Small but significant: Insights and new perspectives of exosomes in cardiovascular disease

Abstract Cardiovascular diseases (CVDs) are a major health problem worldwide, and health professionals are still actively seeking new and effective approaches for CVDs treatment. Presently, extracellular vesicles, particularly exosomes, have gained its popularity for CVDs treatment because of their function as messengers for inter‐ and extra‐cellular communications to promote cellular functions in cardiovascular system. However, as a newly developed field, researchers are still trying to fully understand the role of exosomes, and their mechanism in mediating cardiac repair process. Therefore, a comprehensive review of this topic can be timely and favourable. In this review, we summarized the basic biogenesis and characterization of exosomes and then further extended the focus on the circulating exosomes in cellular communication and stem cell‐derived exosomes in cardiac disease treatment. In addition, we covered interactions between the heart and other organs through exosomes, leading to the diagnostic characteristics of exosomes in CVDs. Future perspectives and limitations of exosomes in CVDs were also discussed with a special focus on exploring the potential delivery routes, targeting the injured tissue and engineering novel exosomes, as well as its potential as one novel target in the metabolism‐related puzzle.

a high demand for developing new therapies in CVDs treatment.
Recently, the utilization of paracrine principle to regenerate ischaemic tissues by mediating cellular communications for CVDs treatment has gained its popularity, due to its effectiveness of recovering cardiac function after the initial infarction. 5 Among those paracrine signalling factors playing a role in cardiac repair, extracellular vesicles (EVs) specifically draw increasing interest because of their ability in regulation of cellular communication resulting in the promotion of cellular function.
Extracellular vesicles were first considered as cellular membrane debris without biological significance, until Rapose reported that EVs played a role in the simulation of immune response. 6 Since then, studies have shown the importance of EVs in cellular communication, and the identification of EVs gained growing interests. Both microvesicles and apoptotic bodies were identified as EVs that play a role in the biological processes. 7,8 In 1983, another small-sized EVs were discovered first time in rat reticulocytes and later were named as exosomes by Raposo et al 9 Initially, exosomes were considered cell 'dumpsters' that contain undesirable cellular waste to maintain cellular homeostasis. Later studies indicated that exosomes are involved in intercellular connection 10 and play an important assignment in multiple physiological and pathological processes. 11 As a result, exosomes, as a biocomponent, have been intensively investigated.
The exosome is typically classified as an EV with a diameter of 40-150 nm, 12 which are endosome-derived, and secreted by most, if not all, cells. They normally contain lipids, proteins and various RNA species (including mRNA, miRNAs and lncRNAs), 13 depending on the cell type and the cellular microenvironment ( Figure 1). Additionally, the exosomes released by the same cell line can be heterogeneous due to the different cargo-sorting mechanisms. Through releasing the proteins and nucleic acids, exosomes can mediate local as well as systemic cell-to-cell communications by intervening with cellular physiological change. However, the mechanisms of the exosome-stimulated signalling pathway remain unclear. A previous study indicated that this process involves receptor-ligand interactions and exosomal internalization, resulting in the release of cargo into the cytoplasm of recipient cells. 14 Because of the vital biocomponent with exosome contributing to intercellular and extracellular communications, scientists have been postulating if the exosome may mediate pathological pathways in cardiac tissue. Experiments conducted by Barile 15 and Ibrahim 16 showed that cardiac progenitor cells (CPCs) and human cardiospheres (CSp) generated EVs with enriched anti-apoptotic and proangiogenic miRNAs such as miR-210, miR-132 as well as miR-146a, which can up-regulate angiogenesis of endothelial cells and recover left ventricle (LV) function of the post-ischaemic heart. Similar studies used mouse CPCs and fibroblast-induced pluripotent stem cells (iPSC) to isolate exosomes for cardioprotection and anti-apoptosis.
Another study extracted exosomes from mouse embryonic stem cells (ESCs) and identified that miR-294 is the main driven force to augment CPCs and cardiomyocytes proliferation-based endogenous cardiac repair. 17 All those studies suggest that exosomes hold a great clinical potential in CVDs treatment.
However, despite significant research attention and developments in exosomes and their potential applications in CVDs treatment, there is a lack of an informative summary on therapeutic and diagnostic functions of exosomes in CVDs, especially the inter/extra-cellular communication between the cardiovascular system and other organs mediated through exosomes. The aim of this review is to provide a detailed overlook of the exosomes and their roles in cellular communication, which lead to their ability in diagnosing and treating CVDs. First, an overview of the fundamental biogenesis and F I G U R E 1 Schematic image for cell secreted or circulating exosomes. A, A cell works like a factory secreting factors and extracellular vesicles, and exosomes are secreted like packages with specific proteins and exRNAs. B, TEM analysis on circulating exosomes(red arrows indicating exosomes). Scale bar = 200 nm. C, The distribution on size of exosomes analysed by Nanoparticle tracking analysis (NTA). D, Western blotting analysis on the surface makers as CD63 and CD81 of exosomes characterization of exosome is discussed. Next, we describe the role of exosome in cardiac tissue communication in details with a special focus on stem cell-derived exosomes in infarcted heart, which provides the underlined working mechanism of exosome. We then review the interaction between heart and distant organs through exosomes, together with the diagnostic and therapeutic role of exosomes in CVDs using the most recent studies to showcase the progression of exosome-based therapy in current clinical applications.
Finally, future perspectives and current limitations of exosome in CVDs treatment are discussed. We believe that there is much progress that can be made as the understanding of exosomes are further advanced and hope that this review-by introducing the mechanism of exosome-mediated cellular communication within cardiac tissue or through circulatory system-will help to promote the development exosome-based therapy in CVDs diagnosis and treatment.

| Biogenesis of exosomes
As nano-sized particles, exosomes carry many vital biocomponents that mediate their function and represent a unique class of EVs by virtue of their biogenesis. The biogenesis of exosomes involves different steps. Early endosomes must mature into late endosomes or multivesicular bodies (MVBs); then, the invagination of the plasma membrane allows for the generation of intraluminal vesicles (ILVs) in the lumen of organelles. 18 MVBs further fuse with the plasma membrane to release ILVs into the extracellular matrix; the MVBs are called exosomes at this stage. Rab GTPases 27A and 27B are reported to be the two components that can mediate the fusion and docking of MVBs to the plasma membrane, thus promoting exosome production. 19 The process of sorting cargo within exosomes involves endosomal sorting complexes required for transport (ESCRT) and tetraspaninand lipid-dependent mechanisms. 20 ESCRT-0, ESCRT-I, ESCRT-II and ESCRT-III protein complexes play roles on producing the ILVs that bud into MVBs and in sorting monoubiquitinated proteins via an ESCRTdependent mechanism. For example, the ESCRT-III-associated protein ALIX can affect cargo loading and MVB subtypes. 21 The protein Hrs within ESCRT-0 is involved in exosome secretion. 22 Tetraspanins such as CD82, CD9 and CD63 are factors involved in the ESCRTindependent process of sorting exosome cargo. 23 Studies have indicated that CD9 and CD82 can up-regulate the exosomal release of β-catenin from HEK293 cells. 24 Furthermore, the knockout of CD63 can reduce the secretion of EVs, which proves the key role of CD63 in the exosome biogenesis process. 25  GTPases, depending on their endosomal origin. Moreover, the resulting protein profiles of exosomes from the same cell type can be discriminated, and these differences are always dependent on the microenvironment and the physiological states of the parent cells. 28 Tetraspanins (CD9, CD63 and CD81), 14-3-3 proteins, heat shock proteins (HSPs), Tsg101 and the ESCRT-3-binding protein Alix, which are abundant in EVs, could be used as specific markers. 29 Tetraspanins were previously considered as specific markers for exosomes, but these proteins could be found in apoptotic bodies and microvesicles. 30 Additionally, cellular signalling proteins such as β-catenin, TNFα, Wnt5, TGF-β, delta-like 4 and Notch ligand can be found within exosomes. Cytoskeletal and metabolic proteins (glyceraldehyde 3-phosphate dehydrogenase, GAPDH) have also been identified in cargo. 31 Interestingly, Jeppesen Apart from proteins, lipids are critical in the vesicular transportation; however, the lipid component in the cargo is still unclear. 33 Some lipid molecules that participate in exosome biogenesis, such as lysobisphosphatidic acid, have been found in exosomes. 34 Additional lipid raft molecules, such as cholesterol, sphingolipid ceramide and glycerophospholipids, are also found in the cargo. Furthermore, lipid molecules that can mediate cellular signalling pathways, such as prostaglandins, rearrange within exosomes. 35 The most important components of exosome cargo are cell type-specific mRNAs and miRNAs. miRNAs are well known to play a crucial role in the regulation of multiple biological processes. As a result, those small non-coding RNA molecules encapsulated within exosomes can mediate cell-to-cell communication, which suggests potential subsequent bioapplications. Exosome-encased miRNAs, such as miR-292, miR-20a, miR-17 and miR-22, are involved in CVDs, and miR-21 is abundant in cardiac fibroblast-derived exosomes. 36 These miRNAs have important roles in cell signalling in cardiac tissue. Interestingly, one quantitative and stoichiometric approach was applied to analyse the miRNA content in exosomes regardless of the source; the results indicated that most individual exosomes in standard preparations do not carry biologically significant numbers of miRNAs. 37 Hence, the accurate role of miRNAs in exosomes needs to be further exploited. Compared to miRNAs, lncRNAs are more tissue-specific and have been identified as critical mediators of cardiac remodelling and valuable diagnostic markers. For example, the exosomal lncRNAs could take part in the mediating of ageing-induced cardiac dysfunction.

| Isolation and characterization of exosome
Traditional exosome isolation methods comprise ultracentrifugation, density sucrose or iodixanol gradient centrifugation, precipitation, immune-affinity capture and so on. However, the distinctions among different EV subgroups need to be standardized resulting from the slight diversity in physical properties and composition. Thus, although the specific subtype of EVs known as exosomes has attracted a large amount of attention, the definitive characterization of exosomes has proven to be elusive due to the heterogeneity of EV species and the assortment of non-specific isolation techniques. 12 Notably, Jeppesen et al 32 recently reported a more accurate method for exosome extraction that used high-resolution density gradient fractionation to separate small EVs from non-vesicular material and direct immunoaffinity capture to specifically isolate exosomes from other types of small EVs.
The characterization of exosomes has been a challenge due to their nano-scale size. Additional creative and advanced approaches need to be applied to characterize exosomes. Antibody-based methods are popular, whereby proteins such as CD9, CD63 and CD81 can be applied as specific markers because of the endosomal pathway.
Additionally, the expression of phosphatidylserine in exosomes allows other methods to also be used to characterize exosome populations. 38  tify exosomes among all vesicles. Flow cytometer may be able to detect the existence of exosome but fails to quantify the particles because of swarming effects. 40 Other techniques, such as atomic force microscopy, 41 Raman microspectroscopy, 42 small-angle X-ray scattering 43 and field emission scanning electron microscopy, 41 have been also applied to detect exosomes. To date, it is widely accepted that isolated exosomes need to be characterized for their specific markers (CD9, CD63, CD81), their sizes (NTA) along with their morphologies (TEM) together in order to confirm their identity.

| Exosomes in cardiac cell-cell communication
The production of exosomes allows cells to communicate by interacting with or taking up exosomes from other cells in cardiac tissue ( Figure 2), given the fact that the various RNA species, lipidS and proteins within exosomes can involve in the transcription and translation process resulting in the regulation of cellular proliferation and function. 44 In cardiac tissue, studies have shown that exosomes can mediate the communication between endothelial cells, smooth muscle cells, cardiomyocytes, monocytes, dendritic cells and fibroblasts. 45,46 For example, activated macrophage-derived exosomes containing miR-155 have been shown to decrease the fibroblast proliferation and promote fibroblasts inflammation, when cardiac tissue is injured. 47 In addition, exosomes derived from mature dendritic cells increase endothelial inflammation and atherosclerosis via the membrane TNF-α-mediated NF-kB pathway. 48 Other evidence showed that the crosstalk between cardiomyocytes and endothelial cells involves cardiac exosomes.

F I G U R E 2
Exosomes mediated effect on cardiac microenvironment. Cardiac-related exosomes could have several effects on recipient cells. At the original cardiac tissue site, the microenvironment could be regulated by the complex interaction with exosomes derived from surrounding cells, including cardiomyocytes, macrophages, cardiac fibroblasts and other cardiac cells. The secretion of exosomes (exRNAs, proteins, etc) may directly participate in extracellular matrix (ECM) remodelling involved in the inflammatory process in the local area, cell apoptosis, fibroblasts differentiation, cardiac hypertrophy, new vessel formation and so on HIF-1α can be found in the exosomes released by cardiomyocytes under hypoxic conditions; this protein can up-regulate the expression of Hsp20 49 and promote angiogenesis by increasing the expression of vascular endothelial growth factor receptor-2 in endothelial cells. 50 Another finding showed that cardiac fibroblast-derived exosomes contain abundant miRNA fragments (such as miR-21*) that are normally eradicated during the miRNA biogenesis process can induce cardiomyocyte hypertrophy to achieve the crosstalk between cardiac fibroblasts and cardiomyocytes. 10 Acute myocardial infarction (AMI) leads the pathological alteration within the cardiac tissue, resulting in the changes in the number and cargo of exosomes. A recent study showed the numbers of MVBs and exosomes increased after AMI, 51 and their actions were likely exerted by mediating cell-cell signalling via the transport of bioactive proteins between cells, 52 resulting in the direct activation of target cells. 53,54 Additionally, these miRNAs and proteins delivered by circulating EVs derived from red blood cells, platelets and white blood cells can mediate the activation of plaque macrophage through vascular cell adhesion molecule-1 (VCAM-1) inhibition during atherosclerosis progression. 55 The transcription factor, Snail within the exosomes released after the infarction, could induce endothelial-to-mesenchymal transition, which aggregates the process of fibrosis formation. 56 In another study, Qiao

| Interplay between the heart and distant organs through exosomes
In addition to their role in cell-cell communication, exosomes play a role in interaction between cardiac organs and distant organs ( Figure 3). There is an evidence indicating an active role for exosomes in the communication between the heart and bone marrow. For example, myocardial miRNAs could be delivered by cardiac exosomes and recruit circulating progenitor cells into an infarcted area for cardiac repair, which indicates that exosomes may mediate the functional crosstalk between ischaemic heart and bone marrow. 60

| E XOSOME S A S D IAG NOS TIC TOOL S IN C ARDIOVA SCUL AR DIS E A S E S
Since pathological changes to the cardiac tissue after CVDs can alter the content of exosomes, Sluijter et al 64 proposed that exosomes from different sources may be useful biomarkers to diagnose various CVDs. Given the cell types and status of the original cells decide the contents of exosome (ie miRNAs, proteins, IncRNAs), exosomes are widely generated and derived from multiply types of cells and actively participate in a wide range of cardiovascular processes, both physiological and pathological. 65 A significant amount of evidence has shown that exosomes seem to be associated with myocardial ischaemia and that exosome levels correlate with the severity of myocardial injury. In hypoxic or ischaemic environments, large numbers of exosomes with unique miRNA are released into plasma; thus, exosome levels are elevated in patients with CVDs or AMI. 64 In one study, the expression of circulating exosomal miR-133a originating mainly from infarcted and peri-infarcted myocardium was significantly elevated in patients with acute coronary syndrome. Furthermore, the level of serum miR-133a was elevated within 2 hours after the onset of chest pain, before creatine kinase and troponin were increased. 66 In addition, Cheow et al 67  Some of these miRNAs may be transported by EVs, especially under pathological conditions. 68 Thus, the number and contents of exosomes are considered early and disease-specific biomarkers for CVDs. 69 The determination of exosomal contents is crucial for clinicians to rapidly diagnose and identify diseases, prevent disease progression and improve prognosis.
Apart from their ability to reflect the physiological and pathological alteration within the cardiac tissue, 70

| E XOSOME S A S THER APEUTI C S IN C ARDIOVA SCUL AR DIS E A S E S
In addition to their capability to reflect early pathological changes working as a diagnostic tool, exosomes can also contribute to the cardiac tissue repair because of their ability to intervene the physiological and pathological process of cells. Often, after myocardial injury, the loss of cardiomyocytes cannot be rescued in current clinical setting. Recently, considerable effort has been made to develop cellbased cardiac repair therapies aiming to promote cardiomyocytes proliferation and reactivation. 75

| Cardiac resident cells derived exosome
Cardiac resident cells include CPC, Sca-1 + CPCs and side popula- CPC-derived exosome has also been used for myocardial infarction treatment, in which CPC-derived exosomes can inhibit cardiomyocytes apoptosis and improve cardiac function. 15 A recent report has demonstrated exosome derived from CXCR4-overexpressing CPCs might improve heart function by transferring exogenous proteins and mRNA to the target cells. 84 In another study, pregnancy-associated plasma protein-A (PAPP-A, also known as pappalysin-1) plays a key role in CPCs exosome-mediated cardioprotection by protolytic cleaving insulin-like growth factor-binding protein to promote the release of insulin-like growth factor-1, which active the intercellular ERK1/2 and Akt. 85

| Stem cell-derived exosome
Similarly, the therapeutic effects of exosome derived from iPSCs and iPSC-derived cardiomyocytes (iCMs) have been regarded as one possible opportunity to repair damaged tissue and restore cardiac function. Jung and colleagues summarized that exosomes derived from iCMs inherit the protective molecules to salvage the injured heart. 86 Importantly, some researchers have investigated that EVs derived from iPSCs are safer and more effective for cardiac function preservation than cells themselves. 87 Lai et al 81  Vunjak-Novakovic and colleagues recently showed that implanted hydrogel patches can deliver purified EVs originating from iCMs to the heart over an extended period of time, and this treatment significantly improved cardiac recovery following ischaemic injury because of the improved retention rate. 105 Han et al 106 have encapsulated the human umbilical cord MSC-derived exosomes in antioxidant peptide, which could enhance therapeutic effects with better target. Nevertheless, the development of targeted exosome delivery approaches with enhanced retention still need to be further explored. Also, those delivery approaches should be able to be incorporated with a minimally invasive surgical approach such as CT or ultrasound guide tube pericardiostomy to reduce the risk associated with the treatment.
The predictability and control over the cargos of exosomes needs to be addressed in order to achieve consistent therapeutic effect.
The components of exosomes, namely proteins, lipids, mRNAs and miRNAs, are determined by the source and types of the cells as well as the status of cells during the release process. 107 Given the exosome-mediated beneficial effects heavily depend on the content of exosome, it is important to understand the cells that release exosome.
For example, MSC-exosomes are abundant with miR-21a-5p, which could down-regulate the level of targeted pro-apoptotic genes 108 ; meanwhile, exosomes derived from hypoxia elicited MSCs with high miR-125b expression primarily reduce cell death to protect heart. 80 Especially, it has been reported that miR-106a-363 cluster was overexpressed in hypoxia iCMs-derived exosomes when compared to those in normal one. 109

| CON CLUS ION
In summary, the promise and excitement surrounding exosomes in cardiovascular research can be manifested daily by newly F I G U R E 4 Schematic of tissue engineering application in exosomes treatment. Exosomes themselves or sourced cells can be genetically altered with miRNAs, lncRNAs or tRNAs to express targeted genes using CRISPER/ Cas9 or gene delivery method. Exosomes themselves or engineered exosomes could be played as therapeutic vehicles to transport biological substance or chemical compounds and further embedded in scaffolds and delivered as injectable scaffold, patch or 3D tissue construct to enhance the function of exosomes. In addition, exosomes could be engineered with targeted peptides to further improve retention and efficacy when delivery intravenously reported studies. Exosomes may act as messengers and regulate the communication between the heart and other organs, such as the kidney or brain, or the formation of thrombosis in the distal vein. Exosomes might, therefore, offer tools to predict the progression of disease or work as a natural nano-scale delivery system carrying cargo with a specific, targeted function, or they may serve as therapeutic targets. Although the field of exosomes still has much to be explored, the exploration and specific application of exosomes in cardiovascular disease and potential treatment will continue to be a rapidly advancing focus for cardiovascular researchers.  Figure 1A.

CO N FLI C T O F I NTE R E S T
All authors declare that they have no conflicts of interests.