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1.
Arch Microbiol ; 206(6): 244, 2024 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-38702412

RESUMO

Aggregatibacter actinomycetemcomitans is an opportunistic Gram-negative periodontopathogen strongly associated with periodontitis and infective endocarditis. Recent evidence suggests that periodontopathogens can influence the initiation and progression of oral squamous cell carcinoma (OSCC). Herein we aimed to investigate the effect of A. actinomycetemcomitans-derived extracellular vesicles (EVs) on OSCC cell behavior compared with EVs from periodontopathogens known to associate with carcinogenesis. EVs were isolated from: A. actinomycetemcomitans and its mutant strains lacking the cytolethal distending toxin (CDT) or lipopolysaccharide (LPS) O-antigen; Porphyromonas gingivalis; Fusobacterium nucleatum; and Parvimonas micra. The effect of EVs on primary and metastatic OSCC cells was assessed using cell proliferation, apoptosis, migration, invasion, and tubulogenesis assays. A. actinomycetemcomitans-derived EVs reduced the metastatic cancer cell proliferation, invasion, tubulogenesis, and increased apoptosis, mostly in CDT- and LPS O-antigen-dependent manner. EVs from F. nucleatum impaired the metastatic cancer cell proliferation and induced the apoptosis rates in all OSCC cell lines. EVs enhanced cancer cell migration regardless of bacterial species. In sum, this is the first study demonstrating the influence of A. actinomycetemcomitans-derived EVs on oral cancer in comparison with other periodontopathogens. Our findings revealed a potential antitumorigenic effect of these EVs on metastatic OSCC cells, which warrants further in vivo investigations.


Assuntos
Aggregatibacter actinomycetemcomitans , Apoptose , Proliferação de Células , Vesículas Extracelulares , Neoplasias Bucais , Aggregatibacter actinomycetemcomitans/genética , Vesículas Extracelulares/metabolismo , Neoplasias Bucais/microbiologia , Neoplasias Bucais/patologia , Humanos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Movimento Celular , Fusobacterium nucleatum/fisiologia , Carcinoma de Células Escamosas/microbiologia , Carcinoma de Células Escamosas/patologia , Porphyromonas gingivalis/genética
2.
Function (Oxf) ; 5(3): zqae012, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38706963

RESUMO

Acute kidney injury (AKI) is a heterogeneous syndrome, comprising diverse etiologies of kidney insults that result in high mortality and morbidity if not well managed. Although great efforts have been made to investigate underlying pathogenic mechanisms of AKI, there are limited therapeutic strategies available. Extracellular vesicles (EV) are membrane-bound vesicles secreted by various cell types, which can serve as cell-free therapy through transfer of bioactive molecules. In this review, we first overview the AKI syndrome and EV biology, with a particular focus on the technical aspects and therapeutic application of cell culture-derived EVs. Second, we illustrate how multi-omic approaches to EV miRNA, protein, and genomic cargo analysis can yield new insights into their mechanisms of action and address unresolved questions in the field. We then summarize major experimental evidence regarding the therapeutic potential of EVs in AKI, which we subdivide into stem cell and non-stem cell-derived EVs. Finally, we highlight the challenges and opportunities related to the clinical translation of animal studies into human patients.


Assuntos
Injúria Renal Aguda , Vesículas Extracelulares , Injúria Renal Aguda/terapia , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/patologia , Humanos , Vesículas Extracelulares/transplante , Vesículas Extracelulares/metabolismo , Animais , Técnicas de Cultura de Células/métodos , MicroRNAs/metabolismo , MicroRNAs/genética
3.
Reprod Fertil Dev ; 362024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38713808

RESUMO

Context Extracellular vesicles (EVs) derived from the oviductal fluid (oEVs) play a critical role in various reproductive processes, including sperm capacitation, fertilisation, and early embryo development. Aims To characterise porcine oEVs (poEVs) from different stages of the estrous cycle (late follicular, LF; early luteal, EL; mid luteal, ML; late luteal, LL) and investigate their impact on sperm functionality. Methods poEVs were isolated, characterised, and labelled to assess their binding to boar spermatozoa. The effects of poEVs on sperm motility, viability, acrosomal status, protein kinase A phosphorylation (pPKAs), tyrosine phosphorylation (Tyr-P), and in in vitro fertility were analysed. Key results poEVs were observed as round or cup-shaped membrane-surrounded vesicles. Statistical analysis showed that poEVs did not significantly differ in size, quantity, or protein concentration among phases of the estrous cycle. However, LF poEVs demonstrated a higher affinity for binding to sperm. Treatment with EL, ML, and LL poEVs resulted in a decrease in sperm progressive motility and total motility. Moreover, pPKA levels were reduced in presence of LF, EL, and ML poEVs, while Tyr-P levels did not differ between groups. LF poEVs also reduced sperm penetration rate and the number of spermatozoa per penetrated oocyte (P Conclusions poEVs from different stages of the estrous cycle play a modulatory role in sperm functionality by interacting with spermatozoa, affecting motility and capacitation, and participating in sperm-oocyte interaction. Implications The differential effects of LF and LL poEVs suggest the potential use of poEVs as additives in IVF systems to regulate sperm-oocyte interaction.


Assuntos
Ciclo Estral , Vesículas Extracelulares , Capacitação Espermática , Motilidade dos Espermatozoides , Espermatozoides , Animais , Feminino , Vesículas Extracelulares/metabolismo , Masculino , Espermatozoides/metabolismo , Espermatozoides/fisiologia , Ciclo Estral/metabolismo , Ciclo Estral/fisiologia , Motilidade dos Espermatozoides/fisiologia , Suínos , Capacitação Espermática/fisiologia , Oviductos/metabolismo , Oviductos/fisiologia , Interações Espermatozoide-Óvulo/fisiologia , Tubas Uterinas/metabolismo , Tubas Uterinas/fisiologia , Fosforilação
4.
Am J Reprod Immunol ; 91(5): e13861, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38716765

RESUMO

BACKGROUND: Maternal-fetal immunology is intricate, and the effects of mRNA-S maternal vaccination on immune regulation at the maternal-fetal interface require further investigation. Our study endeavors to elucidate these immunological changes, enhancing our comprehension of maternal and fetal health outcomes. By analyzing immune profiles and cytokine responses, we aim to provide valuable insights into the impact of mRNA-S vaccination on the delicate balance of immune regulation during pregnancy, addressing critical questions in the field of reproductive pharmacology. OBJECTIVES: This investigation sought to examine the prospective influence of mRNA-S-based vaccines and extracellular vesicles (EVs) containing the Spike (S) protein at the maternal-fetal interface. Our primary emphasis was on evaluating their effects on maternal decidua cells and fetal chorion trophoblast cells (hFM-CTCs). METHODS: We validated the generation of EVs containing the S protein from small human airway epithelial cell lines (HSAECs) following mRNA-S vaccine exposure. We assessed the expression of angiotensin-converting enzyme 2 (ACE2) gene and protein in fetal membranes and the placenta, with specific attention to decidual cells and fetal membrane chorion cells. To assess cellular functionality, these cells were exposed to both recombinant S protein and EVs loaded with S proteins (eSPs). RESULTS: Our findings revealed that cells and EVs subjected to mRNA-S-based vaccination exhibited altered protein expression levels of S proteins. At the feto-maternal interface, both placental and fetal membrane tissues demonstrated similar ACE-2 expression levels. Among individual cellular layers, syncytiotrophoblast cells in the placenta and chorion cells in the fetal membrane exhibited elevated ACE-2 expression. Notably, EVs derived from HSAECs activated the MAPK pathway in decidual cells. Additionally, decidual cells displayed a substantial increase in gene expression of chemokines like CXCL-10 and CXCL-11, as well as proinflammatory cytokines such as IL-6 in response to eSPs. However, the levels of Ccl-2 and IL-1ß remained unchanged in decidual cells under the same conditions. Conversely, hFM-CTCs demonstrated significant alterations in the proinflammatory cytokines and chemokines with respect to eSPs. CONCLUSION: In conclusion, our study indicates that mRNA-S-based maternal vaccination during pregnancy may influence the maternal-fetal interface's COVID-19 interaction and immune regulation. Further investigation is warranted to assess safety and implications.


Assuntos
Vesículas Extracelulares , Trofoblastos , Humanos , Feminino , Gravidez , Trofoblastos/imunologia , Vesículas Extracelulares/imunologia , Vesículas Extracelulares/metabolismo , Decídua/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Citocinas/metabolismo , Vacinação , Enzima de Conversão de Angiotensina 2/metabolismo , Enzima de Conversão de Angiotensina 2/genética , Troca Materno-Fetal , SARS-CoV-2/imunologia , COVID-19/prevenção & controle , COVID-19/imunologia , Linhagem Celular , Vacinas contra COVID-19/imunologia , RNA Mensageiro/metabolismo , RNA Mensageiro/genética
5.
Nat Commun ; 15(1): 3884, 2024 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-38719909

RESUMO

Only a minority of cancer patients benefit from immune checkpoint blockade therapy. Sophisticated cross-talk among different immune checkpoint pathways as well as interaction pattern of immune checkpoint molecules carried on circulating small extracellular vesicles (sEV) might contribute to the low response rate. Here we demonstrate that PD-1 and CD80 carried on immunocyte-derived sEVs (I-sEV) induce an adaptive redistribution of PD-L1 in tumour cells. The resulting decreased cell membrane PD-L1 expression and increased sEV PD-L1 secretion into the circulation contribute to systemic immunosuppression. PD-1/CD80+ I-sEVs also induce downregulation of adhesion- and antigen presentation-related molecules on tumour cells and impaired immune cell infiltration, thereby converting tumours to an immunologically cold phenotype. Moreover, synchronous analysis of multiple checkpoint molecules, including PD-1, CD80 and PD-L1, on circulating sEVs distinguishes clinical responders from those patients who poorly respond to anti-PD-1 treatment. Altogether, our study shows that sEVs carry multiple inhibitory immune checkpoints proteins, which form a potentially targetable adaptive loop to suppress antitumour immunity.


Assuntos
Antígeno B7-1 , Antígeno B7-H1 , Vesículas Extracelulares , Receptor de Morte Celular Programada 1 , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/imunologia , Receptor de Morte Celular Programada 1/metabolismo , Humanos , Antígeno B7-1/metabolismo , Antígeno B7-H1/metabolismo , Antígeno B7-H1/imunologia , Animais , Camundongos , Linhagem Celular Tumoral , Feminino , Neoplasias/imunologia , Neoplasias/patologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Inibidores de Checkpoint Imunológico/farmacologia , Tolerância Imunológica , Camundongos Endogâmicos C57BL , Masculino , Microambiente Tumoral/imunologia
7.
Int J Biol Sci ; 20(7): 2727-2747, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38725857

RESUMO

Phenotypic switching (from contractile to synthetic) of vascular smooth muscle cells (VSMCs) is essential in the progression of atherosclerosis. The damaged endothelium in the atherosclerotic artery exposes VSMCs to increased interstitial fluid shear stress (IFSS). However, the precise mechanisms by which increased IFSS influences VSMCs phenotypic switching are unrevealed. Here, we employed advanced numerical simulations to calculate IFSS values accurately based on parameters acquired from patient samples. We then carefully investigated the phenotypic switching and extracellular vesicles (EVs) secretion of VSMCs under various IFSS conditions. By employing a comprehensive set of approaches, we found that VSMCs exhibited synthetic phenotype upon atherosclerotic IFSS. This synthetic phenotype is the upstream regulator for the enhanced secretion of pro-calcified EVs. Mechanistically, as a mechanotransducer, the epidermal growth factor receptor (EGFR) initiates the flow-based mechanical cues to MAPK signaling pathway, facilitating the nuclear accumulation of the transcription factor krüppel-like factor 5 (KLF5). Furthermore, pharmacological inhibiting either EGFR or MAPK signaling pathway blocks the nuclear accumulation of KLF5 and finally results in the maintenance of contractile VSMCs even under increased IFSS stimulation. Collectively, targeting this signaling pathway holds potential as a novel therapeutic strategy to inhibit VSMCs phenotypic switching and mitigate the progression of atherosclerosis.


Assuntos
Receptores ErbB , Vesículas Extracelulares , Fatores de Transcrição Kruppel-Like , Músculo Liso Vascular , Miócitos de Músculo Liso , Estresse Mecânico , Vesículas Extracelulares/metabolismo , Receptores ErbB/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Humanos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Líquido Extracelular/metabolismo , Fenótipo , Animais , Aterosclerose/metabolismo , Sistema de Sinalização das MAP Quinases , Transdução de Sinais
8.
Front Immunol ; 15: 1388769, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38726003

RESUMO

Background: Newer 3D culturing approaches are a promising way to better mimic the in vivo tumor microenvironment and to study the interactions between the heterogeneous cell populations of glioblastoma multiforme. Like many other tumors, glioblastoma uses extracellular vesicles as an intercellular communication system to prepare surrounding tissue for invasive tumor growth. However, little is known about the effects of 3D culture on extracellular vesicles. The aim of this study was to comprehensively characterize extracellular vesicles in 3D organoid models and compare them to conventional 2D cell culture systems. Methods: Primary glioblastoma cells were cultured as 2D and 3D organoid models. Extracellular vesicles were obtained by precipitation and immunoaffinity, with the latter allowing targeted isolation of the CD9/CD63/CD81 vesicle subpopulation. Comprehensive vesicle characterization was performed and miRNA expression profiles were generated by smallRNA-sequencing. In silico analysis of differentially regulated miRNAs was performed to identify mRNA targets and corresponding signaling pathways. The tumor cell media and extracellular vesicle proteome were analyzed by high-resolution mass spectrometry. Results: We observed an increased concentration of extracellular vesicles in 3D organoid cultures. Differential gene expression analysis further revealed the regulation of twelve miRNAs in 3D tumor organoid cultures (with nine miRNAs down and three miRNAs upregulated). MiR-23a-3p, known to be involved in glioblastoma invasion, was significantly increased in 3D. MiR-7-5p, which counteracts glioblastoma malignancy, was significantly decreased. Moreover, we identified four miRNAs (miR-323a-3p, miR-382-5p, miR-370-3p, miR-134-5p) located within the DLK1-DIO3 domain, a cancer-associated genomic region, suggesting a possible importance of this region in glioblastoma progression. Overrepresentation analysis identified alterations of extracellular vesicle cargo in 3D organoids, including representation of several miRNA targets and proteins primarily implicated in the immune response. Conclusion: Our results show that 3D glioblastoma organoid models secrete extracellular vesicles with an altered cargo compared to corresponding conventional 2D cultures. Extracellular vesicles from 3D cultures were found to contain signaling molecules associated with the immune regulatory signaling pathways and as such could potentially change the surrounding microenvironment towards tumor progression and immunosuppressive conditions. These findings suggest the use of 3D glioblastoma models for further clinical biomarker studies as well as investigation of new therapeutic options.


Assuntos
Vesículas Extracelulares , Glioblastoma , MicroRNAs , Organoides , Microambiente Tumoral , Humanos , Glioblastoma/imunologia , Glioblastoma/patologia , Glioblastoma/metabolismo , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/imunologia , Organoides/imunologia , MicroRNAs/genética , Microambiente Tumoral/imunologia , Transdução de Sinais , Células Tumorais Cultivadas , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/metabolismo , Regulação Neoplásica da Expressão Gênica , Linhagem Celular Tumoral , Técnicas de Cultura de Células em Três Dimensões/métodos
9.
PLoS One ; 19(5): e0301761, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38718025

RESUMO

Tracking small extracellular vesicles (sEVs), such as exosomes, requires staining them with dyes that penetrate their lipid bilayer, a process that leaves excess dye that needs to be mopped up to achieve high specificity. Current methods to remove superfluous dye have limitations, among them that they are time-intensive, carry the risk of losing sample and can require specialized equipment and materials. Here we present a fast, easy-to-use, and cost-free protocol for cleaning excess dye from stained sEV samples by adding their parental cells to the mixture to absorb the extra dye much like sponges do. Since sEVs are considered a next-generation drug delivery system, we further show the success of our approach at removing excess chemotherapeutic drug, daunorubicin, from the sEV solution.


Assuntos
Vesículas Extracelulares , Vesículas Extracelulares/química , Vesículas Extracelulares/metabolismo , Humanos , Daunorrubicina/economia , Corantes/química , Coloração e Rotulagem/métodos , Coloração e Rotulagem/economia
10.
Sci Rep ; 14(1): 10054, 2024 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-38698053

RESUMO

ß-Thalassaemia is one of the most common genetic diseases worldwide. During the past few decades, life expectancy of patients has increased significantly owing to advance in medical treatments. Cognitive impairment, once has been neglected, has gradually become more documented. Cognitive impairment in ß-thalassaemia patients is associated with natural history of the disease and socioeconomic factors. Herein, to determined effect of ß-thalassaemia intrinsic factors, 22-month-old ß-thalassaemia mouse was used as a model to assess cognitive impairment and to investigate any aberrant brain pathology in ß-thalassaemia. Open field test showed that ß-thalassaemia mice had decreased motor function. However, no difference of neuronal degeneration in primary motor cortex, layer 2/3 area was found. Interestingly, impaired learning and memory function accessed by a Morris water maze test was observed and correlated with a reduced number of living pyramidal neurons in hippocampus at the CA3 region in ß-thalassaemia mice. Cognitive impairment in ß-thalassaemia mice was significantly correlated with several intrinsic ß-thalassaemic factors including iron overload, anaemia, damaged red blood cells (RBCs), phosphatidylserine (PS)-exposed RBC large extracellular vesicles (EVs) and PS-exposed medium EVs. This highlights the importance of blood transfusion and iron chelation in ß-thalassaemia patients. In addition, to improve patients' quality of life, assessment of cognitive functions should become part of routine follow-up.


Assuntos
Disfunção Cognitiva , Modelos Animais de Doenças , Hipocampo , Talassemia beta , Animais , Talassemia beta/patologia , Talassemia beta/complicações , Talassemia beta/genética , Disfunção Cognitiva/etiologia , Disfunção Cognitiva/patologia , Camundongos , Hipocampo/patologia , Hipocampo/metabolismo , Masculino , Neurônios/metabolismo , Neurônios/patologia , Sobrecarga de Ferro/patologia , Sobrecarga de Ferro/metabolismo , Sobrecarga de Ferro/complicações , Vesículas Extracelulares/metabolismo , Eritrócitos/metabolismo , Eritrócitos/patologia , Células Piramidais/metabolismo , Células Piramidais/patologia , Aprendizagem em Labirinto
12.
Sci Rep ; 14(1): 12274, 2024 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-38806540

RESUMO

Cranial irradiation used to control brain malignancies invariably leads to progressive and debilitating declines in cognition. Clinical efforts implementing hippocampal avoidance and NMDAR antagonism, have sought to minimize dose to radiosensitive neurogenic regions while normalizing excitatory/inhibitory (E/I) tone. Results of these trials have yielded only marginal benefits to cognition, prompting current studies to evaluate the potential of systemic extracellular vesicle (EV) therapy to restore neurocognitive functionality in the irradiated brain. Here we tested the hypothesis that EVs derived from inhibitory but not excitatory neuronal cultures would prove beneficial to cognition and associated pathology. Rats subjected to a clinically relevant, fractionated cranial irradiation paradigm were given multiple injections of either GABAergic- or glutamatergic-derived EV and subjected to behavioral testing. Rats treated with GABAergic but not glutamatergic EVs showed significant improvements on hippocampal- and cortical-dependent behavioral tasks. While each treatment enhanced levels of the neurotrophic factors BDNF and GDNF, only GABAergic EVs preserved granule cell neuron dendritic spine density. Additional studies conducted with GABAergic EVs, confirmed significant benefits on amygdala-dependent behavior and modest changes in synaptic plasticity as measured by long-term potentiation. These data point to a potentially more efficacious approach for resolving radiation-induced neurological deficits, possibly through a mechanism able to restore homeostatic E/I balance.


Assuntos
Irradiação Craniana , Vesículas Extracelulares , Neurônios GABAérgicos , Animais , Vesículas Extracelulares/metabolismo , Ratos , Irradiação Craniana/efeitos adversos , Neurônios GABAérgicos/metabolismo , Neurônios GABAérgicos/efeitos da radiação , Masculino , Hipocampo/efeitos da radiação , Hipocampo/metabolismo , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Neurônios/efeitos da radiação , Neurônios/metabolismo , Ácido Glutâmico/metabolismo , Plasticidade Neuronal/efeitos da radiação , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Comportamento Animal/efeitos da radiação
13.
Sci Rep ; 14(1): 12272, 2024 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-38806562

RESUMO

Recently, Lactobacillus johnsonii N6.2-derived extracellular vesicles (EVs) were shown to reduce apoptosis in human beta cell lines and stimulate insulin secretion in human islets. Our goal was to identify a physiologically relevant environmental condition that induces a hypervesiculation phenotype in L. johnsonii N6.2 and to evaluate if transcriptional changes are involved in this process. Culturing this strain in the presence of 0.2% bovine bile, which mimics a stressor encountered by the bacterium in the small intestine, resulted in approximately a 100-fold increase in EVs relative to cells grown in media without bile. Whole transcriptome analysis of cells grown with bile revealed upregulation of several peptidoglycan hydrolases as well as several genes involved in fatty acid utilization. These results suggest that the hypervesiculation phenotype may be the result of increased cell wall turnover combined with increased accumulation of phospholipids, in agreement with our previous proteomic and lipidomics results. Additionally, EVs isolated from L. johnsonii N6.2 grown in presence of bile maintained their immunomodulatory properties in host-derived ßlox5 pancreatic and THP-1 macrophage cell lines. Our findings suggest that in L. johnsonii N6.2 vesiculogenesis is significantly impacted by the expression of cell wall modifying enzymes and proteins utilized for exogenous fatty acid uptake that are regulated at the transcriptional level. Furthermore, this data suggests that vesiculogenesis could be stimulated in vivo using small molecules thereby maximizing the beneficial interactions between bacteria and their hosts.


Assuntos
Bile , Vesículas Extracelulares , Lactobacillus johnsonii , Vesículas Extracelulares/metabolismo , Humanos , Lactobacillus johnsonii/metabolismo , Bile/metabolismo , Animais , Linhagem Celular , Bovinos , Células THP-1 , Parede Celular/metabolismo , Perfilação da Expressão Gênica
14.
Sci Rep ; 14(1): 12267, 2024 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-38806574

RESUMO

Extracellular vesicles (EVs) are lipid-membrane enclosed structures that are associated with several diseases, including those of genitourinary tract. Urine contains EVs derived from urinary tract cells. Owing to its non-invasive collection, urine represents a promising source of biomarkers for genitourinary disorders, including cancer. The most used method for urinary EVs separation is differential ultracentrifugation (UC), but current protocols lead to a significant loss of EVs hampering its efficiency. Moreover, UC protocols are labor-intensive, further limiting clinical application. Herein, we sought to optimize an UC protocol, reducing the time spent and improving small EVs (SEVs) yield. By testing different ultracentrifugation times at 200,000g to pellet SEVs, we found that 48 min and 60 min enabled increased SEVs recovery compared to 25 min. A step for pelleting large EVs (LEVs) was also evaluated and compared with filtering of the urine supernatant. We found that urine supernatant filtering resulted in a 1.7-fold increase on SEVs recovery, whereas washing steps resulted in a 0.5 fold-decrease on SEVs yield. Globally, the optimized UC protocol was shown to be more time efficient, recovering higher numbers of SEVs than Exoquick-TC (EXO). Furthermore, the optimized UC protocol preserved RNA quality and quantity, while reducing SEVs separation time.


Assuntos
Vesículas Extracelulares , Ultracentrifugação , Ultracentrifugação/métodos , Humanos , Vesículas Extracelulares/metabolismo , Biomarcadores/urina , Urina/citologia , Urina/química , Feminino
15.
Biotechnol J ; 19(5): e2400128, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38797724

RESUMO

Small extracellular vesicles (sEVs) are nanosized vesicles enclosed in a lipid membrane released by nearly all cell types. sEVs have been considered as reliable biomarkers for diagnostics and effective carriers. Despite the clear importance of sEV functionality, sEV research faces challenges imposed by the small size and precise imaging of sEVs. Recent advances in live and high-resolution microscopy, combined with efficient labeling strategies, enable us to investigate the composition and behavior of EVs within living organisms. Here, a modified sEVs was generated with a near infrared fluorescence protein mKate2 using a VSVG viral pseudotyping-based approach for monitoring sEVs. An observed was made that the mKate2-tagged protein can be incorporated into the membranes of sEVs without altering their physical properties. In vivo imaging demonstrates that sEVs labeled with mKate2 exhibit excellent brightness and high photostability, allowing the acquisition of long-term investigation comparable to those achieved with mCherry labeling. Importantly, the mKate2-tagged sEVs show a low toxicity and exhibit a favorable safety profile. Furthermore, the co-expression of mKate2 and rabies virus glycoprotein (RVG) peptide on sEVs enables brain-targeted visualization, suggesting the mKate2 tag does not alter the biodistribution of sEVs. Together, the study presents the mKate2 tag as an efficient tracker for sEVs to monitor tissue-targeting and biodistribution in vivo.


Assuntos
Vesículas Extracelulares , Vesículas Extracelulares/química , Vesículas Extracelulares/metabolismo , Animais , Camundongos , Humanos , Proteínas Luminescentes/química , Proteínas Luminescentes/metabolismo , Encéfalo/metabolismo , Encéfalo/diagnóstico por imagem , Distribuição Tecidual
16.
Gut Microbes ; 16(1): 2359515, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38808455

RESUMO

The intestinal microbiota, consisting of an estimated 10^10-10^11 organisms, regulate physiological processes involved in digestion, metabolism, and immunity. Surprisingly, these intestinal microorganisms have been found to influence tissues that are not directly in contact with the gut, such as adipose tissue, the liver, skeletal muscle, and the brain. This interaction takes place even when intestinal barrier function is uncompromised. An increasing body of evidence suggests that bacterial membrane vesicles (bMVs), in addition to bacterial metabolites such as short-chain fatty acids, are able to mediate effects of the microbiota on these host tissues. The ability of bMVs to dissipate from the intestinal lumen into systemic circulation hereby facilitates the transport and presentation of bacterial components and metabolites to host organs. Importantly, there are indications that the interaction between bMVs and tissues or immune cells may play a role in the etiology of (chronic metabolic) disease. For example, the gut-derived bMV-mediated induction of insulin resistance in skeletal muscle cells and pro-inflammatory signaling by adipocytes possibly underlies diseases such as type 2 diabetes and obesity. Here, we review the current knowledge on bMVs in the microbiota's effects on host energy/substrate metabolism with a focus on etiological roles in the onset and progression of metabolic disease. We furthermore illustrate that vesicle production by bacterial microbiota could potentially be modulated through lifestyle intervention to improve host metabolism.


Assuntos
Bactérias , Microbioma Gastrointestinal , Humanos , Microbioma Gastrointestinal/fisiologia , Animais , Bactérias/metabolismo , Bactérias/classificação , Bactérias/genética , Doenças Metabólicas/microbiologia , Doenças Metabólicas/metabolismo , Vesículas Extracelulares/metabolismo
17.
Biomolecules ; 14(5)2024 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-38785998

RESUMO

Small extracellular vesicles (sEVs) have emerged as promising therapeutic agents and drug delivery vehicles. Targeted modification of sEVs and their contents using genetic modification strategies is one of the most popular methods. This study investigated the effects of p53 fusion with arrestin domain-containing protein 1 (ARRDC1) and CD63 on the generation of sEVs, p53 loading efficiency, and therapeutic efficacy. Overexpression of either ARRDC1-p53 (ARP) or CD63-p53 (CDP) significantly elevated p53 mRNA and protein levels. The incorporation of ARRDC1 and CD63 significantly enhanced HEK293T-sEV biogenesis, evidenced by significant increases in sEV-associated proteins TSG101 and LAMP1, resulting in a boost in sEV production. Importantly, fusion with ARRDC1 or CD63 substantially increased the efficiency of loading both p53 fusion proteins and its mRNA into sEVs. sEVs equipped with ARP or CDP significantly enhanced the enrichment of p53 fusion proteins and mRNA in p53-null H1299 cells, resulting in a marked increase in apoptosis and a reduction in cell proliferation, with ARP-sEVs demonstrating greater effectiveness than CDP-sEVs. These findings underscore the enhanced functionality of ARRDC1- and CD63-modified sEVs, emphasizing the potential of genetic modifications in sEV-based therapies for targeted cancer treatment.


Assuntos
Apoptose , Vesículas Extracelulares , Tetraspanina 30 , Proteína Supressora de Tumor p53 , Humanos , Tetraspanina 30/metabolismo , Tetraspanina 30/genética , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/genética , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , Células HEK293 , Linhagem Celular Tumoral , Proliferação de Células , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Proteína 1 de Membrana Associada ao Lisossomo
18.
Anal Chim Acta ; 1309: 342699, 2024 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-38772652

RESUMO

Extracellular vesicles (EVs) are cell-released, nucleus-free particles with a double-membrane structure that effectively prevents degradation of internal components by a variety of salivary enzymes. Saliva is an easily accessible biofluid that contains a wealth of valuable information for disease diagnosis and monitoring and especially reflect respiratory and digestive tract diseases. However, the lack of efficient and high-throughput methods for proteomic analysis of salivary biomarkers poses a significant challenge. Herein, we designed a salivary EV amphiphile-dendrimer supramolecular probe (SEASP) array which enables efficient enrichment and in situ detection of EVs protein biomarkers. Detergent Tween-20 washing of SEASP arrays removes high abundance of heteroproteins from saliva well. This array shows good analytical performance in the linear range of 10 µL-150 µL (LOD = 0.4 µg protein, or 10 µL saliva), exhibiting a good recovery (80.0 %). Compared to ultracentrifugation (UC), this procedure provides simple and convenient access to high-purity EVs (1.3 × 109 particles per mg protein) with good physiological status and structure. Coupling with mass spectrometry based proteomic analysis, differentially expressed proteins as selected asthma biomarkers have been screened. Then, we validated the proteomics primary screening results through clinical samples (100 µL each) using the SEASP array. Utilizing the dual antibody fluorescence technology, SEASP enables the simultaneous high-throughput detection of two proteins. Therefore, the EVs marker protein CD81 could be used as an internal standard to normalize the number of EVs, which was stably expressed in EVs. Proteomics and array results suggested that HNRNPU (P = 4.9 * 10-6) and MUC5B (P = 4.7 * 10-11) are promising protein biomarkers for infantile asthma. HNRNPU and MUC5B may be associated with disease onset and subtypes. The SEASP arrays provide a significant advancement in the field of salivary biomarker. The array enables high-throughput in situ protein detection for highly viscous and complex biological samples. It provides a rapid, low-cost, highly specific screening procedure and experimental basis for early disease screening and diagnosis in the field of liquid biopsy.


Assuntos
Vesículas Extracelulares , Proteômica , Saliva , Saliva/química , Humanos , Vesículas Extracelulares/química , Vesículas Extracelulares/metabolismo , Proteômica/métodos , Biomarcadores/análise , Ensaios de Triagem em Larga Escala , Asma/diagnóstico , Asma/metabolismo
19.
FASEB J ; 38(10): e23672, 2024 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-38775929

RESUMO

Cardiovascular disease (CVD) is a leading global cause of mortality, difficult to predict in advance. Evidence indicates that the copy number of mitochondrial DNA (mtDNAcn) in blood is altered in individuals with CVD. MtDNA released into circulation may act as a mediator of inflammation, a recognized factor in the development of CVD, in the long distance. This pilot study aims to test if levels of mtDNAcn in buffy coat DNA (BC-mtDNA), in circulating cellfree DNA (cf-mtDNA), or in DNA extracted from plasma extracellular vesicles (EV-mtDNA) are altered in CVD patients and if they can predict heart attack in advance. A group of 144 people with different CVD statuses (50 that had CVD, 94 healthy) was selected from the LifeLines Biobank according to the incidence of new cardiovascular event monitored in 6 years (50 among controls had heart attack after the basal assessment). MtDNAcn was quantified in total cf-DNA and EV-DNA from plasma as well as in buffy coat. EVs have been characterized by their size, polydispersity index, count rate, and zeta potential, by Dynamic Light Scattering. BC-mtDNAcn and cf-mtDNAcn were not different between CVD patients and healthy subjects. EVs carried higher mtDNAcn in subject with a previous history of CVD than controls, also adjusting the analysis for the EVs derived count rate. Despite mtDNAcn was not able to predict CVD in advance, the detection of increased EV-mtDNAcn in CVD patients in this pilot study suggests the need for further investigations to determine its pathophysiological role in inflammation.


Assuntos
Doenças Cardiovasculares , Ácidos Nucleicos Livres , Variações do Número de Cópias de DNA , DNA Mitocondrial , Vesículas Extracelulares , Humanos , DNA Mitocondrial/genética , DNA Mitocondrial/sangue , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/genética , Masculino , Ácidos Nucleicos Livres/sangue , Ácidos Nucleicos Livres/genética , Feminino , Projetos Piloto , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/sangue , Pessoa de Meia-Idade , Estudos de Casos e Controles , Idoso , Estudos Prospectivos
20.
J Extracell Vesicles ; 13(5): e12454, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38760878

RESUMO

Extracellular vesicles (EVs) are emerging as a promising drug delivery vehicle as they are biocompatible and capable of targeted delivery. However, clinical translation of EVs remains challenging due to the lack of standardized and scalable manufacturing protocols to consistently isolate small EVs (sEVs) with both high yield and high purity. The heterogenous nature of sEVs leading to unknown composition of biocargos causes further pushback due to safety concerns. In order to address these issues, we developed a robust quality-controlled multi-stage process to produce and isolate sEVs from human embryonic kidney HEK293F cells. We then compared different 2-step and 3-step workflows for eliminating protein impurities and cell-free nucleic acids to meet acceptable limits of regulatory authorities. Our results showed that sEV production was maximized when HEK293F cells were grown at high-density stationary phase in semi-continuous culture. The novel 3-step workflow combining tangential flow filtration, sucrose-cushion ultracentrifugation and bind-elute size-exclusion chromatography outperformed other methods in sEV purity while still preserved high yield and particle integrity. The purified HEK293F-derived sEVs were thoroughly characterized for identity including sub-population analysis, content profiling including proteomics and miRNA sequencing, and demonstrated excellent preclinical safety profile in both in-vitro and in-vivo testing. Our rigorous enrichment workflow and comprehensive characterization will help advance the development of EVs, particularly HEK293F-derived sEVs, to be safe and reliable drug carriers for therapeutic applications.


Assuntos
Vesículas Extracelulares , Humanos , Vesículas Extracelulares/metabolismo , Células HEK293 , Proteômica/métodos , Fluxo de Trabalho , Ultracentrifugação/métodos , MicroRNAs/metabolismo
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