Methods for the identification and characterization of extracellular vesicles in cardiovascular studies: from exosomes to microvesicles.
Extracellular vesicles (EVs) are nanosized vesicles with a lipid bilayer that are released from cells of the cardiovascular system, and are considered important mediators of intercellular and extracellular communications. Two types of EVs of particular interest are exosomes and microvesicles, which have been identified in all tissue and body fluids and carry a variety of molecules including RNAs, proteins, and lipids. EVs have potential for use in the diagnosis and prognosis of cardiovascular diseases and as new therapeutic agents, particularly in the setting of myocardial infarction and heart failure. Despite their promise, technical challenges related to their small size make it challenging to accurately identify and characterize them, and to study EV-mediated processes. Here, we aim to provide the reader with an overview of the techniques and technologies available for the separation and characterization of EVs from different sources. Methods for determining the protein, RNA, and lipid content of EVs are discussed. The aim of this document is to provide guidance on critical methodological issues and highlight key points for consideration for the investigation of EVs in cardiovascular studies.
Extracellular vesicles as next generation immunotherapeutics.
Seminars in cancer biology
Extracellular vesicles (EVs) function as a mode of intercellular communication and molecular transfer to elicit diverse biological/functional response. Accumulating evidence has highlighted that EVs from immune, tumour, stromal cells and even bacteria and parasites mediate the communication of various immune cell types to dynamically regulate host immune response. EVs have an innate capacity to evade recognition, transport and transfer functional components to target cells, with subsequent removal by the immune system, where the immunological activities of EVs impact immunoregulation including modulation of antigen presentation and cross-dressing, immune activation, immune suppression, and immune surveillance, impacting the tumour immune microenvironment. In this review, we outline the recent progress of EVs in immunorecognition and therapeutic intervention in cancer, including vaccine and targeted drug delivery and summarise their utility towards clinical translation. We highlight the strategies where EVs (natural and engineered) are being employed as a therapeutic approach for immunogenicity, tumoricidal function, and vaccine development, termed immuno-EVs. With seminal studies providing significant progress in the sequential development of engineered EVs as therapeutic anti-tumour platforms, we now require direct assessment to tune and improve the efficacy of resulting immune responses - essential in their translation into the clinic. We believe such a review could strengthen our understanding of the progress in EV immunobiology and facilitate advances in engineering EVs for the development of novel EV-based immunotherapeutics as a platform for cancer treatment.
In-Depth Proteomic Analysis of Blood Circulating Small Extracellular Vesicles.
Methods in molecular biology (Clifton, N.J.)
Circulating small extracellular vesicles (sEVs), also called exosomes, are key players in the investigation of cell-cell communication mechanisms and in the identification of new potential biomarkers. These particles can carry proteins, DNA, mRNA, miRNA, lipids and metabolites that are transported all over the human body, potentially reaching all the cells. In particular, proteins, which are well-known biological actors in cell signalling, will be discussed in this context. In this article, we present a mass spectrometry approach for the in-depth characterization of the sEVs proteome. The protocols include strategies for the isolation and purification of sEVs, for the extraction of proteins and the purification of sEVs proteins by the immunodepletion of the most abundant plasmatic proteins. Finally, bioinformatic analysis for the extraction of the most important biological features associated with the proteomic content of sEVs is reported.
Impact of extracellular vesicles on the pathogenesis, diagnosis, and potential therapy in cardiopulmonary disease.
Frontiers in pharmacology
Cardiopulmonary diseases span a wide breadth of conditions affecting both heart and lung, the burden of which is globally significant. Chronic pulmonary disease and cardiovascular disease are two of the leading causes of morbidity and mortality worldwide. This makes it critical to understand disease pathogenesis, thereby providing new diagnostic and therapeutic avenues to improve clinical outcomes. Extracellular vesicles provide insight into all three of these features of the disease. Extracellular vesicles are membrane-bound vesicles released by a multitude, if not all, cell types and are involved in multiple physiological and pathological processes that play an important role in intercellular communication. They can be isolated from bodily fluids, such as blood, urine, and saliva, and their contents include a variety of proteins, proteases, and microRNA. These vesicles have shown to act as effective transmitters of biological signals within the heart and lung and have roles in the pathogenesis and diagnosis of multiple cardiopulmonary diseases as well as demonstrate potential as therapeutic agents to treat said conditions. In this review article, we will discuss the role these extracellular vesicles play in the diagnosis, pathogenesis, and therapeutic possibilities of cardiovascular, pulmonary, and infection-related cardiopulmonary diseases.
The Pathophysiological Role of Platelet-Derived Extracellular Vesicles.
Seminars in thrombosis and hemostasis
Platelets are very abundant in the blood, where they play a role in hemostasis, inflammation, and immunity. When activated, platelets undergo a conformational change that allows the release of numerous effector molecules as well as the production of extracellular vesicles, which are circulating submicron vesicles (10 to 1,000 nm in diameter) released into the extracellular space. Extracellular vesicles are formed by the budding of platelet and they carry some of its contents, including nucleic acids, surface proteins, and organelles. While platelets cannot cross tissue barriers, platelet-derived extracellular vesicles can enter the lymph, bone marrow, and synovial fluid. This allows the transfer of diverse contents carried by these platelet-derived vesicles to cell recipients and organs inaccessible to platelets where they can perform many functions. This review highlights the importance of these platelet-derived extracellular vesicles under different physiological and pathophysiological conditions.
Immunomodulatory potential of mesenchymal stem cell-derived extracellular vesicles: Targeting immune cells.
Frontiers in immunology
Various intractable inflammatory diseases caused by disorders of immune systems have pressed heavily on public health. Innate and adaptive immune cells as well as secreted cytokines and chemokines are commanders to mediate our immune systems. Therefore, restoring normal immunomodulatory responses of immune cells is crucial for the treatment of inflammatory diseases. Mesenchymal stem cell derived extracellular vesicles (MSC-EVs) are nano-sized double-membraned vesicles acting as paracrine effectors of MSCs. MSC-EVs, containing a variety of therapeutic agents, have shown great potential in immune modulation. Herein, we discuss the novel regulatory functions of MSC-EVs from different sources in the activities of innate and adaptive immune cells like macrophages, granulocytes, mast cells, natural killer (NK) cells, dendritic cells (DCs) and lymphocytes. Then, we summarize the latest clinical trials of MSC-EVs in inflammatory diseases. Furthermore, we prospect the research trend of MSC-EVs in the field of immune modulation. Despite the fact that the research on the role of MSC-EVs in regulating immune cells is in infancy, this cell-free therapy based on MSC-EVs still offers a promising solution for the treatment of inflammatory diseases.
Empagliflozin-Pretreated Mesenchymal Stem Cell-Derived Small Extracellular Vesicles Attenuated Heart Injury.
Oxidative medicine and cellular longevity
Objective:Small extracellular vesicles derived from mesenchymal stem cells (MSCs) play important roles in cardiac protection. Studies have shown that the cardiovascular protection of sodium-glucose cotransporter 2 inhibitor (SGLT2i) is independent of its hypoglycemic effect. This study is aimed at investigating whether small extracellular vesicles derived from MSCs pretreated with empagliflozin (EMPA) has a stronger cardioprotective function after myocardial infarction (MI) and to explore the underlying mechanisms. Methods and Results:We evaluated the effects of EMPA on MSCs and the effects of EMPA-pretreated MSCs-derived small extracellular vesicles (EMPA-sEV) on myocardial apoptosis, angiogenesis, and cardiac function after MI in vitro and in vivo. The small extracellular vesicles of control MSCs (MSC-sEV) and EMPA-pretreated MSCs were extracted, respectively. Small extracellular vesicles were cocultured with apoptotic H9c2 cells induced by HO or injected into the infarcted area of the Sprague-Dawley (SD) rat myocardial infarction model. EMPA increased the cell viability, migration ability, and inhibited apoptosis and senescence of MSCs. In vitro, EMPA-sEV inhibited apoptosis of H9c2 cells compared with the control group (MSC-sEV). In the SD rat model of MI, EMPA-sEV inhibited myocardial apoptosis and promoted angiogenesis in the infarct marginal areas compared with the MSC-sEV. Meanwhile, EMPA-sEV reduced infarct size and improved cardiac function. Through small extracellular vesicles (miRNA) sequencing, we found several differentially expressed miRNAs, among which miR-214-3p was significantly elevated in EMPA-sEV. Coculture of miR-214-3p high expression MSC-derived small extracellular vesicles with H9c2 cells produced similar protective effects. In addition, miR-214-3p was found to promote AKT phosphorylation in H9c2 cells. Conclusions:Our data suggest that EMPA-sEV significantly improve cardiac repair after MI by inhibiting myocardial apoptosis. miR-214-3p at least partially mediated the myocardial protection of EMPA-sEV through the AKT signaling pathway.
A Versatile Design-Enabled Analysis of Circulating Extracellular Vesicles in Disease Diagnosis.
Advanced healthcare materials
Circulating extracellular vesicles (EVs) are considered as potential biomarkers for treatment and diagnosis of many diseases. Most of the existing methods for the EV analysis only have a single function and thus reveal limited information carried by EVs. Herein, a phosphatidylserine-targeting peptide-facilitated design that enables the versatile analysis of circulating EVs for varying requirement is proposed. In the design, DNA probes are inserted into the EV membrane through hydrophobic interactions, and accelerate the removal of protective shielding from DNA-gated metal-organic framework, thereby releasing a large number of methylene blue molecules to amplify the electrochemical signal. Electrochemical results demonstrate equally high sensitivities toward the quantification of EVs derived from different cell sources using an indiscriminative DNA probe. More importantly, the probe can be endowed with extended function for more accurate classification of cell-specific features through the identification of specific EV biomarkers, and demonstrates the potential use in the diagnosis of cardiovascular in a principle-of-proof study for clinical application. Therefore, the method provides a versatile design for the identification of EV features, and may address the needs of clinical diagnosis in the future.
Extracellular Vesicles in Kidney Diseases: Moving Forward.
Extracellular vesicles (EVs) are evolving as novel cell mediators, biomarkers, and therapeutic targets in kidney health and disease. They are naturally derived from cells both within and outside the kidney and carry cargo which mirrors the state of the parent cell. Thus, they are potentially more sensitive and disease-specific as biomarkers and messengers in various kidney diseases. Beside their role as novel communicators within the nephron, they likely communicate between different organs affected by various kidney diseases. Study of urinary EVs (uEVs) can help to fill current knowledge gaps in kidney diseases. However, separation and characterization are challenged by their heterogeneity in size, shape, and cargo. Fortunately, more sensitive and direct EV measuring tools are in development. Many clinical syndromes in nephrology from acute to chronic kidney and glomerular to tubular diseases have been studied. Yet, validation of biomarkers in larger cohorts is warranted and simpler tools are needed. Translation from in vitro to in vivo studies is also urgently needed. The therapeutic role of uEVs in kidney diseases has been studied extensively in rodent models of AKI. On the basis of the current exponential growth of EV research, the field of EV diagnostics and therapeutics is moving forward.
Therapeutic potential of extracellular vesicles in neurodegenerative disorders.
Handbook of clinical neurology
Neurodegenerative disorders are characterized by complex multifactorial pathogeneses, thus posing a challenge for standard therapeutic approaches that tend to focus only on one underlying disease aspect. For systemically administered drugs, the blood-brain barrier (BBB) is yet another major obstacle to overcome. In this context, naturally occurring extracellular vesicles (EVs) with intrinsic ability to cross the BBB have been investigated as therapeutics for various diseases, including Alzheimer's and Parkinson's diseases. EVs are cell-derived, lipid membrane-enclosed vesicles carrying a broad spectrum of biologically active molecules, which play a crucial role in intercellular communication. In a therapeutic context, mesenchymal stem cell (MSC)-derived EVs are in the spotlight because they reflect the therapeutic properties of their parental cells and, thus, hold promise as independent cell-free therapeutics. On the other hand, EVs can be used as drug delivery vehicles by modifying their surface or content, e.g., by decorating the surface with brain-specific ligands or loading the EVs with therapeutic RNAs or proteins, thus further enhancing the EV's targeting and therapeutic potency, respectively. Although EVs have been deemed safe for use in humans, some obstacles remain that prevent their progression into clinics. This review scrutinizes the promises and challenges of EV-based treatments for neurodegenerative disorders.
Extracellular vesicles of immune cells; immunomodulatory impacts and therapeutic potentials.
Clinical immunology (Orlando, Fla.)
Extracellular vesicles (EVs) are a diverse collection of lipid bilayer-membrane-bound particles which are released from cells into the extracellular space and biologic fluids. In multicellular organisms, these vesicles facilitate the exchange of bioactive compounds such as RNA, DNA, proteins, various metabolites, and lipids between the cells. EVs are produced and released by almost all eukaryotic cells including immune cells and can have immunomodulating effects by either stimulation or suppression of their activities. This immune-modulating feature may provide a promising strategy for treating immune-mediated diseases such as cancer, neurodegenerative diseases, autoimmune disorders and graft-versus-host disease. Moreover, immune cell-derived EVs have received attention as potential biomarkers for being used as diagnostic tools and preventive strategies such as for developing vaccines. In this review, we focus on the EVs produced by different immune cell types, their effects on the immune system, and highlight their potential applications for immunotherapy.