Extracellular vesicle-mediated bidirectional communication between heart and other organs

Abstract

In recent years, a wealth of studies has identified various molecular species released by cardiac muscle under physiological and pathological conditions that exert local paracrine and/or remote endocrine effects. Conversely, humoral factors, principally produced by organs such as skeletal muscle, kidney, or adipose tissue, may affect the function and metabolism of normal and diseased hearts. Although this cross communication within cardiac tissue and between the heart and other organs is supported by mounting evidence, research on the role of molecular mediators carried by exosomes, microvesicles, and apoptotic bodies, collectively defined as extracellular vesicles (EVs), is at an early stage of investigation. Once released in the circulation, EVs can potentially reach any organ where they transfer their cargo of proteins, lipids, and nucleic acids that exert potent biological effects on recipient cells. Although there are a few cases where such signaling was clearly demonstrated, the results from many other studies can only be tentatively inferred based on indirect evidence obtained by infusing exogenous EVs in experimental animals or by adding them to cell cultures. This area of research is in rapid expansion and most mechanistic interpretations may change in the near future; hence, the present review on the role played by EV-carried mediators in the two-way communication between heart and skeletal muscle, kidneys, bone marrow, lungs, liver, adipose tissue, and brain is necessarily limited. Nonetheless, the available data are already unveiling new, intriguing, and ample scenarios in cardiac physiology and pathophysiology.

INTRODUCTION

In a review article on cardiac endocrine function published in this journal 15 years ago, Clerico et al. (1) hypothesized the existence of a means of “cross-talk between cardiac endocrine function and other integrated systems of the body.” At that time, the role of cardiac muscle as a source of potent natriuretic factors was well defined (2); however, virtually nothing was known regarding possible two-way communication between heart and other organs. In more recent years, a wealth of studies identified a variety of peptides and proteins, collectively named myokines/cardiokines (3–5), as well as of lipids and nucleic acids released by cardiac muscle under physiological and pathological conditions, which exert local paracrine and/or remote endocrine effects (6, 7). For instance, an intriguing line of research is focusing on the modulation of systemic metabolism by cardiac factors, many of which have not been identified (8–10). On the other hand, humoral factors, principally released by organs such as skeletal muscle, kidney, or adipose tissue, may affect function and metabolism of normal and diseased hearts (6, 11). A typical example of myokine released by both skeletal and cardiac muscle is follistatin-like protein 1, a protective factor and possibly a mediator of metabolic function (12–15).

To add a further layer of complexity to this network, studies published over the past 10 years have uncovered a wealth of intraorgan and interorgan signals mediated by factors enclosed in vesicles known as extracellular vesicles (EVs) (Fig. 1). Virtually all cell types can release EVs, and cardiac tissue is no exception (16–20).

Figure 1.

Schematic representation of extracellular vesicle production by viable (exosomes and microvesicles) and apoptotic cells (apoptotic bodies).

EV-mediated intracardiac cell-to-cell communication has been recently covered by extensive review articles (21, 22). Cardiomyocytes represent only ∼30% of cardiac cells (23, 24); hence, they need to communicate with neighboring fibroblasts, endothelial cells, macrophages, smooth muscle cells, and pericytes. Such constant communication ensures heart homeostasis (25). The flow of information inside cardiac tissue is a complex and highly regulated process, in which EVs play a substantial role. EVs can be released and taken up by cardiomyocytes (17), cardiac fibroblasts (16, 26–28), endothelial cells (18, 20, 29, 30), and vascular smooth muscle cells (19) under normal and pathological conditions. These vesicles function as vectors of cell-cell signaling. A large array of proteins and nucleic acids derived from cardiomyocyte mitochondria, cytosol, and plasma membrane have been identified in exosomes and may influence biological functions when delivered to other cells (31). Some examples are the Wnt-binding protein, a major player in the cardiac development signaling pathway (32), inflammatory factors such as IL-6 (33), IL-1β (34), and TNF-α (192), the metabolic transporters/enzymes GLUT1, GLUT4, and lactate dehydrogenase (29), and the heat shock proteins, such as Hsp20 (36) as well as a wide variety of miRNAs (26, 30, 37). The specific cargo carried by EVs is determined during their biogenesis and by stimuli affecting the parent cells (31). For instance, growth factor stimulation of cultured cardiomyocytes can induce a change in EV miRNA content (38).

Regarding the communication between heart and other organs, although many review articles, including a very recent position paper by the European Society of Cardiology Working Group (39), have covered the endocrine signals mediated by various free molecules, none of them has extensively discussed the current body of knowledge of interorgan signals carried by EVs. Here, we aimed at gathering representative literature on the role played by EV-carried mediators in the communication between the heart and other organs. In a few cases, such a role was clearly demonstrated however, in many others, it can be inferred based on indirect evidence obtained by infusing exogenous EVs in experimental animals or by adding them to cell cultures. The signaling described in the following paragraphs is schematically summarized in Fig. 2.

Figure 2.

Extracellular vesicle-carried putative mediators of cell-cell communication between heart and other organs. ATIRa, angiotensin II type I receptors; ATMSC, adipose tissue mesenchymal stem cells; BMSC, bone marrow stem cells.

Extracellular Vesicles: Definition and Characteristics

EVs constitute a highly heterogeneous population of vesicles and are broadly classified as exosomes, microvesicles, and apoptotic bodies (5, 40) (Fig. 1). Despite attempts to further categorize EVs based on their size, origin, or function, major overlaps of these features do not allow a clear distinction among them (40, 41). Therefore, the International Society for Extracellular Vesicles (ISEV) consensus recommendation is to use “extracellular vesicle” as the “generic term for particles naturally released from the cell that are delimited by a lipid bilayer and cannot replicate (42),” which we will use in the present review; however, in some cases we will cite the same term used by the authors in their articles.

Dimensions and genesis of EVs vary considerably. Exosomes are 50–150-nm vesicles (43, 44), whereas microvesicles range between 50 and 1,000 nm (43, 45). Apoptotic bodies are typically larger in size and can reach 5,000 nm in diameter (46). The formation of exosomes starts in the endosomal system, where early endosomes mature into multivesicular bodies. This process involves double invagination of the plasma membrane (47, 48). The first invagination forms a cup-shaped structure composed of cell surface proteins and hydrophilic proteins. This results in the genesis of early endosomes (48, 49). The second, inward invagination of the endosomal limiting membrane produces multivesicular bodies containing intraluminal vesicles, i.e., the future exosomes (43, 50). The process ends with either multivesicular bodies content degradation by lysosomes or autophagosomes or with their fusion with the plasma membrane and release of intraluminal vesicles as exosomes (40, 51, 52).

Different from exosomes, microvesicles are directly shed from the cell membrane. A Ca²⁺-dependent enzymatic system, including aminophospholipid translocases, scramblases, and calpain, alters the organization of phospholipids in the cell membrane. Together with a rearrangement of the underlying actin cytoskeleton, the cell membrane is induced to bend outward (53–55). This favors membrane budding and formation of microvesicles (55).

Finally, apoptotic bodies are released by cells undergoing programmed cell death, which causes plasma membrane blebbing and nuclear fragmentation (56–58). Apoptotic bodies are then removed by phagocytic cells to prevent spillover and damage to the surrounding cells or tissues, hence their role in cell-cell interactions is poorly understood (59, 60).

Most EVs do not display a tropism for a specific cell type. One study demonstrated that fibroblast-derived exosomes uptake by cardiomyocytes depends on actin and temperature as cell treatment with cytochalasin D (an inhibitor of actin polymerization) or incubation of cells at 4°C decrease exosome uptake (16). Interestingly, three-dimensional (3-D) reconstruction of confocal image z-stacks demonstrated that exosomes were not simply attached to the cardiomyocyte surface but completely internalized by the cell (16). In other cases, exosomes can either bind to cell surface plasma membrane receptors and trigger signaling cascades (61, 62) or merge with the plasma membrane and release their cargo directly into the cytosol (63). Another mechanism of EV cargo release is the docking of Cx43-containing channels localized on both cell and exosome surfaces leading to the vesicle unloading into the target cells (64).

Exosomes and microvesicles convey messages encrypted in proteins (enzymes, biogenesis factors, chaperones, etc.), lipids, sugars, and nucleic acids (40). Over the last decade, EV proteins and nucleic acids have drawn remarkable interest as potential disease biomarkers or therapeutic targets (65–70), whereas EV lipids have not been widely studied (37, 71). The composition of EVs depends on the condition of the mother cells and may change depending on physiologic conditions such as normal aging, or in response to pathological processes like chronic or acute stress, inflammation, and fibrosis (72–74).

EV-MEDIATED COMMUNICATION BETWEEN HEART AND OTHER ORGANS

Mounting evidence supports a role for EVs in the communication between heart and other organs. Once released in the blood at a given site, EVs can virtually reach and target any organ (75) and transport a variety of different cargoes, including lipids, peptides, proteins, and nucleic acids (40). Interestingly, microvesicles and exosomes can cross also the blood-brain (76) and the maternal-fetal barrier (77). Bryl-Górecka et al. (78) found an inverse relationship between coronary flow rate and levels of circulating, protein loaded EVs in plasma, identified as endothelial cell microvesicles. Understanding the source of circulating EVs is a major challenge. For instance, tetraspanins CD9, CD63, and CD81, heat shock proteins HSP70 and HSP90, class I and II major histocompatibility complexes, and flotillin-1 are all particularly abundant in cardiac exosomes, yet they cannot be considered heart-specific (79, 80). Since EVs content changes depending on alterations of the parent cell, understanding their source may be less complicated under pathological than physiological conditions. A good example is represented by EVs carrying the cardiac hormone brain natriuretic peptide (BNP) during heart failure (72) or angiotensin II type I receptors during cardiac pressure overload (73). Nonetheless, identifying the EVs origin should be possible also under physiological conditions, based on the characterization of cell-specific protein cargos (7, 81).

EVs in Heart-Skeletal Muscle Cross Talk

Skeletal muscle is the most obvious organ to start with, as its cross-communication with cardiac muscle via myokines has been actively studied and might modulate their respective contractile functions (3, 10, 14, 82). Skeletal muscle is an important source of EVs (83) and, in fact, a rapid increase in circulating EVs has been observed during exercise (84) and following hindlimb ischemia (85). Fluorescently labeled muscle-derived exosomes injected in mice are taken up by the heart (86). Similarly, cardiosphere-derived exosomes are taken up by skeletal muscle (65).

The association between heart failure and abnormalities in skeletal muscle morphology and biochemistry is well known (87, 88). Possible mediators of the negative impact on skeletal muscle are myocardium-derived miRNAs released in the circulation during heart failure progression (89). The so called myomiRs, i.e., miR-1, miR-133, miR-208, and miR-499 (90–92), that strongly affect skeletal muscle, are abundantly represented in cardiac muscle and in the circulation after myocardial damage (93). As the EVs carry a significant amount of circulating myomiRs (94), they possibly play a role in heart-skeletal muscle communication. Aminzadeh et al. (65) found that cardiosphere-derived exosome treatment had a beneficial effect in mdx mice (a model of Duchenne muscular dystrophy) by increasing dystrophin expression, perhaps due to transfer of miR-148a in skeletal muscle cells. Conversely, the administration of circulating EVs isolated from the mdx mice decreased apoptosis in cultured C2C12 myocytes (95).

Pironti et al. (73) found that exosomes released from the heart of mice subjected to pressure overload are enriched with angiotensin II type 1 receptor and are transported to skeletal muscle, cardiomyocytes, and mesenteric resistance vessels. Importantly, trafficking and sorting of these receptors into exosomes required the multifunctional adaptor protein, β-arrestin (73).

Bei et al. (96) reported that intramyocardial injections of EVs collected from exercised mice had a better protective effect on the heart against acute ischemia/reperfusion injury compared with EVs from nonexercised controls. Correspondingly, exercise-associated EVs exerted an antiapoptotic effect in H₂O₂-treated H9C2 cardiomyocytes, mediated by the activation of ERK1/2 and HSP27 signaling (96). Homme et al. (85) recently reported that remote limb ischemic conditioning causes the release of exosomes from skeletal muscle that contain musclin, CD71, VEGF, and β₂-adrenergic receptor (β₂-AR) and alleviated heart failure in mice. Whitham et al. (97) also found that some myokine candidates could be enclosed in muscle-derived EVs; however, the validation of their function warrants further studies.

EVs in Heart-Kidney Cross Talk

Cardiac natriuretic peptides are long known to be secreted by degranulation (98). However, Gao et al. (72) found BNP and nephrin (a renal marker) enrichment in exosomes isolated from mouse serum after myocardial infarction, supposedly released by ischemic myocardium and by kidneys, respectively. Those authors suggested that exosomes may serve as circulating endocrine signals between the two organs (72). The BNP positive effects on kidneys are well known (99). Other organs may intervene in cardiac and renal EVs-mediated protection under pathological conditions. Recently, Lindoso et al. investigated the effects of adipose mesenchymal stromal cell-derived EVs in a rat model of deoxycorticosterone acetate-induced hypertension. They reported that these EVs can preserve renal filtration and prevent cardiac tissue fibrosis by maintaining blood pressure within normal levels (100). The authors hypothesized that both of these positive effects were due to vessel protection (100).

Circulating exosomal miRNAs have been recently profiled in patients with urinary albumin excretion, a marker of cardiovascular risk and renal damage in hypertension (101). In albuminuric subjects, one study identified a signature of 29 dysregulated circulating miRNAs, with miR-26a being the only one downregulated (101). Interestingly, human podocytes exposed to TGF-β1 in vitro did not show miR-26a downregulation at cellular level, but only in exosomes, suggesting a selective sorting of miRNAs (101). It has recently emerged that miR-26a is a regulator of podocyte homeostasis and plays an active role in the actin cytoskeleton maintenance (102). miR-26a is involved in cardiovascular repair mechanisms, such as cardiac angiogenesis and endothelial cell growth (103). Wang et al. (104) tested exosome-encapsulated miR-26a delivery in mice with uremic cardiopathy, a multifactorial complication of chronic kidney disease principally characterized by hemodynamic overload, circulating uremic toxins, metabolic acidosis, inflammation, increased oxidant stress, mineral bone disease, and insulin resistance (104, 105). Exogenous miR-26a in uremic mice diminished cardiac fibrosis and improved heart function, most probably by ameliorating insulin resistance (104).

Several circular RNAs are known to be altered during cardiovascular complications in chronic kidney disease (106). The circRNAs are believed to be principally transported by exosomes (107). Whether this kind of kidney-heart communication plays a pathogenic or rather a protective role remains an intriguing question.

EVs in Heart-Bone Marrow Cross Talk

The effects of the bone marrow-derived mesenchymal stem cells transplanted into infarcted myocardium have been the object of intensive investigations and represent a controversial topic. The initial hypothesis was that transplanted bone marrow-derived mesenchymal stem cells could induce cardiac repair by assuming a functional cardiomyocyte phenotype (108, 109). However, subsequent studies pointed to the paracrine release of bioactive molecules as an alternative reparative mechanism (110–112). These findings prompted research on exosomes released by bone marrow-derived mesenchymal stem cells and their cardioprotective/reparative effects. Yu et al. (113) reported diminished cardiac infarct size in rats after injection of bone marrow-derived mesenchymal stem cell-derived exosomes enriched with GATA-4 in the peri-infarct region. They also observed overexpression of miR-19a and miR-451 in the myocardium of treated rats (113). Recently, bone marrow-derived mesenchymal stem cell-derived exosomes have been reported to attenuate pressure overload-induced left ventricular hypertrophy and fibrosis in mice (66), as well as pulmonary pressure and right ventricular hypertrophy in rats with pulmonary artery hypertension (114). The same type of exosomes stimulates neovascularization and reduces the inflammatory response, thus improving heart function after ischemic injury (115). Mesenchymal stem cell-derived exosomes released after ischemic preconditioning were enriched with miR-22 and diminished apoptosis when delivered intramyocardially in infarcted mice (116). If added to fibroblasts isolated from patients with diabetes, these exosomes activate Akt, ERK, and STAT3 signaling pathways that are essential for wound healing and induce the expression of several growth factors, such as IL-6, hepatocyte growth factor, insulin-like growth factor-1, nerve growth factor, and stromal-derived growth factor-1 (117). These exosome-induced mechanisms of wound healing need deeper investigation to elucidate their possible role also in cardiac repair.

Interestingly, Sun et al. found a potential EV-mediated interorgan communication in patients with multiple myeloma. Exosomes extracted from the serum of these patients contained 10,106 differentially expressed circular RNAs compared with the control group (70). By correlation analysis they showed that the expression level of circ-G042080 was positively correlated with multiple myoloma-related myocardial damage and could interfere with myocardial damage processes through a downstream miRNA/TLR4 axis (70).

Possible EV-mediated signaling from heart to bone marrow is suggested by several studies. Cheng et al. (118) found that, soon after ischemic injury, myocardium releases a remarkable quantity of EVs loaded with cardiomyocyte-derived miRNAs, which travel with peripheral blood to target other tissues and preferentially bone marrow. MiR-1, miR-208, and miR-499 and, in part, miR-133 suppress chemokine receptor CXCR4 expression and mediate progenitor cell mobilization in bone marrow (118).

EVs in Heart-Adipose Tissue Cross Talk

Adipose tissue influences cardiovascular homeostasis by releasing adipocytokines (11). In obesity, overproduction of proinflammatory adipocytokines and decreased expression of anti-inflammatory adipocytokines are associated with cardiovascular diseases (119). Vice versa, proinflammatory and oxidative signals from stressed myocardium or vessels may modify the adipose tissue biological environment (11). Besides adipocytokines, adipose tissue is a major source of circulating miRNAs, a large fraction of which are carried by exosomes (120). Thomou et al. (120) proposed that the majority of circulating exosomes are derived from adipose tissue. However, another group found that under physiological conditions, putative adipose tissue exosomes were present at a low level in the blood stream (121). Aside from these conflicting reports, there is evidence that exosomes mediate the cross communication between heart and adipose tissue. This may even explain, at least in part, the “obesity paradox,” i.e., the better prognosis of cardiovascular diseases in patients with obesity compared with their slimmer matches (122). In fact, a recent major study by Crewe et al. (123) demonstrated that, in obesity, the stressed adipocytes release EVs transporting damaged mitochondrial particles, which enter the blood stream and are taken up by cardiomyocytes, where they induce the production of reactive oxygen species. This leads to the activation of an ischemic preconditioning-like adaptation mechanism possibly responsible for the attenuation of cardiac injury during ischemia (123).

Another study found that adiponectin, the most abundant circulating adipocytokine, stimulates exosome production from T cadherin-expressing cells in the heart, aorta, and muscle (124). This causes an impoverishment of ceramides in those cells, as ceramides are incorporated into exosomes that are then released in the circulation (125). Such a mechanism might be considered protective since excessive ceramide in cells is correlated with an elevated risk of atherosclerosis, cardiomyopathy, heart failure, and myocardial infarction (124–126). Fang et al. (127) proposed additional molecular players in the EV-mediated signaling between adipose tissue and cardiomyocytes. They found that peroxisome proliferator-activated receptor-gamma (PPARγ) activation in adipocytes causes the release of circulating miR-200a carried by exosomes that targets cardiomyocytes (127). MiR-200a downregulates TSC1, thus removing its inhibition of mammalian target of rapamycin (mTOR) signaling and consequently inducing cardiac hypertrophy (127). Other in vitro experiments demonstrated that hypertrophic adipocyte-derived exosomes are enriched with miR-802-5p that, if delivered to rat cardiomyocytes to target HSP60, causes unfolded protein response activation, oxidative stress, inflammation, and ultimately insulin resistance (128). Gan et al. (129) found significantly elevated levels of miR-130b-3p in EVs from diabetic rat adipocytes and from plasma of patients with diabetes compared with healthy controls. When the adipocyte EVs were delivered to the myocardium, detrimental effects after ischemia/reperfusion were observed. EV-treated hearts displayed significantly increased miR-130b-3p levels and AMPKα1/α2, Birc6, and Ucp3, all identified as miR-130b-3p direct downstream targets (129).

Recently, Guarnieri et al. (130) obtained data suggesting the involvement of exosome-mediated signaling in the development of cardiac fibrosis in mice with adipose-specific deletion of the human antigen R (Hur, an RNA binding protein). Adipo-HuR^−/− mice displayed an interesting co-overexpression of genes related to exosomes formation/secretion and inflammation-related genes in subcutaneous white adipose tissue (130).

The link between atherosclerosis and adipose tissue-derived exosomes has been extensively investigated. One study demonstrated how these exosomes play a role in accelerating atherosclerosis by inducing M1 proinflammatory polarization of macrophages and so favoring the progression of atherosclerosis in APOE^−/− mice (131). Another study reported that adipose tissue-derived mesenchymal stem cell-derived exosomes protects endothelial cells against atherosclerosis by restraining microRNA-342-5p expression (132).

Six weeks of intravenous administration of exosomes isolated from brown adipose tissue led to improved cardiac function and amelioration of metabolic syndrome in high-fat diet-fed mice (133). This beneficial effect can be attributed indirectly to the remodeled metabolism (reduced body weight, lowered blood glucose, and alleviated lipid accumulation); however, based on the fact that circulating exosomes can be internalized by cardiomyocytes (16, 17, 86), the authors hypothesized that there may be an additional direct effect on the heart (133). Further studies are warranted to test this hypothesis. On the other hand, exposure of cultured adipose tissue-derived mesenchymal stem cells to serum exosomes obtained from infarcted mice enhanced stem cell proliferation through the activation of ERK1/2 (72). Those exosomes, likely released by heart and kidney, were enriched in MiR-1956, identified as the possible messenger that stimulates angiogenesis and paracrine VEGF signaling in adipose tissue stem cells by downregulating Notch-1 (72).

Although exosomes from adipose tissue-derived mesenchymal stem cells have received less attention, several recent studies have documented their protective effects on cardiomyocytes during various types of stress (134, 135). Their administration to rats with myocardial ischemia/reperfusion injury significantly attenuated myocardial necrosis and apoptosis by 1) limiting Bcl-2 downregulation, 2) preventing Bax upregulation and caspase 3 activation, and 3) removing the inhibition of Wnt/β-catenin signaling (136). Other authors found that these exosomes attenuate cardiac damage after myocardial infarction by activating the S1P/SK1/S1PR1 signaling and promoting macrophage M2 polarization (137). Their positive effects during acute myocardial ischemic injury were enhanced by miRNA overexpression (miR-126 and miR-93-5p) (138, 139). Interestingly, adiponectin seems to be an effective stimulator of exosomes release by adipose tissue-derived mesenchymal stem cells (140). Additional adipocytokines such as omentin-1 have also been found enclosed in EVs released by adipose tissue (141) or other organs. For instance, lipocalin 2 has been found in neutrophil exosomes (142) and angiopoietin-like protein 2 in EVs released by vascular endothelial cells (143).

EVs in Heart-Lung Cross Talk

The lung is a major “consumer” of circulating exosomes (144). Even though heart and lung form a functional unit, EV-mediated communication among these two organs is relatively understudied. The published studies are mainly focused on EVs role in pulmonary arterial hypertension (114, 145–148). Circulating endothelial EVs were found elevated in adults with congenital atrial and ventricular septal defects, especially if complicated with pulmonary arterial hypertension (147). These EVs may enhance inflammatory processes leading to endothelial dysfunction and impaired vasodilatation via P38 MAPK-dependent pathways (147). Interestingly, EVs are not increased in children with congenital heart defects with or without associated pulmonary arterial hypertension (149). EVs isolated from blood and lungs of mice with monocrotaline-induced pulmonary arterial hypertension caused right ventricular hypertrophy and pulmonary vascular remodeling when injected into healthy mice (145). A recent study showed that pulmonary vascular endothelial cell-derived exosomes can prevent, through miR-107 transfer, the transdifferentiation of pericytes into a profibrotic phenotype (148). Mir-107 interfered with the HIF-1α/Notch1/PDGFRβ/YAP1/Twist1 signaling pathway (148). Deng et al. (146) found an upregulation of miR-143-3p in mouse lung and right ventricle after hypoxic stimuli causing pulmonary vasoconstriction, as well as in pulmonary artery smooth muscle cells of patients with pulmonary arterial hypertension. Mir-143-3p has also been detected in cardiac myocytes and fibroblasts. Furthermore, extracellular miR-143-3p was found to be transported by exosomes (146). Exosomal miRNA-143-3p activated migration and proliferation of pulmonary arterial endothelial cells (146).

EVs in Heart-Liver Cross Talk

The pathological condition known as cirrhotic cardiomyopathy attests the close relationship between heart and liver (150). Half of the patients with cirrhosis undergoing liver transplantation develop cardiac dysfunction, and 7%–21% of deaths after liver transplantation are caused by heart failure (150). Other associations between cardiac and liver diseases include: the increased risk of cardiac arrhythmias during nonalcoholic fatty liver disease (151, 152), the aberrant hepatic lipid metabolism accompanying hypertrophic cardiomyopathy (153), and the fatty liver as a cardiovascular disease risk factor (154–156). Nonetheless, virtually no information is available regarding EVs as part of the heart-liver communication under normal and pathological conditions. One study found that heart-liver communication involves phospholipase A₂-mediated signaling (157) that modulates hepatic inflammation and lipid metabolism. Phospholipase A₂ functions as a proinflammatory autocrine/paracrine factor (157, 158) and, in myocardium, it is stored in secretory granules (81, 158). Interestingly, there is evidence that lipid-associated proteins and bioactive lipids including the whole set of phospholipases A₂, C, and D are enclosed in exosomes released by 2H3 mast cells (159). The comprehension of EVs role in heart-liver communication is certainly a very interesting topic for future research and might have important therapeutical implications.

EVs in Heart-Brain Cross Talk

Secondary cardiac abnormalities are common in neurological patients (160) and, conversely, cardiac dysfunction has been shown to elevate the risk of stroke (161). Heart-brain communication is an area of very intensive investigation, yet it is only recent that the role of EVs in heart-brain cross talk has come to the attention of the scientific community (162). Of note, 47% of postcardiac surgery patients display increased permeability of the blood-brain barrier (163), which is disrupted by stroke as well (164). EVs can bidirectionally cross the blood-brain barrier under physiological conditions (76) that is enhanced after barrier damage (35). In fact, the level of circulating EVs is elevated during ischemic or hemorrhagic stroke (165, 166) and is associated with endothelial dysfunction and coagulation activation, leading to thrombosis (160, 167). In the brain, EVs normally originate from neurons, astrocytes, microglia, and neural stem cells, and yet after stroke, their major source is represented by endothelial cells (168). Stroke-associated elevation of circulating microvesicles may mediate brain damage-induced cardiac dysfunction (160). These EVs cause endothelial dysfunction by decreasing NO synthesis as the result of endothelial NO synthase inhibition and by raising caveolin-1 levels (160). Moreover, microvesicles bind coagulation factors and activate them via two procoagulants, namely phosphatidylserine and tissue factor that are incorporated in the outer leaflet of their membrane (169, 170). Furthermore, microvesicles contain P-selectin glycoprotein ligand-1, which interacts with the P-selectin exposed by endothelial cells or platelets upon vascular damage. Such interactions activate tissue factor that triggers thrombosis (160, 171). These mechanisms may, at least in part, explain the results of the recently concluded clinical study PROSCIS-B, which reported an association between high levels of leukocyte- and endothelial cells-derived microvesicles after stroke and worse cardiovascular outcome within 3 years (172).

Several other miRNAs abundantly present in EVs have been shown to play a role both in cardiac and cerebral pathophysiology (173). Long et al. (174) found a significant downregulation of circulating levels of miR-126 and miR-30a in patients with stroke and proposed that they are transported by exosomes, which, in turn, would very likely target and influence other organs, including the heart. MiR-126 was later found to be concentrated in EVs (175), including neuronal EVs (176). Endothelial cells-derived miR-126 knockout mice showed more severe cardiac dysfunction and cardiomyocyte hypertrophy after ischemic stroke compared with control stroke mice (177). MiR-126 is also known to be downregulated in heart failure and atrial fibrillation (178).

Another important miRNA in this context is miR-210. It is transported by mesenchymal stem cell-derived EVs and is a relevant player in angiogenesis within the brain (179) and heart (180). MiR-210 enhances the expression of hepatocyte growth factor, a known stimulator of angiogenesis, neurogenesis, and synaptogenesis (181).

The miR-17–92 cluster, carried also by mesenchymal stem cell-derived EVs (182), is a critical proliferation regulator of cardiomyocytes in neonatal and adult hearts (183) and of neural progenitor cells after stroke (184). Moreover, miR-17–92 has a direct protective effect in the ischemic heart and brain by targeting the PI3K/AKT and the MAPK/ERK pathways (173, 185).

MiR-124, primarily expressed in the brain and involved in the activation of the protective PI3K/AKT and MAPK/ERK pathways (186), was significantly decreased within 24 h after stroke and negatively correlated with the infarct volume (187). Consistent with these findings, the level of circulating EV-carried miR-124-3p declined during acute ischemic stroke (186). As for the heart, miR-124 is significantly upregulated during myocardial infarction and its inhibition reduces cardiomyocyte apoptosis (188).

Zhao et al. (189) investigated alterations of circular RNA expression in exosomes isolated from the brain extracellular space of mice with traumatic brain injury. Cardiac muscle contraction and calcium signaling were two of the 10 most upregulated functions affected by circular RNA, as predicted by KEGG pathway analysis (189). These findings are still very preliminary, nonetheless they can provide the reference for future research on the role of exosome carried circular RNA in the brain-heart communication axis.

CONCLUSIONS AND PERSPECTIVES

Research on EVs-mediated communication between heart and other organs is still at its early stage and the related literature is speedily growing. Therefore, the present review is necessarily limited and most of the mechanistic interpretations we reported here are obviously destined to obsolescence. For instance, other subcellular components like mitochondrial damage-associated molecular patterns (DAMS) (190), lysosomes, peroxisomes, cytoskeletal proteins, etc. might function as messengers carried by EVs. In the light of the available body of knowledge described in the aforementioned paragraphs and summarized in Fig. 2 and Table 1, it can be concluded that at least two formidable challenges await investigators: 1) the precise identification of organs and, in particular, cell types that produce or take up specific circulating EVs, in vivo, and 2) the comprehension of the real impact of EV-carried signals on organ functions, in vivo, relative to nervous and other paracrine/endocrine signals. Addressing the first challenge will require well-designed strategies in proper animal models and novel techniques to label organ-specific EVs. The second challenge will perhaps require the generation of genetic animal models with organ-specific impairment of EVs production. Another important open question is whether EVs produced in vitro may function as a new generation of vectors, alternative to viral or synthetic nanovectors, for organ-directed biological therapies (67–69, 191). Similarly, a major challenge is posed by isolation and purity of EVs from circulation, which warrants more accurate and robust methods for measuring EV number and biodistribution and to separate functionally and morphologically distinct subpopulations.

Table 1.

Extracellular vesicle-mediated communication between heart and other organs

EV Content	Source Organ	Recipient Organ	Effect and Proposed Mechanisms of Action	Trigger for EV Release	Ref.
Group 1
Musclin	Skeletal muscle	Heart	Cardioprotection, adrenergic signaling, decrease in oxidative stress, mitigated endothelial dysfunction and fibrosis, and subsequently better cardiac performance.	Remote limb ischemic conditioning	85
VEGF
β2-AR
MiR-1			Decreased apoptosis; caspase-9, possible target of miR-133a.	Duchenne muscular dystrophy	95*
MiR-133a
MiR-206
Numerous myokines:			Unknown	Exercise	97**
Cathepsin D
Calcium and integrin-binding protein
α2-Antiplasmin etc.
Unknown			Reduced myocardial I/R injury, decreased cardiomyocyte apoptosis by activation of ERK1/2 and HSP27 signaling.	Exercise	96
Group 2
Angiotensin II type 1 receptor	Heart	Skeletal muscle	Blood pressure response reconstitution by modulation of vascular responses and cardiac ERK signaling. (ERK phosphorylation).	Pressure overload	73
MiR-148a			Restored expression of dystrophin in mdx hearts.	(In vivo administration)	65*
MiR-1			Unknown	Myocardial damage	93**
MiR-133a
MiR-133b
MiR-206
Group 3
MiR-26a	Kidney	Heart	Cardiac function improvement, diminished fibrosis, and increased insulin sensitivity by inactivation of multiple targets such as GSK-3β, PTEN, CTGF and collagen I, and most importantly FoxO1. miR-26a intervention reduces CTGF abundance and restricts collagen deposition in the heart.	Exosomal MiR-26a levels are decreased in uremic cardiomyopathy and albuminuria	104*
Circular RNAs			Unknown	Chronic kidney disease	106**
Nephrin			Unknown	MI	72**
BNP	Heart	Kidney	Unknown
Group 4
Damaged mitochondria	Adipose tissue	Heart	Mediation of the ROS-signal, attenuation of cardiac injury during ischemia by ischemic preconditioning-like adaptation mechanism.	Mitochondrial stress	123
MiR-802-5p			Insulin resistance, increased oxidative stress by targeting HSP60.	Adipocyte hypertrophy	128*
MiR-130b-3p			Worsened myocardial infarction/reperfusion effects by targeting AMPKα1/α2, Birc6, and Ucp3.	Diabetes	129*
Mir-200a			Cardiac hypertrophy by removing TSC1 inhibition of mTOR signaling.	PPARγ activation in adipocytes	127
Numerous proteins (genes: Cav1, Gpd, Ugp2, Pgd, Etfa, Cs, etc.)	Brown adipose tissue		Amelioration of the metabolism leading to hepatic and cardiac function restoration, decreased expression of the two inflammatory genes in the liver, (TNFα and IL-1β). reduced cardiomyocyte hypertrophy.	-	133*
Unknown	ATMSC		Endothelial cell protection against atherosclerosis by restraining miR-324-5p expression.	-	132*
Mir-126			Reduced cardiac fibrosis, cell apoptosis, suppressed inflammatory cytokine expression and promoted angiogenesìs possibly by targeting Spred1 and PI3KR2 and enhancing the VEGF signaling pathway.	ATMSC have been transfected with miR-126	138*
MiR-93-5p			Suppressed autophagy and inflammation after MI, infarction-induced TLR4 expression and NF-κB p65 phosphorylation, decreased inflammatory factors: IL-6, IL-1β, and TNF-α in MI patients.	ATMSC have been transfected with miR-93-5p	139*
Unknown			Reduction of apoptosis, induced by oxidative stress	-	134*
Unknown			Protection against I/R-induced myocardial injury by limiting Bcl-2 downregulation, preventing Bax upregulation and caspase 3 activation, and removing the inhibition of Wnt/β-catenin signaling	-	136*
Unknown	Heart	ATMSC	Proliferation of ATMSC through the activation of ERK1/2	MI	72*
BNP			Unknown
MiR-1956			Angiogenesis stimulation and paracrine VEGF signaling in ATMSC by downregulating Notch-1
Group 5
MiR‐19a	BMSC	Heart	Downregulation of two genes: PTEN and BIM, followed by increased ERK and Akt phosphorylation and subsequently cardiomyocyte survival and reduced apoptosis.	BMSC have been transduced by GATA4	113*
MiR‐451
MiR-10a			Cardiac hypertrophy prevention, apoptosis attenuation, fibrosis, and cardiac function preservation during pressure overload. (Proposed mechanism: increase in the Bcl-2/Bax ratio and decrease of cleaved caspase-3 expression).	-	66*
MiR-16
MiR-21
MiR-29a
MiR-199b
MiR-22			Apoptosis reduction by direct targeting of methyl CpG binding protein 2, diminished cardiac fibrosis	Ischemic preconditioning	116*
Unknown			The mean pulmonary artery pressure and mean right ventricle pressure amelioration, decrease the right ventricle hypertrophy	-	114*
Unknown			Cardioprotection through angiogenesis and anti-inflammation, (promoted tube formation of HUVECs and decreased the proliferation of spleen lymphocytes). Improvement in cardiac function and amelioration of fibrosis after myocardial infarction.	-	115*
Circ-G042080	Bone marrow		Myocardial damage by the circ-G042080/hsa-miR-4268/TLR4 ceRNA axis. increased LC3 and Beclin1 levels and a decreased P62, increased number of autophagic vesicles leading to autophagic death in cardiomyocytes	Multiple myeloma	70
MiR-1	Heart	Bone marrow	Chemokine receptor CXCR4 expression suppression and progenitor cell mobilization in bone marrow	MI	118
Mir-208
Mir-499
Group 6
Phospholipase A2	Heart	Liver	Regulation of inflammation and lipid metabolism in the liver	-	157, 159**
Group 7
miR-143-3p	Heart	Lung	Possibly take part in pulmonary artery hypertension pathogenesis	Hypoxia	146**
miR-10b	Lung	Heart	Bone marrow progenitor cell differentiation to endothelial progenitor cells that contribute to pulmonary hypertensive changes and RV hypertrophy in normal mice.	Monocrotaline treatment	145*
miR-698
miR-743b
Diverse protein species: low-density lipoprotein receptor-related protein 2, alanyl-tRNA synthetase, myeloperoxidase, etc.
Group 8
Phosphatidylserine and tissue factor	Brain	Heart	Initiation of the extrinsic coagulation pathways and promotion of the assembly of clotting enzymes leading to thrombin generation	Ischemic stroke	160, 172
P-selectin glycoprotein ligand-1			Thrombosis, by the concentration of the tissue factor activity at the thrombus
MiR-126			Unknown		174–177**

ATIRa, angiotensin II type I receptors; ATMSC, adipose tissue mesenchymal stem cells; BIM, B-cell lymphoma-2-like 11 gene; BMSC, bone marrow stem cells; CTGF, connective tissue growth factor; EV, extracellular vesicle; GSK-3β, glycogen synthase kinase 3β; HUVECs, human umbilical vein endothelial cells; MI, myocardial infarction; mTOR, mammalian target of rapamycin; PPAR, peroxisome proliferator-activated receptor; PTEN, phosphatase and tensin homolog; ROS, reactive oxygen species.

*Indirect evidence obtained by exogenous EV infusion in experimental animals or exposure of cultured cells; **presumed mechanism, requires further studies.

The identification and mapping of a new bidirectional communication network between the heart as a hub and other organs, mediated by circulating EV, might open at least two intriguing perspectives. One is the addition of a major chapter of integrative physiology to the classical knowledge of communications mediated by nervous systems, hormones, and hormone-like mediators, which may inspire similar research on other “hubs.” The second perspective is the possible exploitation of EV cardioprotective messengers for therapeutic use: molecules indicated in Table 1 and represented in Fig. 2 might be potentially used as curative biological agents if properly purified and delivered through effective vectors.

The new scenarios that are opening before us are ample and intriguing and are calling for intensive investigation that will keep physiologists and pathophysiologists busy for several more years.

GRANTS

This work was supported by National Heart, Lung, and Blood Institute Grants HL151345 (to F. A. Recchia) and HL135177 (to M. Khan) and American Heart Association Transformational Project Award 20TPA35490355 (to M. Khan).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

K.G. prepared figures; K.G. drafted manuscript; K.G., M.K., and F.A.R. edited and revised manuscript; K.G., M.K., and F.A.R. approved final version of manuscript.