Exosomes are small membrane-bound vesicles of endocytic origin that are actively secreted. The potential of exosomes as effective communicators of biological signaling in myocardial function has previously been investigated, and a recent explosion in exosome research not only underscores their significance in cardiac physiology and pathology, but also draws attention to methodological limitations of studying these extracellular vesicles. In this review, we discuss recent advances and challenges in exosome research with an emphasis on scientific innovations in isolation, identification, and characterization methodologies, and we provide a comprehensive summary of web-based resources available in the field. Importantly, we focus on the biology and function of exosomes, highlighting their fundamental role in cardiovascular pathophysiology to further support potential applications of exosomes as biomarkers and therapeutics for cardiovascular diseases. EVs are gaining popularity in clinics as therapeutic drug delivery vehicles that transfer bioactive molecules such as proteins, genes, microRNAs (miRNAs), viruses, and other therapeutic agents to treat diseases and halt disease progression. Despite our limited but growing knowledge of EV biology and technology, it is imperative to understand their fundamental role in cardiovascular pathophysiologies to engineer the complex mechanisms of EV cargo sorting in pursuit of designing next generation EV-based therapeutic delivery systems.The last decade has seen a sharp increase in the number of scientific publications describing the physiological, pathological, and therapeutic functions of extracellular vesicles (EVs). According to the minimal information for studies of extracellular vesicles guidelines set by the International Society for Extracellular Vesicles (ISEV) for EV studies,We discuss recent advances and challenges in exosome research with an emphasis on scientific innovations in isolation, identification, and characterization methodologies, and we provide a comprehensive summary of web-based resources available in the field. Importantly, we focus on the biology and function of exosomes, highlighting their fundamental role in cardiovascular pathophysiology to further support potential applications of exosomes as biomarkers and therapeutics for cardiovascular diseases.2 There is no gold standard for EV nomenclature that currently exists. However, a general consensus is that some EVs that are released directly from a cell’s plasma membrane are often called microvesicles or ectosomes. These EVs display sizes ranging from several nanometers to microns. Internal vesicles generated in multivesicular endosomal compartments are termed as exosomes, which are secreted by fusion of the multivesicular body membrane with plasma membrane. Exosomes are classically defined by their size  and contain endosome-associated proteins.2 However, the term exosomes is more commonly used in the literature, as well as in this article, for a heterogenous population of small-size EVs isolated by either ultracentrifugation methods or size-exclusion filters without demonstrating their intracellular origin. Exosomes themselves are also considered to be a very heterogenous population. Interestingly, an abundant population of nonmembranous (nonvesicular) nanoparticles known as exomeres (≈35 nm) was recently discovered.4 Microvesicles are larger (≈100 nm and larger) plasma membrane–derived ectosomes. Apoptoic bodies originate during apoptosis, and are also large vesicles (≈50–500 nm).9 In this review, we focus on exosomes and microvesicles as major groups of EVs and as potential therapeutic vehicles to treat cardiovascular disease. A sketch of exosome biogenesis and their basic characteristics is provided in Figure 10 and apoptosomes.11EV populations released from cells as well as found in circulating biofluids are known to be heterogeneous in their origin, size, and composition.Given growing interest in the role of exosomes in cardiovascular physiology and pathology, here we summarize the most recent findings on the role of exosomes as signaling mediators, biomarkers, and potential therapeutic targets that are relevant to cardiovascular medicine. Furthermore, this review provides a comprehensive update on recent methodological advances in the study of exosomes and intends to be valuable to researchers currently active in exosome research, as well as to recent scientific newcomers.However, before exploring different aspects of exosomes, it is worth explaining why exosomes earned so much interest in the cardiovascular field:Exosomes, a subgroup of EVs, are nanosized membrane particles actively released by cells under both physiological and pathological conditions.Exosomes mediate intercellular transport of cytosolic cargo.Exosomes offer great potential to the clinical field, with applications in both diagnostics and therapeutics.12Small size and quantity, varying physicochemical properties, and complexity of the surrounding biological fluid make exosomes difficult to obtain in relatively pure preparations. Separation of exosomes from other extracellular entities and vesicle subtypes represents a significant challenge mainly because of the lack of universal or unequivocally specific exosome-based markers.13 Because there is no single optimal separation method for exosome isolation or characterization fulfilling experimental and clinical needs, choosing a suitable method for exosome studies could be a challenging decision.A plethora of different isolation methods has been described that impact various degrees of EV purity, integrity, and active state, as well as yields Table .13 Becau27The choice of separation method should be guided by the type of exosome-containing matrix and required degree of exosome yield, purity, integrity, and concentration that is dictated by the downstream applications and scientific question to be addressed.28 By contrast, for exosome isolation from biofluids, in most cases, dilution of biological fluids may be advisable and may enhance the recovery of vesicles.28 Additionally, one should be aware that vesicles recovered from biofluids will not only have a mixed cellular origin, but also will likely be contaminated to various degrees with nonexosomal structures  and more advanced isolation methods should be used to separate these components from exosomes.1Regardless of the type of starting material , similar preanalytical elementary considerations apply. For example, it is important to ensure that separated exosomes are indeed from extracellular space rather than from intracellular space or soluble nanoparticles released as a result of exosome-containing matrix harvest, processing, and storage. However, each exosome-containing matrix presents specific biophysical and chemical characteristics, thereby requiring additional sample collection and processing considerations. For instance, it is especially important for cellular studies to avoid contamination of cell-derived vesicles by serum exosomes, microbes , or dead cell–derived vesicles.Finally, the choice of isolation procedures is additionally influenced by the ultimate goal of exosome research question. The type of study—discovery, clinical, or preparative research—will mandate a different level of yield, purity, and concentration depending on the application, thereby requiring specific isolation approach. In some cases, combination of several methods or specific modifications to existing methods may offer better outcomes when addressing specific types of scientific questions.A variety of exosome isolation methods are currently in use, each with its own advantages and disadvantages. While centrifugation-based and size-based exosome isolation methods are traditionally followed by wide range of researchers, rapid advances in modern science and technology have started to yield many new techniques for improving exosome isolation, both quantitatively and qualitatively.In Table To date, isolating exosomes directly from tissues remains a major challenge mainly attributed to the lack of a suitable exosome isolation procedure as well as the complexities associated with isolating exosomes with preserved biochemical properties from the intact extracellular milieu.29 or frozen postmortem,29 and central nervous system.30 However, it should be noted that tissue homogenization may lead to physical damage and compromise the extracellular environment with intracellular vesicles and exosome mimetics.30By using tissue homogenization, exosomes have been isolated from tissues of the mouse brain, freshly removed31 In this study, the purification of exosomes was achieved by gentle dissociation of the tissue using collagenase type III to free the brain extracellular space, followed by sequential low-speed centrifugations and sucrose cushion ultracentrifugation.31In 2017, Vella et al reported an efficient protocol for the enrichment of exosomes from postmortem brain tissues stored at −80°C which provided significant advantages over previously described protocols.32In relation to the cardiac research field, Loyer et al recently demonstrated that small and large EVs can be isolated directly from sham-operated and infarcted mouse heart tissue as well as human heart tissue of patients undergoing aortic valve replacement by tissue disruption and different centrifugation steps.34A notable challenge in the advancement of exosome-based therapeutics into clinical applications lies in developing sterile, scalable, reproducible, and efficient protocols for clinical-grade exosome production with minimal batch-to-batch variation.35 In that study, isolation and purification of exosomes were achieved by filtration and ultracentrifugation using closed and semiclosed systems followed by exosomes analyzed by NanoSight and flow cytometry and tested for endotoxin and sterility to ensure their potential clinical applicability.35 Similar developments are currently emerging to successfully upscale good manufacturing practice−grade manufacturing methods for the treatment of cardiovascular and other diseases.36The process and feasibility of generating good manufacturing practice−grade exosomes for therapies was recently demonstrated using bioreactor-based culture system.Genuine and unique markers that distinguish different EV subpopulations and allow for precise analyses of their subtypes are still not well-established. Nevertheless, a common feature of all EVs is a lipid bilayer membrane that surrounds a rich cargo of selective biomolecules from its originating cells. Thus, the EV cargos are partially reflective of their cellular origin.An important criterion for EV subtype classification is to determine EV protein markers, which can be membrane-bound or present in the EV transmembrane or as intraluminal soluble proteins.2 Interestingly, small EVs are selectively enriched with unique proteins such as TSG101 (tumor susceptibility 101), syntenin-1, EHD4 (Eps15 homology domain protein 4), annexin XI, and ADAM10 , and could be distinguishable from larger EVs.2 Conversely, actinin-4, mitofilin, and GP96 (heat shock protein 96) were shown to be unique or enriched in large EVs.2 Additionally, Jeppesen et al recently performed extensive proteomic profiling of small EVs and nonvesicular fractions, and identified annexin A1 as a specific marker for microvesicles that are shed directly from the plasma membrane.3Biochemical studies by Kowal et al revealed some of the common proteins shared by several EV subtypes that include: actin, ezrin, moesin, HSC70 (heat shock cognate 71 kDa), HSP70 (heat shock protein 70), flotillin-1, MHCI (major histocompatibility complex class I), MHCII (major histocompatibility complex class II), annexin II, CD63 (cluster of differentiation 63), and CD9 (cluster of differentiation 9).37 because of their increased enrichment in exosomes; these markers are not to be considered as canonical exosome markers.12 Indeed, using global integrated proteogenomic analysis of neuroblastoma cell–derived EVs, Keerthikumar et al identified enrichment of Alix, TSG101, CD63, and CD9 in exosomes subpopulations as compared with ectosomes.38 Similarly, another study supported that CD81 (cluster of differentiation 81) might specifically be utilized as an exosomal marker.39 Together, these studies indicate that selectively enriched proteins could represent specific EV subtypes and serve as biomarkers for both investigations as well as for diagnostics.Although numerous proteomic studies have implicated Alix (programmed cell death 6-interacting protein), TSG101, CD63, and CD9 as exosomal markers40 the presence of RNA in EVs was only demonstrated in the last 2 decades.While the occurrence of proteins in EVs was reported together with the discovery of EVs,41 Several years later, Nolte-’t Hoen et al found a large variety of small noncoding RNA (ncRNA) species in immune cell–derived vesicles, including small nucleolar RNA, Y RNA, mitochondrial RNA, and vault RNA as well as mRNA (mostly fragmented).42 According to Wei et al, exosomes are enriched in small ncRNAs, such as miRNAs and precisely processed tRNA and Y RNA fragments.43 Even though exosomal RNA composition is an expanding and promising area of research, recent studies suggest that massive exosome uptake would be required to ensure functional impact of transferred RNA on recipient cells as there seems to be <1 copy of nonribosomal RNA per exosome.43 However, studies of the cardiovascular system showed that also small miRNAs can be shuttled between cardiac cell types  via exosomes.45In 2007, Valadi et al were the first to report the existence of mRNAs and miRNAs in EVs derived from mast cells and the functional transfer of RNA to EV-targeted cells.46 One of the first observations of this phenomenon was made by Kogure et al where a 1198-nucleotide ultraconserved lncRNA referred to as TUC339 was identified in exosomes derived from hepatocellular carcinoma and its enrichment was dramatically altered within EVs compared with that of cells of their origin.46 Additionally, in these studies Kogure et al demonstrated that hepatocellular carcinoma cell–derived EVs can be taken up by other hepatocellular carcinoma cells resulting in intercellular transfer of ultraconserved lncRNA with subsequent modulation of cell growth and spread.46 In hypoxic cardiomyocytes, RNA sequencing of lncRNAs revealed strong differences in cargo of exosomes in contrast to larger microvesicles or even complete cells.47 Recently, Li et al showed for the first time the presence of another type of endogenous lncRNA species, circular RNA (circRNA), in exosomes derived from MHCC-LM3 liver cancer cells.48 RNA sequencing (RNA-seq) analyses revealed that circRNAs were highly enriched in exosomes compared with parental cells. Interestingly, the authors found that serum exosomal circRNAs may distinguish patients with cancer from healthy individuals demonstrating its substantial translational potential as a circulating biomarker for cancer diagnosis.48Interestingly, long ncRNAs (lncRNAs) are often detected in EVs and thought to act as messengers in intercellular communication.49 These observations open up an interesting line of investigation into the extent and purpose of RNA modifications in EVs and their roles in cardiovascular physiology and pathology.50Recent studies provide evidence that EV RNAs are not just linear RNA molecules, but rather they can be modified. Specifically there is evidence for EV-specific tRNA modification, perhaps indicating a role for posttranscriptional modification in the sorting of some RNA species into EVs. Moreover, the RNA-binding protein YBX1 (Y box binding protein 1), which is required for the sorting of selected miRNAs into exosomes, plays a role in the sorting of highly abundant small ncRNA species, including tRNAs, Y RNAs, and vault RNAs.53 EVs are often enriched in eicosanoids, fatty acids, and cholesterol, and their lipid composition can be modified by in vitro manipulation.53 EVs also contain metabolizing enzymes involved in lipid synthesis so they can represent an autonomous production unit of bioactive lipids.To a large extent, EVs constitute of membrane lipids organized in a bilayer structure. In addition to the lipid carriers such as fatty acid binding proteins embedded in the bilayer, EVs also shuttle various bioactive lipids, which are captured from the cytosol during exosome formation.56 For instance, Hough et al demonstrated that chronic airway inflammation may be driven by alterations in the composition of lipid mediators within airway EVs of human subjects with asthma.57 Data of diagnostic relevance was recently shown by Pieragostino et al, who reported a high number of acid sphingomyelinase–enriched exosomes in multiple sclerosis, opening a window for novel therapeutic approaches in the treatment of this disease that attacks the central nervous system.58Compared to their parental cells, EVs are 2 to 3 times enriched in cholesterol, sphingomyelin, phosphatidylserine, and glycosphingolipid, and the accumulation of these EV-components in cells might modulate recipient cell homeostasis.60As lipid bilayers are indispensable components of EVs, technological developments in EV lipidomics may further contribute to developing a reliable and reproducible method for the standardization and characterization of EV samples and/or EV subtypes. Osteikoetxea et al and Visnovitz et al proposed the use of total lipid content and the protein-to-lipid ratio as additional parameters to total protein content and particle counts for quantifying and characterizing EV preparations. Authors have also proposed that protein-to-lipid ratio, lipid bilayer order (which reflects the degree of lipid packing), and selected lipid composition can also discriminate among EV subpopulations of a different size.51The lipid composition of EVs attracts substantial attention not only because of the proposed role of these molecules in various biological processes but also because of their potential to serve as biomarkers for a variety of diseases, including cancer. Skotland et al recently investigated the lipid content of exosomes purified from the urine of 15 patients with prostate cancer and 13 healthy volunteers, and identified molecular lipid species in urinary exosomes as potential prostate cancer biomarkers.61 Adeno-associated virus (AAV) is one of the most actively investigated gene therapy vehicles because of the vector’s good safety profile in clinical trials. However, one significant obstacle to AAV-based gene therapy is the potential blocking of AAV by naturally existing anti-AAV neutralizing antibodies. Recently, it was shown that AAV capsids may be incorporated into EVs during virus vector production and therefore, exosome-associated AAV (Exo-AAV) can be isolated from conditioned medium of the packaging cell line.62 Exo-AAV vectors outperformed conventional AAV vectors in transduction, both in vitro and in vivo, and were more resistant to neutralizing antibodies compared with standard AAV.64 For example, Hudry et al demonstrated that Exo-AAV is capable of efficient transduction of cells in the central nervous system after systemic injection in mice.65 Importantly, first attempts to test the use of Exo-AAV as superior agents for delivering genes  to cardiomyocytes and to the heart were made in our laboratory and showed that Exo-AAV9 outperformed conventional AAV vectors in preserving cardiac function post–myocardial infarction, even in the presence of neutralizing antibodies66 (and unpublished data).Exosomes are also produced and secreted by virus-infected cells and are believed to be involved in cell-to-cell communication between infected and uninfected cells.68 Additionally, several studies demonstrated that cells release vesicles containing mitochondrial DNA.72 For example, Sansone et al identified the full mitochondrial genome packaged in cancer-associated fibroblast-derived EVs and in circulating EVs from patients with hormonal therapy–resistant metastatic breast cancer.72 Additionally, authors demonstrated that transfer of mitochondrial DNA through EVs acts as an oncogenic signal promoting an exit from dormancy of therapy-induced cancer stem-like cells and leading to endocrine therapy resistance in breast cancer.72EVs have been previously shown to deliver genomic DNA to target cells.73 Using EV membrane isolation and mass spectrometry analysis, Jang et al discovered enrichment of mitochondrial membrane proteins in the melanoma tissue–derived EVs, compared with nonmelanoma-derived EVs. Specifically, EVs with mitochondrial inner membrane proteins MT-CO2  and COX6c (cytochrome c oxidase subunit 6C) were detected in the plasma of melanoma patients as well as in ovarian and breast cancer patients. Importantly, this subpopulation of EVs was shown to contain active mitochondrial enzymes.73Recent studies also suggest that in addition to mitochondrial DNA, mitochondrial proteins are also secreted via EVs.3 Therefore, some precautions, such as determining whether or not the detected DNA or protein is truly carried by EVs rather than being an artefactual contaminant, are advisable for such studies.Although exosomes are thought to be mediators of extracellular DNA secretion, Jeppesen et al recently provided evidence that double-stranded DNA and DNA-binding histones are not carried by exosomes or any other type of small EVs.74 Exosomes have been shown to be internalized by recipient cells by a variety of endocytic pathways, such as clathrin-dependent endocytosis and clathrin-independent pathways , macropinocytosis, phagocytosis, and lipid raft–mediated internalization.75 Interestingly, it seems possible that a heterogeneous population of vesicles may gain entry into a cell via >1 route. For functional implications, it is important that the vesicle is uptaken and not simply fated for the lysosome thus destroying the signal. Importantly, the vesicle has to be uptaken unpackaged and the signal should be delivered more specifically.Exosomes transfer their molecular cargo to recipient cells to regulate cell function. Several types of interactions may be involved in the functional communication of exosomes with cells, including: release of exosome contents in the extracellular space; exosome-binding to the recipient cell surface; exosome–plasma membrane fusion; direct transfer of cargo through connexin 43–containing channels formed by the docking of exosomes and target cells; and uptake by endocytosis.76 while other studies demonstrate that vesicular uptake is a highly specific process between a specific cell type and EVs.77Contradictory opinions as to the mode of exosome internalization exist within the literature. Results from some studies show that exosomes can be taken up by essentially every cell type investigated,76 In another study, tetraspanin 8–containing lymph node stroma–derived EVs were most effectively internalized by endothelial cells and pancreatic cells but to a lower degree by parental lymph node stroma cells.78 Näslund et al demonstrated that exosomes from breast milk can be taken up via monocyte-derived dendritic cells partly via MUC1 /DC-SIGN (dendritic cell–specific intercellular adhesion molecule-3–grabbing nonintegrin) interaction, whereas plasma exosomes lacking MUC1 are unable to enter these cells.79 Moreover, results from our laboratory indicate that injection of mouse hindlimb ischemic tissue with exosomes derived from human CD34+ stem cells resulted in the most efficient exosome uptake by endothelial cells as compared with smooth muscle cells and fibroblasts.77Zech et al showed that pancreatic adenocarcinoma–derived exosomes bind to, and are taken up by, all leukocyte subpopulations both in vivo and in vitro. However, these leukocyte subpopulations differed in tumor–exosome uptake, which was highest for peritoneal exudate cells and lowest for granulocytes.80 A labeling approach is very important, especially from a clinical drug development perspective, to gain more information about pharmacokinetics and biodistribution of EVs. Here, we discuss examples of such strategies.A major challenge for the development and optimization of exosome-based diagnosis and treatment is being able to visualize clinical and preclinical in vivo transfer of exosomes to recipient cells. To address this problem, a few groups have recently developed novel approaches based on genetic modification of the cell of their origin, which then secrete EVs containing labeled components.81 Fusion of luciferase to a protein transmembrane domain similarly allowed its secretion in EVs and subsequent analysis of distant bioluminescent cells in vivo.82 Authors also designed an intracellular probe to fluorescently label mRNA secreted in EVs and tools to detect EV-associated transcripts encoding luciferase signal in target cells.82Genetic fusion of fluorescent protein  to a consensus palmitoylation sequence induced its localization at the plasma membrane, as well as in secreted EV subpopulations. Bound or internalized EVs were then visualized by intravital microscopy of tumor tissue in animals with tumors expressing this fusion protein.83 where the authors generated a novel transgenic rat model in which green fluorescent protein (GFP)–tagged human CD63 (CD63-GFP) is under control of the CAG  promoter, resulting in high expression of GFP in various body tissues. Indeed, exogenous human CD63-GFP was detected on EVs isolated from 3 body fluids of animals: blood serum, breast milk, and amniotic fluid. Additionally, in vitro analyses demonstrated transfer of serum-derived CD63-GFP–expressing EVs into recipient rat embryonic fibroblasts, where the EVs localized in endocytic organelles. These findings suggest that transgenic animal model developed by Yoshimura et al may provide significant information for understanding of the intercellular transfer as well as mother-to-child transfer of EVs in vivo.Another elegant approach to elucidate the intercellular EV transfer in vivo was proposed by Yoshimura et al,84 The authors have successfully live-visualized single endogenous vesicles in the whole zebrafish embryo using a fluorescent reporter, CD63-pHluorin. This model will allow investigation of the composition of EVs and the molecular mechanisms controlling their biogenesis, fate and function in receiving cells.In a recent article, Verweij et al provided a unique model to track in vivo interorgan communication by endogenous exosomes at the individual vesicle level with high spatiotemporal accuracy.87 Recently, it has been shown that certain miRNAs are differentially expressed in mouse induced pluripotent stem cells (iPSCs) and iPSC-derived EVs.85 Indeed, using quantitative polymerase chain reaction–based method 33 miRNAs were detected only in iPSC-derived EVs, which suggests their selective enrichment during EV production and release by iPSCs.85 Similar phenomenon was previously observed by Shurtleff et al, who performed Illumina-based small RNA-seq on libraries prepared from purified exosomes and their parent cell of origin.87 They found at least 134 miRNAs that are selectively enriched in exosomes as compared with parent cells.87 Similarly, Squadrito et al showed that several miRNAs are differentially enriched in macrophage-derived exosomes as compared with their parent cells.86 The same strong differences in the cargo between different extracellular vesicles was shown for lncRNAs.47Interestingly, EV contents are not always reflective of the cell of their origin.88 Other studies reported that the neutral sphingomyelinase 2 might in part regulate the sorting process of specific miRNAs into small EVs.89 In addition, posttranscriptional modifications such as 3´-end uridylation were previously shown to play a role in defining the fate of miRNAs to be loaded into EVs.90 Villarroya-Beltri et al described another mechanism where the researchers discovered that RNA-binding proteins (RBPs) recognize specific GGAG motifs in the 3´-end of miRNAs and regulate miRNA packaging into exosomes.91 Specifically, they identified that binding of hnRNPA2B1 to exosomal miRNAs is dependent on its sumoylation state.91 Another example of RNA-binding protein–dependent miRNA sorting is the identification of Y-box protein 1 as a candidate exosomal miR-223 sorting protein.87Nonetheless, the underlying mechanisms of the active sorting of specific miRNAs to EVs and the significance of miRNA transfer to recipient cells are still poorly understood. Some studies propose that RISC (RNA-induced silencing complex)–associated proteins are involved in the loading process of miRNAs into EVs as RNA-induced silencing complex proteins were found to be colocalizing with multivesicular bodies where exosomes are sorted for packaging and release.86These findings open the way for a great tool to control the specific miRNA content in EVs by either mutagenesis of sequence motifs or modulation of hnRNPA2B1 sumoylation and expression of RNA-binding proteins. However, it remains unclear whether these proteins only control a specific set of miRNAs or are globally involved in the RNA sorting process, nor is it confirmed if RNA-binding proteins play an actual active role or are just passively attached to EV sorting process. Indeed, direct and persuasive evidence for a physiological role of EV-borne miRNAs has so far proven elusive. Alternatively, it seems possible that vesicles may be a convenient carrier to eliminate unnecessary or inhibitory RNA molecules from cells.95Recently, several studies have investigated the molecular composition of different EV subpopulations from a single cell type. Interestingly, these direct comparisons evaluated that vesicular subtypes have at least partly different protein, lipid, and RNA content, and might exert different biological functions on target cells.93 Authors demonstrated that the population of EVs contained in the medium floatation density fraction showed superior activity in promoting renal protection from injury in vitro.93 Tauro et al used magnetic beads coated with anti-A33 and anti-EpCAM  antibodies to isolate 2 molecularly distinct populations of exosomes, EpCAM- and A33-Exos, being released from the apical and basolateral side of human colon carcinoma cells, respectively.96 These findings suggest that vesicles with different molecular structure can be released from different cell regions  and that the expression of plasma membrane molecules can be used to separate EVs, which would be coisolated using other purification methods.The results obtained by Collino et al demonstrated heterogeneity in quantity and composition  of subpopulations distributed at different densities.The purpose of this section is to list and make readers aware of a variety of available web-based resources that integrate our knowledge of exosome biology and provide databases of different vesicular molecules. Best practices for biological sample collection and analysis of submicron particles, including exosomes and other EVs, have been discussed in several position papers and statements from ISEV which we also summarize in this section.http://microvesicles.org) serves as an online, community-annotated compendium of molecular data  pertaining to EVs from >500 independently published studies.97 It catalogs recent studies on all classes of EVs, including apoptotic bodies, exosomes, large dense-core vesicles, microparticles, and microvesicles. EV researchers can query molecules of interest or browse by organism, vesicle type, molecular content type, or sample material.Vesiclepedia (http://www.exocarta.org) is a database manually curated by EV biologists, which serves as a free web-based compendium of molecules identified in exosomes derived from multiple cell types.101 The current version of ExoCarta hosts ≈41 860 protein, 4946 mRNA, and 1116 lipid entries catalogued from 286 exosomal studies from 10 different species (including 6514 molecules identified in Homo sapiens and 1714 in Mus musculus), and annotated with ISEV minimal experimental requirements for definition of EVs.101 ExoCarta can be queried using the gene symbol, protein name, and miRNA ID, or it can be browsed as a group based on the organism, molecular content type, sample material, and gene symbol. Furthermore, ExoCarta features dynamic protein–protein interaction networks and biological pathways of exosomal proteins.101ExoCarta (http://evpedia.info) is an integrated and comprehensive proteome, transcriptome, and lipidome database of prokaryotic and eukaryotic EVs.103 EVpedia provides a broad array of tools, such as (1) search and browse tools for vesicular proteins, mRNAs, miRNAs, lipids, and metabolites, as well as publications and principal investigators; (2) a database for identification of orthologous vesicular components; (3) tools for bioinformatic analyses including Gene Ontology enrichment analysis and network analysis; and (4) personalized function to deposit the user’s own datasets, results, and literature of interest.39 Recently, EVpedia has been further improved . Now EVpedia includes 14 192 publications, 722 551 vesicular components from 503 high-throughput studies  and has been globally accessed >19 500 000 times from >88 countries. In addition, about 2445 members from 78 countries have participated in developing EVpedia.EVpedia (http://www.exoRBase.org) is a web-accessible database that is a repository of circRNA, lncRNA, and mRNA derived from RNA-seq data analyses of exosomes derived from human blood. The first release of exoRBase featured 58 330 circRNAs, 15 501 lncRNAs, and 18 333 mRNAs.104 Furthermore, 77 experimental validations from 18 published articles are also included.104 exoRBase contains the integration and visualization of RNA expression profiles based on normalized RNA-seq data from healthy individuals as well as from patients with different diseases.exoRBase  and is the result of collaboration with University of California Irvine (USA), University of Gothenburg (Sweden), and Pohang University of Science and Technology (South Korea). The goal of the course is to provide the basic knowledge about EV history, nomenclature, biogenesis, and EV cargo, as well as the release and uptake mechanisms, collection, and processing before isolation, different isolation methods, characterization. and quantification techniques.The ISEV Education Committee has produced a massive open online course for researchers interested in the field of EVs. The first ISEV massive open online course, http://evtrack.org) is an open-source knowledgebase that integrates methodological parameters of EV-related publications with the goal of motivating researchers, reviewers, editors, and funders to put experimental guidelines into practice and facilitating standardization of EV research through systematic reporting on EV biology and methodology.105EV-TRACK (https://exrna-atlas.org) is the data repository of the Extracellular RNA Communication Consortium106 and provides access to small RNA–seq and quantitative polymerase chain reaction–derived extracellular RNA (exRNA) profiles from multiple human and mouse biofluids . A suite of web-accessible analysis and visualization tools enables users to analyze exRNA-seq profiles from the Atlas, process and analyze their own exRNA-seq data, and contribute their data and analyses to the Atlas. In an attempt to minimize experimental variability, all exRNA-seq datasets are uniformly processed using the exceRpt (extra-cellular RNA processing toolkit) pipeline107 and Extracellular RNA Communication Consortium–developed quality metrics are uniformly applied to these datasets. The latest release of the Atlas  contains 2270 exRNA-seq and 3039 quantitative polymerase chain reaction profiles from 19 different studies that cover 23 health conditions, and additionally combines the exceRpt pipeline with computational deconvolution step to facilitate interpretation of exRNA profiling studies and our understanding of exRNA communication.108exRNA Atlas  that must be considered to develop best practice models for EV therapies and translating EVs into the clinic.In the position statement by Lener et al, basic and clinical researchers summarize recent developments and the current knowledge on EV-based therapeutics in clinical trials.http://www.evflowcytometry.org) is a collaborative initiative involving specialists in the fields of EVs, cytometry, and vascular biology to develop more robust flow cytometric methods for the study of submicron particles, including exosomes and other EVs.112 To accomplish that, 3 major societies, the International Society for Advancement of Cytometry (ISAC), the International Society on Thrombosis and Haemostasis (ISTH), and ISEV joined forces in 2015 and committed to improve tools, technologies, and standards for EV analysis.The EV Flow Cytometry Working Group  and chemokines CCL-2 and CCL-6 .32 Interestingly, Ribeiro-Rodrigues et al demonstrated in vitro and in vivo that cardiosomes produced under ischemic conditions stimulate cardiac angiogenesis.114 Moreover, they provided evidence that these ischemic cardiosomes are specifically enriched in miR-222 and miR-143 and account for the angiogenic process. More recently, another study identified miR-217 as a promising therapeutic target for congestive heart failure mediating cardiac hypertrophy and cardiac fibrosis processes through regulation of PTEN. Importantly, miR-217 was contained in cardiosomes which were shown to enhance proliferation of fibroblasts in vitro.115Exosomes secreted by cardiomyocytes (cardiosomes) exert diverse autocrine and paracrine effects dependent on the conditions of release and therefore, exosomal contents, as well as the type, of a target cell. In their study, Yu et al demonstrated that HIF-1α (hypoxia-inducible factor 1α) is involved in the regulation of hypoxia-induced autocrine effects of TNF-α (tumor necrosis factor α) in cardiomyocytes, mediated by the exosomal pathway.45 Another study has shown that stimulation of cardiac fibroblast with angiotensin II resulted in secretion of exosomes that further induced angiotensin II production and angiotensin II receptor expression in cardiomyocytes and promoted myocyte hypertrophy.116Cardiac fibroblast–derived exosomes have been shown to regulate diverse aspects of cardiomyocyte biology such as hypertrophy. For example, Bang et al demonstrated that passenger strand miR, miR-21-3p/miR-21 is significantly enriched in cardiac fibroblast–derived exosomes and regulates hypertrophic crosstalk between cardiomyocytes and cardiac fibroblasts.117 Interestingly, the 16-kDa N-terminal prolactin fragment, which is associated with peripartum cardiomyopathy, stimulated secretion of exosomal miR-146a from endothelial cells.118 Furthermore, these endothelial cell–derived EVs were shown to be taken up by cardiomyocytes, in which exosomal miR-146a mediated posttranscriptional gene silencing of target genes Erbb4, Notch1, and Irak1, and reduced cardiomyocyte metabolic activity.118 In another study, EVs secreted by Krüppel-like factor 2–transduced or shear-stress–stimulated human umbilical vein endothelial cells were shown to be enriched in miR-143/145 and to induce atheroprotective phenotype in cocultured smooth muscle cells.44 Furthermore, Liu et al provided evidence that clinical and experimental atherosclerotic conditions can promote the packaging of functional miR-92a-3p into endothelial microvesicles, thus promoting angiogenic responses in the recipient endothelial cells by a THBS1 (thrombospondin 1)–dependent mechanism.119 Recently, Davidson et al demonstrated that endothelial cell–derived exosomes can confer resistance to simulated ischemia and reperfusion injury in cardiomyocytes via the activation of the ERK1/2  MAPK (mitogen-activated protein kinase) signaling pathway, and may contribute to ischemic preconditioning.120 In another study, it was shown that endothelial exosomal Mst1 (macrophage stimulating 1) inhibits autophagy and promotes apoptosis in cardiomyocytes under diabetic conditions, and inhibits glucose metabolism by disrupting the binding between Daxx (death domain associated protein) and GLUT4 (glucose transporter type 4).121There is an extensive literature demonstrating that cardiac endothelial cells are an important source of exosomes within healthy and diseased heart. Endothelial cell–derived exosomes containing miR-214 were shown to suppress senescence and stimulate angiogenic program in target mouse and human endothelial cells.122 Cardiac exosomes of endothelial and cardiomyocyte origin locally produced after myocardial infarction were shown to be taken up by monocytes infiltrating the ischemic heart and to subsequently increase their proinflammatory response.32 In this context, endothelium-derived EVs were enriched in VCAM-1 (vascular cell adhesion protein 1), localized to the spleen, and interacted with and mobilize splenic monocytes in myocardial infarction.123In cardiovascular disease, exosome-mediated communication between endothelial and immune cells is emerging as a key player at different stages of disease development.124Recently in their study, Qiao et al compared the treatment effects of cardiac exosomes from healthy donor hearts to those of cardiac exosomes from failing hearts. They demonstrated that pathological condition altered the miRNA composition of cardiac-derived exosomes and impaired their regenerative activities. Importantly, they identified miR-21-5p as a key factor contributing to exosome-mediated heart repair, enhancing angiogenesis and cardiomyocyte survival through the phosphatase and tensin homolog/Akt pathway.125 Kapustin et al reported that coagulation protein prothrombin can bind to the surface of vascular smooth muscle cell–derived exosomes and can also be loaded into exosomes by cellular internalization therefore regulating coagulation and calcification.126Interestingly, recent studies have identified exosomes as mediators of multiple forms of cardiovascular calcification, in both hyperphosphatemic and inflammatory milieus. Exosomes released by smooth muscle cells, valvular interstitial cells, endothelial cells, and macrophages may promote or inhibit mineralization, depending on the phenotype of their parental cells and extracellular environment to which they are secreted.+ T cells and improve wound healing postmyocardial infarction.127 It has been also reported recently that activated macrophages secrete mir-155–enriched exosomes inhibiting target cardiac fibroblast proliferation and enhancing inflammation during cardiac injury.128Another potentially important source of exosomes in the heart are immune cells such as dendritic cells or macrophages which are able to mediate immune responses on vesicle secretion. For example, Liu et al reported that exosomes derived from dendritic cells that have infiltrated infarcted myocardium could act as activators of CD4130 In a similar study, activation of the hypoxia-inducible factor pathway in pericytes was essential for the promotion of endothelial cell angiogenesis in an exosome-dependent manner.129 Conversely, inflammation-induced endothelial cell–derived EVs regulated expression of angiogenic genes in pericytes.131 Taken together, these results suggest that pericytes and endothelial cell communication essentially contribute to remodeling events and the flow of information via exosomes between pericytes and endothelial cells is bidirectional.Communication between pericytes and other cardiac cell types forms another major crosstalk in myocardial remodeling. When stimulated with hypoxic conditions, pericyte-derived conditioned media modulated proliferation of cardiac fibroblasts and macrophage.132Immortalization of cell lines to boost engineered EV production is a topic of great clinical interest as large amounts of EV are needed for clinical applications, eg, after myocardial infarction. Indeed, recently it was shown that genetic manipulation of β-catenin can converted deleterious fibroblasts into therapeutically-beneficial engineered novel cell entities that can produce EVs in high amount and strong functional efficacy.133 These data indicated that functional AT1Rs are carried by EVs elsewhere in circulation, providing a new paradigm to the traditional G protein–coupled receptor trafficking at a whole organism level.133 Interestingly, AT1R knockout mice, when administered with AT1R-enriched exosomes, showed improvement in blood pressure responsiveness to angiotensin II, providing functional evidence that exosomes can significantly mediate myocardial signaling and function.133 Li et al investigated the role of coronary serum exosomes and showed that exosomes from patients with myocardial ischemia had lower levels of miR-939-5p compared with healthy patients which promoted endothelial angiogenesis through the inducible nitric oxide synthase–nitric oxide pathways.134 Importantly, authors demonstrated that cardiomyocytes, but not cardiac fibroblasts or endothelial cells, were the source of bioactive exosomes in coronary serum.134 Another recent study found that long-term exercise–derived circulating exosomes conveyed cardioprotective signals and identified exosomal miR-342-5p as a novel cardioprotective bioactive factor against myocardial ischemia/reperfusion injury in rats.135Communication mediated by cardiac exosomes has also been described at distant microenvironments. In this line, recent investigations by Pironti et al performed in mice subjected to pressure overload revealed myocardial secretion of AT1Rs (angiotensin II receptor type 1) via exosomes into the circulation.136 Furthermore, exosomes mediate the transfer of muscle-specific miRNAs to bone marrow mononuclear cells, where muscle-specific miRNAs downregulate CXCR4 (C-X-C motif chemokine receptor 4) expression and mediate progenitor cell mobilization.136A growing interest in understanding the signal exchanges between myocardium and bone marrow led to the conclusion that exosomes may play an important role in these regulatory mechanisms. Interestingly, Cheng et al showed that circulating myocardial microRNAs are released after acute myocardial infarction into the circulation in exosomes and mediate functional crosstalk between the ischemic heart and the bone marrow.There are several major challenges in studying cell type–specific exosomes and their function in recipient cells locally or in distant target tissues, which we highlight within this paragraph.All currently used methods for exosome isolation and characterization have different advantages and disadvantages, and although these techniques are commonly used in practice, some issues are still unsolved Tables  and 2. IRapid advances in the identification of universal exosome-specific markers as well as parental cell–specific exosome components would enable the enrichment of this particular EV subtype through flow sorting or antibody-based isolation strategies ultimately enhancing our understanding of exosome biology. Importantly, such experimental progress would possibly allow researchers to costain parental cell–specific markers along with EV-specific markers to determine and compare different EV subpopulations secreted by investigated cell type.Mechanisms through which exosomes are loaded with selective molecules are still unclear. Existing evidence on exosome packaging and intracellular trafficking after internalization are mainly obtained from in vitro studies of specific cell types in the culture, which do not truly reflect physiological function. Studies using in vivo models of exosome trafficking are more reliable and will likely identify molecules involved in exosome biogenesis and uptake by recipient cells.137 do not represent the true physiology of cells in the myocardium and thus, often lead to poor interpretation of exosome biological functions. Furthermore, investigations of exosomes in vitro often involve inhibitors of exosome secretion enabling the study specific mechanisms regulating exosome release and clearance. One of the major limitations of these inhibitors is that they are known to affect the entire cellular secretome and other unrelated exosome and nonexosome pathways. Small molecule inhibitors or inducers of exosome-specific proteins that can be effectively used in vivo can shed better understanding of exosome biology in vivo. A major limitation in studying exosome distribution in vivo is the lack of suitable methodologies to investigate cell-specific exosome types secreted within the local tissue environment or into the circulation.Primary cultures of cardiomyocytes and cardiac fibroblasts, or other cardiac cells do not represent pure cultures, and typically have other unwanted cell types as contaminants. Moreover, commercially available cardiac cell lines as well as iPSC-derived cardiac cellsExosomes represent a precious biomedical tool as their molecular content is a fingerprint of the releasing cell type and its physiological state, and because they are detected in easily accessible body fluids such as blood and urine Figure .152 For instance, the amount of circulating microvesicles was found to be elevated in patients with cardiovascular risk factors such as diabetes.153 In the context of exosomal content reflecting different pathophysiological stages, recent studies showed that miR-1 and miR-133a serum levels are increased in patients with acute myocardial infarction, Takotsubo cardiomyopathy, and unstable angina pectoris.154 In vitro experiments confirmed exosomal origin of these miRNAs as stimulation of H9C2 myoblasts with a calcium ionophore led to elevated miR-133a expression within exosomes with the highest rate when cells were dead. This observation led to the conclusion that miR-133a secretion is connected to cardiac injury and cardiomyocyte death. Furthermore, a cardioprotective miR-214 originating from human endothelial cell–derived exosomes was identified in plasma of coronary artery disease patients and was shown to be upregulated in a mouse model of ischemic injury regulating cardiac survival.156 Interestingly, other studies showed that exosomal miRNAs can be used as biomarkers in early stages of acute myocardial infarction as well as in the prognosis of late stages in heart failure development. Matsumoto et al indeed identified miR-192, miR-194, and miR34a as predictors for heart failure development and ventricular remodeling.157Recently, several studies have shown that circulating EVs isolated from patients with cardiovascular disease differ not only in their molecular content compared with healthy subjects, but also in EV counts.These findings exemplify future prospects for exosomes to serve as diagnostic and prognostic markers. Furthermore, as elevated exosome levels have been shown to correlate with the severity of cardiovascular disorders, a potential approach would be to block exosome formation or secretion, thereby preventing signaling pathways mediated by exosomes.161In recent years, attention has been focused on the potential of stem cells to regenerate and repair the injured heart. A large number of studies, including one of the first investigations from our laboratory, are building a consensus that exosomes constitute a key paracrine component that mediate the reparative function of stem cells in the heart.165 cardiac stem cells,168 and endothelial progenitor cells.158 Besides multipotent/unipotent adult stem cell–derived exosomes, vesicles released by pluripotent stem cells  have been similarly investigated in the field of cardiac regenerative medicine. Exosomes mediate their cardioprotective effect often by acting directly on cardiac endothelial cells and promoting angiogenesis.170 Interestingly, vesicles purified from other biological sources, such as body fluids  and somatic cells  were also shown to represent a promising therapeutic option for various cardiovascular diseases.174Cells extensively studied for exosome-based cardiac repair include mesenchymal stem cells (MSCs),175 Additionally, in 2018, the European Society of Cardiology Working Group published a position paper providing a set of recommendations for the analysis and translational application of exosomes with the focus on the diagnosis and therapy of the ischemic heart.176Latest research and debates on cardiac repair and regenerative potential of exosomes from a variety of biological sources were discussed in the recent review.To use exosomes as therapeutics, vesicles can either be exogenously reengineered and modified to serve as a drug delivery tool, or can be used as the active drug themselves.177 These vesicles can either be isolated directly from body fluids and tissue explants or primary cell types prepared from them can be indirectly used to collect exosomes from conditioned media after cell culture  and alleviated cardiac fibrosis with improved cardiac function in a rat model of ischemia/reperfusion injury.178 Similarly, exosomes isolated from bone marrow–derived MSCs cultured under hypoxia were enriched with miR-125-5p and hypoxia exosomes treatment facilitated cardiac repair in a mouse model of myocardial infarction.179Cell manipulations aimed at enhancing beneficial properties of exosomes may include introduction of microenvironmental stimuli such as cell culture at lower oxygen levels Figure . Hypoxia180 Metabolic labeling involves supplementing cell culture medium with nonnative metabolites to introduce functional groups, such as azides, alkynes, thiols, methacryloyls, and ketones, to exosomes, which allows subsequent bio-orthogonal reactions to be performed.180Using genetic or metabolic engineering of cells or through cellular uptake of exogenous material, exosomes can be modified for function Figure . Genetic181 As an example, vesicle membrane can be permeabilized to allow active loading of molecules into the exosomes. Analogous approach uses lipophilic or amphiphilic molecules that can insert into the exosome membrane via hydrophobic interactions with the phospholipid bilayer.180 Exosome surface can also be modified chemically .180 In addition, exogenous material can be introduced to exosomes via liposomes or micelles that fuse with cytoplasmic membranes.Exosomes themselves can be chemically or biologically modified to broaden or enhance their therapeutic properties Figure .180,181 182 Using that approach, they repaired critical-sized calvarial bone defects and found that exosome/tricalcium phosphate incorporated scaffolds possessed better osteogenesis activity than pure biomaterial.182 In a similar fashion, exosomes obtained from human iPSC-derived MSCs integrated with acellular tissue patch for articular cartilage regeneration retained EVs better and positively regulated both chondrocytes and human bone marrow–derived MSCs in vitro.Combining exosomes with biomaterial scaffolds provides a promising strategy for efficient delivery of exosomes to a specific tissue type as well as to retain exosomes durably at the site of injury Figure . Zhang eWhile exosome-scaffolds may offer increased therapeutic retention at sites of tissue damage, it may also offer reduced off-target effects, which will need further investigation. Nevertheless, such scaffold-based exosome targeting approaches could provide new means of enhanced therapeutic efficacy for application of exosomes in cardiology.183 Owing to their nature, exosomes with a robust membrane may provide a protection against enzymatic and nonenzymatic degradation of their molecular content,183 and could have lower tumorigenicity and immunogenicity compared with direct transplantation of cells.85It is worth emphasizing that nonliving yet bioactive exosomes can exert unique biological activity reflective of their cellular origin that may be potentially applied for cardiovascular therapeutics. Exosomes are smaller, less complex, and more stable than parental cells, and are relatively easy to modify, manufacture, and store.160 Therefore, major challenge in developing exosome-based therapy is to determine whether tailored vesicles targeting specific cell types within the tissue  can be obtained and whether the short half-life of vesicles can be extended.160 It is crucial to assess the safety of exosome-based therapy in clinical settings, investigating their biodistribution, toxicity, thrombogenicity, and half-life times in vivo.184 In this line, the use of large animal models (over rodent models) should be encouraged for preclinical testing of the therapeutic efficacy of exosomes with careful considerations for clinical translation. Furthermore, careful exosome dose and dosage regimen must be determined along with the development of optimal procedures for efficient vesicle administration. Before bringing exosomes to clinic, it is also important to ensure that large-scale supply of exosomes is feasible in recommended safety level meeting the clinical requirement. It will be important to identify a robust source to obtain massive exosome production to increase the overall exosome yield with clinical-grade purity .Exosomes are internalized by cells relatively quickly in and around sites of delivery, often entering nonspecific cell types as well as systemic circulation.https://www.clinicaltrials.gov found 95 interventional clinical trials (both recruiting and not yet recruiting) involving exosomes or extracellular vesicles or microvesicles. The bulk of these exosome clinical trials are occurring within the United States and Europe. Interestingly, few of these studies involve cardiovascular interventions .Exosomes should go through proper clinical trials to be proven safe and effective before being marketed to patients. A search on 151 Even though the primary end point was not reached, this clinical trial confirmed the efficacy of injected dendritic cell–derived exosomes in enhancing natural killer cell immune responses in patients with advanced nonsmall cell lung cancer.151 Unfortunately, vast majority of exosome clinical trials are still in progress and respective study outcomes are not yet available online or published. Therefore, whether clinical trials involving administration of exosomes will become successful remain to be seen in the near future.As exosomes become recognized for their benefits as cell-free therapeutics, exosome clinical trials are increasing in number. An example of a published and completed clinical phase II trial can be found in the work done by Besse et al as a continuation of 2 previous phase I trials.Exosomes show great potential to improve both diagnosis and therapy within cardiovascular medicine, but many questions remain unanswered.Because the outcome of any exosome experiment can be biased by choices made , it is critical to comprehensively study protocols for exosome isolation. Furthermore, standardizing the morphological, biochemical, and biological characterization methods for exosome preparations, preferably by using a combination of several methods Table , can impWhile exosome-based biomarkers offer great potential in the development of unique signatures for various cardiovascular diseases, a greater understanding of exosome biology is needed before we can draw any strong conclusions. Our knowledge of exosome synthesis, cargo-loading, and uptake pathways are limited, and as our knowledge of the basic science increases, our ability to make use of exosomes in diagnostics and therapeutics should be improved.Studies conducted so far have indicated that exosomes derived from various types of progenitor cells exert cardioprotective and regenerative effects in different experimental settings, yet translation to clinical practice would require further exploration of this phenomenon. Chemical or biological modification strategies offer exciting opportunity to extend therapeutic capability of exosomes beyond their native function, most likely by improving their half-life and efficiency of cargo delivery to target tissues, and maintaining progress particularly in this area may substantially advance the field.Exosome-based therapeutics hold great promise for the development of therapy aimed at regenerating and repairing heart after pathological damage. However, recent excitement and enthusiasm over these powerful cell-free extracellular components should not mask the significant research efforts that still need to be taken to advance our understanding of cardiovascular exosomes and to translate them into the clinic. Despite these challenges, the emerging field of exosome signaling offers many opportunities for cardiovascular medicine.The authors thank Jill Gregory, Associate Director of Instructional Technology at Icahn School of Medicine, for the help with illustrations Figures  and 3.This work was supported by National Institutes of Health grants R01HL140469, R01HL124187, and R01HL148786 and New York State Stem Cell Science C32562GG (to S.S.), and by the German Research Foundation (DFG Th903-20-1) and the European Research Council Consolidator grant Longheart (to T.T.).Dr Thum has filed and licensed patents in the field of noncoding RNAs; is founder and shareholder of Cardior Pharmaceuticals GmbH; and reports speaker fees or other support from Boehringer Ingelheim, Novo Nordisk, Amicus Therapeutics, Sanofi-Genzyme, and Takeda (outside the scope of this review). Dr Sahoo has filed patents in the field of exosomes for cell and gene therapy. The other authors report no conflicts.


Of increasing prevalence, diabetes is characterised by elevated blood glucose and chronic inflammation that precedes the onset of multiple secondary complications, including those of the kidney and the eye. As the leading cause of end stage renal disease and blindness in the working population, more than ever is there a demand to develop clinical interventions which can both delay and prevent disease progression. Connexins are membrane bound proteins that can form pores (hemichannels) in the cell membrane. Gated by cellular stress and injury, they open under pathophysiological conditions and in doing so release ‘danger signals’ including adenosine triphosphate into the extracellular environment. Linked to sterile inflammation via activation of the nod-like receptor protein 3 inflammasome, targeting aberrant hemichannel activity and the release of these danger signals has met with favourable outcomes in multiple models of disease, including secondary complications of diabetes. In this review, we provide a comprehensive update on those studies which document a role for aberrant connexin hemichannel activity in the pathogenesis of both diabetic eye and kidney disease, ahead of evaluating the efficacy of blocking connexin-43 specific hemichannels in these target tissues on tissue health and function.  Impacting almost 10% of adults, diabetes is a global healthcare concern that affects an estimated 463 million people worldwide. With the prevalence of diabetes expected to rise to 700 million people by 2045 , retinopIn diabetes, complications develop in response to sustained hyperglycaemia and low-grade systemic inflammation, the latter of which is heightened in type 2 diabetes mellitus (T2DM), where coupled with obesity, increased adipose tissue secretes inflammatory mediators that exacerbate a state of pre-existing inflammation ,16,17,18Connexins are a family of membrane bound proteins involved in the transfer of small molecules and ions between two cells (gap junctions) and between cells and their immediate environment (hemichannels), highlighted in Instrumental to the pathogenesis of diabetes and its complications, targeted anti-inflammatory therapy has been suggested for both prevention and treatment of diabetes and has been extensively reviewed ,55,56. KThe NLRP3 inflammasome has been referred to as the ‘grumpy old man of inflammation’  and is lDespite these encouraging observations, blanket blockade of a complex integral part of the innate immune response has been met with concern. Activated by both damage-associated molecular patterns (DAMPs) and pathogen associated molecular patterns (PAMPs), the NLRP3 inflammasome mediates both sterile and non-sterile inflammation . ConsequWhilst evident that there is much to learn in our quest to develop new interventions that successfully (a) target sterile inflammation and (b) do so in the absence of serious side effects, recent FDA approval of SGLT2 inhibitors has perhaps been the most significant step forward in managing and improving outcomes in patients with nephropathy  and cardWith evidence that Cx43 hemichannel mediated communication contributes to the pathogenesis and progression of tissue damage in secondary complications of diabetes ,107,108,+/− mouse induced with NTS-GN, proteinuria, blood urea nitrogen (BUN) and serum creatinine levels are reduced [Diabetic nephropathy is widely regarded as a glomerular disease, where proteinuria is the predominant early clinical marker . Signs o reduced . Similar reduced . This pr reduced . Mediate reduced . Paracri reduced .+/− UUO mouse model [+/− UUO mouse where disassembly of the adherens  and tight  junction complexes were significantly blunted as compared to wild-type control [Although a glomerular disease in origin, advanced stages of nephropathy are characterised by severe tubule interstitial inflammation and fibrosis . Work wise model . Blockinse model . Not sur control . Whilst In contrast to the observations above, Sun et al. recently suggested that Cx43 expression is downregulated in diabetic kidney disease and that overexpression of Cx43 using short hairpin RNAs attenuates renal fibrosis and reduces epithelial-to-mesenchymal transition (EMT) in a carboxyl-terminal signal transduction-dependent manner in leptin receptor-deficient type 2 diabetic (db/db) mice and in rat kidney NRK-52E cells treated with high (30 mM) glucose . They atPeptide 5 is a connexin peptidomimetic that mimics a portion of the 2nd extracellular loop of Cx43  and has +/− model [N-cadherin, fibronectin and collagen IV as compared to cells uncoated control. Interestingly, co-incubation of TGFβ1 treated cells with Peptide 5 significantly blocked the increase in hemichannel mediated dye uptake and ultimately restored expression of markers of tubular injury to levels representative of control cells cultured on plastic. Moreover, Peptide 5 blocked TGFβ1 induced secretion of collagen I [+/− mouse [Instrumental to cell adhesion and maintenance of polarity, disassembly of cell junction complexes is linked to partial EMT, events which predispose inflammation and fibrosis , the lat/− model , we hypollagen I , corrobo/− mouse  and high+/− UUO mouse presents with decreased fibroblast activation and diminished macrophage infiltration as compared to wild type UUO control [+/− UUO mouse [Building on our published observations with Peptide 5, we recently assessed the efficacy of Danegaptide in conferring protection in an in vitro model of tubular injury. A Cx43 gap junction modifier ,134, we  control , we empl control . Soluble control . However control ,170), in control ). In supUO mouse , work byUO mouse . The finDiabetic retinopathy affects around one third of people with diabetes and is the primary contributor to blindness in the working age population , often rLoss of vision in retinopathy is associated with breakdown of the retinal pigment epithelium (RPE), where periods of sustained hyperglycemia drive inflammation and apoptosis through increased secretion of key inflammatory mediators, growth factors and hypoxia-inducible factors . This inIn targeting this damage, it is important to understand how it manifests itself. Interestingly, the series of events which drive proliferative diabetic retinopathy are of a similar aetiology to those which we see in late-stage diabetic kidney disease . As withBoth in vitro  and in vSince the NLRP3 inflammasome is an integral mediator of our innate immune response, the link between aberrant Cx43 hemichannel mediated ATP release and activation of the NLRP3 inflammasome was further explored in vitro, where NLRP3 complex assembly, caspase 1 activation and IL1β secretion were blocked in treated ARPE-19 cells co-incubated with Peptide 5 . SimilarWhilst the evidence of a role for Cx43 hemichannels in driving the pathology of diabetic retinopathy is undeniable, loss of tissue function in response to altered GJIC has also been reported. Under conditions of glycaemic injury, Cx43 gap junction coupling is reduced in retinal capillaries from people with diabetes ,186, ratRecent statistics from Eurostat suggest that 28% of Europeans will be aged 65yr and older by 2060, with estimates predicting the average UK life expectancy of women to be 91yr and men 88yr by 2030 . With th


Porcine reproductive and respiratory syndrome virus (PRRSV) affects the global swine industry and causes disastrous economic losses each year. The genome of PRRSV is an enveloped single-stranded positive-sense RNA of approximately 15 kb. The PRRSV replicates primarily in alveolar macrophages of pig lungs and lymphatic organs and causes reproductive problems in sows and respiratory symptoms in piglets. To date, studies on how PRRSV survives in the host, the host immune response against viral infections, and pathogenesis, have been reported. PRRSV vaccines have been developed, including inactive virus, modified live virus, attenuated live vaccine, DNA vaccine, and immune adjuvant vaccines. However, there are certain problems with the durability and effectiveness of the licensed vaccines. Moreover, the high variability and fast-evolving populations of this RNA virus challenge the design of PRRSV vaccines, and thus effective vaccines against PRRSV have not been developed successfully. As is well known, viruses interact with the host to escape the host’s immune response and then replicate and propagate in the host, which is the key to virus survival. Here, we review the complex network and the mechanism of PRRSV–host interactions in the processes of virus infection. It is critical to develop novel antiviral strategies against PRRSV by studying these host–virus interactions and structures to better understand the molecular mechanisms of PRRSV immune escape. Porcine reproductive and respiratory syndrome virus (PRRSV) has undoubtedly become a substantial financial issue that has affected pig production and caused substantial economic losses for the swine industry worldwide since its outbreak in the 1980s . Annual As a member of the Nidovirales order in the Arteriviridae family, PRRSV is an enveloped virus with an average diameter of 55 nm; it is a positive-stranded RNA virus with an approximately 15 kb genome with a 5′ cap and a 3′ poly A tail ,6. ExcepThe PRRSV is mainly divided into two genotypes: type 1 (represented by the European strain Lelystad Virus) and type 2 (represented by the North American strain VR-2332), and both PRRSV genotypes have only 50–60% nucleotide identity . In addiNumerous studies have been performed to learn more about the biochemistry of the PRRSV. The process of the virus entry into its host cell is the first crucial step in the infection and the focus of basic research . PRRSV hIn the early stages, PRRSV enters the macrophage via a receptor-mediated method ,57. PRRSThe genome released by the virus was identified as the template used to translate into pp1a and pp1ab, and after that the proteases hydrolyze and cleave polyproteins into mature nonstructural proteins as described above . The virThe complex networks of virus–host interactions are essential in the overall process of PRRSV entry and infection. The viral structural proteins interact with host receptors to mediate viral entry as described above. The interactions of host and viral nonstructural proteins can exert an influence on the replication and transcription of viral genomes. The virus’s invasion causes a series of immune responses, and then virions escape from the host immune system to favor their own replication by interacting with the host. Only by better understanding the molecular mechanism of PRRSV immune evasion and modulation can we design more effective vaccines. Further studies of these host–virus interactions are critical to the development of novel antiviral strategies against PRRSV.Innate immunity is the first line of defense without memory and specificity, and interferons (IFNs) are one of the earliest cytokines and major elements of the host fighting with virus invasion, as shown in In highly pathogenic (HP) PRRSV-infected swine, the cytokines including interleukin (IL)-1, IL-6, IL-8, IL-10, and tumor necrosis factor (TNF)-α are significantly increased ,70. The Tripartite motif (TRIM) proteins, as critical components of the innate immune system, play significant roles in fighting virus invasion for mammalian cells, and can regulate multiple cellular processes including transcription-dependent antiviral responses such as cytokine-mediated or autophagy-mediated antiviral modulation ,74,75. TMicroRNAs (miRNAs) are small non-coding RNA molecules containing about 21 nucleotides in length, and they play key roles in the complex networks of PRRSV–host interactions ,77. MorePRRSV depends on host factors to complete genome replication and interacts with host molecules for its survival and reproduction. The host immune system will take some measures to suppress the virus replication during virus invasion. Besides the abovementioned host factors, other cellular components are also involved in interacting with PRRSV. The cellular protein nucleoporin 62 (Nup62) interacts with nsp1β, leading to inhibition of host antiviral protein expression, revealing a new strategy of immune escape . In addiIn conclusion, a complex network of PRRSV–host interactions exists throughout the virus cycle, including virus entry, replication, and infection, as shown in Since the outbreak of PRRSV in the 1980s, efforts have been made vehemently to find effective antiviral strategies and vaccines to control its damage to the swine industry. However, for a variety of reasons, this has become a huge challenge. Modified-live virus (MLV) vaccines and inactivated vaccines against PRRSV are available and used worldwide . Both noClassically attenuated viruses are created by passaging viruses in cultured cells and are effective for many viruses. However, many safety issues exist for these empiric attenuations . FurtherMany viral proteins, such as nsp1α, nsp1β, nsp2, nsp4, nsp7, nsp10, and nsp11, play an essential role in suppressing the host innate immune response. These key proteins can be used as antiviral targets for attenuated vaccines. For example, the nsp4 D185N mutant exhibits a slower replication rate and a higher ability to induce IFN-I expression compared with WT PRRSV . AdditioFurthermore, the identification of miRNAs that target the PRRSV genome provides alternative targets for gene editing . With thSubunit peptide vaccines reproducing the B and T epitopes are a novel antivirus strategy . CytotoxThe review of the PRRSV–host interactions network helps us to understand the function of critical host factors involved in virus infection. More research is still needed to complete this complex network and provide the basis for the development of antiviral drugs and vaccines.PRRSV is undoubtedly one of the most economically devastating swine pathogen, and has aroused sustained concern. In spite of constant efforts of the pathogenicity and immunology of PRRSV, it has always been a substantial challenge to design effective vaccines and drugs. MLV vaccines exist some safety issues. Compared with MLV vaccines, DIVA vaccines can distinguish PRRSV-specific antibodies produced by natural infection with WT viruses or by vaccination, and has become an important aim of many current research efforts. The acquisition of gene-edited pigs will be a potential antiviral strategy, but it’s a problem in acceptability of genetically modified foods. Structural information allows us to better understand the mechanisms of viral infection. A structure-based vaccine design approach may improve the antigenicity and create immunogens capable of eliciting robust neutralizing and protective immune responses.The complex network of PRRSV–host interactions exists throughout the virus cycle. Only by studying the function of the PRRSV–host interactions in the overall process of PRRSV entry and infection, can we understand mechanisms of PRRSV replication and propagation, thereby providing the basis for antiviral strategies. Some host factors and viral proteins play important roles in the PRRSV–host network, but lack of structural information prevents us from understanding the mechanisms of infection thoroughly, Therefore, these host factors and viral proteins need to be explored to provide structural bases and new insights for treatments against PRRSV.


While current therapies for chronic HBV infection work well to control viremia and stop the progression of liver disease, the preferred outcome of therapy is the restoration of immune control of HBV infection, allowing therapy to be removed while maintaining effective suppression of infection and reversal of liver damage. This “functional cure” of chronic HBV infection is characterized by the absence of detectable viremia (HBV DNA) and antigenemia (HBsAg) and normal liver function and is the goal of new therapies in development. Functional cure requires removal of the ability of infected cells in the liver to produce the hepatitis B surface antigen. The increased observation of transaminase elevations with new therapies makes understanding the safety and therapeutic impact of these flares an increasingly important issue. This review examines the factors driving the appearance of transaminase elevations during therapy of chronic HBV infection and the interplay of these factors in assessing the safety and beneficial nature of these flares. Exposure to the hepatitis B virus (HBV) causes a predominantly hepatotropic infection, which in its acute state commonly causes liver inflammation and dysfunction and in its chronic state fibrosis, cirrhosis and hepatocellular carcinoma (HCC) ,2,3. WhiThe current challenge in the treatment of chronic HBV infection is to restore immune control allowing suppression of viral infection to be maintained in the absence of therapy, thereby allowing liver damage to regress and the risk of developing hepatocellular carcinoma to decline. This “functional cure” of chronic HBV infection requires the reconstitution of immune control and is characterized by the absence of circulating HBV surface antigen (HBsAg) without therapy ,18,19,20Since the clearance of hepatocytes with integrated HBV DNA from the liver is a critical milestone in achieving functional cure, the resulting damage in the liver is also expected to result in elevations in serum concentrations of liver enzymes. This creates a clinical challenge as liver enzyme flares are known to occur with a variety of other liver diseases where they are associated with hepatoxicity. Given the current focus on achieving a functional cure of HBV with new therapeutic agents, this review explores how liver enzyme flares during the treatment of chronic HBV infection affect liver function and the potential utility of these flares to predict positive therapeutic outcomes.During acute  infection, HBV infection appears infrequently in individual hepatocytes where HBsAg is located primarily in the cytoplasm . These cIn chronic HBV infection, numerous studies have confirmed the heterogenous pattern of infected hepatocytes in chronic HBV infection  where inAlthough HBV infection is not directly cytopathic, HBV infection of hepatocytes is followed by extensive metabolic reprogramming. These effects are summarized in Alanine aminotransferase (ALT), also known as glutamic–pyruvic transaminase (GPT), is a cytosolic enzyme involved in gluconeogenesis that catalyzes the amination of α-ketoglutarate from alanine to produce pyruvate and glutamate. In humans, two distinct ALT isozymes are produced from distinct but related genes, ALT1 and the more recently characterized ALT2 , which bAspartate aminotransferase (AST), also known as glutamic oxaloacetic transaminase (GOT), catalyzes the reversible transfer of an amino group between aspartate and glutamate. There are two AST isozymes dervied from distinct genes which produce the ALT forms found either in the cytoplasm (AST1) or mitochondria (AST2) . These iγ-glutamyl transferase (GGT) cleaves the γ-glutamyl moiety from glutathione and transfers this moiety to a variety of acceptors (including water to form glutamate). There are two GGT genes (GGT 1 and GGT 2), each producing GGT precursor protein; however, only the GGT1 precursor undergoes autocleavage to become catalytically active . GGT GG.In subjects with previously characterized viral hepatitis, elevations of ALT, AST and GGT in the blood are driven by release of these enzymes from cells in the liver. This release can occur via rupture of plasma membrane blebs formed in response to cellular stress or inflammation , from ce9 copies/mL (2.66 × 108 IU/mL) [Definitions of clinically significant transaminase elevations including flares range from >2–5x the upper limit of normal (ULN) ,78,79,808 IU/mL) . ALT ele8 IU/mL) ,94 conco8 IU/mL)  and inte8 IU/mL) .From the initial establishment of HBV infection in the liver and its spread during the evolution of chronic HBV infection, elevations in liver enzymes are driven by hepatocyte damage and loss from: (1) the increased apoptotic sensitivity in hepatocytes resulting from HBV infection, (2) inflammation driven by the innate immune response to HBV infection and (3) specific targeting of infected hepatocytes by the HBV specific adaptive immune response, both B-cell  and T-ceThe control of liver enzyme elevations is an important consideration during the management of chronic HBV infection as they can lead to hepatic decompensation, which is of special concern in patients with advanced fibrosis and cirrhosis who have only a fraction of the normal reservoir of hepatic function. As such, transaminase elevations, even if they may be beneficial, are generally recognized as an important indicator to initiate antiviral therapy to control liver dysfunction and the progression of liver disease. Current treatment guidelines with approved therapies ,71,72 inThe guanine nucleoside analog entecavir (ETV), the adenosine nucleotide analog tenofovir disoproxil fumarate (TDF) and its more recent derivative tenofovir alafenamide (TAF) are the currently recommended NUC therapies  for the Transaminase elevations often accompany removal of NUC therapy in HBeAg negative patients ,122. WitPegylated interferon (pegIFN) is a multifunctional immunotherapy that stimulates both innate ,130 and Thymosin alpha 1 is synthetic form of a naturally occurring thymic peptide hormone that acts primarily as a T-cell agonist ,141,142.These oligonucleotide-based compounds are designed to target the cleavage of HBV mRNA via the RISC complex (in the case of RNAi) or via RNAse H (in the case of antisense). The specificity and activity of both these classes of oligonucleotides is strictly dependent on the perfect homology between the single-stranded (antisense) or double-stranded (RNAi) oligonucleotide and the target region of the mRNA. While in vivo data have been consistent with this mechanism, clinical studies with a variety of these compounds have indicated that off-target stimulation of innate immunity is playing a role in the clinical responses observed ,145. LimNucleic acid polymers are broad spectrum antiviral agents  that selThe benefit/risk of transaminase flares in the presence of cirrhosis is an important consideration as cirrhotic patients represent a small but significant proportion of patients of HBV monoinfection , with faHowever, recent data have demonstrated that transaminase flares are beneficial in cirrhotic patients who achieved HBV suppression on NUC therapy, with increased HBeAg seroconversion, reduced incidence of ascites, esophageal varices, splenomegaly and death or liver transplantation and were not accompanied by biochemical flare or hepatic decompensation . MoreoveWith the overall goal of functional cure requiring the removal of hepatocytes with integrated HBV DNA and active cccDNA, transaminase flares are likely an essential milestone in this process and will be required for the achievement of HBsAg loss with currently approved therapies and therapies in development. The current clinical data indicate that with maintenance of viral suppression, transaminase flares appear to be universally host-mediated, do not negatively impact liver function and are associated with both improvement in virologic status and liver function and correlated with increased rates of functional cure of HBV, even with cirrhosis. As such, viral suppression during experimental therapies (such as NAPs) which yield both high rates of transaminase flares and functional cure should be accompanied by NUCs such as ETV, TDF or TAF to ensure safety during therapy.However, higher-frequency monitoring of transaminases, GGT as well as markers of liver function should continue to be both essential data gathered during clinical trials for agents in HBV and be reported in detail to increase the available clinical database, especially during treatment of cirrhotic patients. Of important note here is GGT; although it is not considered as hepato-specific as ALT and AST, its elevation in the blood can signal effective targeting of infected cells in the bile duct epithelium and endothelial and smooth muscle cells of hepatic blood vessels. The reporting of changes in this liver enzyme should be included not only to indicate the targeting of non-hepatocyte reservoirs of HBV infection in the liver but also to better exclude a link between GGT elevations and other potential safety concerns.In the interim, the current clinical experience with transaminase flares indicates a common approach for interpreting these events with all antiviral agents . Transam