Extracellular Vesicles Subtypes Research Articles

Microvesicles derived from dermal myofibroblasts modify the integrity of the blood and lymphatic barriers using distinct endocytosis pathways

AbstractMicrovesicles (MVs) are a subtype of extracellular vesicles that can transfer biological information from their producer cells to target cells. This communication can in turn affect both normal and pathological processes. Mounting evidence has revealed that dermal wound myofibroblasts (Wmyo) produce MVs, which can transfer biomolecules impacting receptor cells such as human dermal microvascular endothelial cells (HDMECs). While the effects of MVs on HDMECs are generally well described in the literature, little is known about the transport of MVs across the HDMEC barrier, and their potential effect on the barrier integrity remains unknown. Here, we investigated these roles of Wmyo‐derived MVs on two sub‐populations of HDMECs, blood endothelial cells (BECs) and lymphatic endothelial cells (LECs). Using an in vitro model to mimic the endothelial barrier, we showed that MVs crossed the LEC barrier but not the BEC barrier. In addition, we demonstrated that MVs were able to influence the cell‐cell junctions of HDMECs. Specifically, we observed that after internalization via the predominantly caveolin‐dependent pathway, MVs induced the opening of junctions in BECs. Conversely, in LECs, MVs mainly use the macropinocytosis pathway and induce closure of these junctions. Moreover, proteins in the MV membrane were responsible for this effect, but not specifically those belonging to the VEGF family. Finally, we found that once the LEC barrier permeability was reduced by MV stimuli, MVs ceased to cross the barrier. Conversely, when the BEC barrier was rendered permeable following stimulation with MVs, they were subsequently able to cross the barrier via the paracellular pathway. Taken together, these results suggest that the study of Wmyo‐derived MVs offers valuable insights into their interaction with the HDMEC barrier in the context of wound healing. They highlight the potential significance of these MVs in the overall process.

Circulating plasma-derived extracellular vesicles expressing bone and kidney markers are associated with neurocognitive impairment in people living with HIV.

Although effective antiretroviral therapy (ART) has improved the life expectancy of people with HIV (PWH), the prevalence of milder forms of HIV-associated neurocognitive disorders (HAND) persist, and it is associated with systemic and neuro-inflammatory processes that could impact other organ systems. However, the complex signaling mechanisms between the bone-kidney systems and the brain in HAND remain unknown. Extracellular vesicles (EVs) play a potential role in inter-organ communication and are involved in regulating cell activity in distant tissues. In this study, we examined whether levels of EVs from bone-and kidney-related cells associate with cognitive dysfunction and explored the relationship between kidney-bone EV axis in PWH experiencing cognitive deficits. EV subtypes were characterized in plasma from 61 PWH with either cognitive impairment (CI, n = 53) or normal cognition (NC, n = 8) based on the American Academy of Neurology criteria for HIV-associated dementia (HAD, n = 11), minor cognitive motor disorder (MCMD, n = 25) or asymptomatic neurocognitive impairment (ANI, n = 17) by spectral flow cytometry. EVs were profiled with markers reflecting bone and kidney cell origin. A support vector machine learning-based model was employed for analyses of EV phenotypes to predict the cognitive dysfunction. Plasma-EVs expressing osteocalcin, sclerostin, and nephrin were significantly higher in the cognitive impairment group compared to the normal cognition group. EVs bearing kidney cell markers correlated significantly with bone-derived EVs. A machine learning-based model, comprised of osteocalcin+, nephrin+, and CD24+ EVs predicted cognitive impairment in PWH on ART. Our study reveals that neurocognitive impairment in PWH is associated with increased levels of plasma EVs enriched with the bone markers osteocalcin and sclerostin and the kidney marker nephrin, suggesting that these EV subtypes may be novel candidate biomarkers for disease-spanning neurocognitive dysfunction. Moreover, the relationship between bone-derived EVs with kidney-derived EVs may suggest their role in mediating inter-organ crosstalk in the pathogenesis of HIV-associated cognitive impairment.

The Triple Adipose-Derived Stem Cell Exosome Technology as a Potential Tool for Treating Triple-Negative Breast Cancer.

Extracellular vesicles are pivotal mediators in intercellular communication, facilitating the exchange of biological information among healthy, pathological and tumor cells. Between the diverse subtypes of extracellular vesicles, exosomes have unique properties and clinical and therapeutical applications. Breast cancer ranks as one of the most prevalent malignancies across the globe. Both the tumor core and its surrounding microenvironment engage in a complex, orchestrated interaction that facilitates cancer's growth and spread. The most significant PubMed literature about extracellular vesicles and Adipose-Derived Stem Cell Exosomes and breast cancer was selected in order to report their biological properties and potential applications, in particular in treating triple-negative breast cancer. Adipose-Derived Stem Cell Exosomes represent a potential tool in targeting triple-negative breast cancer cells at three main levels: the tumor core, the tumor microenvironment and surrounding tissues, including metastases. The possibility of impacting triple-negative breast cancer cells with engineered Adipose-Derived Stem Cell Exosomes is real. The opportunity to translate our current in vitro analyses into a future in vivo scenario is even more challenging.

Exosome-Mediated Antigen Delivery: Unveiling Novel Strategies in Viral Infection Control and Vaccine Design.

Exosomes are small subtypes of extracellular vesicles (EVs) naturally released by different types of cells into their environment. Their physiological roles appear to be multiple, yet many aspects of their biological activities remain to be understood. These vesicles can transport and deliver a variety of cargoes and may serve as unconventional secretory vesicles. Thus, they play a crucial role as important vectors for intercellular communication and the maintenance of homeostasis. Exosome production and content can vary under several stresses or modifications in the cell microenvironment, influencing cellular responses and stimulating immunity. During infectious processes, exosomes are described as double-edged swords, displaying both beneficial and detrimental effects. Owing to their tractability, the analysis of EVs from multiple biofluids has become a booming tool for monitoring various pathologies, from infectious to cancerous origins. In this review, we present an overview of exosome features and discuss their particular and ambiguous functions in infectious contexts. We then focus on their properties as diagnostic or therapeutic tools. In this regard, we explore the capacity of exosomes to vectorize immunogenic viral antigens and their function in mounting adaptive immune responses. As exosomes provide interesting platforms for antigen presentation, we further review the available data on exosome engineering, which enables peptides of interest to be exposed at their surface. In the light of all these data, exosomes are emerging as promising avenues for vaccine strategies.

High-Performance Gel-Free and Label-Free Size Fractionation of Extracellular Vesicles with Two-Dimensional Electrophoresis in a Microfluidic Artificial Sieve.

Extracellular vesicles (EVs) are cell-derived particles that exhibit diverse sizes, molecular contents, and clinical implications for various diseases depending on their specific subpopulations. However, fractionation of EV subpopulations with high resolution, efficiency, purity, and yield remains an elusive goal due to their diminutive sizes. In this study, we introduce a novel strategy that effectively separates EV subpopulations in a gel-free and label-free manner, using two-dimensional (2D) electrophoresis in a microfluidic artificial sieve. The microfabricated artificial sieve consists of periodically arranged micro-slit-well structures in a 2D array and generates an anisotropic electric field pattern to size fractionate EVs into discrete streams and steer the subpopulations into designated outlets for collection within a minute. Along with fractionating EV subpopulations, contaminants such as free proteins and short nucleic acids can be simultaneously directed to waste outlets, thus accomplishing both size fractionation and purification of EVs with high performance. Our platform offers a simple, rapid, and versatile solution for EV subpopulation isolation, which can potentially facilitate the discovery of biomarkers for specific EV subtypes and the development of EV-based therapeutics.

Lipoprotein apheresis affects the concentration of extracellular vesicles in patients with elevated lipoprotein (a)

Lipoprotein apheresis (LA) is a therapeutic option for hyperlipoproteinemia(a) (hyper-Lp(a)) and atherosclerotic cardiovascular disease (ASCVD). LA improves blood rheology, reduces oxidative stress parameters and improves endothelial function. The underlying molecular mechanisms of LA beneficial effects are unknown, but it has been suggested that LA exhibits multiple activities beyond simply removing lipoproteins. We hypothesized that LA removes not only lipoproteins, but also extracellular vesicles (EVs). To test this hypothesis, we performed a prospective study in 22 patients undergoing LA for hyper-Lp(a) and ASCVD. Different EVs subtypes were measured before and directly after LA, and after 7 days. We used calibrated flow cytometry to detect total particle concentration (diameter >  ~ 100 nm), total lipoproteins concentration (diameter > 200 nm, RI > 1.51), total EV concentration (diameter > 200 nm, RI < 1.41), concentrations of EVs derived from erythrocytes (CD235a+; diameter > 200 nm, RI < 1.41), leukocytes (CD45+; diameter > 200 nm, RI < 1.41) and platelets (CD61+, PEVs; diameter > 200 nm, RI < 1.41). LA reduced the concentrations of all investigated EVs subtypes and lipoproteins. Lp(a) concentration was lowered by 64.5% [(58% – 71%); p < 0.001]. Plasma concentrations of EVs > 200 nm in diameter derived from platelets (CD61 +), leukocytes (CD45+) and erythrocytes (CD235a+) decreased after single LA procedure by 42.7% [(12.8–54.7); p = 0.005], 42.6% [(29.7–54.1); p = 0.030] and 26.7% [(1.0–62.7); p = 0.018], respectively, compared to baseline. All EV subtypes returned to the baseline concentrations in blood plasma after 7 days. To conclude, LA removes not only Lp(a), but also cell-derived EVs, which may contribute to LA beneficial effects.

Emerging Role of Extracellular Vesicles in Intercellular Communication in the Brain: Implications for Neurodegenerative Diseases and Therapeutics.

Extracellular vesicles (EVs) are minute lipid-bilayer sacs discharged by cells, encompassing a diverse array of proteins, nucleic acids, and lipids. The identification of EVs as pivotal agents in intercellular communication has sparked compelling research pathways in the realms of cell biology and neurodegenerative diseases. Utilizing EVs for medicinal reasons has garnered interest due to the adaptability of EV-mediated communication. EVs can be classified based on their physical characteristics, biochemical composition, or cell of origin following purification. This review delves into the primary sub-types of EVs, providing an overview of the biogenesis of each type. Additionally, it explores the diverse environmental conditions triggering EV release and the originating cells, including stem cells and those from the Central Nervous System. Within the brain, EVs play a pivotal role as essential mediators of intercellular communication, significantly impacting synaptic plasticity, brain development, and the etiology of neurological diseases. Their potential diagnostic and therapeutic applications in various brain-related conditions are underscored, given their ability to carry specific cargo. Specially engineered EVs hold promise for treating diverse diseases, including neurodegenerative disorders. This study primarily emphasizes the diagnostic and potential therapeutic uses of EVs in neurological disorders such as Alzheimer's Disease, Huntington's Disease, Parkinson's Disease, Amyotrophic Lateral Sclerosis, and Prions disease. It also summarizes innovative techniques for detecting EVs in the brain, suggesting that EVs could serve as non-invasive biomarkers for early detection, disease monitoring, and prognosis in neurological disorders.

Liver-derived Exosomal miRNA in NAFLD: Mechanisms of Action, Biomarkers, and Therapeutic Applications.

Nonalcoholic fatty liver disease (NAFLD) is of global concern due to its high prevalence worldwide. NAFLD, as one of the most common causes of liver function abnormalities, is associated with obesity, insulin resistance, and type 2 diabetes mellitus, and there are no medications available to treat NAFLD. Extracellular vesicles (EVs) are nanosized, membrane-bound vesicles that deliver biomolecules between cells. Exosomes are a subtype of EVs that mediate intercellular communication by delivering proteins and RNAs. MicroRNAs (miRNAs) are a highly conserved class of small tissue-specific non-coding RNAs that influence the expression of many functionally interacting genes. Hepatic-derived exosomal miRNAs are tightly associated with NAFLD occurrence and progression through multiple mechanisms. In addition, the characterization of miRNAs suggests that they may serve as multifunctional biomarkers for NAFLD, be used as clinical therapeutic targets for NAFLD, and be significant predictors of patient prognosis. Here, we review recent advances in the regulation and function of exosome-derived miRNAs in NAFLD, focusing on miRNAs specifically expressed or enriched in hepatocytes (HCs), hepatic macrophages, hepatic stellate cells (HSCs), and other immune cells in the liver. Finally, we discuss future research directions on exosomal miRNAs as biomarkers for NAFLD's diagnosis and clinical therapeutic targets.

Immunophenotype profile by flow cytometry reveals different subtypes of extracellular vesicles in porcine seminal plasma

BackgroundPorcine seminal plasma (SP) is endowed with a heterogeneous population of extracellular vesicles (sEVs). This study evaluated the immunophenotypic profile by high-sensitivity flow cytometry of eight sEV subpopulations isolated according to their size (small [S-sEVs] and large [L-sEVs]) from four different SP sources, namely three ejaculate fractions (the first 10 mL of the sperm rich fraction [SRF-P1], the remaining SRF [SRF-P2], and the post-SRF [PSRF]) and entire ejaculate (EE).MethodsSeminal EVs were isolated using a size exclusion chromatography-based protocol from six SP pools (five ejaculates/pool) of each SP source and characterized using complementary approaches including total protein (BCA™assay), particle size distribution (dynamic light scattering), morphology (transmission electron microscopy), and purity (albumin by Western blot). Expression of CD9, CD63, CD81, CD44 and HSP90β was analyzed in all sEV subpopulations by high-sensitivity flow cytometry according to MIFlowCyt-EV guidelines, including an accurate calibration, controls, and discrimination by CFSE-labelling.ResultsEach sEV subpopulation exhibited a specific immunophenotypic profile. The percentage of sEVs positive for CD9, CD63, CD81 and HSP90β differed between S- and L-sEVs (P < 0.0001). Specifically, the percentage of sEVs positive for CD9 and CD63 was higher and that for CD81 was lower in S- than L-sEVs in the four SP sources. However, the percentage of HSP90β-positive sEVs was lower in S-sEVs than L-sEVs in the SRF-P1 and EE samples. The percentage of sEVs positive for CD9, CD63, and CD44 also differed among the four SP sources (P < 0.0001), being highest in PSRF samples. Notably, virtually all sEV subpopulations expressed CD44 (range: 88.04–98.50%).ConclusionsThis study demonstrated the utility of high-sensitivity flow cytometry for sEV immunophenotyping, allowing the identification of distinct sEV subpopulations that may have different cellular origin, cargo, functions, and target cells.

Red blood cell transfusion-related dynamics of extracellular vesicles in intensive care patients: a prospective subanalysis

Extracellular vesicles (EVs) accumulate during packed red blood cell (PRBC) storage. To date, the involvement of EVs in transfusion-related immunomodulation (TRIM) has not been prospectively evaluated in intensive care unit (ICU) patients. This was a prospective subanalysis of a recent observational feasibility study in postoperative ICU patients after: (1) open aortic surgery (Aorta), (2) bilateral lung transplantation (LuTx), and (3) other types of surgery (Comparison). Patient plasma was collected three times each before and after leukoreduced PRBC transfusion at 30-min intervals. The total number of EVs and EVs derived from erythrocytes (EryEVs), total platelets (total PEVs), activated platelets, granulocytes (GEVs), monocytes, and myeloid cells in PRBC samples and patient plasma were analyzed by flow cytometry. Statistical analysis was performed by Spearman’s correlation test, linear mixed models and pairwise comparisons by Wilcoxon matched-pairs test. Twenty-three patients (Aorta n = 5, LuTx n = 9, Comparison n = 9) were included in the final analysis. All EV subgroups analyzed were detectable in all PRBCs samples (n = 23), but concentrations did not correlate with storage time. Moreover, all EVs analyzed were detectable in all plasma samples (n = 138), and EV counts were consistent before transfusion. Concentrations of total EVs, EryEVs, total PEVs, and GEVs increased after transfusion compared with baseline in the entire cohort but not in specific study groups. Furthermore, the change in plasma EV counts (total EVs and EryEVs) after transfusion correlated with PRBC storage time in the entire cohort. Extracellular vesicles were detectable in all PRBC and plasma samples. Individual EV subtypes increased after transfusion in the entire cohort, and in part correlated with storage duration. Future clinical studies to investigate the role of EVs in TRIM are warranted and should anticipate a larger sample size.Trial registration: Clinicaltrials.gov: NCT03782623.

Heat Shock Proteins Mediate Intercellular Communications within the Tumor Microenvironment through Extracellular Vesicles

From an evolutive perspective, tumor cells endure successive turnover upon stress conditions and pressure to adapt to new environments. These cells use exceptional communication skills to share biological information to “survive upon every metabolic cost”. The tumor microenvironment (TME) is a miscellaneous collection of cells, factors, and extracellular vesicles (EVs). EVs are small lipid bilayer-delimited particles derived from cells with sizes ranging from 100 to 1000 nm. Exosomes (&lt;160 nm) are the minor subtype of EVs, originating from the endosomal pathways. The TME also contains “giant” vesicles, microvesicles (100–1000 nm, MV), originated from membrane blebbing. EVs can act as intercellular communication mediators, contributing to many biological processes, by carrying different biomolecules, such as proteins, lipids, nucleic acids, and metabolites. EV secretion can promote either tumor cell survival or manage their stress to death. Tumor-derived EVs transfer adaptative stress signaling to recipient cells, reprograming these cells. Heat shock proteins (HSP) are prominent stress response regulators, specifically carried by exosomes. HSP-loaded EVs reprogram tumor and TME cells to acquire mechanisms contributing to tumor progression and therapy resistance. The intercellular communication mediated by HSP-loaded EVs favors the escape of tumor cells from the endoplasmic reticulum stress, hypoxia, apoptosis, and anticancer therapies. Extracellular HSPs activate and deactivate the immune response, induce cell differentiation, change vascular homeostasis, and help to augment the pre-metastatic niche formation. Here we explore EVs’ mechanisms of HSP transmission among TME cells and the relevance of these intercellular communications in resistance to therapy.

The diffusion of normal skin wound myofibroblast‐derived microvesicles differs according to matrix composition

AbstractMicrovesicles (MVs) are a subtype of extracellular vesicles that can transfer biological information over long distances, affecting normal and pathological processes including skin wound healing. However, the diffusion of MVs into tissues can be impeded by the extracellular matrix (ECM). We investigated the diffusion of dermal wound myofibroblast‐derived MVs into the ECM by using hydrogels composed of different ECM molecules such as fibrin, type III collagen and type I collagen that are present during the healing process. Fluorescent MVs mixed with hydrogels were employed to detect MV diffusion using fluorometric methods. Our results showed that MVs specifically bound type I collagen and diffused freely out of fibrin and type III collagen. Further analysis using flow cytometry and specific inhibitors revealed that MVs bind to type I collagen via the α2β1 integrin. These data demonstrate that MV transport depends on the composition of the wound environment.

Vertical transmission of maternal DNA through extracellular vesicles associates with altered embryo bioenergetics during the periconception period

The transmission of DNA through extracellular vesicles (EVs) represents a novel genetic material transfer mechanism that may impact genome evolution and tumorigenesis. We aimed to investigate the potential for vertical DNA transmission within maternal endometrial EVs to the pre-implantation embryo and describe any effect on embryo bioenergetics. We discovered that the human endometrium secretes all three general subtypes of EV - apoptotic bodies (ABs), microvesicles (MVs), and exosomes (EXOs) - into the human endometrial fluid (EF) within the uterine cavity. EVs become uniformly secreted into the EF during the menstrual cycle, with the proportion of different EV populations remaining constant; however, MVs contain significantly higher levels of mitochondrial (mt)DNA than ABs or EXOs. During the window of implantation, MVs contain an eleven-fold higher level of mtDNA when compared to cells-of-origin within the receptive endometrium, which possesses a lower mtDNA content and displays the upregulated expression of mitophagy-related genes. Furthermore, we demonstrate the internalization of EV-derived nuclear-encoded (n)DNA/mtDNA by trophoblast cells of murine embryos, which associates with a reduction in mitochondrial respiration and ATP production. These findings suggest that the maternal endometrium suffers a reduction in mtDNA content during the preconceptional period, that nDNA/mtDNA become packaged into secreted EVs that the embryo uptakes, and that the transfer of DNA to the embryo within EVs occurs alongside the modulation of bioenergetics during implantation.

Vertical transmission of maternal DNA through extracellular vesicles associates with altered embryo bioenergetics during the periconception period.

The transmission of DNA through extracellular vesicles (EVs) represents a novel genetic material transfer mechanism that may impact genome evolution and tumorigenesis. We aimed to investigate the potential for vertical DNA transmission within maternal endometrial EVs to the pre-implantation embryo and describe any effect on embryo bioenergetics. We discovered that the human endometrium secretes all three general subtypes of EV - apoptotic bodies (ABs), microvesicles (MVs), and exosomes (EXOs) - into the human endometrial fluid (EF) within the uterine cavity. EVs become uniformly secreted into the EF during the menstrual cycle, with the proportion of different EV populations remaining constant; however, MVs contain significantly higher levels of mitochondrial (mt)DNA than ABs or EXOs. During the window of implantation, MVs contain an eleven-fold higher level of mtDNA when compared to cells-of-origin within the receptive endometrium, which possesses a lower mtDNA content and displays the upregulated expression of mitophagy-related genes. Furthermore, we demonstrate the internalization of EV-derived nuclear-encoded (n)DNA/mtDNA by trophoblast cells of murine embryos, which associates with a reduction in mitochondrial respiration and ATP production. These findings suggest that the maternal endometrium suffers a reduction in mtDNA content during the preconceptional period, that nDNA/mtDNA become packaged into secreted EVs that the embryo uptakes, and that the transfer of DNA to the embryo within EVs occurs alongside the modulation of bioenergetics during implantation.

The role of exosomes in the pathogenesis of chemotherapy-induced cardiotoxicity: A diagnostic and prognostic approach: exosome and cardiotoxicity

Exosomes are a subtype of natural extracellular vesicles that transfer a great number of biomolecules, released by almost all types of cells, and can be specifically taken up by recipient cells in order to maintain homeostasis and induce a proper response to stress or harsh environmental conditions. Since the heart is part of complex systems which involve various cell types, exosome-mediated communication plays a crucial role in the cardiovascular field. Given the extended studies on the molecular mechanisms mediated by exosome cargos, leading to cardiovascular diseases, we focus on the most important data that report the roles of proteins and microRNAs (miRNAs) transferred by exosomes and whether they possess cardiotoxicity or cardioprotection and also the potential of these biomolecules as therapeutic tools or diagnostic biomarkers.

Extracellular vesicles and co-isolated endogenous retroviruses from murine cancer cells differentially affect dendriticcells.

Cells secrete extracellular vesicles (EVs) and non-vesicular extracellular (nano)particles (NVEPs or ENPs) that may play a role in intercellular communication. Tumor-derived EVs have been proposed to induce immune priming of antigen presenting cells or to be immuno-suppressive agents. We suspect that such disparate functions are due to variable compositions in EV subtypes and ENPs. We aimed to characterize the array of secreted EVs and ENPs of murine tumor cell lines. Unexpectedly, we identified virus-like particles (VLPs) from endogenous murine leukemia virus in preparations of EVs produced by many tumor cells. We established a protocol to separate small EVs from VLPs and ENPs. We compared their protein composition and analyzed their functional interaction with target dendritic cells. ENPs were poorly captured and did not affect dendritic cells. Small EVs specifically induced dendritic cell death. A mixed large/dense EV/VLP preparation was most efficient to induce dendritic cell maturation and antigen presentation. Our results call for systematic re-evaluation of the respective proportions and functions of non-viral EVs and VLPs produced by murine tumors and their contribution to tumor progression.

Exosomes: New Insights into the Pathogenesis of Metabolic Syndrome.

Exosomes are a subtype of extracellular vesicles (EVs) with a diameter of 30~150 nm (averaging ~100 nm) that are primarily produced through the endosomal pathway, and carry various components such as lipids, proteins, RNA, and other small molecular substances. Exosomes can mediate intercellular communication through the bioactive substances they carry, thus participating in different physiological activities. Metabolic syndrome (MS) is a disease caused by disturbances in the body's metabolism, mainly including insulin resistance (IR), diabetes, obesity, non-alcoholic fatty liver disease (NAFLD), hyperlipidemia, and atherosclerosis (AS). Recent studies have shown that exosomes are closely related to the occurrence and development of MS. Exosomes can act as messengers to mediate signaling transductions between metabolic cells in the organism and play a bidirectional regulatory role in the MS process. This paper mainly reviews the components, biogenesis, biological functions and potential applications of exosomes, and exosomes involved in the pathogenesis of MS as well as their clinical significance in MS diagnosis.

Electrophysiological effects of mitochondria‐derived extracellular vesicles: implications for memory dysfunction in neurodegenerative disorders

AbstractBackgroundMitovesicles are a newly identified subtype of small extracellular vesicles (EVs) of mitochondrial origin. We recently developed a method to separate mitovesicles from other EVs after their isolation from the extracellular space of murine and human brain tissues. This method permits the study of the roles of mitovesicles in physiology and disease.Given their origin, we hypothesized that mitochondrial dysfunction, a hallmark of both Alzheimer’s disease (AD) and Down syndrome (DS), could induce alterations in mitovesicles, thus perturbing brain homeostasis and contributing to propagation of pathology. Therefore, we studied mitovesicle components and functionality after their isolation from the brain of a mouse model of DS (hereafter Ts2) as compared to diploid (2N) controls, as well as from frontal cortices of individuals with DS as compared to their age‐matched 2N controls.MethodBrain mitovesicles were isolated and analyzed by Western blotting, electron microscopy, nanotrack analysis, and monoamine neurotransmitter degradation assay as recently described (D’Acunzo, et al., 2022, PMID: 35962195). Coronal hippocampal slices from wild‐type mice were perfused with different subtypes of brain EVs, pretreated or not with clorgiline and pargyline (1 μM each), irreversible inhibitors of the monoamine oxidases type A and B (MAO‐A and MAO‐B), respectively. Long‐term potentiation (LTP) was induced at the level of the Schaeffer collateral fibers through a theta‐burst stimulation (Fà, et al., 2014, PMID: 24643165).ResultMitovesicle number is higher in brains of DS individuals and of Ts2 mice and contain a higher MAO‐B protein amount and enzymatic activity as compared to diploid controls, suggesting a potential mitovesicle‐dependent impairment of neuroamine homeostasis in DS. In agreement with that, Ts2 (but not 2N) mitovesicles stimulated a decreased LTP in recipient hippocampal slices as compared to vehicle control (Fig. 1; mtVs: mitovesicles). This biological activity was abolished when MAO‐B (but not MAO‐A) was blocked. The other EV subtypes of both genotypes, microvesicles and exosomes, did not trigger an effect on LTP. DS changes were found to be sex independent.ConclusionPathogenic mitovesicles are previously undescribed active players of the brain extracellular milieu by regulating LTP via MAO‐B, and potentially contribute to memory formation deficiencies typically found in AD and DS.

Exploring capabilities of elemental mass spectrometry for determination of metal and biomolecules in extracellular vesicles

Extracellular vesicles (EVs) are increasingly recognized as crucial components influencing various pathophysiological processes, such as cellular homeostasis, cancer progression, and neurological disease. However, the lack of standardized methods for EV isolation and classification, coupled with ambiguity in biochemical markers associated with EV subtypes, remains a major challenge. This Trends article highlights the most common approaches for EV isolation and characterization, along with recent applications of elemental mass spectrometry (MS) to analyse metals and biomolecules in EVs obtained from biofluids or in vitro cellular models. Considering the promising capabilities of elemental MS, the article also looks ahead to the potential analysis of EVs at the single-vesicle and single-cell levels using ICP-MS. These approaches may offer valuable insights into individual characteristics of EVs and their functions, contributing to a deeper understanding of their role in various biological processes.Graphical

Double Digital Assay for Single Extracellular Vesicle and Single Molecule Detection.

Extracellular vesicles (EVs) have emerged as a promising source of biomarkers for disease diagnosis. However, current diagnostic methods for EVs present formidable challenges, given the low expression levels of biomarkers carried by EV samples, as well as their complex physical and biological properties. Herein, a highly sensitive double digital assay is developed that allows for the absolute quantification of individual molecules from a single EV. Because the relative abundance of proteins is low for a single EV, tyramide signal amplification (TSA) is integrated to increase the fluorescent signal readout for evaluation. With the integrative microfluidic technology, the technology's ability to compartmentalize single EVs is successfully demonstrated, proving the technology's digital partitioning capacity. Then the device is applied to detect single PD-L1 proteins from single EVs derived from a melanoma cell line and it is discovered that there are ≈2.7 molecules expressed per EV, demonstrating the applicability of the system for profiling important prognostic and diagnostic cancer biomarkers for therapy response, metastatic status, and tumor progression. The ability to accurately quantify protein molecules of rare abundance from individual EVs will shed light on the understanding of EV heterogeneity and discovery of EV subtypes as new biomarkers.