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1.
Sheng Wu Gong Cheng Xue Bao ; 36(4): 632-642, 2020 Apr 25.
Artigo em Chinês | MEDLINE | ID: mdl-32347058

RESUMO

Extracellular vesicles (EVs) refer to bilayer membrane transport vesicles secreted by cells. EVs can take macromolecules from cells and transfer them to receptor cells. Among these macromolecular substances, the most studied are microRNAs (miRNAs). miRNA is non-coding RNA involved in the regulation of gene expression. It has been confirmed that there are different non-coding RNAs in mammalian follicular fluid EVs. EVs carrying miRNA can act as an alternative mechanism for autocrine and paracrine, affecting follicular development. This paper systematically introduced the kinds, characteristics and methods of isolation and identification of EVs, focusing on the effects of EVs and miRNAs on follicular development, including early follicular development, oocyte maturation, follicular dominance and effects on granulosa cell function. At the same time, the authors prospected the future research of EVs and microRNAs in follicular fluid, and provided ideas and directions for the research and application of EVs and miRNA functions in follicular fluid.


Assuntos
Vesículas Extracelulares , Líquido Folicular , MicroRNAs , Oogênese , Animais , Vesículas Extracelulares/metabolismo , Feminino , Líquido Folicular/química , Células da Granulosa/efeitos dos fármacos , MicroRNAs/farmacologia , Oogênese/efeitos dos fármacos
2.
Basic Res Cardiol ; 115(3): 26, 2020 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-32146560

RESUMO

Mesenchymal stromal cells (MSCs) exhibit antiapoptotic and proangiogenic functions in models of myocardial infarction which may be mediated by secreted small extracellular vesicles (sEVs). However, MSCs have frequently been harvested from aged or diseased patients, while the isolated sEVs often contain high levels of impurities. Here, we studied the cardioprotective and proangiogenic activities of size-exclusion chromatography-purified sEVs secreted from human foetal amniotic fluid stem cells (SS-hAFSCs), possessing superior functional potential to that of adult MSCs. We demonstrated for the first time that highly pure (up to 1.7 × 1010 particles/µg protein) and thoroughly characterised SS-hAFSC sEVs protect rat hearts from ischaemia-reperfusion injury in vivo when administered intravenously prior to reperfusion (38 ± 9% infarct size reduction, p < 0.05). SS-hAFSC sEVs did not protect isolated primary cardiomyocytes in models of simulated ischaemia-reperfusion injury in vitro, indicative of indirect cardioprotective effects. SS-hAFSC sEVs were not proangiogenic in vitro, although they markedly stimulated endothelial cell migration. Additionally, sEVs were entirely responsible for the promigratory effects of the medium conditioned by SS-hAFSC. Mechanistically, sEV-induced chemotaxis involved phosphatidylinositol 3-kinase (PI3K) signalling, as its pharmacological inhibition in treated endothelial cells reduced migration by 54 ± 7% (p < 0.001). Together, these data indicate that SS-hAFSC sEVs have multifactorial beneficial effects in a myocardial infarction setting.


Assuntos
Líquido Amniótico/citologia , Cardiotônicos/metabolismo , Movimento Celular , Vesículas Extracelulares/metabolismo , Células-Tronco Mesenquimais/metabolismo , Traumatismo por Reperfusão/metabolismo , Animais , Quimiotaxia , Humanos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Fosfatidilinositol 3-Quinase/metabolismo , Ratos , Traumatismo por Reperfusão/patologia
3.
Adv Exp Med Biol ; 1248: 455-483, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32185722

RESUMO

Originally treated as part of a cellular waste, extracellular vesicles (EVs) are being shown to possess a vast variety of functions, of which exosome is the most studied one. Most cells, such as tumor cells, immunocytes, and fibroblasts can secrete exosomes, especially under certain stresses the amount is much higher, and the contents of exosome represent the status of the donor cells and the tumor microenvironment. As crucial transporters for cells' content exchange, much attention has been raised in the utilities of exosomes to suppress immune response, and to modify a microenvironment favorable for cancer progression. Exosomal immune checkpoints, such as programmed cell death ligand 1 (PD-L1), contribute to immunosuppression and are associated with anti-PD-1 response. Many forms of soluble immune checkpoint receptors have also been shown to influence efficacy mediated by their therapeutic antibodies. Therefore, targeting pro-tumorous exosomes may achieve antitumor effect supplementary to existing therapies. Exosome, itself natural liposome-like structure, allows it to be a potential drug delivery tool.


Assuntos
Exossomos/metabolismo , Sistemas de Liberação de Medicamentos , Exossomos/efeitos dos fármacos , Vesículas Extracelulares/efeitos dos fármacos , Vesículas Extracelulares/metabolismo , Humanos , Tolerância Imunológica , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Neoplasias/metabolismo , Neoplasias/patologia , Microambiente Tumoral/efeitos dos fármacos
4.
Nat Commun ; 11(1): 719, 2020 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-32024826

RESUMO

Lipid overload results in lipid redistribution among metabolic organs such as liver, adipose, and muscle; therefore, the interplay between liver and other organs is important to maintain lipid homeostasis. Here, we show that liver responds to lipid overload first and sends hepatocyte-derived extracellular vesicles (EVs) targeting adipocytes to regulate adipogenesis and lipogenesis. Geranylgeranyl diphosphate synthase (Ggpps) expression in liver is enhanced by lipid overload and regulates EV secretion through Rab27A geranylgeranylation. Consistently, liver-specific Ggpps deficient mice have reduced fat adipose deposition. The levels of several EV-derived miRNAs in the plasma of non-alcoholic fatty liver disease (NAFLD) patients are positively correlated with body mass index (BMI), and these miRNAs enhance adipocyte lipid accumulation. Thus, we highlight an inter-organ mechanism whereby the liver senses different metabolic states and sends corresponding signals to remodel adipose tissue to adapt to metabolic changes in response to lipid overload.


Assuntos
Tecido Adiposo/metabolismo , Vesículas Extracelulares/metabolismo , Hepatócitos/metabolismo , Metabolismo dos Lipídeos/fisiologia , Fígado/metabolismo , Hepatopatia Gordurosa não Alcoólica/sangue , Adipócitos/metabolismo , Adipócitos/patologia , Tecido Adiposo/patologia , Animais , Índice de Massa Corporal , Dieta Hiperlipídica/efeitos adversos , Vesículas Extracelulares/genética , Farnesiltranstransferase/genética , Humanos , Lipogênese , Fígado/citologia , Fígado/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/sangue , Complexos Multienzimáticos/genética , Hepatopatia Gordurosa não Alcoólica/genética , Proteínas rab27 de Ligação ao GTP/genética , Proteínas rab27 de Ligação ao GTP/metabolismo
5.
PLoS One ; 15(2): e0228948, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32059016

RESUMO

Remote ischemic preconditioning (RIPC) can evoke cardioprotection following ischemia/reperfusion and this may depend on the anesthetic used. We tested whether 1) extracellular vesicles (EVs) isolated from humans undergoing RIPC protect cardiomyoblasts against hypoxia-induced apoptosis and 2) this effect is altered by cardiomyoblast exposure to isoflurane or propofol. EVs were isolated before and 60 min after RIPC or Sham from ten patients undergoing coronary artery bypass graft surgery with isoflurane anesthesia and quantified by Nanoparticle Tracking Analysis. Following EV-treatment for 6 hours under exposure of isoflurane or propofol, rat H9c2 cardiomyoblasts were cultured for 18 hours in normoxic or hypoxic atmospheres. Apoptosis was detected by flow cytometry. Serum nanoparticle concentrations in patients had increased sixty minutes after RIPC compared to Sham (2.5x1011±4.9x1010 nanoparticles/ml; Sham: 1.2x1011±2.0x1010; p = 0.04). Hypoxia increased apoptosis of H9c2 cells (hypoxia: 8.4%±0.6; normoxia: 2.5%±0.1; p<0.0001). RIPC-EVs decreased H9c2 cell apoptosis compared to control (apoptotic ratio: 0.83; p = 0.0429) while Sham-EVs showed no protection (apoptotic ratio: 0.97). Prior isoflurane exposure in vitro even increased protection (RIPC-EVs/control, apoptotic ratio: 0.79; p = 0.0035; Sham-EVs/control, apoptotic ratio:1.04) while propofol (50µM) abrogated protection by RIPC-EVs (RIPC-EVs/control, Apoptotic ratio: 1.01; Sham-EVs/control, apoptotic ratio: 0.94; p = 0.602). Thus, EVs isolated from patients undergoing RIPC under isoflurane anesthesia protect H9c2 cardiomyoblasts against hypoxia-evoked apoptosis and this effect is abrogated by propofol. This supports a role of human RIPC-generated EVs in cardioprotection and underlines propofol as a possible confounder in RIPC-signaling mediated by EVs.


Assuntos
Vesículas Extracelulares/fisiologia , Isquemia/metabolismo , Miócitos Cardíacos/fisiologia , Idoso , Anestesia/métodos , Animais , Apoptose/fisiologia , Ponte de Artéria Coronária/métodos , Vesículas Extracelulares/metabolismo , Feminino , Humanos , Hipóxia/metabolismo , Precondicionamento Isquêmico/métodos , Isoflurano/farmacologia , Masculino , Pessoa de Meia-Idade , Miócitos Cardíacos/metabolismo , Propofol/farmacologia , Ratos
6.
Invest Ophthalmol Vis Sci ; 61(2): 30, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-32084266

RESUMO

Purpose: Extracellular vesicles (EVs) contain RNA and protein cargo reflective of the genotype and phenotype of the releasing cell of origin. Adult neural retina EV release, RNA transfer, and proteomic cargo are the focus of this study. Methods: Adult wild-type mouse retinae were cultured and released EV diameters and concentrations quantified using Nanosight. Immunogold transmission electron microscopy (TEM) was used to image EV ultrastructure and marker protein localization. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to analyze retinal cell transcripts present in EVs. Super-resolution microscopy was used to image fluorescent (green) RNA and (red) lipid membrane labeled EVs, released by adult retina, and internalized by isolated retinal cells. Mass spectrometry was used to characterize the proteomes of adult retina and EVs. Results: Adult neural retina released EVs at a rate of 1.42 +/- 0.08 × 108/mL over 5 days, with diameters ranging from 30 to 910 nm. The canonical EV markers CD63 and Tsg101 localized to retinal EVs. Adult retinal and neuronal mRNA species present in both retina and EVs included rhodopsin and the neuronal nuclei marker NeuN. Fluorescently labeled RNA in retinal cells was enclosed in EVs, transported to, and uptaken by co-cultured adult retinal cells. Proteomic analysis revealed 1696 protein species detected only in retinal cells, 957 species shared between retina and EVs, and 82 detected only in EVs. Conclusions: The adult neural retina constitutively releases EVs with molecular cargo capable of intercellular transport and predicted involvement in biological processes including retinal physiology, mRNA processing, and transcription regulation within the retinal microenvironment.


Assuntos
Vesículas Extracelulares/metabolismo , Proteínas do Olho/metabolismo , Neurônios/metabolismo , Transporte Proteico/fisiologia , Transporte de RNA/fisiologia , Retina/metabolismo , Animais , Camundongos , Microscopia Eletrônica de Transmissão , RNA Mensageiro/metabolismo
7.
Adv Exp Med Biol ; 1226: 73-86, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32030677

RESUMO

The term "adipose tissue" represents a multicellular and multifunctional organ involved in lipid storage, in hormone and temperature regulation, and in the protection of bones and vital organs from impact-based damage. Emerging evidence now suggests a more malignant role of adipose tissue in promoting cancer onset and progression via the release of secreted factors such as interleukin-6 (IL6) and extracellular vesicles (EVs). These adipose-source factors subsequently affect various aspects of tumorigenesis and/or cancer progression by either directly enhancing the tumor cell oncogenic phenotype or indirectly by the stimulating adjacent normal cells to adopt a more pro-cancer phenotype. Due to the recent growing interest in the role of IL6 and EVs released by adipose tissue in cancer promotion and progression, we are focusing on the protumorigenic impact of fat tissue via IL6 and EV secretion.


Assuntos
Tecido Adiposo/metabolismo , Carcinogênese , Vesículas Extracelulares/metabolismo , Interleucina-6/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Microambiente Tumoral , Humanos
8.
Ann Hematol ; 99(3): 459-475, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31932899

RESUMO

Acute myeloid leukemia (AML) is a heterogeneous malignant disease characterized by a collection of genetic and epigenetic changes. As a consequence, AML can evolve towards more aggressive subtypes during treatment, which require additional therapies to prevent future relapse. As we have previously detected double-stranded DNA (dsDNA) in tumor-derived extracellular vesicles (EVs), in this current study we attempted to evaluate the potential diagnostic applications of AML EV-dsDNA derived from primary bone marrow and peripheral blood plasma samples. EVs from plasma of 29 pediatric AML patients (at initial diagnosis or during treatment) were isolated by ultracentrifugation, after which dsDNA was extracted from obtained EVs and analyzed for leukemia-specific mutations using next generation sequencing (NGS) and GeneScan-based fragment-length analysis. In 18 out of 20 patients, dsDNA harvested from EVs mirrored the (leukemia-specific) mutations found in the genomic DNA obtained from primary leukemia cells. In the nanoparticle tracking analysis (NTA), a decrease in EV numbers was observed in patients after treatment compared with initial diagnosis. Following treatment, in 75 samples out of the 79, these mutations were no longer detectable in EV-dsDNA. In light of our results, we propose the use of leukemia-derived EV-dsDNA as an additional measure for mutational status and, potentially, treatment response in pediatric AML.


Assuntos
DNA de Neoplasias , Vesículas Extracelulares , Leucemia Mieloide Aguda , Mutação , Adolescente , Criança , Pré-Escolar , DNA de Neoplasias/sangue , DNA de Neoplasias/genética , Vesículas Extracelulares/genética , Vesículas Extracelulares/metabolismo , Feminino , Humanos , Lactente , Leucemia Mieloide Aguda/sangue , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Masculino
9.
Nat Biomed Eng ; 4(1): 52-68, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31937944

RESUMO

A small percentage of the short interfering RNA (siRNA) delivered via passive lipid nanoparticles and other delivery vehicles reaches the cytoplasm of cells. The high doses of siRNA and delivery vehicle that are thus required to achieve therapeutic outcomes can lead to toxicity. Here, we show that the integration of siRNA sequences into a Dicer-independent RNA stem-loop based on pre-miR-451 microRNA-which is highly enriched in small extracellular vesicles secreted by many cell types-reduces the expression of the genes targeted by the siRNA in the liver, intestine and kidney glomeruli of mice at siRNA doses that are at least tenfold lower than the siRNA doses typically delivered via lipid nanoparticles. Small extracellular vesicles that efficiently package siRNA can significantly reduce its therapeutic dose.


Assuntos
Vesículas Extracelulares/metabolismo , MicroRNAs/metabolismo , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/metabolismo , Animais , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos/métodos , Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , MicroRNAs/química , Neurônios Motores/efeitos dos fármacos , Nanopartículas/administração & dosagem , Interferência de RNA , RNA Interferente Pequeno/química
10.
Diabetes Res Clin Pract ; 160: 108010, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31987752

RESUMO

AIMS: MicroRNAs (miRNAs) that circulate in biological fluids are frequently enclosed in extracellular vesicles (EVs). However, urinary EVs and their cargo miRNAs have not been systematically studied according to their EV isolation methods. METHODS: In type 2 diabetes mellitus persons with diabetic nephropathy (n = 4), we compared miRNA species in urine EVs prepared by ultracentrifugation (UC), qEV original size exclusion column (qEV), ExoQuick-TC Plus (ExoQuick), and ultrafiltration using Amicon Ultra centrifugal filter devices (Amicons) 10 K and 100 K. EV miRNAs were profiled by next-generation sequencing (NGS). Additionally, we evaluated the correlations of EV miRNA expression between the urine and serum samples isolated by UC. RESULTS: From each of 100 ml of urine, the UC method yielded the highest number of EV miRNA species (233 ± 37.3), with the ExoQuick yielded the lowest (103 ± 17.4). Urine EV miRNA profiles were highly correlated between UC, qEV, ExoQuick and Amicon 10 K methods. EV miRNA profiles between the urine and serum samples showed variable correlations between the patients (paired sample number = 3, r = 0.39-0.72). CONCLUSIONS: UC, qEV, ExoQuick, and Amicon 10 K are acceptable for urinary EV isolation to profile miRNAs. Urine- and serum-derived EV miRNA profiles have variable correlations depending on specific patients.


Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Vesículas Extracelulares/metabolismo , MicroRNAs/metabolismo , Urina/química , Nefropatias Diabéticas/metabolismo , Feminino , Humanos , Masculino
11.
Nat Cell Biol ; 22(2): 187-199, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31932738

RESUMO

Traditionally viewed as an autodigestive pathway, autophagy also facilitates cellular secretion; however, the mechanisms underlying these processes remain unclear. Here, we demonstrate that components of the autophagy machinery specify secretion within extracellular vesicles (EVs). Using a proximity-dependent biotinylation proteomics strategy, we identify 200 putative targets of LC3-dependent secretion. This secretome consists of a highly interconnected network enriched in RNA-binding proteins (RBPs) and EV cargoes. Proteomic and RNA profiling of EVs identifies diverse RBPs and small non-coding RNAs requiring the LC3-conjugation machinery for packaging and secretion. Focusing on two RBPs, heterogeneous nuclear ribonucleoprotein K (HNRNPK) and scaffold-attachment factor B (SAFB), we demonstrate that these proteins interact with LC3 and are secreted within EVs enriched with lipidated LC3. Furthermore, their secretion requires the LC3-conjugation machinery, neutral sphingomyelinase 2 (nSMase2) and LC3-dependent recruitment of factor associated with nSMase2 activity (FAN). Hence, the LC3-conjugation pathway controls EV cargo loading and secretion.


Assuntos
Autofagossomos/metabolismo , Autofagia/genética , Vesículas Extracelulares/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas de Ligação a RNA/genética , Proteínas Adaptadoras de Transporte Vesicular/deficiência , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Autofagossomos/química , Proteína 7 Relacionada à Autofagia/deficiência , Proteína 7 Relacionada à Autofagia/genética , Proteínas Relacionadas à Autofagia/deficiência , Proteínas Relacionadas à Autofagia/genética , Transporte Biológico , Biotinilação , Vesículas Extracelulares/química , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Células HEK293 , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lisossomos/química , Lisossomos/metabolismo , Proteínas de Ligação à Região de Interação com a Matriz/genética , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas à Matriz Nuclear/genética , Proteínas Associadas à Matriz Nuclear/metabolismo , Proteômica/métodos , Células RAW 264.7 , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/metabolismo , Proteínas de Ligação a RNA/classificação , Proteínas de Ligação a RNA/metabolismo , Receptores Estrogênicos/genética , Receptores Estrogênicos/metabolismo , Esfingomielina Fosfodiesterase/genética , Esfingomielina Fosfodiesterase/metabolismo
12.
Cancer Sci ; 111(3): 857-868, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31930596

RESUMO

Increasing evidence indicates that extracellular vesicles (EVs) play an important role in cancer cell-to-cell communication. The Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1), which is closely associated with nasopharyngeal carcinoma (NPC) pathogenesis, can trigger multiple cell signaling pathways that affect cell progression. Several reports have shown that LMP1 promotes EV secretion, and LMP1 trafficking by EVs can enhances cancer progression and metastasis. However, the molecular mechanism by which LMP1 promotes EV secretion is not well understood. In the present study, we found that LMP1 promotes EV secretion by upregulated syndecan-2 (SDC2) and synaptotagmin-like-4 (SYTL4) through nuclear factor (NF)-κB signaling in NPC cells. Further study indicated that SDC2 interacted with syntenin, which promoted the formation of the EVs, and SYTL4 is associated with the release of EVs. Moreover, we found that stimulation of EV secretion by LMP1 can enhance the proliferation and invasion ability of recipient NPC cells and tumor growth in vivo. In summary, we found a new mechanism by which LMP1 upregulates SDC2 and SYTL4 through NF-κB signaling to promote EV secretion, and further enhance cancer progression of NPC.


Assuntos
Vesículas Extracelulares/metabolismo , Herpesvirus Humano 4/metabolismo , Carcinoma Nasofaríngeo/metabolismo , Neoplasias Nasofaríngeas/metabolismo , Sindecana-2/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas da Matriz Viral/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Feminino , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , NF-kappa B/metabolismo , Transdução de Sinais/fisiologia , Regulação para Cima/fisiologia
13.
Int J Cancer ; 146(4): 1052-1063, 2020 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-31259424

RESUMO

Sorafenib provides survival benefits in patients with advanced renal cell carcinoma (RCC), but its use is hampered by acquired drug resistance. It is important to fully clarify the molecular mechanisms of sorafenib resistance, which can help to avoid, delay or reverse drug resistance. Extracellular vesicles (EVs) can mediate intercellular communication by delivering effector molecules between cells. Here, we studied whether EVs are involved in sorafenib resistance of RCC and its possible molecular mechanisms. Using differential centrifugation, EVs were isolated from established sorafenib-resistant RCC cells (786-0 and ACHN), and EVs derived from sorafenib-resistant cells were uptaken by sensitive parental RCC cells and thus promoted drug resistance. Elevated exogenous miR-31-5p within EVs effectively downregulated MutL homolog 1 (MLH1) expression and thus promoted sorafenib resistance in vitro. Mice experiments also confirmed that miR-31-5p could mediate drug sensitivity in vivo. In addition, low expression of MLH1 was observed in sorafenib-resistant RCC cells and upregulation of MLH1 expression restored the sensitivity of resistant cell lines to sorafenib. Finally, miR-31-5p level in circulating EVs of RCC patients with progressive disease (PD) during sorafenib therapy was higher when compared to that in the pretherapy status. In conclusion, EVs shuttled miR-31-5p can transfer resistance information from sorafenib-resistant cells to sensitive cells by directly targeting MLH1, and thus magnify the drug resistance information to the whole tumor. Furthermore, miR-31-5p and MLH1 could be promising predictive biomarkers and therapeutic targets to prevent sorafenib resistance.


Assuntos
Carcinoma de Células Renais/tratamento farmacológico , Vesículas Extracelulares/metabolismo , MicroRNAs/metabolismo , Proteína 1 Homóloga a MutL/metabolismo , Sorafenibe/farmacologia , Animais , Antineoplásicos/farmacologia , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Vesículas Extracelulares/genética , Humanos , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteína 1 Homóloga a MutL/biossíntese , Proteína 1 Homóloga a MutL/genética , Ensaios Antitumorais Modelo de Xenoenxerto
14.
J Cancer Res Clin Oncol ; 146(2): 467-475, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31734835

RESUMO

PURPOSE: The expression of active tissue factor (TF) on the surface of microvesicles (MVs) is essential for the activation of the coagulation system and transduction of the signaling pathways in cancer cells. In its use as a biomarker for cancer-associated venous thromboembolism (VTE), TF has shown high expression variability. As a contribution to this discussion, we present a study investigating plasma samples from patients with various progressive tumors at high risk for VTE. METHODS: Based on our previous study uncovering microvesicles (MVs), the larger ectosome-like extracellular vesicles (EV), as the major source of TF activity in EV preparations, we now determined TF activity on enriched MVs isolated from plasma of cancer patients and compared it with that on MVs from healthy individuals. RESULTS: We found considerably higher amounts of MVs as well as higher levels of MV-bound TF activities in the plasma of cancer patients. We also show that preparations from plasma of cancer patients have the potency to induce ERK phosphorylation in a human tumor cell line through proteinase-activated receptor two (PAR2) activation. CONCLUSION: We suggest that MVs instead of whole EV preparations, and TF activity rather than its antigenic quantification should be used in clinical studies for identifying patients with progressive tumors at high risk for VTE.


Assuntos
Tromboplastina/metabolismo , Idoso , Estudos de Casos e Controles , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/patologia , Feminino , Humanos , Sistema de Sinalização das MAP Quinases , Masculino , Neoplasias Pancreáticas/sangue , Neoplasias Pancreáticas/patologia , Fosforilação , Tromboplastina/biossíntese , Tromboembolia Venosa/sangue , Tromboembolia Venosa/patologia
15.
Scand J Immunol ; 91(2): e12841, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31833575

RESUMO

Tumour-associated macrophages (TAMs) comprise an important part of the tumour microenvironment and play a key role in malignant tumours progression. Tumour-derived extracellular vesicles (EVs) modulated TAMs polarization and reprogrammed TAMs to influence the progression of various tumours. Here, we hypothesized that diffuse large B cell lymphoma (DLBCL)-derived EVs can regulate macrophages polarization and thus contribute to tumour progression. Our results demonstrated that EVs, released from DLBCL, augment the M2 polarization effects of macrophages. Moreover, conditional medium derived from macrophages by DLBCL-derived EVs stimulation revealed the superior effects of promoting tumour proliferation, migration and invasion. Further analysis demonstrated that DLBCL-derived EVs regulated the metabolism of macrophages by increasing the expression of PGC-1ß protein, thereby reprogramming the macrophage phenotype of promoting tumour progression. In conclusion, our findings signify that the DLBCL-derived EVs mediated the increasing of functional PGC-1ß protein in macrophages to promote the tumour progression.


Assuntos
Vesículas Extracelulares/metabolismo , Linfoma Difuso de Grandes Células B/imunologia , Macrófagos/fisiologia , Proteínas de Ligação a RNA/metabolismo , Carcinogênese , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Reprogramação Celular , Meios de Cultivo Condicionados , Progressão da Doença , Humanos , Ativação de Macrófagos , MicroRNAs/genética , Fenótipo , Microambiente Tumoral , Regulação para Cima
16.
J Dairy Sci ; 103(1): 16-29, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31677838

RESUMO

MicroRNAs (miRNAs) are small gene-regulatory noncoding RNA that are highly enriched in cow milk. They are encapsulated in different extracellular vesicle (EV) subsets that protect them from the extracellular milieu and the harsh conditions of the gastrointestinal tract during digestion. Here, we isolated pellets enriched in 4 different EV subsets, via differential ultracentrifugation of commercial cow milk: 12,000 × g (P12K), 35,000 × g (P35K), 70,000 × g (P70K), and 100,000 × g (P100K). Small RNA sequencing (sRNA-Seq) analyses revealed an unprecedented level of diversity in the complete miRNA repertoire and features of unfractionated cow milk and derived EV subsets. Although 5 miRNA sequences represented more than 50% of all miRNAs, milk EV exhibited heterogeneous content of miRNAs and isomeric variants (termed isomiR): P100K EV were enriched in reference miRNA sequences, and P12K and P35K EV in related isomiR. Incubation of milk EV with human cultured HeLa cells led to cellular enrichment in miRNA miR-223, which was concomitant with decreased expression of a reporter gene placed under the control of miR-223, thereby demonstrating the functionality of miR-223. These results suggest that cow milk EV may transfer their miRNAs to human cells and regulate recipient cell gene expression programming in a manner as complex as that of their miRNA transcriptome. The biological activity and relevance of the different milk EV subsets and bioactive mediators, including small noncoding RNA, in health and disease, warrants further investigation.


Assuntos
Vesículas Extracelulares/química , MicroRNAs/síntese química , Transcriptoma/fisiologia , Ultracentrifugação/veterinária , Animais , Bovinos , Vesículas Extracelulares/genética , Vesículas Extracelulares/metabolismo , Feminino , Regulação da Expressão Gênica , Células HeLa , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Leite/metabolismo , Análise de Sequência de RNA
17.
Am J Physiol Renal Physiol ; 318(2): F486-F495, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31869243

RESUMO

Extracellular vesicles released into urine (uEVs) can represent interesting biomarkers of renal cell damage. CD133, a stem/progenitor cell marker expressed by renal progenitor cells, is highly expressed in uEVs of healthy individuals. In the present study, we evaluated the level of CD133 in the uEVs of patients with acute and chronic glomerular damage by cytofluorimetric analysis. The level of CD133+ uEVs was significantly decreased in pediatric patients with acute glomerulonephritis during the acute phase of renal damage, while it was restored after the subsequent recovery. A similar decrease was also observed in patients with chronic glomerulonephritis. Moreover, CD133+ uEVs significantly declined in patients with type 2 diabetes, used as validation group, with the lowest levels in patients with albuminuria with diabetic nephropathy. Indeed, receiver-operating characteristic curve analysis indicates the ability of CD133+ uEV values to discriminate the health condition from that of glomerular disease. In parallel, a significant decrease of CD133 in renal progenitor cells and in their derived EVs was observed in vitro after cell treatment with a combination of glucose and albumin overload, mimicking the diabetic condition. These data indicate that the level of CD133+ uEVs may represent an easily accessible marker of renal normal physiology and could provide information on the "reservoir" of regenerating cells within tubules.


Assuntos
Antígeno AC133/urina , Nefropatias Diabéticas/urina , Vesículas Extracelulares/metabolismo , Glomerulonefrite/urina , Glomérulos Renais/metabolismo , Células-Tronco/metabolismo , Doença Aguda , Adolescente , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/urina , Estudos de Casos e Controles , Proliferação de Células , Células Cultivadas , Criança , Pré-Escolar , Doença Crônica , Nefropatias Diabéticas/patologia , Nefropatias Diabéticas/fisiopatologia , Regulação para Baixo , Vesículas Extracelulares/patologia , Feminino , Taxa de Filtração Glomerular , Glomerulonefrite/patologia , Glomerulonefrite/fisiopatologia , Humanos , Glomérulos Renais/patologia , Glomérulos Renais/fisiopatologia , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Prognóstico , Regeneração , Reprodutibilidade dos Testes , Células-Tronco/patologia , Urinálise
18.
Platelets ; 31(1): 26-32, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-30585111

RESUMO

Activated platelets contribute to thrombosis and inflammation by the release of extracellular vesicles (EVs) exposing P-selectin, phosphatidylserine (PS) and fibrinogen. P2Y12 receptor antagonists are routinely administered to inhibit platelet activation in patients after acute myocardial infarction (AMI), being a combined antithrombotic and anti-inflammatory therapy. The more potent P2Y12 antagonist ticagrelor improves cardiovascular outcome in patients after AMI compared to the less potent clopidogrel, suggesting that greater inhibition of platelet aggregation is associated with better prognosis. The effect of ticagrelor and clopidogrel on the release of EVs from platelets and other P2Y12-exposing cells is unknown. This study compares the effects of ticagrelor and clopidogrel on (1) the concentrations of EVs from activated platelets (primary end point), (2) the concentrations of EVs exposing fibrinogen, exposing PS, from leukocytes and from endothelial cells (secondary end points) and (3) the procoagulant activity of plasma EVs (tertiary end points) in 60 consecutive AMI patients. After the percutaneous coronary intervention, patients will be randomized to antiplatelet therapy with ticagrelor (study group) or clopidogrel (control group). Blood will be collected from patients at randomization, 48 hours after randomization and 6 months following the index hospitalization. In addition, 30 age- and gender-matched healthy volunteers will be enrolled in the study to investigate the physiological concentrations and procoagulant activity of EVs using recently standardized protocols and EV-dedicated flow cytometry. Concentrations of EVs will be determined by flow cytometry. Procoagulant activity of EVs will be determined by fibrin generation test. The compliance and response to antiplatelet therapy will be assessed by impedance aggregometry. We expect that plasma from patients treated with ticagrelor (1) contains lower concentrations of EVs from activated platelets, exposing fibrinogen, exposing PS, from leukocytes and from endothelial cells and (2) has lower procoagulant activity, when compared to patients treated with clopidogrel. Antiplatelet therapy effect on EVs may identify a new mechanism of action of ticagrelor, as well as create a basis for future studies to investigate whether lower EV concentrations are associated with improved clinical outcomes in patients treated with P2Y12 antagonists.


Assuntos
Protocolos Clínicos , Vesículas Extracelulares/efeitos dos fármacos , Vesículas Extracelulares/metabolismo , Infarto do Miocárdio/complicações , Infarto do Miocárdio/metabolismo , Inibidores da Agregação de Plaquetas/administração & dosagem , Trombose/etiologia , Trombose/prevenção & controle , Biomarcadores , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Feminino , Humanos , Masculino , Infarto do Miocárdio/terapia , Intervenção Coronária Percutânea , Ativação Plaquetária/efeitos dos fármacos , Antagonistas do Receptor Purinérgico P2Y/administração & dosagem
19.
Cancer Sci ; 111(2): 610-620, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31845438

RESUMO

High-grade neuroendocrine lung cancer (HGNEC), which includes small cell lung cancer (SCLC) and large cell neuroendocrine carcinoma (LCNEC) of the lung is a rapidly proliferating, aggressive form of lung cancer. The initial standard chemotherapeutic regimens of platinum doublets are recommended for SCLC and have been frequently used for LCNEC. However, there are currently no molecularly targeted agents with proven clinical benefit for this disease. The deubiquitinating enzyme ubiquitin C-terminal hydrolase-L1 (UCHL1) is a neuroendocrine cell-specific product that is known as a potential oncogene in several types of cancer, but little is known about the biological function of UCHL1 and its therapeutic potential in HGNEC. In this study, we found that preclinical efficacy evoked by targeting UCHL1 was relevant to prognosis in HGNEC. UCHL1 was found to be expressed in HGNEC, particularly in cell lines and patient samples of SCLC, and the combined use of platinum doublets with selective UCHL1 inhibitors improved its therapeutic response in vitro. Immunohistochemical expression of UCHL1 was significantly associated with postoperative survival in patients with HGNEC and contributed towards distinguishing SCLC from LCNEC. Circulating extracellular vesicles (EV), including exosomes isolated from lung cancer cell lines and serum from early-stage HGNEC, were verified by electron microscopy and nanoparticle tracking analysis. Higher levels of UCHL1 mRNA in EV were found in the samples of patients with early-stage HGNEC than those with early-stage NSCLC and healthy donors' EV. Taken together, UCHL1 may be a potential prognostic marker and a promising druggable target for HGNEC.


Assuntos
Carcinoma Neuroendócrino/patologia , Vesículas Extracelulares/genética , Neoplasias Pulmonares/patologia , Carcinoma de Pequenas Células do Pulmão/patologia , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo , Células A549 , Idoso , Idoso de 80 Anos ou mais , Carcinoma Neuroendócrino/tratamento farmacológico , Carcinoma Neuroendócrino/genética , Carcinoma Neuroendócrino/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Vesículas Extracelulares/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Platina/farmacologia , Platina/uso terapêutico , Prognóstico , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Carcinoma de Pequenas Células do Pulmão/genética , Carcinoma de Pequenas Células do Pulmão/metabolismo , Ubiquitina Tiolesterase/antagonistas & inibidores , Regulação para Cima
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