Clinical Prospect of Mesenchymal Stromal/Stem Cell-Derived Extracellular Vesicles in Kidney Disease: Challenges and the Way Forward.

Kidney disease is a growing public health problem worldwide, including both acute and chronic forms. Existing therapies for kidney disease target various pathogenic mechanisms; however, these therapies only slow down the progression of the disease rather than offering a cure. One of the potential and emerging approaches for the treatment of kidney disease is mesenchymal stromal/stem cell (MSC) therapy, shown to have beneficial effects in preclinical studies. In addition, extracellular vesicles (EVs) released by MSCs became a potent cell-free therapy option in various preclinical models of kidney disease due to their regenerative, anti-inflammatory, and immunomodulatory properties. However, there are scarce clinical data available regarding the use of MSC-EVs in kidney pathologies. This review article provides an outline of the renoprotective effects of MSC-EVs in different preclinical models of kidney disease. It offers a comprehensive analysis of possible mechanisms of action of MSC-EVs with an emphasis on kidney disease. Finally, on the journey toward the implementation of MSC-EVs into clinical practice, we highlight the need to establish standardized methods for the characterization of an EV-based product and investigate the adequate dosing, safety, and efficacy of MSC-EVs application, as well as the development of suitable potency assays.

Special considerations for studies of extracellular vesicles from parasitic helminths: A community-led roadmap to increase rigour and reproducibility.

Over the last decade, research interest in defining how extracellular vesicles (EVs) shape cross-species communication has grown rapidly. Parasitic helminths, worm species found in the phyla Nematoda and Platyhelminthes, are well-recognised manipulators of host immune function and physiology. Emerging evidence supports a role for helminth-derived EVs in these processes and highlights EVs as an important participant in cross-phylum communication. While the mammalian EV field is guided by a community-agreed framework for studying EVs derived from model organisms or cell systems [e.g., Minimal Information for Studies of Extracellular Vesicles (MISEV)], the helminth community requires a supplementary set of principles due to the additional challenges that accompany working with such divergent organisms. These challenges include, but are not limited to, generating sufficient quantities of EVs for descriptive or functional studies, defining pan-helminth EV markers, genetically modifying these organisms, and identifying rigorous methodologies for in vitro and in vivo studies. Here, we outline best practices for those investigating the biology of helminth-derived EVs to complement the MISEV guidelines. We summarise community-agreed standards for studying EVs derived from this broad set of non-model organisms, raise awareness of issues associated with helminth EVs and provide future perspectives for how progress in the field will be achieved.

Serum-derived extracellular vesicles: Novel biomarkers reflecting the disease severity of COVID-19 patients.

COVID-19 is characterized by a wide spectrum of disease severity, whose indicators and underlying mechanisms need to be identified. The role of extracellular vesicles (EVs) in COVID-19 and their biomarker potential, however, remains largely unknown. Aiming to identify specific EV signatures of patients with mild compared to severe COVID-19, we characterized the EV composition of 20 mild and 26 severe COVID-19 patients along with 16 sex and age-matched healthy donors with a panel of eight different antibodies by imaging flow cytometry (IFCM). We correlated the obtained data with 37 clinical, prerecorded biochemical and immunological parameters. Severe patients' sera contained increased amounts of CD13+ and CD82+ EVs, which positively correlated with IL-6-producing and circulating myeloid-derived suppressor cells (MDSCs) and with the serum concentration of proinflammatory cytokines, respectively. Sera of mild COVID-19 patients contained more HLA-ABC+ EVs than sera of the healthy donors and more CD24+ EVs than severe COVID-19 patients. Their increased abundance negatively correlated with disease severity and accumulation of MDSCs, being considered as key drivers of immunopathogenesis in COVID-19. Altogether, our results support the potential of serum EVs as powerful biomarkers for COVID-19 severity and pave the way for future investigations aiming to unravel the role of EVs in COVID-19 progression.

Extracellular Vesicles and Acute Kidney Injury: Potential Therapeutic Avenue for Renal Repair and Regeneration.

Acute kidney injury (AKI) is a sudden decline of renal function and represents a global clinical problem due to an elevated morbidity and mortality. Despite many efforts, currently there are no treatments to halt this devastating condition. Extracellular vesicles (EVs) are nanoparticles secreted by various cell types in both physiological and pathological conditions. EVs can arise from distinct parts of the kidney and can mediate intercellular communication between various cell types along the nephron. Besides their potential as diagnostic tools, EVs have been proposed as powerful new tools for regenerative medicine and have been broadly studied as therapeutic mediators in different models of experimental AKI. In this review, we present an overview of the basic features and biological relevance of EVs, with an emphasis on their functional role in cell-to-cell communication in the kidney. We explore versatile roles of EVs in crucial pathophysiological mechanisms contributing to AKI and give a detailed description of the renoprotective effects of EVs from different origins in AKI. Finally, we explain known mechanisms of action of EVs in AKI and provide an outlook on the potential clinical translation of EVs in the setting of AKI.

Harnessing immunomodulatory mechanisms of Trichinella spiralis to design novel nanomedical approaches for restoring self-tolerance in autoimmunity.

The rapid increase in the prevalence of autoimmune diseases in recent decades, especially in developed countries, coincided with improved living conditions and healthcare. Part of this increase could be ascribed to the lack of exposure to infectious agents like helminths that co-evolved with us and display potent immune regulatory actions. In this review we discussed many investigations, including our own, showing that Trichinella spiralis via its excretory-secretory products attenuate Th1/Th17 immunopathological response in autoimmunity and potentiate the protective Th2 and or regulatory T cell response, acting as an effective induction of tolerogenic dendritic cells (DCs), and probably mimicking the autoantigen in some diseases. A recent discovery of T. spiralis extracellular vesicles (TsEVs) suggested that inducing a complex regulation of the immune response requires simultaneous delivery of different signals in nano-sized packages. Indeed, different artificial nanomedical approaches discussed here suggested that co-delivery of multiple signals via nanoparticles is the most promising strategy for the treatment of autoimmune diseases. Although a long way is ahead of us before we could completely replicate natural nano-delivery systems which are both safe and potent in restoring self-tolerance, a clear path is being opened from a careful examination of parasite-host interactions.

Extracellular Vesicles and Renal Fibrosis: An Odyssey toward a New Therapeutic Approach.

Renal fibrosis is a complex disorder characterized by the destruction of kidney parenchyma. There is currently no cure for this devastating condition. Extracellular vesicles (EVs) are membranous vesicles released from cells in both physiological and diseased states. Given their fundamental role in transferring biomolecules to recipient cells and their ability to cross biological barriers, EVs have been widely investigated as potential cell-free therapeutic agents. In this review, we provide an overview of EVs, focusing on their functional role in renal fibrosis and signaling messengers responsible for EV-mediated crosstalk between various renal compartments. We explore recent findings regarding the renoprotective effect of EVs and their use as therapeutic agents in renal fibrosis. We also highlight advantages and future perspectives of the therapeutic applications of EVs in renal diseases.

Extracellular Vesicles as Innovative Tool for Diagnosis, Regeneration and Protection against Neurological Damage.

Extracellular vesicles (EVs) have recently attracted a great deal of interest as they may represent a new biosignaling paradigm. According to the mode of biogenesis, size and composition, two broad categories of EVs have been described, exosomes and microvesicles. EVs have been shown to carry cargoes of signaling proteins, RNA species, DNA and lipids. Once released, their content is selectively taken up by near or distant target cells, influencing their behavior. Exosomes are involved in cell-cell communication in a wide range of embryonic developmental processes and in fetal-maternal communication. In the present review, an outline of the role of EVs in neural development, regeneration and diseases is presented. EVs can act as regulators of normal homeostasis, but they can also promote either neuroinflammation/degeneration or tissue repair in pathological conditions, depending on their content. Since EV molecular cargo constitutes a representation of the origin cell status, EVs can be exploited in the diagnosis of several diseases. Due to their capability to cross the blood-brain barrier (BBB), EVs not only have been suggested for the diagnosis of central nervous system disorders by means of minimally invasive procedures, i.e., "liquid biopsies", but they are also considered attractive tools for targeted drug delivery across the BBB. From the therapeutic perspective, mesenchymal stem cells (MSCs) represent one of the most promising sources of EVs. In particular, the neuroprotective properties of MSCs derived from the dental pulp are here discussed.

Trichinella spiralis muscle larvae release extracellular vesicles with immunomodulatory properties.

AIMS: Extracellular vesicles (EVs) represent a newly discovered but universal communication tool between cells or organisms. However, few data exist on nematode EVs and none for Trichinella spiralis. Here, we aimed to investigate whether T spiralis muscle larvae produce EVs, whether they carry immunomodulatory proteins and whether they have a role in immunomodulation as a component of excretory-secretory muscle larvae products (ES L1). METHODS AND RESULTS: EVs were enriched from conditioned medium of T spiralis muscle larvae. Transmission electron microscopy images showed T spiralis EVs to be 30-80 nm in size, and Western blot confirmed the presence of two out of three glycoproteins with the immunodominant epitope characteristic for muscle larvae of the genus Trichinella. Using a peripheral blood mononuclear cell (PBMC) stimulation assay, it was shown that these EVs elevated production of IL10 and IL6. CONCLUSION: T spiralis muscle larvae produce EVs. Those EVs carry immunomodulatory proteins and have the capacity independently to induce regulatory responses in the same way as the T spiralis excretory-secretory muscle larvae products from which they were isolated.

Surface glycans contribute to differences between seminal prostasomes from normozoospermic and oligozoospermic men.

Background: Extracellular vesicles (EVs), released from the plasma membrane or intracellular compartments, have a specific composition related to their parent cells, but they can, additionally, be modified by the extracellular environment. Although glycans are known to contribute to EV composition and may have biomedical importance as biomarkers and recognition signals, they have not been extensively investigated. In this study, seminal prostasomes, i.e. EVs from seminal plasma (SP) of normo- and oligozoospermic men, were analyzed in order to detect possible changes in their surface glycans under altered physiological conditions. Methods: Prostasomes were isolated from pooled SP by differential centrifugation and gel filtration, followed by glycobiochemical characterization using lectin/immune-transmission microscopy and ion-exchange chromatography. Results: Within the frame of overall similarity in protein composition, surface glycans specifically contributed to the differences between the examined groups of prostasomes in terms of presentation of sialylated and mannosylated moieties. These changes did not affect their anti-oxidative capacity, but implied a possible influence on the accessibility of galectin-3 to its ligands on the prostasomal surface. Conclusions: Subtle differences in the presentation of surface molecules may be helpful for differentiation among vesicles sharing the same physical properties. In addition, this may point to some unexpected regulatory mechanisms of interaction of distinct populations of vesicles with their binding partners.

Nano-sized CA125 antigen glycocamouflage: Mucin - Extracellular vesicles alliance to watch?

Mucin 16 (MUC16) is a transmembrane type mucin and its released extracellular portion is designated as CA125 antigen. It is considered to be part of a supramolecular glycoprotein complex having a complicated epitope map and extreme structural heterogeneity. Starting from the initial transmembrane localization of MUC16/CA125 antigen and its alternative routes of release by shedding or putative secretion, CA125 antigen from human amniotic fluid soluble and extracellular vesicles (EVs)-containing fractions were characterized aiming at the possible glycosylation-associated mode of distribution as a factor contributing to the reported conflicting structural data. Ultracentrifugation, sucrose density gradient centrifugation, ion-exchange chromatography and TEM were used for analysis. The results indicated that the smeared abundantly glycosylated high molecular mass CA125-immunoreactive species, which follow the wheat germ agglutinin-binding pattern, were shared across amniotic fluid soluble and particulate fractions. A lower molecular mass glycoprotein-like CA125-immunoreactive species which follows the peanut agglutinin-binding pattern and was specifically associated with the EVs-enriched fraction was observed. CA125 presentation in the particulate amniotic fluid fraction was found to be shaped by a complex interactome partially involving lactose-sensitive galectin-3 binding. The MUC16 - EVs alliance as well as heterogeneous mucin/macromolecular complexes, at membranes or extracellularly, may represent cryptic pools of distinct CA125 species.

Ion-exchange chromatography purification of extracellular vesicles.

Despite numerous studies, isolating pure preparations of extracellular vesicles (EVs) has proven challenging. Here, we compared ion-exchange chromatography (IEC) to the widely used sucrose density gradient (SDG) centrifugation method for the purification of EVs. EVs in bulk were isolated from pooled normal human amniotic fluid (AF) by differential centrifugation followed by IEC or sucrose density gradient separation. The purity of the isolated EVs was evaluated by electrophoresis and lectin blotting/immuno blotting to monitor the distribution of total proteins, different EVs markers, and selected N-glycans. Our data showed efficient separation of negatively charged EVs from other differently charged molecules, while comparative profiling of EVs using SDG centrifugation confirmed anion-exchange chromatography is advantageous for EV purification. Finally, although this IEC-based method was validated using AF, the approach should be readily applicable to isolation of EVs from other sources as well.

Evidence-Based Clinical Use of Nanoscale Extracellular Vesicles in Nanomedicine.

Recent research has demonstrated that all body fluids assessed contain substantial amounts of vesicles that range in size from 30 to 1000 nm and that are surrounded by phospholipid membranes containing different membrane microdomains such as lipid rafts and caveolae. The most prominent representatives of these so-called extracellular vesicles (EVs) are nanosized exosomes (70-150 nm), which are derivatives of the endosomal system, and microvesicles (100-1000 nm), which are produced by outward budding of the plasma membrane. Nanosized EVs are released by almost all cell types and mediate targeted intercellular communication under physiological and pathophysiological conditions. Containing cell-type-specific signatures, EVs have been proposed as biomarkers in a variety of diseases. Furthermore, according to their physical functions, EVs of selected cell types have been used as therapeutic agents in immune therapy, vaccination trials, regenerative medicine, and drug delivery. Undoubtedly, the rapidly emerging field of basic and applied EV research will significantly influence the biomedicinal landscape in the future. In this Perspective, we, a network of European scientists from clinical, academic, and industry settings collaborating through the H2020 European Cooperation in Science and Technology (COST) program European Network on Microvesicles and Exosomes in Health and Disease (ME-HAD), demonstrate the high potential of nanosized EVs for both diagnostic and therapeutic (i.e., theranostic) areas of nanomedicine.

Biological properties of extracellular vesicles and their physiological functions.

In the past decade, extracellular vesicles (EVs) have been recognized as potent vehicles of intercellular communication, both in prokaryotes and eukaryotes. This is due to their capacity to transfer proteins, lipids and nucleic acids, thereby influencing various physiological and pathological functions of both recipient and parent cells. While intensive investigation has targeted the role of EVs in different pathological processes, for example, in cancer and autoimmune diseases, the EV-mediated maintenance of homeostasis and the regulation of physiological functions have remained less explored. Here, we provide a comprehensive overview of the current understanding of the physiological roles of EVs, which has been written by crowd-sourcing, drawing on the unique EV expertise of academia-based scientists, clinicians and industry based in 27 European countries, the United States and Australia. This review is intended to be of relevance to both researchers already working on EV biology and to newcomers who will encounter this universal cell biological system. Therefore, here we address the molecular contents and functions of EVs in various tissues and body fluids from cell systems to organs. We also review the physiological mechanisms of EVs in bacteria, lower eukaryotes and plants to highlight the functional uniformity of this emerging communication system.

Isolation of urinary extracellular vesicles from Tamm- Horsfall protein-depleted urine and their application in the development of a lectin-exosome-binding assay.

Urine is a readily available source of relatively large quantities of extracellular vesicles (EVs). However, the isolation of urinary EVs (uEVs) is complicated by the presence of Tamm-Horsfall protein (THP), which polymerizes and co-precipitates as a contaminant. This may make glycan analysis of uEVs difficult since THP is heavily glycosylated. To facilitate glycosylation analysis and address the need for elimination of non-uEV glycans, we present a modification of the uEV isolation procedure and use the isolated uEVs in the development of a lectin-exosome binding assay. Salt precipitation was employed to remove THP under conditions originally described for its separation from urine, followed by differential centrifugation. The quality of the isolated uEVs was examined by electron microscopy, SDS-PAGE, and immunoblotting. The uEVs were subsequently immobilized on solid phase and probed with labeled plant lectins using the lectin-exosome binding assay. Our results indicate that the isolated uEVs had preserved structural integrity and reacted with labeled plant lectins in a selective, carbohydrate-dependent manner. The basic lectin binding pattern of uEVs obtained by our method can be used as a reference for assessing the composition of their surface glycans in different physiological and pathological conditions.

On chip immuno-affinity profiling of cancer- and benign hyperplasia-associated free prostate specific antigen.

Prostate specific antigen (PSA) exhibits pronounced heterogeneity in both primary structure and glycan composition, resulting in the existence of different molecular forms. Investigation of PSA structure is a demanding task facing limitations due to inadequate sensitivity of analytical techniques and low concentrations of the different forms. This study aimed to profile free PSA (fPSA), especially lower molecular mass species lacking detailed classification, in normal seminal plasma and in sera from subjects with benign hyperplasia (BPH) or cancer of the prostate (PCa) as samples of known clinical relevance. fPSA forms were separated from complex proteomes on chips with immobilized anti-fPSA antibody followed by detection using surface-enhanced laser desorption/ionization time of flight mass spectrometry. At least 39 fPSA-immunoreactive species, ranging from 3-29 kDa were detected in seminal plasma. General fPSA profiles in seminal plasma and sera were similar, but differed in the abundance and presence of particular peaks/clusters of the lower molecular mass species. No striking difference in fPSA forms was observed between BPH and PCa samples, but some distinct peaks varied in intensity and frequency within or between groups. Obtained data verify fPSA heterogeneity that might be important for better exploration of all their molecular and marker potentials.

Molecular heterogeneity of gelatin-binding proteins from human seminal plasma.

Defining the molecular characteristics of seminal plasma proteins is essential for understanding their function in physiological and pathological conditions. Starting from the predicted importance of human seminal plasma gelatin-binding proteins, comprising fibronectin (FN) and FN-related molecules, for male fertility, this study aims at gaining insight into their immuno-glycobiochemical properties. Human seminal plasma from subjects with normal semen parameters were separated on a gelatin-Sepharose column and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting using antibodies against distinct FN forms. Heterogeneity of the isolated molecular species was examined by protein chip arrays combined with surface-enhanced laser desorption/ionization time of flight mass spectrometry, on normal, metal and hydrophobic surfaces. Carbohydrate composition was investigated using mannose-, fucose- and sialic acid-specific plant lectins and galectin-1. The results obtained indicated a pattern of isolated proteins corresponding to that of known FN fragments, as confirmed by immunoreactivity. Among them heparin-binding ability was preferentially associated with low molecular mass species. As for posttranslational modifications, phosphorylation and glycosylation of distinct fragments were revealed. Lectin binding to fragments containing the gelatin-binding domain, particularly with Ricinus communis agglutinin I, was stronger than to fragments containing the cell-binding site of FN. A low level of sialylation and distinctive concanavalin A- and Lens culinaris agglutinin-reactive species were also observed. Galectin-1 did not interact with the isolated preparation. Resolving the molecular heterogeneity of normal human seminal plasma FN and gaining initial insight into possible similarities/differences with known FN molecular species may be considered a prerequisite step preceding challenging the clinical usefulness of these molecular properties.

Fibronectin pattern in benign hyperplasia and cancer of the prostate.

Fibronectin (FN) is a multifunctional glycoprotein involved in cell-matrix interactions. It exhibits a complex pattern of forms differing in respect to aminoacid and oligosaccharide composition. In this study we examined glycobiochemical and functional properties of the FN in benign prostatic hyperplasia (BPH) and prostatic cancer (PCa), attempting to resolve disease-related differences. Two BPH sera pools and three PCa sera pools were used as the FN source. The affinity-purified molecule was characterized by SDS-PAGE, immuno- and lectin blot, lectin-affinity chromatography and adhesion assay. BPH FN existed as intact molecule, giving the main immunoreactive band at 220 kDa. In contrast, PCa FN comprised three main immunoreactive fragments of 140, 110 and 90 kDa. As for glycosylation the ratio of altogether lectin-reactive PCa FN was different from that of BPH FN manifested as a decrease of Con A- and an increase of LCA-reactive moieties. Fibroblasts adhered to both FN preparations in a concentration dependent manner, but with a significantly lower efficiency to PCa FN. The results obtained showing distinct structural characteristics of PCa FN compared to BPH FN could be important for modulation of its ligand and recognition properties expressed as gain or loss of functions or as specific markers of its origin.

Glycosylation of urinary prostate-specific antigen in benign hyperplasia and cancer: assessment by lectin-binding patterns.

OBJECTIVES: In the present study, we examined the glycosylation of urinary prostate-specific antigen (PSA) from benign prostatic hyperplasia (BPH) and prostate cancer (PCa) subjects, specifically looking at alterations in its oligosaccharide chain as a potential biomarker of these pathophysiological conditions. DESIGN AND METHODS: First morning urine voids were collected from subjects with PCa and BPH before initiation of any treatment. Urinary PSA was characterized by ion-exchange chromatography, followed by lectin affinity chromatography on the columns using immobilized plant lectins. RESULTS: Four isoforms of urinary PSA from both BPH and PCa samples were separated by ion-exchange chromatography. The elution profiles from lectin-affinity columns reflected molecular heterogeneity of PSA isoforms and the main differences observed were in the reactivity to Ulex europaeus agglutinin, Aleuria aurantia agglutinin, Phaseolus vulgaris erythroagglutinin and Phaseolus vulgaris leukoagglutinin. CONCLUSIONS: The observed differences in the lectin reactivities between BPH PSA and PCa PSA may be of clinical importance in the evaluation of prostate health.