Keywords

1 Circulating Exosomes and Exosomal Cargos

Numerous studies demonstrated that exosomes in the early phase are formed into a structure which is regarded as a multivesicular body (MVB) through endocytic invagination [1, 2]. Subsequently, the MVB fuses with the cytoplasmic membrane and is secreted with its cargos of lipids, proteins, functional mRNAs, and microRNAs (miRNAs, miRs) into the extracellular environment. The Rab-family GTPases, Annexins, SNAREs, and Endosomal Sorting Complexes Required for Transport (ESCRT) associated proteins are essentially involved in the formation and secretion of exosomes [2, 3]. Some of the exosomes are eventually released into the circulation, known as circulating exosomes [4]. Circulating exosomes could arrive in distant tissues via blood circulation, thus directly communicating with target cells and rapidly regulating intracellular signalings.

In various physiological and pathological conditions, different patterns of proteins, lipids, and non-coding RNAs such as miRNAs can be detected in the circulation [5, 6]. The cell-free non-coding RNAs could be stably present in blood circulation via being packaged into exosomes [7]. The circulating exosomes can be uptaken by recipient cells, whereby transferring the composite cargos or activating the signaling pathways [8,9,10,11]. Particularly, the various types of cargos loaded in exosomes and the signaling diversity are closely related to the different tissue and cell types from which exosomes are originated [12,13,14,15]. Among the diverse exosomal cargos, miRNAs can effectively regulate the target genes and influence the biological functions of target cells. miRNAs are a large group of small (18–25 nucleotides in length) noncoding RNAs that regulate target gene expressions at post transcriptional level [16, 17]. It has been increasingly reported that exosomal components, especially miRNAs, play important roles in regulating cardiac function and protecting the heart against acute myocardial infarction (AMI) and ischemia reperfusion injury (IRI) [18, 19]. For example, exosomes derived from chemokine receptor CXCR4-overexpressing mesenchymal stem cells (MSCs) were reported to activate the IGF-1/PI3K/Akt signaling pathway in cardiomyocytes, thereby reducing myocardial apoptosis, promoting angiogenesis, decreasing ventricular remodeling, and protecting cardiac function after MI [20]. Since it is difficult to obtain cardiac tissue samples from patients, detecting changes of circulating exosomes from peripheral blood might be useful strategy to attain information about the pathophysiological processes of cardiovascular diseases [21,22,23] as well as to guide the treatment for patients [24,25,26].

2 Circulating Exosomes in Cardiovascular Pathophysiology

Intercellular communication is one of the essential mechanisms for cells exerting their biological functions in all multicellular organisms. Almost all cells exchange messages by direct interaction or the secretion of signaling molecules. Studies have revealed that circulating exosomes can mediate comprehensive interactions among various cell types and exert biological functions by transmitting exosomal cargos to recipient cells [2, 27]. Exosomes were proved to be secreted from the injured heart and participate in cardiovascular pathophysiology [28,29,30]. Although real success has been achieved in experimental studies of exosomes in cardiovascular physiological and pathological progresses, the molecular mechanisms remain incompletely understood [2, 31, 32].

Exosomes derived from cardiomyocytes are initially found under the hypoxia and reoxygenation condition, which may contain biological molecules such as HSP70 [33,34,35]. Likewise, exosomes function as messenger of intercellular communication among cardiomyocytes, fibroblasts, smooth muscle cells, and endothelial cells, and participate in the regulation of cardiac regeneration, ventricular remodeling, and angiogenesis in cardiovascular diseases [31]. Due to the perfect peculiarity as carriers of signal molecules, circulating exosomes deliver both protective and detrimental information [36,37,38,39]. Circulating exosomes generally regulate cardiovascular pathophysiology, such as cardiomyocyte hypertrophy, apoptosis, and angiogenesis (Fig. 17.1).

Fig. 17.1
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Circulating exosomes regulate cardiomyocyte hypertrophy, apoptosis, and angiogenesis

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2.1 Cardiomyocyte Hypertrophy

Various forms of stress in the heart can contribute to activate cardiac myocyte hypertrophy [40, 41]. The general cardiac hypertrophy is characterized by myocyte enlargement and the re-expression of embryonic genes. Cardiomyocyte hypertrophy is a common response upon the increased heart hemodynamic state (such as high blood pressure or valvular stenosis), myocardial injury, and neurohormonal stress in the compensatory period. Early compensatory cardiac hypertrophy can be adapted to the enhanced post-ventricular load and maintain normal cardiac output. However, sustained cardiac hypertrophy will eventually lead to cardiac ventricular dilatation, reverse remodeling, and heart failure [40].

Circulating exosomes were reported to be involved in the regulation of pathological cardiac hypertrophy. Circulating exosomes loaded with miR-1 and miR-133a were found to be significantly increased in the serum of patients with AMI [42].miR-1 and miR-133 are preferentially expressed in skeletal muscle and cardiac tissue and are involved in the pathogenesis of cardiac hypertrophy [43]. It was previously demonstrated that miR-133a via targeting RhoA, Cdc42, and NELF-A/WHSC2, while miR-1 via targeting Ras GTPase-activating protein (RasGAP), Cdk9, Rheb, and fibronectin, could inhibit cardiac hypertrophy [42, 44,45,46].

It was previously demonstrated that fibroblast-derived exosomes enriched with miR-21-3p were able to induce cardiomyocyte hypertrophy via targeting SH3 domain-containing protein 2 (SORBS2) and PDZ and LIM domain 5 (PDLIM5). Inhibition of miR-21-3p resulted in reduced cardiac hypertrophy in Angiotensin II-treated animals [47]. In addition to circulating miR-29 and miR-30 that have been identified as possible biomarkers for left ventricle hypertrophy, the relevance of circulating miR-21 in the diagnosis and prognosis of cardiac hypertrophy deserves further investigation [48].

2.2 Cardiomyocyte Apoptosis

Cardiomyocyte apoptosis is a significant issue underlying ischemic cardiac diseases [49], and occurs with dilated cardiomyopathy [50] and aging-related cardiac dysfunction [51]. Myocardial ischemic injury is associated with a shared characteristic patterns of cell death and metabolic changes which could result in irreversible myocardial injury [52, 53]. Apoptosis is involved in the whole process of myocardial ischemic injury, which could range from the initial phase after myocardial infarction to reperfusion stage [54, 55]. However, the specific molecular mechanisms underlying cardiomyocyte apoptosis are not fully understood.

Inhibition of miR-155 was previously demonstrated to inhibit cardiomyocyte apoptosis and cardiac dysfunction in lipopolysaccharide (LPS)-treated mice, via targeting Pea15a. Furthermore, increased circulating miR-155 was found to be associated with cardiac dysfunction in sepsis patients [56]. In this regard, the increased circulating miRNA-155, whether packaged in circulating exosomes or not, deserves further investigation in sepsis-induced cardiac dysfunction [56]. Notably, plasma exosomes isolated from healthy human and rats were recently demonstrated to be able to protect against cardiomyocyte apoptosis and ischemia reperfusion injury, indicating that endogenous circulating exosomes at baseline have protective effect for the heart [57].

2.3 Angiogenesis

Angiogenesis is a biological process of growing new vessels from the existing vascular structure and promoting endothelial cell proliferation to form vascular network. Many factors, such as fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) can stimulate the formation of new vessels. Exosomes were reported to participate in the regulation of angiogenesis which is an essential process contributing to cardiac repair after injury. The CD34-positive stem cell-derived exosomes enriched with angiogenesis-related miR-126 and miR-130a were found to be significantly reduced in the peripheral blood of patients with chronic heart failure [58]. miR-126 and miR-130a were previously reported to stimulate angiogenesis by down-regulating the angiogenic negative regulator SPRED1 and HOXA5, respectively [59,60,61]. SPRED1, the member of Sprouty protein family, blocks angiogenesis through negatively regulating the VEGF-C/VEGFR-3 signaling [62]. HOXA5 also suppresses angiogenesis by upregulating the anti-angiogenic gene Thrombospondin-2. Besides that, HOXA5 also downregulates many pro-angiogenic genes including VEGFR2, Ephrin A1, HIF1alpha, and COX-2 [63].

3 Circulating Exosomes in Myocardial Ischemia Reperfusion Injury

The early reperfusion of the myocardium is considered as an important intervention in the treatment of myocardial ischemia which can efficiently attenuate further damage to the myocardium [64]. However, some infarct areas could be expanded when the blood flow regains after ischemia, which is known as myocardial ischemia reperfusion injury (MIRI) [65]. Ultimately, MIRI can lead to ventricular remodeling and even progressive heart failure [66, 67]. MIRI is associated with a complexity of multiple pathophysiological features [68], such as calcium overload, accumulation of oxygen free radicals, endothelial dysfunction, immune activation, mitochondrial dysfunction, cardiomyocyte apoptosis and autophagy, platelet aggregation, and microembolization [69,70,71,72,73,74]. However, the molecular mechanisms underlying MIRI are not completely understood.

Circulating exosomes can be markedly altered after MIRI and may serve as extracellular messengers through endocytosis, membrane fusion, and cell-receptor interaction to facilitate cell-cell communication [32]. Mounting evidence has shown that exosomes, especially stem cell-derived exosomes, have protective effects against MIRI [19, 28, 75, 76]. Mesenchymal stem cell-derived exosomes were demonstrated to promote cardiomyocyte viability and prevent adverse remodeling after MIRI, by enhancing the generation of ATP, reducing oxidative stress, and activating the PI3K/Akt pathway [28]. More interestingly, circulating exosomes isolated from healthy human and rats were also proved to be able to transmit signals to the heart and provide protective effects against MIRI [57]. The exosomes packed with HSP70, could activate Toll-like receptor 4 (TLR4) signaling and induce ERK1/2 and p38MAPK activation and subsequent HSP27 phosphorylation in cardiac myocytes (Fig. 17.2) [57]. Increasing evidence suggests that the activation of ERK1/2 and/or PI3K/AKT signaling pathways are crucial for the cardioprotective effects [77, 78]. HSP70, a member of small HSP family, can be loaded in exosomes [33] and is present in the circulation of normal individuals [79]. Moreover, the HSPs, especially HSP27 which is abundant in the myocardium, can be generated upon adverse stresses (e.g. heat) thus offering protective effects for the heart [80]. These studies highly suggest that circulating exosomes may provide a promosing non-cellular approach for the treatment of MIRI.

4 Circulating Exosomes in Myocardial Infarction

Myocardial infarction (MI) is occurred when the flow of oxygen-rich blood is blocked in a section of myocardium, which is frequently caused by atherosclerosis-related coronary artery luminal occlusion and plaque rupture [81]. Simultaneously, MI is usually associated with a dramatic decrease of myocardial contractility and reduction of cardiac output [82]. In addition, MI may cause arrhythmia, cardiogenic shock, and heart failure. In pathophysiological aspects, cardiomyocyte apoptosis and necrosis are the essential causes of cardiomyocyte damage and loss in MI [83]. In the late stage, severe MI will ultimately progress to adverse cardiac remodeling and heart failure [84]. In these cases, controlling excessive inflammatory response, inhibiting cardiomyocyte death, preventing ventricular fibrosis, and facilitating angiogenesis are considered as potential therapeutic strategies for improving the prognosis of MI patients.

It has been reported that exosomes are highly involved in the pathophysiological processes of MI [20, 29]. Some exosomes derived from stem cells such as embryonic stem cells (ESCs), mesenchymal stem cells (MSCs), and cardiac progenitor cells (CPCs) were proved to improve cardiac function after MI, likely by reducing cardiomyocyte apoptosis, inhibiting myocardial fibrosis, and promoting angiogenesis [75, 85, 86]. However, some exosomes may exacerbate myocardial injury after MI and also be associated with vascular damage and cardiovascular risk [87, 88]. For example, exosomes containing HSP60, released from highly differentiated adult cardiomyocytes in an anoxic condition, are detrimental to cardiomyocytes during acute MI [34, 89]. Extracellular HSP60 was shown to cause cardiomyocyte apoptosis through activating TLR4 [90]. Nonetheless, HSP20 contained in circulating exosomes derived from cardiomyocytes was identified as a novel cardiokine which may promote myocardial neovascularization via activating vascular endothelial growth factor receptor 2 (VEGFR2) after MI [91].

Intriguingly, circulating miRNAs that are changed upon MI could also be packaged in the exosomes (Fig. 17.2). It was found that miR-1 and miR-208 which might be contained in exosomes were significantly increased in the serum of rats with AMI and in the urine of AMI patients [92]. Equally, the cardiac muscle-specific miRNAs including miR-208b and miR-499 were shown to be increased in the circulation of MI patients [93, 94]. As well, circulating p53-responsive miR-192, miR-194, and miR-34a, particularly enriched in exosomes, were significantly increased in the early stage of MI [95]. Notably, the miR-194 and miR-34a levels were correlated with left ventricle end-diastolic dimension 1 year after MI, indicating that circulating miR-194 and miR-34a might serve as predictors for heart failure development in MI patients [95].

Fig. 17.2
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Circulating exosomes contribute to the pathogenesis of myocardial infarction (MI) and myocardial ischemia reperfusion injury (MIRI)

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5 Circulating Exosomes in Other Cardiovascular Diseases

5.1 Atherosclerosis

Atherosclerosis, the primary cause of MI, is a chronic inflammatory-immune disease of vasculature [96]. Atherosclerosis is associated with the thickening of vessel walls and the formation and deposition of lipid plaques in the cerebral, aortic, and peripheral arteries, which can be regulated by multiple cellular and molecular mechanisms. It was previously reported that high shear-stress or the shear-responsive transcription factor Krüppel-like factor 2 (KLF2) can induce vascular endothelial cells to secret exosomes enriched with miR-143 and miR-145 and subsequently regulate the target genes such as CAMK2d and ELK1 in smooth muscle cells [97], thus may regulate proliferation and de-differentiation of smooth muscle cells [98]. In addition, extracellular vesicles derived from KLF2-expressing endothelial cells can attenuate atherosclerosis formation in vivo [97]. Equally important, macrophage-derived exosomes from both atherosclerotic plaques and the peripheral blood were demonstrated to participate in the development of atherosclerosis [99, 100]. The atherosclerotic patients have higher levels of leucocyte-derived extracellular vesicles in the circulation compared to healthy participants [101]. Furthermore, the circulating exosomes originated from macrophage foam cells were proved to promote smooth muscle cell adhesion and migration in atherosclerotic lesion through activating the ERK and AKT pathways [101].

5.2 Hypertension

The renin-angiotensin system (RAS), principally composed of renin, angiotensinogen, angiotensin-converting enzyme (ACE), angiotensin II (Ang II), and Ang II type 1 and type 2 receptors (AT1R and AT2R), plays key roles in the development of hypertension. It was previously reported that the AT1R-enriched exosomes were secreted from cardiomyocytes into the serum of mice undergoing cardiac pressure overload, thus regulating the blood pressure under hemodynamic stress [102]. Moreover, exogenously delivered AT1R-enriched exosomes were demonstrated to be uptaken by recipient cells such as smooth muscle cells and endothelial cells, which contributed to the regain of blood pressure response induced by Ang II in AT1R knockout mice [102]. Thus, the circulating exosomes containing AT1R, released from cardiomyocytes during pressure overload, may play important roles in regulating the blood pressure in detrimental conditions such as hypertension and heart failure.

5.3 Sepsis Cardiomyopathy

Sepsis cardiomyopathy is common in clinic and is predominantly caused by systemic bacterial infection. Although the pathogenesis of sepsis cardiomyopathy is quite complex, the out-of-control immuno-inflammatory response, oxidative stress, cardiomyocyte apoptosis, and mitochondrial dysfunction are recognized as critical mechanisms. The platelet-derived extracellular vesicles isolated from septic patients were previously shown to induce vascular cell apoptosis through the NADPH oxidase-dependent release of superoxide [103]. The nitric oxide (NO) and bacterial toxin were proved to be positive factors for the secretion of platelet-derived exosomes. The circulating exosomes may further induce endothelial cell apoptosis via generating the peroxinitrite radical and activating Caspase 3 [104]. Further studies will be needed to investigate the potential of circulating exosomes and exosomal cargos in the diagnosis and prognosis of sepsis cardiomyopathy.

6 Perspective and Future Directions

Cardiovascular diseases are one of the major threats to human health [105, 106]. To date, a detailed understanding is available for stem cell transplantation in the treatment of myocardial injury and heart failure, however, there are still many problems in stem cell therapy such as ethical issue, limited source, low viability in local damaged myocardium, and immune rejection [107,108,109]. Although the induced pluripotent stem cells (iPSCs) are more likely to survive in the damaged myocardium compared to mesenchymal stem cells (MSCs) [110], iPSCs-associated tumorigenesis remains a critical issue. Initially, it is thought that stem cells can differentiate into cardiomyocytes and promote cardiac regeneration and repair. Nevertheless, subsequent detection revealed few new cardiomyocytes derived from transplanted stem cells, suggesting that stem cells are likely to promote the process of myocardial regeneration and angiogenesis by other mechanisms [111]. Circulating exosomes enriched with various types of bioactive molecules can be changed not only in the number but also in the composite cargos upon cardiac injury, which may influence cardiomyocyte function and contribute to cardiac regeneration and repair [57, 112]. In particular, compared with stem cell therapy, exosome-based therapeutic strategy would also decrease the risk of disordered differentiation and tumorigenesis induced by stem cells [75, 112, 113].

Circulating exosomes can mediate local and distant cell communication through the horizontal transfer of their contents such as miRNAs and proteins or the activation of signaling pathways in the target cells [12, 36]. Notably, the exosomal contents can be selectively enriched or modified by bioengineering, thus providing desirable effects in the treatment of cardiovascular diseases [114]. Moreover, given the particular lipid bilayer structure, exosomes can be used as a new drug carrier though it remains to be solved whether and how the delivered exosomes would reach the specific target tissues and cells to exert their biological therapeutic effects [115,116,117]. Also importantly, exosomes are naturally secreted into the extracellular environments, which may faultlessly overcome immunogenicity compared with other developed delivery devices. Last but not least, more preclinical and clinical studies will be needed to investigate the potential of circulating exosomes as biomarkers for the diagnosis, risk stratification, treatment, and prognosis of cardiovascular diseases [118, 119].