Membrane-enclosed Particles Research Articles

Differential and targeted vesiculation: pathologic cellular responses to elevated arterial pressure.

Extracellular vesicles are small membrane-enclosed particles released during cell activation or injury. They have been investigated for several decades and found to be secreted in various diseases. Their pathogenic role is further supported by the presence of several important molecules among their cargo, including proteins, lipids, and nucleic acids. Many studies have reported enhanced and targeted extracellular vesicle biogenesis in diseases that involve chronic or transient elevation of arterial pressure resulting in endothelial dysfunction, within either the general circulatory system or specific local vascular beds. In addition, several associated pathologic processes have been studied and reported. However, the role of elevated pressure as a common pathogenic trigger across vascular domains and disease chronicity has not been previously described. This review will therefore summarize our current knowledge of the differential and targeted biogenesis of extracellular vesicles in major diseases that are characterized by elevated arterial pressure leading to endothelial dysfunction and propose a unified theory of pressure-induced extracellular vesicle-mediated pathogenesis.

An Optimized Procedure for Proteomic Analysis of Extracellular Vesicles Using In-Stage Tip Digestion and DIA LC-MS/MS: Application to Liquid Biopsy in Cancer.

Utilizing biofluids to identify cancer biomarkers has received considerable attention in the past decade. In this regard, serum and urine are convenient biofluids to noninvasively recapitulate information usually indicated by traditional tissue biopsies. In particular, we are interested in exploring the extracellular vesicle (ECV)-containing compartment of these fluids as a targeted source for cancer biomarker discovery. ECVs are membrane-enclosed particles, comprising of various fractions including exosomes, microvesicles, and apoptotic bodies. In both physiological and pathological states such as cancer, ECVs carry a rich load of molecular and protein cargoes, which aid in mediating intercellular communication between cells from various tissue types. Here we successfully enriched ECVs using a simple, low-cost, optimized method that we have developed; it is generalizable for the analysis of ECVs from multiple sample types. Such procedures are necessary as ECVs are nanoparticles that contain a treasure trove of large numbers of biomarkers each present at very low levels. Sample processing procedures can enrich for these vesicles and allow for the enhanced detection of proteins in downstream applications such as comprehensive proteomics methods using data-independent acquisition (DIA) and LC-MS/MS.

Extracellular vesicles package dsDNA to aggravate Crohn\u2019s disease by activating the STING pathway

Crohn’s disease (CD) is an intestinal immune-dysfunctional disease. Extracellular vesicles (EVs) are membrane-enclosed particles full of functional molecules, e.g., nuclear acids. Recently, EVs have been shown to participate in the development of CD by realizing intercellular communication among intestinal cells. However, the role of EVs carrying double-strand DNA (dsDNA) shed from sites of intestinal inflammation in CD has not been investigated. Here we isolated EVs from the plasma or colon lavage of murine colitis and CD patients. The level of exosomal dsDNA, including mtDNA and nDNA, significantly increased in murine colitis and active human CD, and was positively correlated with the disease activity. Moreover, the activation of the STING pathway was verified in CD. EVs from the plasma of active human CD triggered STING activation in macrophages in vitro. EVs from LPS-damaged colon epithelial cells were also shown to raise inflammation in macrophages via activating the STING pathway, but the effect disappeared after the removal of exosomal dsDNA. These findings were further confirmed in STING-deficient mice and macrophages. STING deficiency significantly ameliorated colitis. Besides, potential therapeutic effects of GW4869, an inhibitor of EVs release were assessed. The application of GW4869 successfully ameliorated murine colitis by inhibiting STING activation. In conclusion, exosomal dsDNA was found to promote intestinal inflammation via activating the STING pathway in macrophages and act as a potential mechanistic biomarker and therapeutic target of CD.

Mesenchymal Stem Cell-Derived Extracellular Vesicles to the Rescue of Renal Injury.

Acute kidney injury (AKI) and chronic kidney disease (CKD) are rising in global prevalence and cause significant morbidity for patients. Current treatments are limited to slowing instead of stabilising or reversing disease progression. In this review, we describe mesenchymal stem cells (MSCs) and their constituents, extracellular vesicles (EVs) as being a novel therapeutic for CKD. MSC-derived EVs (MSC-EVs) are membrane-enclosed particles, including exosomes, which carry genetic information that mimics the phenotype of their cell of origin. MSC-EVs deliver their cargo of mRNA, miRNA, cytokines, and growth factors to target cells as a form of paracrine communication. This genetically reprograms pathophysiological pathways, which are upregulated in renal failure. Since the method of exosome preparation significantly affects the quality and function of MSC-exosomes, this review compares the methodologies for isolating exosomes from MSCs and their role in tissue regeneration. More specifically, it summarises the therapeutic efficacy of MSC-EVs in 60 preclinical animal models of AKI and CKD and the cargo of biomolecules they deliver. MSC-EVs promote tubular proliferation and angiogenesis, and inhibit apoptosis, oxidative stress, inflammation, the epithelial-to-mesenchymal transition, and fibrosis, to alleviate AKI and CKD. By reprogramming these pathophysiological pathways, MSC-EVs can slow or even reverse the progression of AKI to CKD, and therefore offer potential to transform clinical practice.

Exosomes: Innocent Bystanders or Critical Culprits in Neurodegenerative Diseases.

Extracellular vesicles (EVs) are nano-sized membrane-enclosed particles released by cells that participate in intercellular communication through the transfer of biologic material. EVs include exosomes that are small vesicles that were initially associated with the disposal of cellular garbage; however, recent findings point toward a function as natural carriers of a wide variety of genetic material and proteins. Indeed, exosomes are vesicle mediators of intercellular communication and maintenance of cellular homeostasis. The role of exosomes in health and age-associated diseases is far from being understood, but recent evidence implicates exosomes as causative players in the spread of neurodegenerative diseases. Cells from the central nervous system (CNS) use exosomes as a strategy not only to eliminate membranes, toxic proteins, and RNA species but also to mediate short and long cell-to-cell communication as carriers of important messengers and signals. The accumulation of protein aggregates is a common pathological hallmark in many neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis, and prion diseases. Protein aggregates can be removed and delivered to degradation by the endo-lysosomal pathway or can be incorporated in multivesicular bodies (MVBs) that are further released to the extracellular space as exosomes. Because exosome transport damaged cellular material, this eventually contributes to the spread of pathological misfolded proteins within the brain, thus promoting the neurodegeneration process. In this review, we focus on the role of exosomes in CNS homeostasis, their possible contribution to the development of neurodegenerative diseases, the usefulness of exosome cargo as biomarkers of disease, and the potential benefits of plasma circulating CNS-derived exosomes.

Nucleic acid delivery with extracellular vesicles.

Extracellular vesicles (EVs) are membrane-enclosed particles, heterogeneous in size, shape, contents, biogenesis and structure. They are released by eukaryotic and prokaryotic cells and exert (patho-)physiological roles as mediators for transmitting molecular information from the producer (donor) to a recipient cell. This review focuses on the potential of EVs for delivering nucleic acids, as particularly problematic cargoes with regard to stability/protection and uptake efficacy. It highlights important properties of EVs for nucleic acid delivery and discusses their physiological and pathophysiological roles with regard to various cellular RNA species. It then describes the application of EVs for delivering a broad selection of nucleic acids/oligonucleotides, in particular giving a comprehensive overview of preclinical in vivo studies and the various strategies explored. In this context, different techniques for EV loading are discussed, as well as other important technical aspects related to EV preparation, characterization and in particular, the various approaches of artificial EV modification.

Mesenchymal Stem Cell-derived Extracellular Vesicles for Skin Wound Healing.

Skin is vulnerable to various external insults such as burn, severe injury, or inflammation, which necessitates a better strategy for wound repair. Mesenchymal stem cells (MSCs) can self-renew and differentiate into various supporting tissues including cartilage, bone, muscle, and adipose tissue. Along with their unique multipotent capacity, they secrete various paracrine mediators such as growth factors, cytokines, and membrane-enclosed particles called extracellular vesicles (EVs). Herein, we discussed the general traits of EVs such as cell-to-cell communicator, and highlighted the recent preclinical outcomes, with a focus on the application of MSC-derived EVs in wound repair. This chapter provides insights into developing novel strategies for skin wound healing in a cell-free manner.

Mass-Spectrometry Based Proteome Comparison of Extracellular Vesicle Isolation Methods: Comparison of ME-kit, Size-Exclusion Chromatography, and High-Speed Centrifugation.

Extracellular vesicles (EVs) are small membrane-enclosed particles released by cells under various conditions specific to cells’ biological states. Hence, mass-spectrometry (MS) based proteome analysis of EVs in plasma has gained much attention as a method to discover novel protein biomarkers. MS analysis of EVs in plasma is challenging and EV isolation is usually necessary. Therefore, we compared differences in abundance, subtypes, and contamination for EVs isolated by high-speed centrifugation, size exclusion chromatography (SEC), and peptide-affinity precipitation (PAP/ME kit) for subsequent MS-based proteome analysis. Successful EV isolation was evaluated by nanoparticle-tracking analysis, immunoblotting, and transmission electron microscopy, while EV abundance, EV subtypes, and contamination was evaluated by label-free tandem MS. High-speed centrifugation and SEC isolates showed high EV abundance at the expense of contamination by non-EV proteins and lipoproteins, respectively. These two methods also resulted in EVs of a similar type, however, with smaller EVs in SEC isolates. PAP isolates had a relatively low EV abundance and high contamination. We consider high-speed centrifugation and SEC suitable as EV isolation for MS biomarker studies, where the choice between the two should depend on the scientific questions and whether the focus is on larger or smaller EVs or a combination of both.

Mesenchymal Stem Cell-Derived Extracellular Vesicles: A Novel Cell-Free Therapy for Sepsis.

Sepsis remains a serious and life-threatening disease with high morbidity and mortality. Due to the unique immunomodulatory, anti-inflammatory, anti-apoptotic, anti-microbial, anti-oxidative, and reparative properties, mesenchymal stem cells (MSCs) have been extensively used in preclinical and clinical trials for diverse diseases and have shown great therapeutic potential in sepsis. However, concerns remain regarding whether MSCs can become tumorigenic or have other side effects. Extracellular vesicles (EVs) are a heterogeneous group of membrane-enclosed particles released from almost any cell and perform an important role in intercellular communication. Recently, it has emerged that EVs derived from MSCs (MSC-EVs) appear to exert a therapeutic benefit similar to MSCs in protecting against sepsis-induced organ dysfunction by delivering a cargo that includes RNAs and proteins to target cells. More importantly, compared to their parent cells, MSC-EVs have a superior safety profile, can be safely stored without losing function, and possess other advantages. Hence, MSC-EVs may be used as a novel alternative to MSC-based therapy in sepsis. Here, we summarize the properties and applications of MSC-EVs in sepsis.

Comprehensive palmitoyl-proteomic analysis identifies distinct protein signatures for large and small cancer-derived extracellular vesicles

ABSTRACT Extracellular vesicles (EVs) are membrane-enclosed particles that play an important role in cancer progression and have emerged as a promising source of circulating biomarkers. Protein S-acylation, frequently called palmitoylation, has been proposed as a post-translational mechanism that modulates the dynamics of EV biogenesis and protein cargo sorting. However, technical challenges have limited large-scale profiling of the whole palmitoyl-proteins of EVs. We successfully employed a novel approach that combines low-background acyl-biotinyl exchange (LB-ABE) with label-free proteomics to analyse the palmitoyl-proteome of large EVs (L-EVs) and small EVs (S-EVs) from prostate cancer cells. Here we report the first palmitoyl-protein signature of EVs, and demonstrate that L- and S-EVs harbour proteins associated with distinct biological processes and subcellular origin. We identified STEAP1, STEAP2, and ABCC4 as prostate cancer-specific palmitoyl-proteins abundant in both EV populations. Importantly, localization of the above proteins in EVs was reduced upon inhibition of palmitoylation in the producing cells. Our results suggest that this post-translational modification may play a role in the sorting of the EV-bound secretome and possibly enable selective detection of disease biomarkers.

Proteomic profiling of extracellular vesicles reveals additional diagnostic biomarkers for myocardial infarction compared to plasma alone

Extracellular vesicles (EVs) are submicron, membrane-enclosed particles that are released from cells in various pathophysiological states. The molecular cargo of these vesicles is considered to reflect the composition of the cell of origin, and the EV proteome is therefore a potential source of biomarkers for various diseases. Our aim was to determine whether EVs isolated from plasma provide additional diagnostic value or improved pathophysiological understanding compared to plasma alone in the context of myocardial infarction (MI). A panel of proximity extension assays (n = 92) was employed to analyze EV lysates and plasma from patients with MI (n = 60) and healthy controls (n = 22). After adjustment for multiple comparisons, a total of 11 dysregulated proteins were identified in EVs of MI patients compared to the controls (q < 0.01). Three of these proteins: chymotrypsin C (CTRC), proto-oncogene tyrosine-protein kinase SRC (SRC) and C-C motif chemokine ligand 17 (CCL17) were unaltered in the corresponding plasma samples. As biomarkers for MI, rudimentary to no evidence exists for these proteins. In a separate group of patients with varying degrees of coronary artery disease, the decrease in EV-associated (but not plasma-related) SRC levels was confirmed by ELISA. Confirmation of the presence of SRC on EVs of different sizes and cellular origins was performed with ELISA, flow cytometry and nanoparticle tracking analysis. In conclusion, the data revealed that despite a similarity in the EV and plasma proteomes, analysis of isolated EVs does indeed provide additional diagnostic information that cannot be obtained from plasma alone.

Extracellular Vesicles and Their Potential Use in Monitoring Cancer Progression and Therapy: The Contribution of Proteomics.

Extracellular Vesicles (EVs) are small membrane-enclosed particles released by cells and able to vehiculate information between them. The term EVs categorizes many and different vesicles based on their biogenesis and release pathway, such as exosomes (Exo), ectosomes, or shedding microvesicles (SMVs), apoptotic blebs (ABs), and other EVs subsets, generating a heterogeneous group of components able to redistribute their cargo into the entire organism. Moreover EVs are becoming increasingly important in monitoring cancer progression and therapy, since they are able to carry specific disease biomarkers such as Glypican-1, colon cancer-associated transcript 2, CD63, CD24, and many others. The importance of their biological role together with their heterogeneity prompted researchers to adopt and standardize purification methods able to isolate EVs for characterizing their cargo. In this way, mass spectrometry (MS)-based proteomics approaches are emerging as promising tool for the identification and quantification of EVs protein cargoes, but this technique resulted to be deeply influenced by the low quality of the isolation techniques. This review presents the state-of-the-art of EVs isolation, purification, and characterization for omics studies, with a particular focus to their potential use in monitoring cancer progression and therapy.

Protein Composition Reflects Extracellular Vesicle Heterogeneity.

Extracellular vesicles (EVs) are membrane-enclosed particles that are released by virtually all cells from all living organisms. EVs shuttle biologically active cargo including protein, RNA, and DNA between cells. When shed by cancer cells, they function as potent intercellular messangers with important functional consequences. Cells produce a diverse spectrum of EVs, spanning from small vesicles of 40-150nm in diameter, to large vesicles up to 10μm in diameter. While this diversity was initially considered to be purely based on size, it is becoming evident that different classes of EVs, and different populations within one EV class may harbor distinct molecular cargo and play specific functions. Furthermore, there are considerable cell type-dependent differences in the cargo and function of shed EVs. This review focuses on the most recent proteomic studies that have attempted to capture the EV heterogeneity by directly comparing the protein composition of different EV classes and EV populations derived from the same cell source. Recent studies comparing protein composition of the same EV class(es) derived from different cell types are also summarized. Emerging approaches to study EV heterogeneity and their important implications for future studies are also discussed.

Proteomic Profiling of Extracellular Vesicles during Acute Cellular Rejection

Purpose Non-invasive tests for the diagnosis of acute cellular rejection (ACR) represents an unmet need in the surveillance of cardiac allograft recipients. Extracellular vesicles (EVs) are sub-micron, membrane-enclosed particles released into the circulation upon tissue injury or stress, and T-cell derived EVs have been shown to play a role in ACR pathophysiology. The molecular cargo of EVs are thought to reflect the state of their cell of origin and are therefore considered a potential source of biomarkers. The aim of this study was to profile immune-related proteins in isolated EVs from cardiac transplantation patients to identify novel biomarkers for ACR. Methods Plasma was collected serially from 8 cardiac allograft recipients before, during and after biopsy-proven ACR grade ≥2R (2 [25%] women, mean age 45.2 ±21.2). EVs were isolated from plasma using acoustic seed trapping. The levels of 92 low-abundant immune-related proteins were assayed in plasma and EV lysates using a panel of proximity extension assays. Proteins that were dysregulated during ACR were identified by repeated measures ANOVA, adjusting for multiple comparisons using a false discovery rate approach. The presence of EV proteins on leukocyte-, endothelial- and platelet-derived EVs was confirmed by co-staining of isolated EVs with antibodies specific for CD16, CD31 and CD41a, respectively, and analyzed by flow cytometry. Results 48 proteins were defined as detected (i.e. above the limit of detection in >50% of samples) in the EV lysates. The levels of EV-proteins were overall poorly correlated with those in plasma: only 16.7 % of EV-proteins showed a significant correlation between plasma and EV lysates. The mean levels of 20 EV-proteins were numerically elevated during ACR. One of these, TNF-related weak inducer of apoptosis (TWEAK), was significantly increased (q Conclusion The EV proteome is perturbed during ACR and differs substantially from that in corresponding plasma samples. The analysis of isolated EVs can provide additional diagnostic and pathophysiological information over plasma alone. TWEAK is a vesicle-associated protein with potential utility as a biomarker for ACR.

Micropatterned Viral Membrane Clusters for Antiviral Drug Evaluation.

The function of biological nanoparticles, such as membrane-enveloped viral particles, is often enhanced when the particles form higher-order supramolecular assemblies. While there is intense interest in developing biomimetic platforms that recapitulate these collective properties, existing platforms are limited to mimicking individual virus particles. Here, we present a micropatterning strategy to print linker molecules selectively onto bioinert surfaces, thereby enabling controlled tethering of biomimetic viral particle clusters across defined geometric patterns. By controlling the linker concentration, it is possible to tune the density of tethered particles within clusters while enhancing the signal intensity of encapsulated fluorescent markers. Time-resolved tracking of pore formation and membrane lysis revealed that an antiviral peptide can disturb clusters of the membrane-enclosed particles akin to the targeting of individual viral particles. This platform is broadly useful for evaluating the performance of membrane-active antiviral drug candidates, whereas the micropatterning strategy can be applied to a wide range of biological nanoparticles and other macromolecular entities.

Picornavirus infection induces temporal release of multiple extracellular vesicle subsets that differ in molecular composition and infectious potential.

Several naked virus species, including members of the Picornaviridae family, have recently been described to escape their host cells and spread infection via enclosure in extracellular vesicles (EV). EV are 50–300 nm sized lipid membrane-enclosed particles produced by all cells that are broadly recognized for playing regulatory roles in numerous (patho)physiological processes, including viral infection. Both pro- and antiviral functions have been ascribed to EV released by virus-infected cells. It is currently not known whether this reported functional diversity is a result of the release of multiple virus-containing and non-virus containing EV subpopulations that differ in composition and function. Using encephalomyocarditis virus infection (EMCV, Picornaviridae family), we here provide evidence that EV populations released by infected cells are highly heterogeneous. Virus was contained in two distinct EV populations that differed in physical characteristics, such as sedimentation properties, and in enrichment for proteins indicative of different EV biogenesis pathways, such as the plasma membrane resident proteins Flotillin-1 and CD9, and the autophagy regulatory protein LC3. Additional levels of EV heterogeneity were identified using high-resolution flow cytometric analysis of single EV. Importantly, we demonstrate that EV subsets released during EMCV infection varied largely in potency of transferring virus infection and in their kinetics of release from infected cells. These data support the notion that heterogeneous EV populations released by virus-infected cells can exert diverse functions at distinct time points during infection. Unraveling the compositional, temporal and functional heterogeneity of these EV populations using single EV analysis technologies, as employed in this study, is vital to understanding the role of EV in virus dissemination and antiviral host responses.

Retraction: Engineering of extracellular vesicles as drug delivery vehicles.

[This retracts the article DOI: 10.21037/sci.2017.08.07.].

Engineering of extracellular vesicles as drug delivery vehicles.

Extracellular vesicles (EVs) are secreted membrane-enclosed nano-sized particles (40-1,000 nm) that deliver biological information between cells. The molecular composition of these subcellular particles includes growth factor receptors, ligands adhesion proteins, mRNA, miRNAs, lncRNA and lipids that are derived from donor cells. A number of studies demonstrated that stem cell-derived EVs are the key mediator of tissue repair and regeneration in multiple animal disease models. In addition, the composition of these particles is known to be altered in cancer and disease pathology suggesting them for useful in diagnostic and therapeutic purposes. Their endogenous origin and biological properties offer benefits over conventional drug delivery systems (DDS), such as liposome, synthetic nanoparticles and prompted the further application of EVs as drug delivery vehicles for chemical drugs, genetic materials and proteins. The contents of EVs can be efficiently modified by chemical, biological or physical means. Thus, EVs can be an innovative DDS as it can overcome physical and biological barriers and safely deliver therapeutic drugs to target tissues. In this minireview, we summarized current progress on the strategies of drug loading onto EVs; ex vivo and in vivo loading.

Delivery of Biomolecules via Extracellular Vesicles: A Budding Therapeutic Strategy.

Extracellular vesicles (EVs) are membrane-enclosed particles that are secreted by nearly all cells and play an important role in intercellular communication by transporting protein and nucleic acids between cells. EV-mediated processes shape phenomena as diverse as cancer progression, immune function, and wound healing. The natural role of EVs in encapsulating and delivering cargo to modify cellular function highlights the potential to use these particles as therapeutic delivery vehicles. In this chapter, wedescribe emerging strategies for EV engineering and consider how different approaches to EV production, purification, and design may impact the efficacy of EV-based therapeutics.

From glioblastoma to endothelial cells through extracellular vesicles: messages for angiogenesis.

Glioblastoma has one of the highest mortality rates among cancers, and it is the most common and malignant form of brain cancer. Among the typical features of glioblastoma tumors, there is an aberrant vascularization: all gliomas are among the most vascularized/angiogenic tumors. In recent years, it has become clear that glioblastoma cells can secrete extracellular vesicles which are spherical and membrane-enclosed particles released, in vitro or in vivo, by both normal and tumor cells; they are involved in the regulation of both physiological and pathological processes; among the latter, cancer is the most widely studied. Extracellular vesicles from tumor cells convey messages to other tumor cells, but also to normal stromal cells in order to create a microenvironment that supports cancer growth and progression and are implicated in drug resistance, escape from immunosurveillance and from apoptosis, as well as in metastasis formation; they are also involved in angiogenesis stimulation, inducing endothelial cells proliferation, and other pro-angiogenic activities. To this aim, the present paper assesses in detail the extracellular vesicles phenomenon in the human glioblastoma cell line U251 and evaluates extracellular vesicles ability to promote the processes required to achieve the formation of new blood vessels in human brain microvascular endothelial cells, highlighting that they stimulate proliferation, motility, and tube formation in a dose-response manner. Moreover, a molecular characterization shows that extracellular vesicles are fully equipped for angiogenesis stimulation in terms of proteolytic enzymes (gelatinases and plasminogen activators), pro-angiogenic growth factors (VEGF and TGFβ), and the promoting-angiogenic CXCR4 chemokine receptor.