Introduction

Type 2 diabetes mellitus is a chronic metabolic disorder that has a strong association with the development of cardiovascular diseases, referred to as diabetic heart disease (DHD) [1]. DHD is a broad definition encapsulating a cluster of conditions, including coronary artery disease, heart failure, cardiac autonomic neuropathy, peripheral arterial disease and diabetic cardiomyopathy [2,3,4,5,6]. A cohort study conducted by Shah et al. demonstrated that heart failure and peripheral arterial disease are the most common initial manifestations of cardiovascular disease in type 2 diabetic individuals [7]. Additionally, the Framingham study has shown that diabetic women had a higher risk of develo** cardiovascular disease and congestive heart failure than diabetic males [4]. The Framingham study also implied the direct association between diabetes and the development of cardiomyopathy independent of any associated comorbidities [2].

Multiple studies have identified several molecular alterations in response to diabetes that underlie the development of diabetes-induced comorbidities [8,9,10,11,12,13,14,15,16]. Among these mechanisms, microRNAs (miRNAs) have been extensively studied. Results have shown a clear link between miRNA dysregulation and DHD [9, 10, 12,13,14, 17,18,19,20,21]. Of note, circulating miRNAs under DHD conditions have been demonstrated to have the potential to act as biomarkers [22,23,24]. Additionally, these circulating miRNAs can also affect the progression of disease under diabetic conditions[25,26,27,28]. Further, in addition to being circulated in naïve form, miRNAs can also be encapsulated into exosomes. Exosomes are a common nanovesicle that can encapsulate miRNAs and transport them in circulation [29, 30]. miRNAs isolated from plasma exosomes showed higher stability than miRNAs isolated directly from plasma [29]. Exosomes are efficiently engulfed by recipient cells, allowing miRNAs to exert their effects [31,32,33].This review will first provide an overview of the biogenesis and function of exosomes and miRNAs. Next it will focus on the role of exosomes in cardiovascular disease and the known role of exosomes and the miRNAs encapsulated within them in DHD.

Exosomes

Exosome biogenesis

Fig. 1
figure 1

Exosome biogenesis. The invagination of the cell membrane results in the formation of the early endosome (A). Various budding into the endosomal lumen, initiates the process of forming exosomes/intraluminal vesicles in the late sorting endosome (B). Cargo is shuttled between the trans-golgi network (C), endoplasmic reticulum (D)and the late sorting endosome (B). Cumulatively, this leads to the formation of a multivesicular body (E) with fully formed intraluminal vesicles/exosomes in it. The multivesicular body can be further processed within the cell by the lysosome (F) or autophagosome (G) to breakdown the components of the multivesicular body into the cell. Otherwise, the multivesicular body can be docked (H) and fused to the cell membrane to release the exosomes (I) into the extracellular space. Exosomes can be characterized using different surface markers (J) that are consistently expressed by them. Exosomal functionality (K) is based on the varying cargo within the exosomes. Image Created with Biorender.com with valid license

Full size image

Biogenesis of exosomes begins in the endosomal system, where the early endosomes mature into late endosomes [34]. During the maturation process, the endosomal membrane undergoes further invagination [34], resulting in the formation of intraluminal vesicles (ILVs) or exosomes within the organelles [34]. The accumulation of exosomes in the endosome results in the formation of multivesicular bodies (MVBs) [35]. The MVBs then fuse with the plasma membrane to release exosomes into the extracellular space [36]. The Ras associated binding protein (Rab) family of small GTPases is involved in intracellular vesicle transportation through cytoskeletal interactions and docking of the vesicles to the target compartments [36, 37]. The soluble N-ethylmaleimide-sensitive fusion attachment protein (SNAP) receptors (SNAREs) play a role in the docking and fusion of the MVB to the plasma membrane [38]. The exosomes released will have surface markers from their origin cells [39] (Summarized in Fig. 1).

Exosome cargo sorting

The endosomal sorting complex required for transport (ESCRT) plays an essential role in forming ILVs in the endosome [40, 41]. Colombo et al. demonstrated that ESCRTs were involved in forming and secreting a heterogeneous subpopulation of exosomal vesicles [41]. They further showed that specific ESCRT components were involved in cargo loading in exosome biogenesis [41]. Other studies demonstrated an ESCRT-independent pathway of exosome biogenesis, as silencing of ESCRTs still resulted in the production of exosomes [37]. Tetraspanins such as CD63, CD9 and CD81 play an essential role in ESCRT-independent cargo loading into exosomes [42]. Tetraspanins organize the exosomal membrane into tetraspanin enriched domains [42, 43]. This formation is through the interaction between the exosome tetraspanins and other cellular tetraspanins, cytosolic proteins and lipids [42, 43]. Therefore sorting exosome cargos into exosomes occurs in both ESCRT-dependent and independent pathways [37, 44].

Interestingly, despite being a nanoparticle, exosomes can contain a variety of cargo. Studies have demonstrated the presence of various DNA molecules [45], RNAs such as miRNAs, circular RNAs and messenger RNAs [46], and proteins [47]. Multiple studies have also shown the functional importance of these cargos [32, 48, 49]. Exosome cargos are altered depending on the physiological or pathological conditions of the parent cells [49, 50]. Many studies have demonstrated selective chaperoning of miRNAs into exosomes under pathological conditions [51, 52]. As exosomes prolong the miRNA stability in circulation, they can also be considered a potential biomarker or a therapeutic target for various diseases [49, 50] (Summarized in Fig. 1).

Exosome uptake

Exosome surface markers and cell surface markers determine the selectivity of exosome uptake by the recipient cells [48, 53, 54]. Exosomes can be taken up by nearby recipient cells (juxtracrine uptake), cells further away from donor cells (paracrine uptake), as well as by cells in distant tissues (endocrine) by travelling through the systemic circulation [23, 48, 55,56,57,58,59]. The uptake of exosomes by the selected host cells can occur through either the phagolysosomes, utilizing exosomal surface proteins, or phosphatidylserine receptors [48, 54, 55, 60]. Tian et al. demonstrated that exosomes were internalized through the endocytosis pathway, where the intracellular exosomes were trapped in vesicles and actively transported [53]. In contrast, Feng et al. demonstrated efficient exosome uptake by phagocytic cells, where the exosomes were targeted to large phagolysosomes [61]. They further concluded that the type of exosomal uptake was dependent on the type of cargos and surface markers expressed in the exosomes [61].

Once internalized, the exosomes release their cargo via the phagolysosome or by being localized in the late endosome [53, 61]. Tian et al. demonstrated that exosomal proteins separate from the exosomes approximately 3 h post-internalization, while exosome lipids were re-cycled into the plasma membrane [53]. Interestingly, Montecalvo et al. demonstrated that exosomes dock, bind and then fuse with the recipient cells, [62] thereby “injecting” the exosomal miRNAs into the recipient cells [62]. This injection allows the exosomal miRNAs to reach the recipient cell cytosol to initiate its functional processes [62]. Concerning the endocytosis exosomal uptake pathway, Tian et al. demonstrated that following endocytosis, exosomes were stored in lysosomes where the exosomal proteins were stored and utilized [55]. However, while they were able to determine successful miRNA transfer, they could not determine the exact pathway or mechanism of content release for exosomal RNAs [55] (Summarized in Fig. 1).

miRNAs

miRNAs are short non-coding RNAs (~ 22 nucleotides) which post-transcriptionally alters protein expression by inhibiting the translation of messenger RNA. Under conditions such as diabetes, the alterations that occur at the gene coding regions lead to changes in miRNA expression [63, 64]. These changes induce pathophysiological alterations such as accelerated cardiomyocyte death, vascular remodelling, fibrosis, arrhythmias and stem cells dysfunction [8, 9, 12, 65, 66], all of which play a critical role in the progression of DHD [13, 67,68,69].

miRNA biogenesis

The biogenesis of of miRNAs is initiated in the nucleus where the introns of protein-coding genes or independent genes are initially transcribed by RNA polymerase II/III to form primary miRNA transcripts (pri-miRNA) [70, 71]. The RNase III protein Drosha then processes the pri-miRNA with its essential cofactor DiGeorge syndrome critical region 8 (DGCR8), a microprocessor complex subunit to generate ~ 70 nucleotides long precursor miRNAs (pre-miRNAs) [72].

In the presence of the Ran-GTP cofactor, Exportin-5 binds specifically to pre-miRNAs, facilitating nuclear export of the pre-miRNAs into the cytoplasm[73]. The final stages of miRNA processing occur in the cytoplasm, where the pre-miRNA was then cleaved by the enzyme Dicer to generate the ~ 22 nucleotide miRNA duplex [70]. In order to generate mature miRNA, the miRNA duplex needs to be unwound [74]. Once unwound, the mature miRNAs are bound to argonaute proteins to maintain their stability within the cytoplasm [75].

miRNA function

Mature miRNAs bind with RNA-induced silencing complexes (RISCs) to identify their messenger RNA (mRNA) targets[76, 77]. If the complementarity between the miRNA-RISC complex and the target mRNA is partial, the translation of the target mRNA leads to protein repression [78]. If there is exact complementarity between the complex and the target mRNA, the target mRNA undergoes cleavage and degradation [78, 79]. This feature of the miRNA allows it to target multiple genes and hence regulating multiple different pathological processes in its host cells [78,79,80,81,82].

Physiological and pathological roles of miRNA

As miRNAs have a diverse range of targets, they control both physiological and pathological processes [83]. Physiologically, miRNAs regulate processes such as development, cardiac hypertrophy and neuronal function[83,84,85]. We along with others have demonstrated the crucial role of miRNAs in various physiological and pathological processes (reviewed elsewhere)[86,87,88,89,90,91,92]. For instance, studies have demonstrated that multiple different miRNAs, including miRNA-26b, -27a, -143 and -150, were upregulated in exercise-induced cardiac hypertrophy [93]. While another study showed significant modulation of miRNA-195, -125b, -199a, and -124 in response to pathological cardiac hypertrophy and heart failure [81], suggesting that different miRNAs respond to physiological or pathological hypertrophy. Furthermore, the cardiac-specific loss of miRNAs by deletion of DGCR8 results in the development of dilated cardiomyopathy and heart failure [85].

Studies have demonstrated alterations in plasma miRNA expression in response to type 2 diabetes [10, 12, 23]. In addition to their effects on host cells, miRNAs are also released into the circulation, affecting remote cells/tissues [94]. Once released into circulation, miRNAs are either bound to proteins, high-density lipoproteins or encapsulated in micro- or nanovesicles such as exosomes [94]. This encapsulation prevents miRNA degradation, increasing their stability in the circulation, allowing miRNAs to have various paracrine effects [29, 94]. Further, this stability also allows miRNAs to function as biomarkers for various conditions [50, 95, 96].

Exosomes in CVD

Pathophysiological role of exosomes in CVD

Fig. 2
figure 2

Function of exosomes under DHD conditions. Studies have been conducted on exosomes isolated from DHD biological fluids as well as from culture media under DHD conditions. Exosomes released from patients with DHD has been demonstrated to increase calcification, plaque rupture, adverse remodeling and blood pressure in the vasculature. This was mimicked in the in vitro experiments where diabetic exosomes increased endothelial cell apoptosis, while decreasing proliferation, angiogenesis and migration. Similarly, DHD exosomes in vivo resulted in increased apoptosis, fibrosis and adverse remodeling of the heart as well as reduced cell survival. Just as in in vitro experiments, DHD exosomes promoted cardiomyocyte apoptosis while reducing cardiomyocyte survival

Full size image

Multiple studies have demonstrated exosomes to have various roles in CVD [31, 49, 57, 97,98,99]. Emmanueli et al. [23] demonstrated the potential of plasma exosomes as a biomarker in for recovery of patients post coronary artery bypass graft (CABG). They demonstrated increased density of the plasma exosomes post-CABG[23]. Importantly, these exosomes had increased expression levels of cardiac-specific miR-1, miR-24, miR-133a and miR-133b, both at 24- and 48-h post-surgery [23]. They also showed that positive correlation between alteration in the miRNA cargos within the exosomes and the expression of cardiac troponin I, a known biomarker for myocardial damage [23]. Another study demonstrated increased brain/head glycogen phosphorylase (PYGB) in circulating exosomes as an early and sensitive biomarker for cardiac injury [100]. The alteration of PYGB expression in exosomes isolated from rodents under doxorubicin-induced cardiac injury was observed before any alterations in the circulating cTn-I expressions [100], suggesting that in addition to miRNAs, exosomes can host protein cargos with biomarker potential. miRNA profiling of plasma exosomes from hypertensive rats showed exosomes could have a crucial role in hypertension [101]. Liu et al. demonstrated differential regulation of 27 exosomal miRNAs in spontaneously hypertensive rats compared to the control normotensive rats [101]. They attributed the differential miRNA expression to discriminatory packaging of the exosomal cargos under pathological conditions [101]. Further, another study demonstrated the role of circulating exosomal miRNA-194 that was upregulated in obesity to play a role in the development of cardiac injury and mitochondrial dysfunction [25]. Interestingly, treatment with a miRNA-194 sponge to downregulate the circulating miRNA levels attenuated obesity-related cardiac dysfunction in vivo [25] (Summarized in Fig. 2).

Therapeutic potential of exosomes in CVD

Table 1 Pre-clinical studies showing the efficacy of exosomes in improving cardiovascular function
Full size table

Exosomes have demonstrated both pro-and anti-angiogenic potential depending on the microenvironment [102, 103]. Using cardiosphere-derived cells (CDC), Ahmed et al. demonstrated that the CDCs treated with GW4869, a known exosome production blocker, lost their beneficial effects [104]. Results showed that CDC derived exosomes promoted angiogenesis and cardiac regeneration in scarred and infarcted hearts through activation of miRNA-146a [104]. This was supported by impaired protection against oxidative stress after injection of exosomes with miRNA-146a knockdown [104]. These data suggest that exosomes have therapeutic potential through multiple pro-angiogenic cargos.

Similarly, Gray et al. demonstrated an improved angiogenic potential of exosomes secreted by cardiac progenitor cells (CPC), extensively tested in CVD [105]. The exosomes produced by CPC under hypoxic conditions had increased angiogenic potential[105]. When added to the cultured endothelial cells, they increased the formation of networks compared to the exosomes produced from normoxic conditions[172]. However, the role of exosomal miRNA-1 and − 133a in circulation would need further study to determine the biomarker potential of exosomal miRNAs in diabetic cardiomyopathy.

Like other diseases, exosomes modified for their gene contents either through knockdown or overexpression have been used to treat of DCM. Induction of type 1 diabetes in transgenic mice overexpressed with HSP20 resulted in the production of beneficial exosomes compared to wild-type controls [99]. Notably, the study also showed that these exosomes, when transferred to the adjacent cells, induced beneficial effects, including angiogenesis, reduced oxidative stress, amelioration of fibrosis and apoptosis in the mouse diabetic heart [99]. Another study showed that cardiomyocyte-derived exosomes exhibited systemic effects and local effects. This study showed that both in vitro cellular stretch and in vivo pressure overload promote cardiomyocytes to produce exosomes enriched with angiotensin II type 1 receptor (AT1R [173]. These enriched exosomes were transferred to mesenteric vasculature and skeletal muscle to modulate peripheral vascular resistance and blood pressure [173].

The presented evidence shows that diabetes induces the release of exosomes from various cells, which plays a crucial role in the pathogenesis of the disease. On the other hand, treatments using exosomes derived from various types of stem cells or genetically modified exosomes with miRNA or genes showed therapeutic benefits in DHD. Under pathological conditions, exosomes released into circulation exhibit specific markers that can indicate the pathological state, making them a potential biomarker[23, 100]. In support of this, studies showed that exosomal miRNA expression was a better diagnostic marker for cardiac events in patients with coronary artery disease than freely circulating miRNAs [51]. This altered expression was primarily due to the selective sorting of miRNAs into exosomes in their host cells prior to release [51] (Pathophysiological role summarized in Fig. 2).

Conclusion and future direction

This review summarised the current knowledge regarding the role of exosomes in CVD and DHD. Exosomes originating from cardiovascular cells provide a means for cell to cell communication by transferring miRNA, protein and cytokine cargos. Alterations in the exosomal cargo sorting in the host cells determine the physiological or pathological responses in the recipient cells. There are still some challenges that need to be overcome before its translation into routine clinical practice. For instance, multiple studies have demonstrated the potential therapeutic effects and biomarker capabilities of exosomal miRNAs. However, only a few have looked into the pathophysiological role of miRNA cargos in the DHD [23, 110, 174, 175]. While these studies provided evidence for the possible involvement of multiple miRNAs within the exosome cargos in the protective/destructive effects exhibited by the exosomes on their recipient cells [32, 175], they are only a fraction of the associated miRNAs with diabetes [8,9,10, 17, 176]. Additionally, most of these studies were either assessing individual disease conditions or had yet to delve into the complex molecular pathways affected by exosomal miRNA cargos [31, 33, 99, 110]. Therefore, further comprehensive analysis is essential in determining the full potential of exosomes as biomarkers and therapeutics for DHD.

Another caveat of the studies using exosomes is the lack of standardised isolation protocols for exosomes. This is primarily due to the requirement of a specific size range of exosomes. Due to the vast difference in the isolation procedures, different studies alternate between naming the vesicles as either exosome, extracellular vesicles or microvesicles. Although exosomes can be identified due to the expression of specific surface markers, studies have shown that these can be altered in response to diabetes or other associated comorbidities. Therefore, establishing a standardised protocol for isolating exosomes from tissues, cells and body fluids is crucial before translating these findings to the clinic.