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Circulating cell-free nucleic acids are considered a promising source of biomarkers for diseases and cancer. Liquid biopsy biomarkers for brain tumours represent a major, still unmet, clinical need. In plasma, nucleic acids can be free or be associated with extracellular vesicles (EVs). Here we report an easy and reproducible method to analyse cell-free nucleic acids in plasma and EVs by conventional flow cytometry easy to translate into the clinics. Nucleic acids associated with the EVs or present in plasma samples are stained by Pyronin Y, which is a fluorescent dye that is preferably binding double-stranded nucleic acids. Fluorescent staining of EVs isolated from cell-conditioned media is suitable for DNA and RNA detection by flow cytometry. The nucleic acids are partially protected from degradation by the EVs' membrane. Additionally, DNA and RNA can be stained in plasma samples and plasma-derived EVs. Remarkably, analysis of plasma from patients and healthy individuals reveals a difference in their nucleic acid profiles. Taken together, our results indicate that the proposed methodology, which is based on conventional direct flow cytometry, is a promising easy tool for plasma nucleic acid analysis.
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Seminal plasma (SP) is rich in extracellular vesicles (EVs), which are still poorly studied, especially in livestock species. To better understand their functional role in both spermatozoa and endometrial epithelial cells, proper characterization of EVs is an essential step. The objective was to phenotypically characterize porcine seminal EVs (sEVs) using cryogenic electron microscopy (cryo-EM), which allows visualization of EVs in their native state. Porcine ejaculates are released in fractions, each containing SP from different source. This allows characterization sEVs released from various male reproductive tissues. Two experiments were performed, the first with SP from the entire ejaculate (n:6) and the second with SP from three ejaculate fractions (n:15): the first 10 mL of the sperm-rich ejaculate fraction (SRF-P1) with SP mainly from the epididymis, the remainder of the SRF (SRF-P2) with SP mainly from the prostate, and the post-SRF with SP mainly from the seminal vesicles. The sEVs were isolated by size exclusion chromatography and 1840 cryo-EM sEV images were acquired using a Jeol-JEM-2200FS/CR-EM. The size, electron density, complexity, and peripheral corona layer were measured in each sEV using the ImageJ software. The first experiment showed that sEVs were structurally and morphologically heterogeneous, although most (83.1%) were small (less than 200 nm), rounded, and poorly electrodense, and some have a peripheral coronal layer. There were also larger sEVs (16.9%) that were irregularly shaped, more electrodense, and few with a peripheral coronal layer. The second experiment showed that small sEVs were more common in SRF-P1 and SRF-P2, indicating that they originated mainly from the epididymis and prostate. Large sEVs were more abundant in post-SRF, indicating that they originated mainly from seminal vesicles. Porcine sEVs are structurally and morphologically heterogeneous. This would be explained by the diversity of reproductive organs of origin.
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Microscopia Crioeletrônica , Vesículas Extracelulares , Sêmen , Animais , Vesículas Extracelulares/ultraestrutura , Vesículas Extracelulares/metabolismo , Masculino , Microscopia Crioeletrônica/métodos , Suínos , Espermatozoides/ultraestrutura , Glândulas Seminais/ultraestruturaRESUMO
Extracellular vesicles (EVs) have been involved in metabolic syndrome, although their specific role in the development of the pathology is still unknown. To further study the role of EVs, we have analysed by Raman tweezers microspectroscopy and mass spectrometry-based lipidomics the small EVs population secreted by fatty (ZF) and lean (ZL) hepatocytes obtained from Zucker rats. We have also explored in vivo and ex vivo biodistribution of these EVs through fluorine-18-radiolabelling using a positron emission tomography imaging. Based on the proportion of proteins to lipids and the types of lipids, our results indicate that within the range of small EVs, primary hepatocytes secrete different subpopulations of particles. These differences were observed in the enrichment of triglyceride species in EVs secreted by ZF hepatocytes. Biodistribution experiments showed accumulation in the brain, heart, lungs, kidney and specially in bladder after intravenous administration. In summary, we show that EVs released by a fatty hepatocytes carry a different lipid signature compared to their lean counterpart. Biodistribution experiment has shown no difference in the distribution of EVs secreted by ZF and ZL hepatocytes but has given us a first view of possible target organs for these particles. Our results might open a door to both pathology studies and therapeutic interventions.
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In vitro maturation (IVM) of oocytes is clinically used in horses to produce blastocysts but current conditions used for horses are suboptimal. We analyzed the composition of equine preovulatory follicular fluid (FF) secretome and tested its effects on meiotic competence and gene expression in oocytes subjected to IVM. Preovulatory FF was obtained, concentrated using ultrafiltration with cut-off of 10 kDa, and stored at -80 °C. The metabolic and proteomic composition was analyzed, and its ultrastructural composition was assessed by cryo-transmission microscopy. Oocytes obtained post-mortem or by ovum pick up (OPU) were subjected to IVM in the absence (control) or presence of 20 or 40 µg/ml (S20 or S40) of secretome. Oocytes were then analyzed for chromatin configuration or snap frozen for gene expression analysis. Proteomic analysis detected 255 proteins in the Equus caballus database, mostly related to the complement cascade and cholesterol metabolism. Metabolomic analysis yielded 14 metabolites and cryo-transmission electron microscopy analysis revealed the presence of extracellular vesicles (EVs). No significant differences were detected in maturation rates among treatments. However, the expression of GDF9 and BMP15 significantly increased in OPU-derived oocytes compared to post-mortem oocytes (fold increase ± SEM: 9.4 ± 0.1 vs. 1 ± 0.5 for BMP15 and 9.9 ± 0.3 vs. 1 ± 0.5 for GDF9, respectively; p < 0.05). Secretome addition increased the expression of TNFAIP6 in S40 regardless of the oocyte source. Further research is necessary to fully understand whether secretome addition influences the developmental competence of equine oocytes.
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Líquido Folicular , Proteômica , Feminino , Cavalos , Animais , Líquido Folicular/química , Líquido Folicular/metabolismo , Secretoma , Meiose , Oócitos/metabolismo , Técnicas de Maturação in Vitro de Oócitos/veterináriaRESUMO
Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly.
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Exossomos , Vesículas Extracelulares , Vesículas Extracelulares/metabolismo , Exossomos/metabolismo , Transporte Biológico , Biomarcadores/metabolismo , FenótipoRESUMO
Cerebrospinal fluid (CSF) is a clear, transparent fluid derived from blood plasma that protects the brain and spinal cord against mechanical shock, provides buoyancy, clears metabolic waste and transports extracellular components to remote sites in the brain. Given its contact with the brain and the spinal cord, CSF is the most informative biofluid for studies of the central nervous system (CNS). In addition to other components, CSF contains extracellular vesicles (EVs) that carry bioactive cargoes (e.g., lipids, nucleic acids, proteins), and that can have biological functions within and beyond the CNS. Thus, CSF EVs likely serve as both mediators of and contributors to communication in the CNS. Accordingly, their potential as biomarkers for CNS diseases has stimulated much excitement for and attention to CSF EV research. However, studies on CSF EVs present unique challenges relative to EV studies in other biofluids, including the invasive nature of CSF collection, limited CSF volumes and the low numbers of EVs in CSF as compared to plasma. Here, the objectives of the International Society for Extracellular Vesicles CSF Task Force are to promote the reproducibility of CSF EV studies by providing current reporting and best practices, and recommendations and reporting guidelines, for CSF EV studies. To accomplish this, we created and distributed a world-wide survey to ISEV members to assess methods considered 'best practices' for CSF EVs, then performed a detailed literature review for CSF EV publications that was used to curate methods and resources. Based on responses to the survey and curated information from publications, the CSF Task Force herein provides recommendations and reporting guidelines to promote the reproducibility of CSF EV studies in seven domains: (i) CSF Collection, Processing, and Storage; (ii) CSF EV Separation/Concentration; (iii) CSF EV Size and Number Measurements; (iv) CSF EV Protein Studies; (v) CSF EV RNA Studies; (vi) CSF EV Omics Studies and (vii) CSF EV Functional Studies.
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Vesículas Extracelulares , Biomarcadores/metabolismo , Encéfalo/metabolismo , Vesículas Extracelulares/metabolismo , Proteínas/metabolismo , Reprodutibilidade dos TestesRESUMO
The human CERS2 gene encodes a ceramide synthase enzyme, known as CERS2 (ceramide synthase 2). This protein is also known as LASS2 (LAG1 longevity assurance homolog 2) and TMSG1 (tumor metastasis-suppressor gene 1). Although previously described as a tumor suppressor for different types of cancer, such as prostate or liver cancer, it has also been observed to promote tumor growth in adenocarcinoma. In this review, we focus on the influence of CERS2 in bladder cancer (BC), approaching the existing literature about its structure and activity, as well as the miRNAs regulating its expression. From a mechanistic point of view, different explanations for the role of CERS2 as an antitumor protein have been proposed, including the production of long-chain ceramides, interaction with vacuolar ATPase, and its function as inhibitor of mitochondrial fission. In addition, we reviewed the literature specifically studying the expression of this gene in both BC and biopsy-derived tumor cell lines, complementing this with an analysis of public gene expression data and its association with disease progression. We also discuss the importance of CERS2 as a biomarker and the presence of CERS2 mRNA in extracellular vesicles isolated from urine.
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Proteínas Supressoras de Tumor , Neoplasias da Bexiga Urinária , Masculino , Humanos , RNA Interferente Pequeno/genética , Proteínas Supressoras de Tumor/genética , Longevidade , Ceramidas/metabolismo , Neoplasias da Bexiga Urinária/genética , Esfingosina N-Aciltransferase/genética , Esfingosina N-Aciltransferase/metabolismoRESUMO
There has been an intense focus to uncover the molecular mechanisms by which fasting triggers the adaptive cellular responses in the major organs of the body. Here, we show that in mice, hepatic S-adenosylmethionine (SAMe)-the principal methyl donor-acts as a metabolic sensor of nutrition to fine-tune the catabolic-fasting response by modulating phosphatidylethanolamine N-methyltransferase (PEMT) activity, endoplasmic reticulum-mitochondria contacts, ß-oxidation, and ATP production in the liver, together with FGF21-mediated lipolysis and thermogenesis in adipose tissues. Notably, we show that glucagon induces the expression of the hepatic SAMe-synthesizing enzyme methionine adenosyltransferase α1 (MAT1A), which translocates to mitochondria-associated membranes. This leads to the production of this metabolite at these sites, which acts as a brake to prevent excessive ß-oxidation and mitochondrial ATP synthesis and thereby endoplasmic reticulum stress and liver injury. This work provides important insights into the previously undescribed function of SAMe as a new arm of the metabolic adaptation to fasting.
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Neoplasias Hepáticas , S-Adenosilmetionina , Camundongos , Animais , S-Adenosilmetionina/metabolismo , Fígado/metabolismo , Neoplasias Hepáticas/metabolismo , Jejum , Trifosfato de Adenosina/metabolismo , Metionina Adenosiltransferase/metabolismo , Fosfatidiletanolamina N-Metiltransferase/metabolismoRESUMO
Several studies have described the contribution of glutamate-transforming microbiota to the development of chronic ailments. For instance, the blood concentration of glutamate is higher in some patients with fibromyalgia, chronic fatigue, and pain. Taking advantage of a naturally occurring strain of Bifidobacterium that is able to transform glutamate in γ-aminobutyric caid (GABA), B. adolescentis IPLA60004, we designed a placebo-controlled intervention to test if the presence of this GABA-producing bifidobacteria in mice was able to impact the concentration of glutamate in the blood in comparison with the administration of other strain of the same species lacking the genes of the glutamate decarboxylase (gad) cluster. Animals were fed every day with 8 log CFU of bacteria in a sterilized milk vehicle for 14 days. Samples from feces and blood were collected during this period, and afterwards animals were sacrificed, tissues were taken from different organs, and the levels of different metabolites were analyzed by ultrahigh-performance liquid chromatography coupled to mass spectrometry. The results showed that both bacterial strains orally administered survived in the fecal content, and animals fed B. adolescentis IPLA60004 showed a significant reduction of their glutamate serum concentration, while a nonsignificant decrease was observed for animals fed a reference strain, B. adolescentis LGM10502. The variations observed in GABA were influenced by the gender of the animals, and no significant changes were observed in different tissues of the brain. These results suggest that orally administered GABA-producing probiotics could reduce the glutamate concentration in blood, opening a case for a clinical trial study in chronic disease patients. IMPORTANCE This work presents the results of a trial using mice as a model that were fed with a bacterial strain of the species B. adolescentis, which possesses different active genes capable of degrading glutamate and converting it into GABA. Indeed, the bacterium is able to survive the passage through the gastric tract and, more importantly, the animals reduce over time the concentration of glutamate in their blood. The importance of this result lies in the fact that several chronic ailments, such as fibromyalgia, are characterized by an increase in glutamate. Our results indicate that an oral diet with this probiotic-type bacteria could reduce the concentration of glutamate and, therefore, reduce the symptoms associated with the excess of this neurotransmitter.
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Bifidobacterium adolescentis , Fibromialgia , Probióticos , Camundongos , Animais , Bifidobacterium adolescentis/metabolismo , Ácido Glutâmico/análise , Ácido Glutâmico/metabolismo , Bifidobacterium/genética , Bifidobacterium/metabolismo , Fezes/microbiologia , Ácido gama-Aminobutírico/análise , Ácido gama-Aminobutírico/metabolismoRESUMO
Hyperactivation of mechanistic target of rapamycin complex 1 (mTORC1) is a feature of many solid tumours and is a key pathogenic driver in the inherited condition Tuberous Sclerosis Complex (TSC). Modulation of the tumour microenvironment by extracellular vesicles (EVs) is known to facilitate the development of various cancers. The role of EVs in modulating the tumour microenvironment and their impact on the development of TSC tumours, however, remains unclear. This study, therefore, focuses on the poorly defined contribution of EVs to tumour growth in TSC. We characterised EVs secreted from TSC2-deficient and TSC2-expressing cells and identified a distinct protein cargo in TSC2-deficient EVs, containing an enrichment of proteins thought to be involved in tumour-supporting signalling pathways. We show EVs from TSC2-deficient cells promote cell viability, proliferation and growth factor secretion from recipient fibroblasts within the tumour microenvironment. Rapalogs (mTORC1 inhibitors) are the current therapy for TSC tumours. Here, we demonstrate a previously unknown intercellular therapeutic effect of rapamycin in altering EV cargo and reducing capacity to promote cell proliferation in the tumour microenvironment. Furthermore, EV cargo proteins have the potential for clinical applications as TSC biomarkers, and we reveal three EV-associated proteins that are elevated in plasma from TSC patients compared to healthy donor plasma.
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Vesículas Extracelulares , Esclerose Tuberosa , Humanos , Proteínas Supressoras de Tumor , Esclerose Tuberosa/metabolismo , Esclerose Tuberosa/patologia , Proteína 2 do Complexo Esclerose Tuberosa , Vesículas Extracelulares/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Microambiente TumoralRESUMO
Extracellular vesicles (EVs) are secreted nanostructures that play various roles in critical cancer processes. They operate as an intercellular communication system, transferring complex sets of biomolecules from cell to cell. The concentration of EVs is difficult to decipher, and there is an unmet technological need for improved (faster, simpler, and gentler) approaches to isolate EVs from complex matrices. Herein, an acoustofluidic concentration of extracellular vesicles (ACEV) is presented, based on a thin-film printed circuit board with interdigital electrodes mounted on a piezoelectric substrate. An angle of 120° is identified between the electrodes and the reference flat of the piezoelectric substrate for simultaneous generation of Rayleigh and shear horizontal waves. The dual waves create a complex acoustic field in a droplet, resulting in effective concentration of nanoparticles and EVs. The ACEV is able to concentrate 20 nm nanospheres within 105 s and four EV dilutions derived from the human prostate cancer (Du145) cell line in approximately 30 s. Cryo-electron microscopy confirmed the preservation of EV integrity. The ACEV device holds great potential to revolutionize investigations of EVs. Its faster, simpler, and gentler approach to EV isolation and concentration can save time and effort in phenotypic and functional studies of EVs.
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Vesículas Extracelulares , Nanosferas , Neoplasias da Próstata , Masculino , Humanos , Microscopia Crioeletrônica , Vesículas Extracelulares/metabolismo , Linhagem CelularRESUMO
BACKGROUND & AIMS: Cholangiocarcinoma (CCA), heterogeneous biliary tumours with dismal prognosis, lacks accurate early diagnostic methods especially important for individuals at high-risk (i.e. those with primary sclerosing cholangitis [PSC]). Here, we searched for protein biomarkers in serum extracellular vesicles (EVs). METHODS: EVs from patients with isolated PSC (n = 45), concomitant PSC-CCA (n = 44), PSC who developed CCA during follow-up (PSC to CCA; n = 25), CCAs from non-PSC aetiology (n = 56), and hepatocellular carcinoma (n = 34) and healthy individuals (n = 56) were characterised by mass spectrometry. Diagnostic biomarkers for PSC-CCA, non-PSC CCA, or CCAs regardless of aetiology (Pan-CCAs) were defined and validated by ELISA. Their expression was evaluated in CCA tumours at a single-cell level. Prognostic EV biomarkers for CCA were investigated. RESULTS: High-throughput proteomics of EVs identified diagnostic biomarkers for PSC-CCA, non-PSC CCA, or Pan-CCA, and for the differential diagnosis of intrahepatic CCA and hepatocellular carcinoma, which were cross-validated by ELISA using total serum. Machine learning-based algorithms disclosed CRP/FIBRINOGEN/FRIL for the diagnosis of PSC-CCA (local disease [LD]) vs. isolated PSC (AUC = 0.947; odds ratio [OR] =36.9) and, combined with carbohydrate antigen 19-9, overpowers carbohydrate antigen 19-9 alone. CRP/PIGR/VWF allowed the diagnosis of LD non-PSC CCAs vs. healthy individuals (AUC = 0.992; OR = 387.5). It is noteworthy that CRP/FRIL accurately diagnosed LD Pan-CCA (AUC = 0.941; OR = 89.4). Levels of CRP/FIBRINOGEN/FRIL/PIGR showed predictive capacity for CCA development in PSC before clinical evidence of malignancy. Multi-organ transcriptomic analysis revealed that serum EV biomarkers were mostly expressed in hepatobiliary tissues, and single-cell RNA sequencing and immunofluorescence analysis of CCA tumours showed their presence mainly in malignant cholangiocytes. Multivariable analysis unveiled EV prognostic biomarkers, with COMP/GNAI2/CFAI and ACTN1/MYCT1/PF4V associated negatively and positively with patients' survival, respectively. CONCLUSIONS: Serum EVs contain protein biomarkers for the prediction, early diagnosis, and prognostication of CCA that are detectable using total serum, representing a tumour cell-derived liquid biopsy tool for personalised medicine. IMPACT AND IMPLICATIONS: The accuracy of current imaging tests and circulating tumour biomarkers for cholangiocarcinoma (CCA) diagnosis is far from satisfactory. Most CCAs are considered sporadic, although up to 20% of patients with primary sclerosing cholangitis (PSC) develop CCA during their lifetime, constituting a major cause of PSC-related death. This international study has proposed protein-based and aetiology-related logistic models with predictive, diagnostic, or prognostic capacities by combining two to four circulating protein biomarkers, moving a step forward into personalised medicine. These novel liquid biopsy tools may allow the (i) easy and non-invasive diagnosis of sporadic CCAs, (ii) identification of patients with PSC with higher risk for CCA development, (iii) establishment of cost-effective surveillance programmes for the early detection of CCA in high-risk populations (e.g. PSC), and (iv) prognostic stratification of patients with CCA, which, altogether, may increase the number of cases eligible for potentially curative options or to receive more successful treatments, decreasing CCA-related mortality.
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Neoplasias dos Ductos Biliares , Carcinoma Hepatocelular , Colangiocarcinoma , Colangite Esclerosante , Neoplasias Hepáticas , Humanos , Colangite Esclerosante/complicações , Carcinoma Hepatocelular/etiologia , Carcinoma Hepatocelular/complicações , Neoplasias dos Ductos Biliares/patologia , Colangiocarcinoma/diagnóstico , Colangiocarcinoma/etiologia , Colangiocarcinoma/metabolismo , Biomarcadores Tumorais , Diagnóstico Precoce , Biópsia Líquida , Ductos Biliares Intra-Hepáticos/patologia , Neoplasias Hepáticas/etiologia , Neoplasias Hepáticas/complicações , Carboidratos , Proteínas NuclearesRESUMO
BACKGROUND AND AIMS: Alcohol-associated liver disease (ALD) accounts for 70% of liver-related deaths in Europe, with no effective approved therapies. Although mitochondrial dysfunction is one of the earliest manifestations of alcohol-induced injury, restoring mitochondrial activity remains a problematic strategy due to oxidative stress. Here, we identify methylation-controlled J protein (MCJ) as a mediator for ALD progression and hypothesize that targeting MCJ may help in recovering mitochondrial fitness without collateral oxidative damage. APPROACH AND RESULTS: C57BL/6 mice [wild-type (Wt)] Mcj knockout and Mcj liver-specific silencing (MCJ-LSS) underwent the NIAAA dietary protocol (Lieber-DeCarli diet containing 5% (vol/vol) ethanol for 10 days, plus a single binge ethanol feeding at day 11). To evaluate the impact of a restored mitochondrial activity in ALD, the liver, gut, and pancreas were characterized, focusing on lipid metabolism, glucose homeostasis, intestinal permeability, and microbiota composition. MCJ, a protein acting as an endogenous negative regulator of mitochondrial respiration, is downregulated in the early stages of ALD and increases with the severity of the disease. Whole-body deficiency of MCJ is detrimental during ALD because it exacerbates the systemic effects of alcohol abuse through altered intestinal permeability, increased endotoxemia, and dysregulation of pancreatic function, which overall worsens liver injury. On the other hand, liver-specific Mcj silencing prevents main ALD hallmarks, that is, mitochondrial dysfunction, steatosis, inflammation, and oxidative stress, as it restores the NAD + /NADH ratio and SIRT1 function, hence preventing de novo lipogenesis and improving lipid oxidation. CONCLUSIONS: Improving mitochondrial respiration by liver-specific Mcj silencing might become a novel therapeutic approach for treating ALD.
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Hepatopatias Alcoólicas , Animais , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatias Alcoólicas/metabolismo , Fígado/metabolismo , Etanol/efeitos adversos , Mitocôndrias/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Mitocondriais/metabolismoRESUMO
Interferon-stimulated gene 15 (ISG15) is a 15-kDa ubiquitin-like modifier that binds to target proteins in a process termed ISGylation. ISG15, first described as an antiviral molecule against many viruses, participates in numerous cellular processes, from immune modulation to the regulation of genome stability. Interestingly, the role of ISG15 as a regulator of cell metabolism has recently gained strength. We previously described ISG15 as a regulator of mitochondrial functions in bone marrow-derived macrophages (BMDMs) in the context of Vaccinia virus (VACV) infection. Here, we demonstrate that ISG15 regulates lipid metabolism in BMDMs and that ISG15 is necessary to modulate the impact of VACV infection on lipid metabolism. We show that Isg15-/- BMDMs demonstrate alterations in the levels of several key proteins of lipid metabolism that result in differences in the lipid profile compared with Isg15+/+ (wild-type [WT]) BMDMs. Specifically, Isg15-/- BMDMs present reduced levels of neutral lipids, reflected by decreased lipid droplet number. These alterations are linked to increased levels of lipases and are independent of enhanced fatty acid oxidation (FAO). Moreover, we demonstrate that VACV causes a dysregulation in the proteomes of BMDMs and alterations in the lipid content of these cells, which appear exacerbated in Isg15-/- BMDMs. Such metabolic changes are likely caused by increased expression of the metabolic regulators peroxisome proliferator-activated receptor-γ (PPARγ) and PPARγ coactivator-1α (PGC-1α). In summary, our results highlight that ISG15 controls BMDM lipid metabolism during viral infections, suggesting that ISG15 is an important host factor to restrain VACV impact on cell metabolism. IMPORTANCE The functions of ISG15 are continuously expanding, and growing evidence supports its role as a relevant modulator of cell metabolism. In this work, we highlight how the absence of ISG15 impacts macrophage lipid metabolism in the context of viral infections and how poxviruses modulate metabolism to ensure successful replication. Our results open the door to new advances in the comprehension of macrophage immunometabolism and the interaction between VACV and the host.
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Citocinas , Metabolismo dos Lipídeos , Ubiquitinas , Vacínia , Citocinas/metabolismo , Interferons , Lipídeos , PPAR gama/metabolismo , Ubiquitinas/genética , Ubiquitinas/metabolismo , Vacínia/genética , Vacínia/metabolismo , Vaccinia virus/genética , AnimaisRESUMO
The analysis of extracellular vesicles (EVs) as a source of cancer biomarkers is an emerging field since low-invasive biomarkers are highly demanded. EVs constitute a heterogeneous population of small membrane-contained vesicles that are present in most of body fluids. They are released by all cell types, including cancer cells and their cargo consists of nucleic acids, proteins and metabolites and varies depending on the biological-pathological state of the secretory cell. Therefore, EVs are considered as a potential source of reliable biomarkers for cancer. EV biomarkers in liquid biopsy can be a valuable tool to complement current medical technologies for cancer diagnosis, as their sampling is minimally invasive and can be repeated over time to monitor disease progression. In this review, we highlight the advances in EV biomarker research for cancer diagnosis, prognosis, and therapy monitoring. We especially focus on EV derived biomarkers for glioblastoma. The diagnosis and monitoring of glioblastoma still relies on imaging techniques, which are not sufficient to reflect the highly heterogenous and invasive nature of glioblastoma. Therefore, we discuss how the use of EV biomarkers could overcome the challenges faced in diagnosis and monitoring of glioblastoma.
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Vesículas Extracelulares , Glioblastoma , Humanos , Glioblastoma/diagnóstico , Glioblastoma/genética , Glioblastoma/metabolismo , Vesículas Extracelulares/genética , Vesículas Extracelulares/metabolismo , Biópsia Líquida/métodos , Biomarcadores Tumorais/metabolismo , PrognósticoRESUMO
Mesenchymal stromal cell-derived extracellular vesicles (MSC-EV) are widely considered as a cell-free therapeutic alternative to MSC cell administration, due to their immunomodulatory and regenerative properties. However, the interaction mechanisms between EV and target cells are not fully understood. The surface glycans could be key players in EV-cell communication, being specific molecular recognition patterns that are still little explored. In this study, we focused on the role of N-glycosylation of MSC-EV as mediators of MSC-EV and endothelial cells' interaction for subsequent EV uptake and the induction of cell migration and angiogenesis. For that, EV from immortalized Wharton's Jelly MSC (iWJ-MSC-EV) were isolated by size exclusion chromatography (SEC) and treated with the glycosidase PNGase-F in order to remove wild-type N-glycans. Then, CFSE-labelled iWJ-MSC-EV were tested in the context of in vitro capture, agarose-spot migration and matrigel-based tube formation assays, using HUVEC. As a result, we found that the N-glycosylation in iWJ-MSC-EV is critical for interaction with HUVEC cells. iWJ-MSC-EV were captured by HUVEC, stimulating their tube-like formation ability and promoting their recruitment. Conversely, the removal of N-glycans through PNGase-F treatment reduced all of these functional activities induced by native iWJ-MSC-EV. Finally, comparative lectin arrays of iWJ-MSC-EV and PNGase-F-treated iWJ-MSC-EV found marked differences in the surface glycosylation pattern, particularly in N-acetylglucosamine, mannose, and fucose-binding lectins. Taken together, our results highlight the importance of N-glycans in MSC-EV to permit EV-cell interactions and associated functions.
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Vesículas Extracelulares , Células-Tronco Mesenquimais , Comunicação Celular , Vesículas Extracelulares/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Células-Tronco Mesenquimais/metabolismo , Polissacarídeos/metabolismoRESUMO
Scar formation during wound repair can be devastating for affected individuals. Our group previously documented the therapeutic potential of novel progenitor cell populations from the non-scarring buccal mucosa. These Oral Mucosa Lamina Propria-Progenitor Cells (OMLP-PCs) are multipotent, immunosuppressive, and antibacterial. Small extracellular vesicles (sEVs) may play important roles in stem cell-mediated repair in varied settings; hence, we investigated sEVs from this source for wound repair. We created an hTERT immortalized OMLP-PC line (OMLP-PCL) and confirmed retention of morphology, lineage plasticity, surface markers, and functional properties. sEVs isolated from OMLP-PCL were analyzed by nanoparticle tracking analysis, Cryo-EM and flow cytometry. Compared to bone marrow-derived mesenchymal stromal cells (BM-MSC) sEVs, OMLP-PCL sEVs were more potent at driving wound healing functions, including cell proliferation and wound repopulation and downregulated myofibroblast formation. A reduced scarring potential was further demonstrated in a preclinical in vivo model. Manipulation of OMLP-PCL sEVs may provide novel options for non-scarring wound healing in clinical settings.
Assuntos
Vesículas Extracelulares , Células-Tronco Mesenquimais , Proliferação de Células , Cicatriz/metabolismo , Vesículas Extracelulares/metabolismo , Humanos , Células-TroncoRESUMO
BACKGROUND: Cholangiocarcinoma (CCA) is still a deadly tumour. Histological and molecular aspects of thioacetamide (TAA)-induced intrahepatic CCA (iCCA) in rats mimic those of human iCCA. Carcinogenic changes and therapeutic vulnerabilities in CCA may be captured by molecular investigations in bile, where we performed bile proteomic and metabolomic analyses that help discovery yet unknown pathways relevant to human iCCA. METHODS: Cholangiocarcinogenesis was induced in rats (TAA) and mice (JnkΔhepa + CCl4 + DEN model). We performed proteomic and metabolomic analyses in bile from control and CCA-bearing rats. Differential expression was validated in rat and human CCAs. Mechanisms were addressed in human CCA cells, including Huh28-KRASG12D cells. Cell signaling, growth, gene regulation and [U-13C]-D-glucose-serine fluxomics analyses were performed. In vivo studies were performed in the clinically-relevant iCCA mouse model. RESULTS: Pathways related to inflammation, oxidative stress and glucose metabolism were identified by proteomic analysis. Oxidative stress and high amounts of the oncogenesis-supporting amino acids serine and glycine were discovered by metabolomic studies. Most relevant hits were confirmed in rat and human CCAs (TCGA). Activation of interleukin-6 (IL6) and epidermal growth factor receptor (EGFR) pathways, and key genes in cancer-related glucose metabolic reprogramming, were validated in TAA-CCAs. In TAA-CCAs, G9a, an epigenetic pro-tumorigenic writer, was also increased. We show that EGFR signaling and mutant KRASG12D can both activate IL6 production in CCA cells. Furthermore, phosphoglycerate dehydrogenase (PHGDH), the rate-limiting enzyme in serine-glycine pathway, was upregulated in human iCCA correlating with G9a expression. In a G9a activity-dependent manner, KRASG12D promoted PHGDH expression, glucose flow towards serine synthesis, and increased CCA cell viability. KRASG12D CAA cells were more sensitive to PHGDH and G9a inhibition than controls. In mouse iCCA, G9a pharmacological targeting reduced PHGDH expression. CONCLUSIONS: In CCA, we identified new pro-tumorigenic mechanisms: Activation of EGFR signaling or KRAS mutation drives IL6 expression in tumour cells; Glucose metabolism reprogramming in iCCA includes activation of the serine-glycine pathway; Mutant KRAS drives PHGDH expression in a G9a-dependent manner; PHGDH and G9a emerge as therapeutic targets in iCCA.
Assuntos
Neoplasias dos Ductos Biliares , Colangiocarcinoma , Animais , Aracnodactilia , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/patologia , Ductos Biliares Intra-Hepáticos/metabolismo , Ductos Biliares Intra-Hepáticos/patologia , Carcinogênese/genética , Colangiocarcinoma/patologia , Contratura , Epigênese Genética , Receptores ErbB/genética , Receptores ErbB/metabolismo , Glucose , Glicina/metabolismo , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Camundongos , Fosfoglicerato Desidrogenase/genética , Proteômica , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Ratos , Serina/metabolismoRESUMO
Cells release membrane-delimited particles into the environment. These particles are called "extracellular vesicles" (EVs), and EVs are present in fluids contacting cells, including body fluids and conditioned culture media. Because EVs change and contribute to health and disease, EVs have become a hot topic. From the thousands of papers now published on EVs annually, one easily gets the impression that EVs provide biomarkers for all diseases, and that EVs are carriers of all relevant biomolecules and are omnipotent therapeutics. At the same time, EVs are heterogeneous, elusive and difficult to study due to their physical properties and the complex composition of their environment. This overview addresses the current challenges encountered when working with EVs, and how we envision that most of these challenges will be overcome in the near future. Right now, an infrastructure is being developed to improve the reproducibility of EV measurement results. This infrastructure comprises expert task forces of the International Society of Extracellular Vesicles (ISEV) developing guidelines and recommendations, instrument calibration, standardized and transparent reporting, and education. Altogether, these developments will support the credibility of EV research by introducing robust reproducibility, which is a prerequisite for understanding their biological significance and biomarker potential.