Abstract

1. The biogenesis of exosomes

The unique role of exosomes was originally recognized in the cancer microenvironment where the cancer cell–released exosomes regulated their invasive, metastatic, and angiogenic potential³⁷. These exosomes determine the delivery of DNA and RNA fragments, enzymes, and growth factors, which all impact the surrounding cellular niche³⁸. Various physiologic factors and cellular conditions influence the release and payload of these exosomes such as intracellular levels of calcium, cellular energetics, membrane phospholipid component, membrane-acting enzymes, cytoskeleton–membrane interactions, and other effectors of exocytosis, hypoxia, and oxidative stresses^39,40. The size of exosomes is between 30-150nm, and their membranes usually contain high levels of sphingomyelin, cholesterol, phosphatidylserine and tetraspanins. They are associated with a variety of transmembrane and cytosolic proteins, including integrins CD9, CD63, and CD81 to form specific complexes⁴¹. Exosomes expressing these complexes are readily internalized by specific target cell ligands⁴². Furthermore, these unique bi-lipid membranes of exosomes not only have the ability to protect the contents from degradation enzymes but also enable targeted delivery of large cargo^43,44 (Figure 1).

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Figure 1

Biogenesis of exosome

Exosomes are compartmentalized into the multivesicular bodies (MVBs), which fuse with the cell membrane and released to the extracellular space. This process prevents exosomes from degradation by the lysosomes. Exosomes comprise of various transmembrane and cytosolic proteins, such as integrins, CD9, CD63, and CD81. Furthermore, exosomes retain donor cells’ proteins, DNA fragments, miRNAs, and non-coding RNAs within the bi-lipid membrane. As such, exosomes preserve the genetic information of donor cells from enzymatic degradation while enabling targeted delivery of specific cargo.

One of the main theories of the exosome uptake mechnisms is that exosomes can be endocytosed or internalized into an endocytic compartment from which they may undergo reverse-fusion to release their cargo into the cytoplasm of the recipient cell. After endocytosis, exosomes may subsequently fuse with the endosomal membrane or be targeted to lysosomes for degradation⁴⁵. Another mechanism is through receptor–ligand-mediated interaction, which leads to exosome internalization^46,47. The third likely mode is by fusion of the exosome membrane with the plasma membrane, releasing the cargo directly into the cytoplasm of the recipient cell⁴⁸. Interestingly, several studies suggest that exosome release is strictly controlled by specific signaling pathways, which consequently regulates cellular miRNA release^49,50. In addition, neutral sphingomyelinase 2, the rate-limiting enzyme in the conversion of sphingomyelin to ceramide, is a critical regulator of exosome biogenesis and secretion⁵¹. Another study demonstrated that Rab27 (GTPases) isoforms play essential roles in the exosome secretion pathway⁵². According to a recent study, molecular packaging of miRNA-loaded exosomes is controlled by the recognition of sequence motifs present in the miRNAs by the sumoylated protein hnRNPA2B1⁵³. Koppers-Lalic and colleagues showed that 3′-end-uridylated miRNA isoforms are selectively packaged into exosomes while 3′-end-adenylated miRNA isoforms remain relatively enriched in the original cells⁵⁴. Exosomes are known to be capable of shuttling RNA between cells and greatly increase the complexity by which these cells communicate. Thus, the potential use of exosomes in a clinical setting includes their role as antigen-presenting vesicles to stimulate anti-inflammatory, anti-fibrotic, anti-immunogenic proliferative, or angiogenic effects^55,56.

2. Exosomes in cardiovascular diseases

CVDs are leading cause of death worldwide, necessitating investigations into novel, safe, and effective therapeutic approaches. Various strategies for the transplantation of human stem cells or stem cell–based therapies to treat cardiac disease have been attempted⁵⁷. In vivo mechanistic studies to date have not been forthcoming clinically because of the difficulty of the performing appropriate assays. Nevertheless, most of the researchers recognize the importance of the paracrine factors from the cells rather than the direct effects of the transplanted cells to repair or regenerate the injured tissue. Recently, many studies have provided evidence regarding the importance of exosomes and their miRNAs in cell–cell communication within the cardiovascular system⁵⁸, specifically, from stem cells to cardiovascular cells^59–61, and from the heart to bone marrow stem cells⁶². Exosomes and their miRNAs are also recognized as important regulators in cardiomyocytes, endothelial cells, vascular smooth muscle cells, platelets, and inflammatory cells, which contribute to the initiation and progression of atherosclerosis⁶³. miRNAs retain strong stability, express tissue-specific pattern, and represent the corresponding body fluids. Particularly, several miRNAs, already identified in exosomes, play important roles in CVDs and stem cell trans-differentiation (Figure 2). Ekstrom and co-workers observed that the exosomes from mast cells carry selective miRNAs to bone marrow CD34+ progenitor cells⁶⁴. Bang et al. reported that miRNAs are involved in the crosstalk between cardiac fibroblasts and cardiomyocytes. They demonstrated that the exosome-derived miRNA-21 is transported to cardiomyocytes, leading to cellular hypertrophy by affecting target genes, SORBS2 and PDLIM5⁵⁸. miRNA-150 delivered to endothelial cells enhances migration by the downregulation of c-Myb⁶⁵. Apoptotic bodies are shown to transfer functional miRNA-126 to endothelial cells inducing CXCL12 expression, which are involved in the mobilization of progenitor cells and, therefore, play an anti-apoptotic role⁶⁶. miRNA-126, miRNA-223, and miRNA-197 expression have been found to risk stratifty the predilection to myocardial infarction (MI)⁶⁷. miRNA-133 is specifically expressed in cardiomyocytes⁶⁸ and those undergoing controlled cardiac hypertrophy⁶⁹. miRNA-133a is considered a strong diagnostic marker for acute MI and coronary artery stenosis⁷⁰. Notably, several studies have shown that miR-133 is involved in direct cardiac reprogramming of adult cardiac fibroblasts^71,72. These pleiotropic properties of the miRNAs contained in the exosomes could be leveraged to treat various forms of CVD.

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Figure 2

Exosomes mediated intercellular communication in heart

Exosomes facilitate communication amongst cardiomyocytes, endothelial cells, and vascular smooth muscle cells in the infarcted area of the heart. Exosomes transfer signaling molecules, such as miRNAs, mRNAs, and proteins to confer paracrine effects on the neighboring cells. In addition, pathological and physiological influence on the heart stimulates exosome secretion. Therefore, cardiac exosomes under pathological conditions could be utilized as ideal markers for diagnostic tools in the clinic.

3. Diagnostic capability of exosomes in heart injury

Biomarkers serve as indicators of normal biological functions, pathologic processes, or pharmacological responses to therapeutic intervention. As the direct assessment of biological states is often too invasive or costly, biomarkers have considerable clinical utility in identifying disease status and evaluating disease risk. Moreover, biomarkers allow for the early detection of pathology and subsequent therapy. As a diagnostic tool, for example, exosomes from prostate cancer cells can be obtained from a simple urine sample, making an exosome-based test essentially noninvasive. Exosome tests may detect several RNA-encoding key biomarkers in prostate cancer, such as PCA-3 and TMPRSS2:ERG. Other tumor markers can be added as they are identified and matched to a patient’s exosomal RNA profile. Initial clinical studies have shown that exosome tests for prostate cancer have a 70% accuracy rate, which is almost comparable to the accuracy of a biopsy⁷³. For the implementation of these tests, the standard disease-specific antigen test could be hugely improved by incorporating exosome tests, which would be useful in the diagnosis and prognosis of diseases and the disease states that are difficult or inherently impossible to diagnose.

As exosomes are formed under specific conditions of stress or injury, circulating exosomes are being considered increasingly as candidates for CVD biomarkers. In particular, patients with atherosclerosis associated with vascular injury, inflammation, and prothrombotic state exhibit elevated plasma exosome levels. Several studies have shown an association between the Framingham risk score, used to predict cardiovascular disease risk, and circulating exosomes^74,75. Their formation and clearance reflect a subtle balance between cell activation and damage; cell survival and apoptosis; and vascular remodeling and angiogenesis. Several investigations have demonstrated the selective packaging of miRNAs in the exosomes and their functional transfer by specific signaling molecules^76,77. In addition, the exosomes facilitate the detection of endogenous processes for myocardial restoration, regeneration or protection⁷⁸. These disease-specific expression patterns of exosomes from body fluids suggest the potential of exosomes as molecular markers and therapeutic targets for the treatment of CVDs.

Intercellular signals between endothelial and vascular smooth muscle cells are critical for the maintenance of blood vessel structure, vascular function, and blood pressure⁷⁹ and for functional vessel formation after myocardial injury⁸⁰. When exposed to high atheroprotective shear stress, endothelial cells secrete exosomes that are enriched in miRNA-143/145. Once transferred to smooth muscle cells, exosomes reduce the expression of miRNA-143/145 targets such as ELK1 and CAMK2D, the two central regulators of the smooth muscle cell phenotype⁸¹. In this respect, exosomes might serve as excellent diagnostic candidates in the clinical setting, given that they reflect the real-time microenvironment conditions of the injured lesions. Genetic information of cells in a target region can be achieved without invasive biopsy, requiring only blood or urine sample collection. This is a remarkable advantage, especially when the sample tissue is difficult to access, such as the human heart. Since exosomes can be collected in a straightforward way and with regularity, real-time tracking of a patient’s disease progression is possible.

4. Therapeutic effects of exosomes derived from the different sources of human stem cells

Due to the natural role in intercellular secretion and transport, exosomes have generated great interest as therapeutic tools. They are natural biologics with autologous origin while they maintain cargo integrity and stability. Furthermore, exosomal membranes contain certain proteins that have binding affinities to specific receptors on the surface of the recipient cells. Thus, they can selectively target cell types of interest and manipulate the molecular components through the miRNA cargo to provide promising therapeutic tools for personalized medicine and gene therapy^35,47. More importantly, the exosomes shuttle the RNAs or exosomal shuttle RNAs (esRNAs) between cells, which serves as a novel and effective means to control cell–cell communication^82,83.

Mesenchymal stem cell (MSC)-derived exosomes have been investigated in the field of cardiac regenerative medicine, especially in myocardial ischemia/reperfusion (I/R) injuries^34,84. Exosomes bearing endothelial differentiation signals were found to effect neovascularization, indicating their usefulness in treating angiogenic defects. Exosomes from human umbilical cord mesenchymal stem cells (hucMSCs) have been reported to promote heart repair following ischemic injury by protecting myocardial cells from apoptosis and promoting cell proliferation and angiogenesis. hucMSCs without exosomes have nearly no effect on cardiac function recovery, suggesting hucMSC-exosome–mediated myocardial repair^85,86. hucMSC-exosomes promoted wound healing and angiogenesis in vivo, and this pro-angiogenic effect was mediated by Wnt4/β-catenin activation in endothelial cells⁸⁷. MSC-exosomes also increased ATP levels, which led to decreased oxidative stress via the PI3K/Akt pathway to enhance myocardial viability and prevent adverse remodeling after I/R injury⁸⁸. Similarly, exosomal heat shock protein-70 (HSP70) stimulated toll-like receptor-4 (TLR4) signaling, leading to the activation of ERK1/2 and p38MAPK and subsequent HSP27 phosphorylation in cardiomyocytes^89,90. Cardiac progenitor cells (CPCs) derived from the adult hearts have also emerged as potential stem cell population for cardioprotection and repair. CPC-derived exosomes conferred a protective preconditioning effect when injected into mouse hearts immediately after the induction of ischemia. They appeared to protect cardiomyocytes from oxidative stress in vitro and IR injury in vivo⁹¹ and exerted anti-fibrotic effects on the fibroblasts and promote angiogenesis⁶⁰.

5. Possible limitations to the use of exosome-based therapeutics

Exosomes would provide a more feasible approach for bench-to-bedside translation compared to the cell-based therapy. However, their stability, off-target effects, and inefficient endocytosis by target cells must be addressed¹¹¹. A parameter affecting the stability of exosomes is the zeta potential, which is a physical property of exosomes that measures the magnitude of electrostatic repulsion or attraction between particles¹¹². Nanoparticles are usually stable at absolute zeta potential values ranging from 31 to 40 mV¹¹³. Recent experiments showed that the zeta potential detected in mouse cardiac fibroblast exosomes and cardiac fibroblast-derived iPSC-exosomes were very low (−23.85 mV vs. −23.93 mV at 37°C). Hence, these exosomes are very unstable and aggregate easily⁹³.

Exosome-based miRNA delivery technique has been considered as attractive tools in several diseases to provide reliable evidence to overcome the inefficient, non-specific delivery of miRNAs. However, this technique needs further optimization to investigate the miRNA biology, long-term biochemical and off-target effects, and pathway analysis³⁵. Exosomes may have only short-term benefit owing to their short half-life. Takahashi and colleagues showed that murine melanoma B16-BL6 cells exosomes disappeared very quickly from the blood circulation with a half-life of approximately 2 min¹¹⁴. Exosomes may also have pro-fibroblast effects. For example, iPSC-derived MSCs exosomes can facilitate cutaneous wound healing by promoting collagen synthesis and angiogenesis⁹⁹. It is established that fibroblasts are important to prevent cardiac rupture after acute MI¹¹⁵. Thus, in the short term, iPSC-exosome treatment might reduce the risk of acute heart rupture; however, in the long-term, increased proliferation of fibroblasts could increase scarring, leading to left ventricular remodeling in the ischemic myocardium.

Cell manipulations aimed at enhancing beneficial properties of exosomes may include hypoxia and stress preconditioning⁶⁰, genetic modification¹¹⁶, and epigenetic reprogramming of exosome-producing cells¹¹⁷. For instance, as shown by Gray et al., the exosomal content depends on the state of the donor cell and disease conditions⁶⁰. The standardization of the conditions under which exosomes are collected is therefore of utmost significance. Although the cargo of these stem cell–derived exosomes can pose beneficial effects on injured heart, the underlying mechanisms are to be uncovered. In addition, many of the proteins in the exosomes are enzymes. Since enzyme activities are catalytic rather than stoichiometric and are influenced by their microenvironment, enzyme-based therapeutic activities of exosomes could be activated or attenuated according to the release of injury-associated substances. The resolution of the disease-precipitating microenvironment would reduce the release of injury-associated substrates, as well as the activity of the exosomal enzymes. Consequently, the efficacy of exosome-based therapeutics could be highly responsive to, but also limited by, the niche and the microenvironment¹¹⁸. Exosomes may also facilitate the uptake of therapeutic proteins or RNAs into injured cells. Although cellular uptake of exosomes has been demonstrated to occur through endocytosis, phagocytosis, and membrane fusion, the mechanism by which these processes are regulated remains to be determined. The efficiency of exosome uptake has been found to correlate directly with intracellular and microenvironmental acidity^119,120.

Many studies demonstrate that the translation of exosomes has great potential in clinical applications. However, this might be complicated by the relative novelty of exosomes with few precedents in the regulation and safety of the biopharmaceuticals. Despite its advantages, exosome delivery may need to address some of the potential complications. In the case of the factors operating at the intracellular level, delivery into the correct cellular compartments while maintaining the stability, integrity, and biological potency of these factors remains challenging and expensive. Certainly, the coming few years will provide many more insights into the biological significance of cell–cell transport via exosomes. Systematic approach to the structural and functional biology of exosomes will create an opportunity to increase our understanding of the role of exosomes in repairing injured myocardium.

6. Conclusion and future perspectives

Until the early 2010s, stem cell transplantation was regarded as a potentially therapeutic alternative in treating ischemic heart disease. Despite the fact that functional cardiomyocytes derived from human pluripotent stem cells improved cardiac morphology and function in animal models of heart failure, they also triggered an arrhythmogenic response^3,48. Teratoma formation has also been observed after the transplantation of an unpurified pluripotent-derived cardiomyocytes^17,102. The derivation of iPSCs appears to have partially addressed the issues related to immune rejection. Nonetheless, pluripotent stem cell-derived cells still pose the same difficulties to engraft in the ischemic myocardium as is noted for adult stem cells. Therefore, there is a critical need for exploiting the powerful regenerative biology of the pluripotent stem cells while avoiding the problems associated with cell transplantation¹²¹. More importantly, the exact mechanism of action of stem cells has not been elucidated. Future studies on paracrine factors such as the exosomes will shed more mechanistic understanding of stem cell therapy.

Among the stem cell secretomes, exosomes appear to be the most suitable vehicle for delivery because of their ability to target the specific lineage related and physiologically relevant cells due to their endosomal origin. Exosomes of different cellular origin bear distinct antigenic proteins of their native cell type¹²². This enables specific binding property to the recipient cell receptors, providing a targeted delivery to the desired cell type. This approach may enable the reality of personalized medicine in the near future, utilizing the exosomes generated from autologous iPSC-derivatives such as the iCMs.

The biotechnological approach to create exosome-based delivery systems used by Alvarez-Erviti et al. was the first demonstration of an exosome-based drug delivery system, which showed efficient in vivo delivery of siRNA¹²³. Another study demonstrated that the successful introduction of exogenous siRNAs into various human exosomes and delivered to human mononuclear blood cells. Plasma exosomes effectively delivered the siRNA into the target cells, causing selective gene silencing of MAPK-1¹²⁴.

6.1. Application of exosomes to personalized medicine

Exosomes could become a game-changer for both diagnostic and therapeutic purposes for chronic diseases with advanced clinical manifestations. Exosomes are natural vehicle to deliver molecules from one cell to another. Researchers are trying to harness this natural ability of the exosomes to deliver therapeutic payloads to the desired cells. Furthermore, gene editing of the native stem cells will produce exosomes with specific cell surface proteins and cargo for targeted delivery of the desired exosomes. Numerous attempts to enhance stem cell biology in the area of reprogramming, transdifferentiation/differentiation, and engraftment of the cells have been made. The results have been quite encouraging. However, significant obstacles remain for clinical translation of pluripotent stem cell-derivatives. Particularly, mutagenic complication, long-term engraftment, mechanism of action, and local physiologic effects must be understood. A more exact and effective approach needs to be developed to translate the pluripotent stem cell biology to enable specific modification of the injury at targeted location. Exosomes from iPSC-derivatives can be applied to enable an autologous therapy by simulating endogenous repair. Thus, the exosomes generated from patient-specific iPSC-derivatives are expected to provide higher therapeutic specificity, quality control, ease of production, and safety than stem cell transplantation. Exosomes from gene-edited patient-specific stem cells can be directed to the specific lesions of each individual patient to promote the salvage of the existing injured cells. (Figure 3). In conclusion, exosomes hold potential for a wide spectrum of beneficial effects on cell function recovery to restore the myocardial injury by simulating and activating the endogenous repair, consisting of the native transfer of proteins, mRNAs, and miRNAs. They will serve as an excellent therapeutic tool for cardioprotection or regeneration of the injured mycordium. Exosomes represent the most feasible approach to translate the enormous potential of pluripotent stem cell biology. Exosome-based treatment approach present an exciting new concept in translational medicine with huge potential to impact the thereapeutic effect of CVDs.

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Figure 3

Personalized therapy with the exosomes generated from iPSC-derived cardiomyocytes (iCMs)

Exosomes from patient-specific iPSC-derivatives provide a platform for personalized therapy by simulating endogenous repair. Exosomes can be directed to specific injured site of each individual patient to promote salvage of the existing injured cardiac cells. Exosomes isolated from the supernatant of iCM culture undergo ultracentrifugation methods, as well as advanced characterization and quantification by transmission electronical microscopy (TEM), Nanosight, and Dynamic Light Scattering (DLS).

Acknowledgment

Non-standard Abbreviations and Acronyms

Footnotes

References