (CD36); accompanied by significant upregulation in fatty acid oxidation (PPARα, CPT1). These events were associated with abrogation of hepatic steatosis and ballooning in HFD-induced NASH. BM-MSCs or BM-MSCs-Exo cotreatment exerted significant anti-apoptotic effects mediated by significant decrease in Bax/Bcl2 ratio. Besides, significant increase in mitochondrial mitophagy genes (Parkin, PINK1, ULK1, BNIP3L, ATG5, ATG7, ATG12) were detected in BM-MSCs or BM-MSCs-Exo cotreated groups. These findings are thought to be modulated through upregulation of miRNA-96-5p which leads to downregulation of its downstream target caspase-2. Being a critical player in NASH development, caspase-2 targeting by miRNA-96-5p could be a promising therapeutic modality to treat NASH.


Assuntos
Cisteína Endopeptidases/metabolismo , Exossomos/metabolismo , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/metabolismo , Hepatopatia Gordurosa não Alcoólica/induzido quimicamente , Animais , Biomarcadores , Cisteína Endopeptidases/genética , Dieta Hiperlipídica/efeitos adversos , Hiperlipidemias , Metabolismo dos Lipídeos , Fígado/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , MicroRNAs/genética , Mitocôndrias , Mitofagia , Compostos Orgânicos/química , Ratos , Ratos Sprague-Dawley , Tetraspanina 30/genética , Tetraspanina 30/metabolismo
9.
Nat Commun ; 12(1): 2536, 2021 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-33953198

RESUMO

Molecular profiling of circulating extracellular vesicles (EVs) provides a promising noninvasive means to diagnose, monitor, and predict the course of metastatic breast cancer (MBC). However, the analysis of EV protein markers has been confounded by the presence of soluble protein counterparts in peripheral blood. Here we use a rapid, sensitive, and low-cost thermophoretic aptasensor (TAS) to profile cancer-associated protein profiles of plasma EVs without the interference of soluble proteins. We show that the EV signature (a weighted sum of eight EV protein markers) has a high accuracy (91.1 %) for discrimination of MBC, non-metastatic breast cancer (NMBC), and healthy donors (HD). For MBC patients undergoing therapies, the EV signature can accurately monitor the treatment response across the training, validation, and prospective cohorts, and serve as an independent prognostic factor for progression free survival in MBC patients. Together, this work highlights the potential clinical utility of EVs in management of MBC.


Assuntos
Neoplasias da Mama/diagnóstico , Neoplasias da Mama/metabolismo , Vesículas Extracelulares/metabolismo , Biomarcadores Tumorais , Neoplasias da Mama/terapia , Linhagem Celular Tumoral , Feminino , Humanos , Glicoproteína IIb da Membrana de Plaquetas/metabolismo , Estudos Prospectivos , Taxa de Sobrevida , Tetraspanina 30/metabolismo
10.
Front Immunol ; 12: 625284, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33790895

RESUMO

The Mas-related G-protein-coupled receptor X2 (MRGPRX2) is prominently expressed by mast cells and induces degranulation upon binding by different ligands. Its activation has been linked to various mast cell-related diseases, such as chronic spontaneous urticaria, atopic dermatitis and asthma. Therefore, inhibition of MRGPRX2 activity represents a therapeutic target for these conditions. However, the exact pathophysiology of this receptor is still unknown. In vitro research with mast cells is often hampered by the technical limitations of available cell lines. The human mast cell types LAD2 and HuMC (human mast cells cultured from CD34+ progenitor cells) most closely resemble mature human mast cells, yet have a very slow growth rate. A fast proliferating alternative is the human mast cell line HMC1, but they are considered unsuitable for degranulation assays due to their immature phenotype. Moreover, the expression and functionality of MRGPRX2 on HMC1 is controversial. Here, we describe the MRGPRX2 expression and functionality in HMC1 cells, and compare these with LAD2 and HuMC. We also propose a model to render HMC1 suitable for degranulation assays by pre-incubating them with latrunculin-B (Lat-B). Expression of MRGPRX2 by HMC1 was proven by RQ-PCR and flowcytometry, although at lower levels compared with LAD2 and HuMC. Pre-incubation of HMC1 cells with Lat-B significantly increased the overall degranulation capacity, without significantly changing their MRGPRX2 expression, phenotype or morphology. The MRGPRX2 specific compound 48/80 (C48/80) effectively induced degranulation of HMC1 as measured by CD63 membrane expression and ß-hexosaminidase release, albeit in lower levels than for LAD2 or HuMC. HMC1, LAD2 and HuMC each had different degranulation kinetics upon stimulation with C48/80. Incubation with the MRGPRX2 specific inhibitor QWF inhibited C48/80-induced degranulation, confirming the functionality of MRGPRX2 on HMC1. In conclusion, HMC1 cells have lower levels of MRGPRX2 expression than LAD2 or HuMC, but are attractive for in vitro research because of their high growth rate and stable phenotype. HMC1 can be used to study MRGPRX2-mediated degranulation after pre-incubation with Lat-B, which provides the opportunity to explore MPRGRX2 biology in mast cells in a feasible way.


Assuntos
Degranulação Celular , Mastócitos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Neuropeptídeos/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Degranulação Celular/efeitos dos fármacos , Linhagem Celular , Humanos , Ligantes , Mastócitos/efeitos dos fármacos , Mastócitos/imunologia , Fenótipo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Receptores de IgE/metabolismo , Transdução de Sinais , Tetraspanina 30/metabolismo , Tiazolidinas/farmacologia , beta-N-Acetil-Hexosaminidases/metabolismo , p-Metoxi-N-metilfenetilamina/farmacologia
11.
PLoS One ; 16(4): e0250852, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33909702

RESUMO

Aristotelia chilensis (Mol.) Stuntz, also known as maqui, is a plant native to Chile without chemical characterization and quantification of the bioactive compounds present in it. HPLC-UV and HPLC-MS/MS studies have shown the presence, at different concentrations, of phenolic and anthocyanin compounds in fruit and leave extracts of the domesticated maqui clones Luna Nueva, Morena, and Perla Negra. The extracts from leaves and unripe fruits of Luna Nueva and Morena clones significantly inhibit platelet aggregation induced by several agonists; the extracts inhibit platelet granule secretion by decreasing the exposure of P-selectin and CD63 at the platelet membrane. Reactive oxygen species formation in platelets is lower in the presence of maqui extracts. Statistical Pearson analysis supports the levels of phenolic and anthocyanin compounds being responsible for the antiaggregant maqui effects. This work is the first evidence of antiplatelet activity from Aristotelia chilensis giving added value to the use of leaves and unripe fruits from this species.


Assuntos
Antocianinas/farmacologia , Elaeocarpaceae/química , Inibidores da Agregação Plaquetária/farmacologia , Polifenóis/farmacologia , Antocianinas/química , Antocianinas/isolamento & purificação , Chile , Cromatografia Líquida de Alta Pressão , Domesticação , Frutas/química , Humanos , Selectina-P/metabolismo , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Folhas de Planta/química , Inibidores da Agregação Plaquetária/química , Inibidores da Agregação Plaquetária/isolamento & purificação , Polifenóis/química , Polifenóis/isolamento & purificação , Espectrometria de Massas em Tandem , Tetraspanina 30/metabolismo
12.
Med Sci Monit ; 27: e929609, 2021 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-33879761

RESUMO

BACKGROUND A lack of physical exercise, a critical aspect of a healthy lifestyle, contributes to several cerebral diseases, such as cognitive impairment, Parkinson disease (PD), and Alzheimer disease (AD). The purpose of the present study was to evaluate the effect of physical exercise on cerebral disease via released extracellular vesicles (EVs). MATERIAL AND METHODS Short-term high-intensity treadmill exercise was applied to assess the effect of physical activity on EVs in the serum and brain tissue. Immunofluorescence staining and western blot analysis were used to analyze biomarkers of EVs, including TSG101, HSC70, and CD63. Nanoparticle tracking analysis (NTA) was used to analyze the size and concentration of EVs. RESULTS Short-term high-intensity exercise increased the number of neuronal EVs in the brain. In the peripheral blood serum, the level of HSC70 showed a temporary increase after exercise and quickly returned to the normal level, whereas the levels of CD63 and TSG101 showed no obvious change in response to physical exercise. In brain tissue, the levels of HSC70 and TSG101 increased dramatically after exercise, while the level of CD63 remained unchanged. The concentration of EVs was significantly increased after exercise, while the mean diameter of the EVs showed no significant change. The levels of ceramide were significantly increased after exercise, and quickly returned to normal levels. CONCLUSIONS These data suggest that the secretion of EVs in the brain and blood is a transitory response to physical exercise and is dependent on ceramide synthesis.


Assuntos
Encefalopatias/prevenção & controle , Sistema Nervoso Central/fisiologia , Vesículas Extracelulares/metabolismo , Condicionamento Físico Animal/fisiologia , Animais , Ceramidas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Teste de Esforço , Proteínas de Choque Térmico HSC70/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Tetraspanina 30/metabolismo , Fatores de Transcrição/metabolismo
13.
Exp Eye Res ; 205: 108496, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33610602

RESUMO

The aim of the study is to clarify the participation of extracellular vesicles (EV) secreted by murine primary retinal pigment epithelial (mpRPE) cells in the cell to cell communication with macrophages (Mps), firstly described by the authors in 2016. In ocular inflammation, Mps act as sources of tumor necrosis factor-α (TNF-α), an activator of RPE cells. TNF-α stimulates the production of monocyte chemotactic protein (MCP-1) by RPE cells, thereby causing greater recruitment of Mps to the sub-RPE space. Murine RAW 264.7 Mps cells were co-cultured with C57BL/6 mouse mpRPE cells, either together or separated in transwells, vertically or horizontally connectable, with 0.40 or 0.03 µm membrane filters. The association of EV with mpRPE or RAW 264.7 was quantified by fluorescence cell sorting (FACS) using Qdot655 streptavidin-conjugated biotinylated EV. Increased levels of CD63+ EV were detected in co-cultures by western blotting or FACS analysis, in accordance with the increased production of nanoparticles (50-150 nm) detected by Nanosight tracking analysis. The gene expressions of cytokines, MCP-1, IL-6, IL-8, and VEGF in mpRPE cells and the corresponding proteins were increased in co-cultures even in transwells, vertically connected with 0.40 µm membrane filters, while the repressed TNF-α protein production was not affected. Most of the CD63+ EVs produced by mpRPE cells in co-cultures were associated with Raw264.7, but not with mpRPE cells. Semi-purified CD63+ EV secreted from mpRPE cells, increased the secretion of MCP-1, IL-6, and VEGF in co-cultures with RAW 264.7. Culture chamber separation horizontally connected with 0.03 µm membrane filters reduced this increased secretion. Collectively, mpRPE derived CD63+ EV partly participate in the sub-retinal innate inflammation. To evaluate the functional damage of RPE cells upon chronic exposure to here defined EVs will be the critical issue to uncover their roles in chronic retinal degenerative diseases.


Assuntos
Comunicação Celular/fisiologia , Vesículas Extracelulares/metabolismo , Imunidade Inata/fisiologia , Macrófagos/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Tetraspanina 30/metabolismo , Animais , Western Blotting , Linhagem Celular , Quimiocina CCL2/metabolismo , Técnicas de Cocultura , Citocinas/genética , Ensaio de Imunoadsorção Enzimática , Exossomos/genética , Citometria de Fluxo , Regulação da Expressão Gênica/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/imunologia , Fator de Necrose Tumoral alfa/metabolismo
14.
Medicine (Baltimore) ; 100(4): e21370, 2021 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-33530152

RESUMO

ABSTRACT: A number of studies have demonstrated that exosomes were involved in important physiological and pathological processes through cell-to-cell communication in cardiovascular disease, which contained nucleic acids, proteins, and lipid contents. In our study, we found that the protein platelet endothelial cell adhesion molecule-1 (PECAM1) was an extracellular vesicle in the blood of high blood pressure patients (HBPP).Isolated the vesicles from the blood of HBPP and health examiners and detected its size and morphology with nanoparticle tracking analysis, then we identified its surface protein CD63, CD81, and the protein expression of PECAM1 in the exosome with western blot. Furthermore, we analyzed the correlation between the expression of PECAM1 and the high blood degree with linear regression analysis.Our results showed that the morphology of extracellular vesicles was more evident in high blood pressure groups than healthy controls, and the protein expression of PECAM1 was also abundant in the vesicles of HBPP, however, there were no extracellular vesicles in the blood samples of healthy controls. Besides, linear regression showed the linear correlation coefficient R = 0.901, P < .01 between the expression of PECAM1 and the systolic blood pressure of the high blood patients. Therefore, the exosome of protein of PECAM1 was a potential risking star in HBPP.


Assuntos
Exossomos/genética , Hipertensão/genética , Molécula-1 de Adesão Celular Endotelial a Plaquetas/sangue , Adulto , Western Blotting , Vesículas Extracelulares/genética , Feminino , Fatores de Risco de Doenças Cardíacas , Humanos , Hipertensão/sangue , Modelos Lineares , Masculino , Pessoa de Meia-Idade , Nanopartículas/metabolismo , Tetraspanina 28/metabolismo , Tetraspanina 30/metabolismo
15.
Transfusion ; 61(4): 1222-1234, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33580979

RESUMO

BACKGROUND: Patients can form antibodies to foreign human leukocyte antigen (HLA) Class I antigens after exposure to allogeneic cells. These anti-HLA class I antibodies can bind transfused platelets (PLTs) and mediate their destruction, thus leading to PLT refractoriness. Patients with PLT refractoriness need HLA-matched PLTs, which require expensive HLA typing of donors, antibody analyses of patient sera and/or crossmatching. An alternative approach is to reduce PLT HLA Class I expression using a brief incubation in citric acid on ice at low pH. METHODS AND MATERIALS: Apheresis PLT concentrates were depleted of HLA Class I complexes by 5 minutes incubation in ice-cold citric acid, at pH 3.0. Surface expression of HLA Class I complexes, CD62P, CD63, phosphatidylserine, and complement factor C3c was analyzed by flow cytometry. PLT functionality was tested by thromboelastography (TEG). RESULTS: Acid treatment reduced the expression of HLA Class I complexes by 71% and potential for C3c binding by 11.5-fold compared to untreated PLTs. Acid-treated PLTs were significantly more activated than untreated PLTs, but irrespective of this increase in steady-state activation, CD62P and CD63 were strongly upregulated on both acid-treated and untreated PLTs after stimulation with thrombin receptor agonist peptide. Acid treatment did not induce apoptosis over time. X-ray irradiation did not significantly influence the expression of HLA Class I complexes, CD62P, CD63, and TEG variables on acid treated PLTs. CONCLUSION: The relatively simple acid stripping method can be used with irradiated apheresis PLTs and may prevent transfusion-associated HLA sensitization and overcome PLT refractoriness.


Assuntos
Ácido Cítrico/efeitos adversos , Antígenos de Histocompatibilidade Classe I/efeitos dos fármacos , Transfusão de Plaquetas/métodos , Imunodeficiência Combinada Severa/induzido quimicamente , Anticorpos/imunologia , Tipagem e Reações Cruzadas Sanguíneas/métodos , Plaquetas/efeitos da radiação , Feminino , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos de Histocompatibilidade Classe I/efeitos da radiação , Teste de Histocompatibilidade/economia , Teste de Histocompatibilidade/métodos , Humanos , Selectina-P/metabolismo , Transfusão de Plaquetas/efeitos adversos , Plaquetoferese/métodos , Tetraspanina 30/metabolismo , Tromboelastografia/métodos , Trombocitopenia/terapia , Regulação para Cima/genética
16.
J Cell Physiol ; 236(6): 4625-4639, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33452697

RESUMO

Sepsis-induced myocardial dysfunction (SIMD), a deadly symptom in sepsis patients, is mainly caused by cardiovascular inflammation. However, it remains unclear how systemic inflammation triggers and aggravates cardiovascular inflammation in the pathogenesis of SIMD. This study found that proinflammatory cytokines and H2 O2 concentrations were significantly induced in SIMD-mice. In particular, a microarray analysis of CD63+ exosomes isolated from sham- and SIMD-monocytes revealed a significant induction of thioredoxin-interacting protein (TXNIP) and NLR family pyrin domain-containing 3 (NLRP3). We proved that oxidative stress caused the disassociation of the TXNIP-TRX2 (thioredoxin 2) complex and the assembly of the TXNIP-NLRP3 complex. In addition, this finding showed that the latter complex could be embedded into CD63+ exosomes and traffic from monocytes to the resident heart macrophages, where it activated caspase-1 and cleaved inactive interleukin 1ß (IL-1ß) and IL-18. Furthermore, using an amplified luminescent proximity homogeneous assay (Alpha) with GST-TXNIP and His-NLRP3, we obtained a small molecule named PSSM1443 that could disrupt the TXNIP-NLRP3 interaction in vitro, impairing NLRP3 downstream events. Of note, after administering PSSM1443 to the SIMD-mice, we found the small molecule could significantly suppress the activation of caspase-1 and the cleavage of pro-IL-1ß and pro-IL-18, reducing inflammation in the SIMD-mice. Collectively, our results reveal that monocyte-derived exosomes harbor the overexpressed TXNIP-NLRP3 complex, which traffics from circulating monocytes to local macrophages and promotes the cleavage of inactive IL-1ß and IL-18 in the macrophages, aggravating cardiovascular inflammation. PSSM1443 functions as an inhibitor of the TXNIP-NLRP3 complex and its administration can decrease inflammation in SIMD-mice.


Assuntos
Anti-Inflamatórios/farmacologia , Proteínas de Transporte/metabolismo , Exossomos/efeitos dos fármacos , Cardiopatias/prevenção & controle , Inflamassomos/metabolismo , Mediadores da Inflamação/metabolismo , Inflamação/prevenção & controle , Macrófagos/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Sepse/tratamento farmacológico , Tiorredoxinas/metabolismo , Animais , Proteínas de Transporte/genética , Técnicas de Cocultura , Modelos Animais de Doenças , Exossomos/genética , Exossomos/imunologia , Exossomos/metabolismo , Cardiopatias/etiologia , Cardiopatias/imunologia , Cardiopatias/metabolismo , Inflamassomos/genética , Inflamação/etiologia , Inflamação/imunologia , Inflamação/metabolismo , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/imunologia , Monócitos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Estresse Oxidativo , Células RAW 264.7 , Sepse/complicações , Sepse/imunologia , Sepse/metabolismo , Tetraspanina 30/metabolismo , Tiorredoxinas/genética
17.
PLoS One ; 16(1): e0231994, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33497388

RESUMO

Exosomes are a type of extracellular vesicles containing mRNA, miRNA, and proteins of origin cells, which can control the characteristics of other cells or surroundings. Despite increasing evidence on oncogenic properties of tumor-derived exosomes, fibrosarcoma-derived exosomes remain largely unrevealed. While the proper extraction and characterization of exosomes is critical in exosomes research, there are various limitations in techniques to measure the size and homogeneity of exosomes. Here, we analyzed exosomes from a fibrosarcoma cell line WEHI-164 compared with a breast cancer cell line MDA-MD-231 as a control. Results from dot blot and western blot analysis demonstrated that GM1 ganglioside, and TSG101, HSC70 and GAPDH proteins were contained in exosomes from the WEHI-164 fibrosarcoma cell line. The existence of tetraspanins such as CD81, CD63 and CD9 was confirmed in the exosomes by ExoView analysis. The results obtained from TEM showed their sphere-like shapes of around 50 to 70 nm in radius. Through DLS, we found out that the mean radius of the exosomes derived from WEHI-164 and MDA-MB-231 cell lines was 94.4 nm and 107.8 nm, respectively, with high homogeneity. When comparing the radius measured by TEM with the radius measured by DLS, it was revealed that the difference between the two methods was about 40 nm. This study has significance in characterizing the molecular properties of exosomes from a fibrosarcoma, which has not been researched much before, and in providing clear evidence that DLS can be used as an efficient, convenient and noninvasive technique to simply check the homogeneity and size of exosomes.


Assuntos
Exossomos/metabolismo , Fibrossarcoma/metabolismo , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Difusão Dinâmica da Luz , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Vesículas Extracelulares/metabolismo , Feminino , Proteínas de Choque Térmico HSC70/metabolismo , Humanos , Integrina beta1/metabolismo , Tetraspanina 28/metabolismo , Tetraspanina 30/metabolismo , Fatores de Transcrição/metabolismo
18.
Nat Commun ; 12(1): 213, 2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33431899

RESUMO

High-fat diet (HFD) decreases insulin sensitivity. How high-fat diet causes insulin resistance is largely unknown. Here, we show that lean mice become insulin resistant after being administered exosomes isolated from the feces of obese mice fed a HFD or from patients with type II diabetes. HFD altered the lipid composition of exosomes from predominantly phosphatidylethanolamine (PE) in exosomes from lean animals (L-Exo) to phosphatidylcholine (PC) in exosomes from obese animals (H-Exo). Mechanistically, we show that intestinal H-Exo is taken up by macrophages and hepatocytes, leading to inhibition of the insulin signaling pathway. Moreover, exosome-derived PC binds to and activates AhR, leading to inhibition of the expression of genes essential for activation of the insulin signaling pathway, including IRS-2, and its downstream genes PI3K and Akt. Together, our results reveal HFD-induced exosomes as potential contributors to the development of insulin resistance. Intestinal exosomes thus have potential as broad therapeutic targets.


Assuntos
Dieta Hiperlipídica , Exossomos/metabolismo , Resistência à Insulina/genética , Fosfatidilcolinas/metabolismo , Regulação para Cima/genética , Tecido Adiposo/metabolismo , Animais , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Dislipidemias/complicações , Dislipidemias/genética , Dislipidemias/patologia , Células Epiteliais/metabolismo , Fígado Gorduroso/complicações , Fígado Gorduroso/genética , Fígado Gorduroso/patologia , Fezes , Regulação da Expressão Gênica , Intolerância à Glucose , Proteínas de Fluorescência Verde/metabolismo , Humanos , Insulina/metabolismo , Interleucina-6/sangue , Intestinos/citologia , Lipídeos/química , Fígado/metabolismo , Fígado/patologia , Ativação de Macrófagos , Camundongos Endogâmicos C57BL , Receptores de Hidrocarboneto Arílico/metabolismo , Transdução de Sinais , Tetraspanina 30/metabolismo , Fator de Necrose Tumoral alfa/sangue
19.
J Leukoc Biol ; 109(4): 753-762, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-32911568

RESUMO

Eosinophils are granulocytes that are elevated in lung mucosa in approximately half of patients with allergic asthma. These highly granulated cells can synthesize and secrete many cytokines, including IL-9 and IL-13. We hypothesized that IL-9 and IL-13 are found as preformed mediators in crystalloid granules and secreted using distinct trafficking pathways. Human eosinophils were purified from peripheral venous blood, adhered to coverslips, and stimulated with platelet activating factor (PAF). Cells were immunolabeled with antibodies to IL-9 or IL-13 and colocalized with markers for secretory organelles, using CD63 for crystalloid granules and transferrin receptor (TfnRc) for vesicles. Fixed cells were imaged using super-resolution microscopy and quantified by colocalization using Pearson's correlation coefficient. IL-9 immunofluorescence increased in a time-dependent manner to PAF, whereas colocalization of IL-9 and CD63 significantly increased from 0.52 to 0.67 after 5 min PAF. Colocalization of IL-9 with TfnRc significantly increased at 60 min of stimulation with PAF (0.54 at 0 min to 0.60 at 60 min). IL-13 showed lower colocalization with CD63 (0.55) than TfnRc (0.63) in unstimulated cells. Upon PAF stimulation, IL-13 intensity transiently decreased at 5 and 60 min, whereas colocalization of IL-13 with CD63 decreased throughout stimulation to 0.43. While colocalization of IL-13 with TfnRc transiently increased to 0.66 at 5 min PAF, it returned to near baseline levels (0.64) after 15 min PAF. Our results suggest that IL-9 and IL-13 are stored in crystalloid granules as well as endosomal structures, and that IL-9 is primarily trafficked to the cell surface via TfnRc+ endosome-like vesicles.


Assuntos
Eosinófilos/metabolismo , Interleucina-9/metabolismo , Receptores da Transferrina/metabolismo , Vesículas Transportadoras/metabolismo , Adolescente , Adulto , Grânulos Citoplasmáticos/metabolismo , Humanos , Fator de Ativação de Plaquetas/metabolismo , Tetraspanina 30/metabolismo
20.
J Invest Dermatol ; 141(3): 523-532.e2, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-32890627

RESUMO

Slac2-b, also known as exophilin-5, is a Rab27b effector protein with a role in exosome transport and is encoded by the EXPH5 gene. We previously described biallelic loss-of-function mutations in EXPH5 in an autosomal recessive form of epidermolysis bullosa simplex. However, how the loss of Slac2-b expression leads to skin fragility and erosions is unknown. In this study, we demonstrate that keratinocytes (KCs) isolated from two different individuals with mutations in EXPH5 have significant defects in cell‒matrix adhesion. EXPH5-mutant KCs also showed increased perinuclear accumulation and significantly reduced trafficking of CD63+ vesicles. These phenotypes were also seen in Slac2-b‒deficient KCs. This was coincident with a reduction in Rab27a protein expression in Slac2-b‒mutant KCs as well as reduced secretion of extracellular vesicles containing extracellular matrix proteins. Live imaging analysis revealed a strong correlation between CD63+ vesicle trafficking to the plasma membrane and focal adhesion dynamics. These findings support a role for Slac2-b in regulating local focal adhesion dynamics to support effective KC adhesion and provide insight into the underlying pathophysiology of inherited skin blistering.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/deficiência , Epiderme/patologia , Epidermólise Bolhosa Simples/patologia , Vesículas Extracelulares/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Biópsia , Adesão Celular/genética , Movimento Celular/genética , Criança , Epidermólise Bolhosa Simples/genética , Humanos , Microscopia Intravital , Queratinócitos/patologia , Masculino , Mutação , Tetraspanina 30/metabolismo , Imagem com Lapso de Tempo , Proteínas rab27 de Ligação ao GTP/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...