Metabolic Profiling as an Approach to Differentiate T-Cell Acute Lymphoblastic Leukemia Cell Lines Belonging to the Same Genetic Subgroup.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive tumor mainly affecting children and adolescents. It is driven by multiple genetic mutations that together define the leukemic phenotype. Interestingly, based on genetic alterations and/or deregulated expression, at least six genetic subgroups have been recognized. The TAL/LMO subgroup is one of the most represented genetic subgroups, characterizing 30-45% of pediatric T-ALL cases. The study of lipid and metabolic profiles is increasingly recognized as a valuable tool for comprehending the development and progression of tumors. In this study, metabolic and lipidomic analysis via LC/MS have been carried out on four T-ALL cell lines belonging to the TAL/LMO subgroup (Jurkat, Molt-4, Molt-16, and CCRF-CEM) to identify new potential metabolic biomarkers and to provide a subclassification of T-ALL cell lines belonging to the same subgroup. A total of 343 metabolites were annotated, including 126 polar metabolites and 217 lipid molecules. The statistical analysis, for both metabolic and lipid profiles, shows significant differences and similarities among the four cell lines. The Molt-4 cell line is the most distant cell line and CCRF-CEM shows a high activity in specific pathways when compared to the other cell lines, while Molt-16 and Jurkat show a similar metabolic profile. Additionally, this study highlighted the pathways that differ in each cell line and the possible enzymes involved using bioinformatic tools, capable of predicting the pathways involved by studying the differences in the metabolic profiles. This experiment offers an approach to differentiate T-ALL cell lines and could open the way to verify and confirm the obtained results directly in patients.

LipidOne: user-friendly lipidomic data analysis tool for a deeper interpretation in a systems biology scenario.

SUMMARY: LC/MS-based analysis techniques combined with specialized lipid tool allow for the qualitative and quantitative determination of thousands of lipid molecules. Some recent bioinformatics tools have been developed to study changes in the lipid profile in case-control experiments and correlate these changes to different enzyme activity or gene expression. However, the existing tools have the limitation to treat only the assembled lipid molecules. In reality, each individual molecule can be considered as an assembly of smaller parts, often called building blocks. These are the result of a myriad of biochemical synthesis and transformation processes that, from a systems biology perspective, should not be ignored. Here, we present LipidOne, a new lipidomic tool which highlights all qualitative and quantitative changes in lipid building blocks both among all detected lipid classes and among experimental groups. Thanks to LipidOne, even differences in lipid building blocks can now be linked to the activity of specific classes of enzymes, transcripts and genes. AVAILABILITY AND IMPLEMENTATION: LipidOne software is freely available at www.dcbb.unipg.it/LipidOne and https://github.com/matteogiulietti/LipidOne. CONTACT: roberto.pellegrino@unipg.it. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Comparison between Sickle Cell Disease Patients and Healthy Donors: Untargeted Lipidomic Study of Erythrocytes.

Sickle cell disease (SCD) is one of the most common severe monogenic disorders in the world caused by a mutation on HBB gene and characterized by hemoglobin polymerization, erythrocyte rigidity, vaso-occlusion, chronic anemia, hemolysis, and vasculopathy. Recently, the scientific community has focused on the multiple genetic and clinical profiles of SCD. However, the lipid composition of sickle cells has received little attention in the literature. According to recent studies, changes in the lipid profile are strongly linked to several disorders. Therefore, the aim of this study is to dig deeper into lipidomic analysis of erythrocytes in order to highlight any variations between healthy and patient subjects. 241 lipid molecular species divided into 17 classes have been annotated and quantified. Lipidomic profiling of SCD patients showed that over 24% of total lipids were altered most of which are phospholipids. In-depth study of significant changes in lipid metabolism can give an indication of the enzymes and genes involved. In a systems biology scenario, these variations can be useful to improve the understanding of the biochemical basis of SCD and to try to make a score system that could be predictive for the severity of clinical manifestations.

Drug-Induced Lysosomal Impairment Is Associated with the Release of Extracellular Vesicles Carrying Autophagy Markers.

Amiodarone is a cationic amphiphilic drug used as an antiarrhythmic agent. It induces phospholipidosis, i.e., the accumulation of phospholipids within organelles of the endosomal-lysosomal system. Extracellular vesicles (EVs) are membrane-enclosed structures released by any type of cell and retrieved in every fluid of the body. EVs have been initially identified as a system to dispose cell waste, but they are also considered to be an additional manner to transmit intercellular signals. To understand the role of EVs in drug-induced phospholipidosis, we investigated EVs release in amiodarone-treated HEK-293 cells engineered to produce fluorescently labelled EVs. We observed that amiodarone induces the release of a higher number of EVs, mostly of a large/medium size. EVs released upon amiodarone treatment do not display significant morphological changes or altered size distribution, but they show a dose-dependent increase in autophagy associated markers, indicating a higher release of EVs with an autophagosome-like phenotype. Large/medium EVs also show a higher content of phospholipids. Drugs inducing lysosomal impairment such as chloroquine and bafilomycin A1 similarly prompt a higher release of EVs enriched in autophagy markers. This result suggests a mechanism associated with amiodarone-induced lysosomal impairment more than a connection with the accumulation of specific undigested substrates. Moreover, the implementation of the lysosomal function by overexpressing TFEB, a master gene regulator of lysosomal biogenesis, prevents the amiodarone-induced release of EVs, suggesting that this could be a feasible target to attenuate drug-induced abnormalities.

Lipidomic analysis of cancer cells cultivated at acidic pH reveals phospholipid fatty acids remodelling associated with transcriptional reprogramming.

Cancer cells need to modulate the biosynthesis of membrane lipids and fatty acids to adapt themselves to an accelerated rate of cell division and survive into an extracellular environment characterised by a low pH. To gain insight this crucial survival process, we investigated the lipid composition of Mel 501 melanoma cells cultured at either physiological or acidic pH and observed the remodelling of phospholipids towards longer and more unsaturated acyl chains at low pH. This modification was related to changes in gene expression profile, as we observed an up-regulation of genes involved in acyl chain desaturation, elongation and transfer to phospholipids. PC3 prostate and MCF7 breast cancer cells adapted at acidic pH also demonstrated phospholipid fatty acid remodelling related to gene expression changes. Overall findings clearly indicate that low extracellular pH impresses a specific lipid signature to cells, associated with transcriptional reprogramming.

Lysosomal Exocytosis, Exosome Release and Secretory Autophagy: The Autophagic- and Endo-Lysosomal Systems Go Extracellular.

Beyond the consolidated role in degrading and recycling cellular waste, the autophagic- and endo-lysosomal systems play a crucial role in extracellular release pathways. Lysosomal exocytosis is a process leading to the secretion of lysosomal content upon lysosome fusion with plasma membrane and is an important mechanism of cellular clearance, necessary to maintain cell fitness. Exosomes are a class of extracellular vesicles originating from the inward budding of the membrane of late endosomes, which may not fuse with lysosomes but be released extracellularly upon exocytosis. In addition to garbage disposal tools, they are now considered a cell-to-cell communication mechanism. Autophagy is a cellular process leading to sequestration of cytosolic cargoes for their degradation within lysosomes. However, the autophagic machinery is also involved in unconventional protein secretion and autophagy-dependent secretion, which are fundamental mechanisms for toxic protein disposal, immune signalling and pathogen surveillance. These cellular processes underline the crosstalk between the autophagic and the endosomal system and indicate an intersection between degradative and secretory functions. Further, they suggest that the molecular mechanisms underlying fusion, either with lysosomes or plasma membrane, are key determinants to maintain cell homeostasis upon stressing stimuli. When they fail, the accumulation of undigested substrates leads to pathological consequences, as indicated by the involvement of autophagic and lysosomal alteration in human diseases, namely lysosomal storage disorders, age-related neurodegenerative diseases and cancer. In this paper, we reviewed the current knowledge on the functional role of extracellular release pathways involving lysosomes and the autophagic- and endo-lysosomal systems, evaluating their implication in health and disease.

Effect of Curcumin on Protein Damage Induced by Rotenone in Dopaminergic PC12 Cells.

Oxidative stress is considered to be a key factor of the pathogenesis of Parkinson's disease, a multifactorial neurodegenerative disorder characterized by reduced dopaminergic neurons in the substantia nigra pars compacta and accumulated protein aggregates. Rotenone is a worldwide-used pesticide that induces the most common features of Parkinson's by direct inhibition of the mitochondrial complex I. Rotenone-induced Parkinson's models, as well as brain tissues from Parkinson's patients, are characterized by the presence of both lipid peroxidation and protein oxidation markers resulting from the increased level of free radical species. Oxidation introduces several modifications in protein structure, including carbonylation and nitrotyrosine formation, which severely compromise cell function. Due to the link existing between oxidative stress and Parkinson's disease, antioxidant molecules could represent possible therapeutic tools for this disease. In this study, we evaluated the effect of curcumin, a natural compound known for its antioxidant properties, in dopaminergic PC12 cells treated with rotenone, a cell model of Parkinsonism. Our results demonstrate that the treatment of PC12 cells with rotenone causes severe protein damage, with formation of both carbonylated and nitrotyrosine-derived proteins, whereas curcumin (10 µM) co-exposure exerts protective effects by reducing the levels of oxidized proteins. Curcumin also promotes proteasome activation, abolishing the inhibitory effect exerted by rotenone on this degradative system.

Curcumin Analogue C1 Promotes Hex and Gal Recruitment to the Plasma Membrane via mTORC1-Independent TFEB Activation.

The monocarbonyl analogue of curcumin (1E,4E)-1,5-Bis(2-methoxyphenyl)penta-1,4-dien-3-one (C1) has been used as a specific activator of the master gene transcription factor EB (TFEB) to correlate the activation of this nuclear factor with the increased activity of lysosomal glycohydrolases and their recruitment to the cell surface. The presence of active lysosomal glycohydrolases associated with the lipid microdomains has been extensively demonstrated, and their role in glycosphingolipid (GSL) remodeling in both physiological and pathological conditions, such as neurodegenerative disorders, has been suggested. Here, we demonstrate that Jurkat cell stimulation elicits TFEB nuclear translocation and an increase of both the expression of hexosaminidase subunit beta (HEXB), hexosaminidase subunit alpha (HEXA), and galactosidase beta 1 (GLB1) genes, and the recruitment of ß-hexosaminidase (Hex, EC 3.2.1.52) and ß-galactosidase (Gal, EC 3.2.1.23) on lipid microdomains. Treatment of Jurkat cells with the curcumin analogue C1 also resulted in an increase of both lysosomal glycohydrolase activity and their targeting to the cell surface. Similar effects of C1 on lysosomal glycohydrolase expression and their recruitment to lipid microdomains was observed by treating the SH-SY5Y neuroblastoma cell line; the effects of C1 treatment were abolished by TFEB silencing. Together, these results clearly demonstrate the existence of a direct link between TFEB nuclear translocation and the transport of Hex and Gal from lysosomes to the plasma membrane.

Extracellular Vesicles as Conveyors of Membrane-Derived Bioactive Lipids in Immune System.

Over the last 20 years, extracellular vesicles (EVs) have been established as an additional way to transmit signals outside the cell. They are membrane-surrounded structures of nanometric size that can either originate from the membrane invagination of multivesicular bodies of the late endosomal compartment (exosomes) or bud from the plasma membrane (microvesicles). They contain proteins, lipids, and nucleic acids—namely miRNA, but also mRNA and lncRNA—which are derived from the parental cell, and have been retrieved in every fluid of the body. As carriers of antigens, either alone or in association with major histocompatibility complex (MHC) class II and class I molecules, their immunomodulatory properties have been extensively investigated. Moreover, recent studies have shown that EVs may carry and deliver membrane-derived bioactive lipids that play an important function in the immune system and related pathologies, such as prostaglandins, leukotrienes, specialized pro-resolving mediators, and lysophospholipids. EVs protect bioactive lipids from degradation and play a role in the transcellular synthesis of prostaglandins and leukotrienes. Here, we summarized the role of EVs in the regulation of immune response, specifically focusing our attention on the emerging role of EVs as carriers of bioactive lipids, which is important for immune system function.

Oncogenic H-Ras Expression Induces Fatty Acid Profile Changes in Human Fibroblasts and Extracellular Vesicles.

Extracellular vesicles (EVs) are lipid bilayer surrounded particles that are considered an additional way to transmit signals outside the cell. Lipids have not only a structural role in the organization of EVs membrane bilayer, but they also represent a source of lipid mediators that may act on target cells. Senescent cells are characterized by a permanent arrest of cell proliferation, but they are still metabolically active and influence nearby tissue secreting specific signaling mediators, including those carried by EVs. Notably, cellular senescence is associated with increased EVs release. Here, we used gas chromatography coupled to mass spectrometry to investigate the total fatty acid content of EVs released by fibroblasts undergoing H-RasV12-induced senescence and their parental cells. We find that H-RasV12 fibroblasts show increased level of monounsaturated and decreased level of saturated fatty acids, as compared to control cells. These changes are associated with transcriptional up-regulation of specific fatty acid-metabolizing enzymes. The EVs released by both controls and senescent fibroblasts show a higher level of saturated and polyunsaturated species, as compared to parental cells. Considering that fibroblasts undergoing H-RasV12-induced senescence release a higher number of EVs, these findings indicate that senescent cells release via EVs a higher amount of fatty acids, and in particular of polyunsaturated and saturated fatty acids, as compared to control cells.

Effects of xylazine and dexmedetomidine on equine articular chondrocytes in vitro.

OBJECTIVE: To assess the effects of xylazine and dexmedetomidine on equine chondrocytes, in vitro. STUDY DESIGN: Prospective, experimental study. STUDY MATERIAL: Equine articular chondrocytes from five male horses. METHODS: Chondrocytes were isolated from healthy equine articular cartilage of the metacarpo/metatarsophalangeal joints. Cell viability was assessed using the WST-8 assay by exposing chondrocytes to xylazine (0.5, 1, 2, 4, 8, 16.6, 25, 50 mg mL-1) or dexmedetomidine (0.001, 0.005, 0.01, 0.05, 0.175, 0.25 mg mL-1) for 15, 30 and 60 minutes. Based on the results of these tests, cells were treated with xylazine (1, 4, 25 mg mL-1) or dexmedetomidine (0.05, 0.175, 0.25 mg mL-1) for 15 minutes to further evaluate: cell viability by neutral red uptake; cell membrane integrity by lactate dehydrogenase release and by fluorescence microscopy with Hoechst 33342 and propidium iodide (PI), and apoptosis by flow cytometry using double staining with annexin V-fluorescein isothiocyanate/PI and by cell morphology. RESULTS: Both drugs reduced cell viability in a dose-dependent manner. Specifically, all xylazine concentrations, except 0.5 mg mL-1 and 1 mg mL-1, significantly reduced cell viability, whereas the effects of dexmedetomidine were evident only at 0.175 mg mL-1 and 0.25 mg mL-1. The highest concentrations of xylazine (25 mg mL-1) and dexmedetomidine (0.25 mg mL-1) caused loss of membrane integrity. Cell morphology and flow cytometry analyses demonstrated signs of late apoptosis in xylazine-treated cells, and signs of late apoptosis and necrosis in dexmedetomidine-treated cells. CONCLUSIONS AND CLINICAL RELEVANCE: This study offers new insights into the potential chondrotoxicity induced by dexmedetomidine and xylazine. Therefore, the intra-articular administration of α2-agonists should be conducted with care, especially for doses of ≥ 4 mg mL-1 of xylazine and 0.175 mg mL-1 and 0.25 mg mL-1 of dexmedetomidine.

Extracellular Vesicles as New Players in Cellular Senescence.

Cell senescence is associated with the secretion of many factors, the so-called "senescence-associated secretory phenotype", which may alter tissue microenvironment, stimulating the organism to clean up senescent cells and replace them with newly divided ones. Therefore, although no longer dividing, these cells are still metabolically active and influence the surrounding tissue. Much attention has been recently focused not only on soluble factors released by senescent cells, but also on extracellular vesicles as conveyors of senescence signals outside the cell. Here, we give an overview of the role of extracellular vesicles in biological processes and signaling pathways related to senescence and aging.

Isolation of Extracellular Vesicles from Agri-Food Wastes: A Novel Perspective in the Valorization of Agri-Food Wastes and By-Products.

Agri-food wastes generated by industrial food processing are valorized through the extraction of biomolecules to obtain value-added products useful for various industrial applications. In the present review, we describe the valuable by-products and bioactive molecules that can be obtained from agricultural wastes and propose extracellular vesicles (EVs) as innovative nutraceutical and therapeutic compounds that could be derived from agriculture residues. To support this idea, we described the general features and roles of EVs and focused on plant-derived extracellular vesicles (PDEVs) that are considered natural carriers of bioactive molecules and are involved in intercellular communication between diverse kingdoms of life. Consistently, PDEVs exert beneficial effects (anti-inflammatory, anti-tumor, and immune-modulatory) on mammalian cells. Although this research field is currently in its infancy, in the near future, the isolation of EVs and their use as nutraceutical tools could represent a new and innovative way to valorize waste from the agri-food industry in an ecofriendly way.

Evidence of Lysosomal ß-Hexosaminidase Enzymatic Activity Associated with Extracellular Vesicles: Potential Applications for the Correction of Sandhoff Disease.

Extracellular vesicles (EVs) can be isolated from biological fluids and cell culture medium. Their nanometric dimension, relative stability, and biocompatibility have raised considerable interest for their therapeutic use as delivery vehicles of macromolecules, namely nucleic acids and proteins. Deficiency in lysosomal enzymes and associated proteins is at the basis of a group of genetic diseases known as lysosomal storage disorders (LSDs), characterized by the accumulation of undigested substrates into lysosomes. Among them, GM2 gangliosidoses are due to a deficiency in the activity of lysosomal enzyme ß-hexosaminidase, leading to the accumulation of the GM2 ganglioside and severe neurological symptoms. Current therapeutic approaches, including enzyme replacement therapy (ERT), have proven unable to significantly treat these conditions. Here, we provide evidence that the lysosomal ß-hexosaminidase enzyme is associated with EVs released by HEK cells and that the EV-associated activity can be increased by overexpressing the α-subunit of ß-hexosaminidase. The delivery of EVs to ß-hexosaminidase-deficient fibroblasts results in a partial cross-correction of the enzymatic defect. Overall findings indicate that EVs could be a source of ß-hexosaminidase that is potentially exploitable for developing therapeutic approaches for currently untreatable LSDs.

PhosphoLipidome Alteration Induced by Clostridioides difficile Toxin B in Enteric Glial Cells.

Clostridioides difficile (C. difficile) is responsible for a spectrum of nosocomial/antibiotic-associated gastrointestinal diseases that are increasing in global incidence and mortality rates. The C. difficile pathogenesis is due to toxin A and B (TcdA/TcdB), both causing cytopathic and cytotoxic effects and inflammation. Recently, we demonstrated that TcdB induces cytopathic and cytotoxic (apoptosis and necrosis) effects in enteric glial cells (EGCs) in a dose/time-dependent manner and described the underlying signaling. Despite the role played by lipids in host processes activated by pathogens, to counter infection and/or induce cell death, to date no studies have investigated lipid changes induced by TcdB/TcdA. Here, we evaluated the modification of lipid composition in our in vitro model of TcdB infection. Apoptosis, cell cycle, cell viability, and lipidomic profiles were evaluated in EGCs treated for 24 h with two concentrations of TcdB (0.1 ng/mL; 10 ng/mL). In EGCs treated with the highest concentration of TcdB, not only an increased content of total lipids was observed, but also lipidome changes, allowing the separation of TcdB-treated cells and controls into different clusters. The statistical analyses also allowed us to ascertain which lipid classes and lipid molecular species determine the clusterization. Changes in lipid species containing inositol as polar head and plasmalogen phosphatidylethanolamine emerged as key indicators of altered lipid metabolism in TcdB-treated EGCs. These results not only provide a picture of the phospholipid profile changes but also give information regarding the lipid metabolism pathways altered by TcdB, and this might represent an important step for developing strategies against C. difficile infection.

Characterization of Nanovesicles Isolated from Olive Vegetation Water.

Edible plant and fruit-derived nanovesicles (NVs) are membrane-enclosed particles with round-shape morphology and signaling functions, which resemble mammalian cell-derived extracellular vesicles. These NVs can transmit cross-kingdom signals as they contain bioactive molecules and exert biological effects on mammalian cells. Their properties and stability in the gastrointestinal tract suggest NVs as a promising nutraceutical tool. In this study, we have demonstrated for the first time the presence of NVs in olive vegetation water (OVW), a waste by-product generated during olive oil production. Biophysical characterization by scanning electron microscopy, cryo-transmission electron microscopy, and nanoparticle tracking analysis revealed the presence in OVW of NVs having size and morphology similar to that of vesicles isolated from edible plants. Integrated lipidomic, metabolomic, and proteomic analyses showed that OVW-NVs carry a set of lipids, metabolites and proteins which have recognized antioxidant and anti-inflammatory activities. The nature of biomolecules identified in OVW-NVs suggests that these vesicles could exert beneficial effects on mammalian cells and could be used in the nutraceutical and food industries. The successful isolation of OVW-NVs and the characterization of their features strengthen the idea that agricultural waste might represent a source of NVs having features similar to NVs isolated from edible plants/fruits.

Lipid Biomarkers in Liquid Biopsies: Novel Opportunities for Cancer Diagnosis.

Altered cellular metabolism is a well-established hallmark of cancer. Although most studies have focused on the metabolism of glucose and glutamine, the upregulation of lipid metabolism is also frequent in cells undergoing oncogenic transformation. In fact, cancer cells need to meet the enhanced demand of plasma membrane synthesis and energy production to support their proliferation. Moreover, lipids are precursors of signaling molecules, termed lipid mediators, which play a role in shaping the tumor microenvironment. Recent methodological advances in lipid analysis have prompted studies aimed at investigating the whole lipid content of a sample (lipidome) to unravel the complexity of lipid changes in cancer patient biofluids. This review focuses on the application of mass spectrometry-based lipidomics for the discovery of cancer biomarkers. Here, we have summarized the main lipid alteration in cancer patients' biofluids and uncovered their potential use for the early detection of the disease and treatment selection. We also discuss the advantages of using biofluid-derived extracellular vesicles as a platform for lipid biomarker discovery. These vesicles have a molecular signature that is a fingerprint of their originating cells. Hence, the analysis of their molecular cargo has emerged as a promising strategy for the identification of sensitive and specific biomarkers compared to the analysis of the unprocessed biofluid.

Protein and Lipid Content of Milk Extracellular Vesicles: A Comparative Overview.

The characterization of the protein and lipid cargo of milk extracellular vesicles from different mammal species is crucial for understanding their biogenesis and biological functions, as well as for a comprehensive description of the nutritional aspects of animal milk for human diet. In fact, milk EVs have been reported to possess relevant biological effects, but the molecules/biochemical pathways underlying these effects have been poorly investigated. The biochemical characterization is an important initial step for the potential therapeutic and diagnostic use of natural or modified milk EVs. The number of studies analysing the protein and lipid composition of milk EVs is limited compared to that investigating the nucleic acid cargo. Here, we revised the literature regarding the protein and lipid content of milk EVs. Until now, most investigations have shown that the biochemical cargo of EVs is different with respect to that of other milk fractions. In addition, even if these studies derived mostly from bovine and human milk EVs, comparison between milk EVs from different animal species and milk EVs biochemical composition changes due to different factors including lactation stages and health status is also beginning to be reported.

Accurate Analysis of Lipid Building Blocks Using the Tool LipidOne.

LC/MS-based analysis techniques combined with specialized lipid platforms allow the qualitative and quantitative determination of thousands of lipid molecules. Each individual molecule can be considered as an assembly of smaller parts, often called building blocks that are the result of a myriad of biochemical synthesis and transformation processes. LipidOne is a new lipidomic tool that automatically highlights all qualitative and quantitative changes in lipid building blocks both among all detected lipid classes and between experimental groups. Thanks to LipidOne, the discovered differences among lipid building blocks can be easily linked to the activity of specific enzymes.

Extracellular Vesicles in Aging: An Emerging Hallmark?

Extracellular vesicles (EVs) are membrane-enclosed particles secreted by cells and circulating in body fluids. Initially considered as a tool to dispose of unnecessary material, they are now considered an additional method to transmit cell signals. Aging is characterized by a progressive impairment of the physiological functions of tissues and organs. The causes of aging are complex and interconnected, but there is consensus that genomic instability, telomere erosion, epigenetic alteration, and defective proteostasis are primary hallmarks of the aging process. Recent studies have provided evidence that many of these primary stresses are associated with an increased release of EVs in cell models, able to spread senescence signals in the recipient cell. Additional investigations on the role of EVs during aging also demonstrated the great potential of EVs for the modulation of age-related phenotypes and for pro-rejuvenation therapies, potentially beneficial for many diseases associated with aging. Here we reviewed the current literature on EV secretion in senescent cell models and in old vs. young individual body fluids, as well as recent studies addressing the potential of EVs from different sources as an anti-aging tool. Although this is a recent field, the robust consensus on the altered EV release in aging suggests that altered EV secretion could be considered an emerging hallmark of aging.