Exosomes

Lampreys are a jawless vertebrate species belonging to an ancient vertebrate lineage that diverged from a common ancestor with humans ~500 million years ago. The sea lamprey (Petromyzon marinus) has a filter feeding ammocoete larval stage that metamorphoses into a parasitic adult, feeding both on teleost and elasmobranch fish. Lampreys are a valuable comparative model species for vertebrate immunity and physiology due to their unique phylogenetic position, unusual adaptive immune system, and physiological adaptions such as tolerance to salinity changes and urea. Peptidylarginine deiminases (PADs) are a phylogenetically conserved enzyme family which catalyses posttranslational deimination/citrullination in target proteins, enabling proteins to gain new functions (moonlighting). The identification of deiminated protein targets in species across phylogeny may provide novel insights into post-translational regulation of physiological and pathophysiological processes. Extracellular vesicles (EVs) are membrane vesicles released from cells that carry cargos of small molecules and proteins for cellular communication, involved in both normal and pathological processes. The current study identified deimination signatures in proteins of both total plasma and plasma-EVs in sea lamprey and furthermore reports the first characterisation of plasma-EVs in lamprey. EVs were poly-dispersed in the size range of 40-500 nm, similar to what is observed in other taxa, positive for CD63 and Flotillin-1. Plasma-EV morphology was confirmed by transmission electron microscopy. Assessment of deimination/citrullination signatures in lamprey plasma and plasma-EVs, revealed 72 deimination target proteins involved in immunity, metabolism and gene regulation in whole plasma, and 37 target proteins in EVs, whereof 24 were shared targets. Furthermore, the presence of deiminated histone H3, indicative of gene-regulatory mechanisms and also a marker of neutrophil extracellular trap formation (NETosis), was confirmed in lamprey plasma. Functional protein network analysis revealed some differences in KEGG and GO pathways of deiminated proteins in whole plasma compared with plasma-EVs. For example, while common STRING network clusters in plasma and plasma-EVs included Peptide chain elongation, Viral mRNA translation, Glycolysis and gluconeogenesis, STRING network clusters specific for EVs only included: Cellular response to heat stress, Muscle protein and striated muscle thin filament, Nucleosome, Protein processing in endoplasmic reticulum, Nucleosome and histone deacetylase complex. STRING network clusters specific for plasma were: Adipokinetic hormone receptor activity, Fibrinogen alpha/beta chain family, peptidase S1A, Glutathione synthesis and recycling-arginine, Fructose 1,6-bisphosphate metabolic process, Carbon metabolism and lactate dehydrogenase activity, Post-translational protein phosphorylation, Regulation of insulin-like growth factor transport and clotting cascade. Overall, for the EV citrullinome, five STRING network clusters, 10 KEGG pathways, 15 molecular GO pathways and 29 Reactome pathways were identified, compared with nine STRING network clusters, six KEGG pathways, two Molecular GO pathways and one Reactome pathway specific for whole plasma; while further pathways were shared. The reported findings indicate that major pathways relevant for immunity and metabolism are targets of deimination in lamprey plasma and plasma-EVs, with some differences, and may help elucidating roles for the conserved PAD enzyme family in regulation of immune and metabolic function throughout phylogeny.

Objectives: Exosomes are small membrane bound vesicles secreted by most cell types. Exosomes contain various functional proteins, mRNAs and microRNAs (miRNAs) that could be used for diagnostic and therapeutic purposes. Currently, a standard method for serum exosome isolation is differential ultracentrifugation, but a search for alternative, less time-consuming and labour extensive exosomal isolation method for use in clinical settings is ongoing. The effect of serum exosome isolation method on obtained miRNA profile is not yet clear. The aim of this study was to determine to which extent selected exosome isolation methods influence the serum exosomal miRNA profile. Design and methods: Exosomes were isolated from blood serum of healthy individuals by ultracentrifuga-tion and ExoQuick Precipitation methods. The expression profile of 375 miRNAs was determined by real time PCR using Exiqon miRCURY LNA™ microRNA Human panel I assays. Results: Although a strong correlation of exosomal miRNA profiles was observed between the two isolation methods, distinct clusters of miRNA levels between the used methods were identified. The detected levels of two miRNAs, miR-92a and miR-486-5p, were significantly influenced by the exosome isolation method used. Conclusions: Both exosome isolation methods are suitable for serum exosomal miRNA profiling. Differences found in miRNA patterns between the two methods indicate that the observed exosomal miRNA profile is slightly affected by the extracellular vesicle isolation method.

Objective
We aimed to identify the differentially expressed proteins (DEPs) and functional differences between exosomes derived from mesenchymal stem cells (MSCs) derived from umbilical cord (UC) or adipose tissue (AD).

Materials And Methods
In this experimental study, the UC and AD were isolated from healthy volunteers. Then, exosomes from UC-MSCs and AD-MSCs were isolated and characterized. Next, the protein compositions of the exosomes were examined via liquid chromatography tandem mass spectrometry (LC-MS/MS), followed by evaluation of the DEPs between UC-MSC and AD-MSC–derived exosomes. Finally, functional enrichment analysis was performed.

Results
One hundred and ninety-eight key DEPs were identified, among which, albumin (ALB), alpha-II-spectrin (SPTAN1), and Ras-related C3 botulinum toxin substrate 2 (RAC2) were the three hub proteins present at the highest levels in the protein-protein interaction network that was generated based on the shared DEPs. The DEPs were mainly enriched in gene ontology (GO) items associated with immunity, complement activation, and protein activation cascade regulation corresponding to 24 pathways, of which complement and coagulation cascades as well as platelet activation pathways were the most significant.

Conclusion
The different functions of AD- and UC-MSC exosomes in clinical applications may be related to the differences in their immunomodulatory activities.

Extracellular vesicles (EVs) are small bilayer lipid membrane vesicles that can be released by most cell types and detected in most body fluids. EVs exert key functions for intercellular communication via transferring their bioactive cargos to recipient cells or activating signaling pathways in target cells. Increasing evidence has shown the important regulatory effects of EVs in cardiovascular diseases (CVDs). EVs secreted by cardiomyocytes, endothelial cells, fibroblasts, and stem cells play essential roles in pathophysiological processes such as cardiac hypertrophy, cardiomyocyte survival and apoptosis, cardiac fibrosis, and angiogenesis in relation to CVDs. In this review, we will first outline the current knowledge about the physical characteristics, biological contents, and isolation methods of EVs. We will then focus on the functional roles of cardiovascular EVs and their pathophysiological effects in CVDs, as well as summarize the potential of EVs as therapeutic agents and biomarkers for CVDs. Finally, we will discuss the specific application of EVs as a novel drug delivery system and the utility of EVs in the field of regenerative medicine.

Exosomes are microvesicles with sizes ranging from 50 to 150 nm. These small vesicles are known to morphologically and functionally resemble virus particles from human immunodeficiency virus type I (HIV-I) and human T-lymphotropic virus type I (HTLV-I). The function of exosomes is to mainly mediate cell-to-cell communication by exchanging various macromolecules including proteins, lipids and nucleic acids in diverse cellular processes. Due to its size and structural simplicity, the transfer of pathogenic or virulent cellular factors across the cells mediated by exosomes is more efficient, hence facilitating the dissemination of viral infections and cancer diseases. The pathogenic role of exo-somes in various cancers such as lung and breast, and their potentials as biomarkers have been previously studied, yet limited information is known for Epstein-Barr virus (EBV)-associated cancers. In this chapter, we discuss current evidences that support the pathogenic roles of exosomes in EBV-related cancers and their potentials as biomarkers in cancer diagnostics and therapy response. Here, we also highlight the potential challenges in the development of exosome-based biomarkers for clinical application.

The last decade has seen a sharp increase in the number of scientific publications describing
physiological and pathological functions of extracellular vesicles (EVs), a collective term covering
various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles,
ectosomes, oncosomes, apoptotic bodies, andmany other names. However, specific issues arise
when working with these entities, whose size and amount often make them difficult to obtain as
relatively pure preparations, and to characterize properly. The International Society for Extracellular
Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines
for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the
collective knowledge in the last four years. An important point to consider is that ascribing
a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information
beyond mere description of function in a crude, potentially contaminated, and heterogeneous
preparation. For example, claims that exosomes are endowed with exquisite and specific activities
remain difficult to support experimentally, given our still limited knowledge of their specific molecular
machineries of biogenesis and release, as compared with other biophysically similar EVs. The
MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to
document specific EV-associated functional activities. Finally, a checklist is provided with summaries
of key points.

The last decade has seen a sharp increase in the number of scientific publications describing
physiological and pathological functions of extracellular vesicles (EVs), a collective term covering
various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles,
ectosomes, oncosomes, apoptotic bodies, andmany other names. However, specific issues arise
when working with these entities, whose size and amount often make them difficult to obtain as
relatively pure preparations, and to characterize properly. The International Society for Extracellular
Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines
for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the
collective knowledge in the last four years. An important point to consider is that ascribing
a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information
beyond mere description of function in a crude, potentially contaminated, and heterogeneous
preparation. For example, claims that exosomes are endowed with exquisite and specific activities
remain difficult to support experimentally, given our still limited knowledge of their specific molecular
machineries of biogenesis and release, as compared with other biophysically similar EVs. The
MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to
document specific EV-associated functional activities. Finally, a checklist is provided with summaries
of key points.

Extracellular vesicles (EVs), including exosomes and microvesicles, are critical mediators of cell-to-cell communication in tissue homeostasis and repair, both in physiological and pathological conditions.
Recently, progress has been achieved in their use in regenerative medicine as transfer agents for active biomolecules. Specifically, EVs are natural carriers of microRNAs (miRNAs), protecting their cargo from plasma ribonucleases and delivering their content to recipient cells. Expression of miRNAs is dysregulated in virtually all forms of cancer. Therefore, EVs-mediated miRNA delivery may represent a valuable tool
for cancer therapeutic intervention, aiming at restoring cancer miRNAs expression to normal levels.
MicroRNAs may act both as tumor suppressors or oncogenes, consequently different alternative approaches for regulating miRNA expression in tumor tissues have been developed. Here, we review the various strategies for miRNA loading into EVs and highlight studies of EVs-mediated miRNA delivery which have been employed for cancer treatment, both in vitro and in vivo. Collectively, these data support the use of EVs in miRNAs/miRNAs antagonist transfer for cancer therapy, but challenges related to EV biology have yet to be further addressed before safe clinical translation.

Exosomes, a subset of extracellular vesicles (EVs), function as a mode of intercellular communication and molecular transfer. Exosomes facilitate the direct extracellular transfer of proteins, lipids, and miRNA/mRNA/DNAs between cells in vitro and in vivo. The immunological activities of exosomes affect immunoregulation mechanisms including modulating antigen presentation, immune activation, immune suppression, immune surveillance, and intercellular communication. Besides immune cells, cancer cells secrete immunologically active exosomes that influence both physiological and pathological processes. The observation that exosomes isolated from immune cells such as dendritic cells (DCs) modulate the immune response has enforced the way these membranous vesicles are being considered as potential immunotherapeutic reagents. Indeed, tumour- and immune cell-derived exosomes have been shown to carry tumour antigens and promote immunity, leading to eradication of established tumours by CD8(+...

The heart is a very complex conglomeration of organized interactions between various different cell types that all aid in facilitating myocardial function through contractility, sufficient perfusion, and cell-to-cell reception. In order to make sure that all features of the heart work effectively, it is imperative to have a well-controlled communication system among the different types of cells. One of the most important ways that the heart regulates itself is by the use of extracellular vesicles, more specifically, exosomes. Exosomes are types of nano-vesicles, naturally released from living cells. They are believed to play a critical role in intercellular communication through the means of certain mechanisms including direct cell-to-cell contact, long-range signals as well as electrical and extracellular chemical molecules. Exosomes contain many unique features like surface proteins/receptors, lipids, mRNAs, microRNAs, transcription factors and other proteins. Recent studies indic...

Retinal Pigment Epithelial cells (RPE) express both GPR143 and myocilin, which interact in a signal
transduction-dependent manner. In heterologous systems, activation of GPR143 with ligand causes
transient recruitment of myocilin to internalized receptors, which appears to be the entry point of
myocilin to the endocytic pathway. In some but not all cells, myocilin also traffics through the multivesicular
body (MVB) and is released on the surface of exosomes in a signal transduction-dependent
fashion. Little is known regarding the role of exosomes in RPE, but they likely serve as a mode of
communication between the RPE and the outer retina. In this study, we used posterior poles with retina
removed from fresh human donor eyes as a model to test the relationship between GPR143, myocilin,
and exosomes in an endogenous system. We isolated exosomes released by RPE using differential
centrifugation of media conditioned by the RPE for 25 min, and then characterized the exosomes using
nanoparticle tracking to determine the number and size of the exosomes. Next, we tested whether ligand
stimulation of GPR143 using L-DOPA altered RPE exosome release. Finally, we investigated whether
myocilin was present on the exosomes released by RPE and whether L-DOPA stimulation of GPR143
caused recruitment of myocilin to the endocytic pathway, as we have previously observed using cultured
cells. Activation of GPR143 halted RPE exosome release, while simultaneously recruiting myocilin to the
endocytic compartment. Together, our results indicate that GPR143 and myocilin function in a signal
transduction system that can control exosome release from RPE

Exosomes are a subset of tiny extracellular vesicles manufactured by all cells and are present in all body fluids. They are produced actively in tumor cells, which are released and utilized to facilitate tumor growth. Their characteristics enable them to assist major cancer hallmarks, leveraged by cancer cells in fostering cancer growth and spread while implementing ways to escape elimination from the host environment. This review updates on the latest progress on the roles of cancer-derived exosomes, of 30-100 nm in size, in deregulating paracrine trafficking in the tumor microenvironment and circulation. Thus, exosomes are being exploited in diagnostic biomarker development, with its potential in clinical applications as therapeutic targets utilized in exosome-based nanoparticle drug delivery strategies for cancer therapy. Ongoing studies were retrieved from PubMed ® and Scopus database and ClinicalTrials.gov registry for review, highlighting how cancer cells from entirely different cell lines rely on genetic information carried by their exosomes for homotypic and heterotypic intercellular communications in the microenvironment to favor proliferation and invasion, while establishing a pre-metastatic niche in welcoming cancer cells' arrival. We will elaborate on the trafficking of tumor-derived exosomes in fostering cancer proliferation, invasion, and metastasis in hematopoietic (leukemia and myeloma), epithelial (breast cancer), and mesenchymal (soft tissue sarcoma and osteosarcoma) cancers. Cancer-derived exosomal trafficking is observed in several types of liquid or solid tumors, confirming their role as cancer hallmark enabler. Their enriched genetic signals arising from their characteristic DNA, RNA, microRNA, and lncRNA, along with specific gene expression profiles, protein, or lipid composition carried by the exosomal cargo shed into blood, saliva, urine, ascites, and cervicovaginal lavage, are being studied as a diagnostic, prognostic, or predictive cancer biomarker. We reveal the latest research efforts in exploiting the use of nanoparticles to improve the overall cancer diagnostic capability in the clinic.