The role of extracellular vesicles in regulating local and systemic inflammation in cardiovascular disease

Abstract

Extracellular vesicles are heterogeneous structures surrounded by cell membranes and carry complex contents including nucleotides, proteins, and lipids. These proteins include cytokines and chemokines that are important for exaggerating local and systemic inflammation in disease. Extracellular vesicles are mainly categorized as exosomes and micro-vesicles, which are directly shed from the endosomal system or originated from the cell membrane, respectively. By transporting several bioactive molecules to recipient cells and tissues, extracellular vesicles have favorable, neutral, or detrimental impacts on their targets, such as switching cell phenotype, modulating gene expression, and controlling biological pathways such as inflammatory cell recruitment, activation of myeloid cells and cell proliferation. Extracellular vesicles mediate these functions via both autocrine and paracrine signaling. In the cardiovascular system, extracellular vesicles can be secreted by multiple cell types like cardiomyocytes, smooth muscle cells, macrophages, monocytes, fibroblasts, and endothelial cells, and affect functions of cells or tissues in distant organs. These effects involve maintaining homeostasis, regulating inflammation, and triggering pathological process in cardiovascular disease. In this review, we mainly focus on the role of micro-vesicles and exosomes, two important subtypes of extracellular vesicles, in local and systemic inflammation in cardiovascular diseases such as myocardial infarction, atherosclerosis and heart failure. We summarize recent findings and knowledge on the effect of extracellular vesicles in controlling both humoral and cellular immunity, and the therapeutic approaches to harness this knowledge to control exacerbated inflammation in cardiovascular diseases.

Graphical abstract

1. Introduction: Extracellular vesicles

EVs are a heterogeneous population of natural particles that are composed of lipids and secreted by cells [1–3]. The EVs membrane structure is similar to the membranes of their parental cells, and their size can vary between 50 nm to 2 μm. There are three main subtypes of EVs: MVs, exosomes and apoptotic bodies. They can be differentiated by their biogenesis, secretion pathways, cargo, function, and size [2–5]. In table 1, we present the properties of EV subtypes. Various cell types can produce distinct repertoires of vesicles, and even EVs derived from the same source can have opposing functions under different circumstances. Moreover, the mechanisms of EV secretion are diverse. MVs can be directly released from the plasma membrane while exosomes largely depend on the fusion of multivesicular bodies (MVBs) with the plasma membrane and then bud off from the cell membrane. In contrast, apoptotic bodies are usually generated during the process of cellular apoptosis. Many cellular signaling pathways have been found to affect secretion of EVs, but the underlying molecular mechanisms are still unexplored.

Table 1:

Properties of EV subpopulations

	MVs	Exosomes	Apoptotic bodies
Physiology & Origin	The physiological state and microenvironment of the donor cell will define the number of MVs produced. MVs are produced by pinching or direct external budding of the cell’s plasma membrane [1–5, 9].	During endosome formation, exosomes form by inward budding of the cell membrane [2, 4, 9].	Apoptotic bodies are surrounded by a monolayer membrane that are secreted by dying cells and have been found in the body fluid such as plasma, cerebrospinal fluid, urine, semen, saliva, bronchial fluid, bile, serum, amniotic fluid, lymph, synovial fluid, gastric acid, breast milk, and tears [157–166].
Size (diameter)	100 nm up to 1 μm [1].	30–150 nm [1].	50 nm up to 5000 nm [1].
Composition	The protein concentrations in the MVs can be 100-fold higher compared to the cell lysate [1, 81, 167, 168]. The MVs contain proteins such as cytoskeletal, heat shock, integrins, phosphorylated and glycosylated proteins (proteins containing post translational modifications) [1, 169–171]	Exosomes have been shown to be involved in interorganelle trafficking of proteins between the mitochondria and endolysosomal system; however, the concentrations of these proteins in exosomes are too low in comparison to cell lysates for these proteins to be considered biomarkers of these EVs [169, 172, 173].	Small amounts of glycosylated proteins, chromatin and organelles are contained in apoptotic bodies. Thus, one would expect to see elevated levels of proteins associated with the mitochondria, golgi apparatus, nucleus, and endoplasmic reticulum in these EVs [5, 172, 174, 175].
Biological purpose	MVs participate in cell–cell communications between neighboring and distant cells. MVs are also responsible to alter the functions of recipient cells [169–171]	Exosomes propagate cell–cell transmission, cell preservation, and tumor development [1, 36, 176].	Apoptotic bodies help in clearing dead cells from the body by phagocytosis without triggering the immune response [1, 10, 15, 177, 178].

The confirmation of biogenesis of various EVs is difficult if not impossible. Moreover, the EV subtypes may share markers. These limitations make it difficult to accurately characterize EVs just based on their origins and markers. According to the 2018 International Society for Extracellular Vesicles [6], “extracellular vesicles” is the best generic terminology in publications. Separation and concentration methods of EVs vary, and researchers select a method that is most suitable for their studies. However, the methods of EV isolation should be reported in publications in detail. Additionally, the following practices have been recommended: 1) Besides choosing the right positive markers of EVs, negative markers are also important. 2) Topology to characterize EVs has been suggested to discern the location of a protein, lipid, or RNA in or on a vesicle. 3) The functional analysis of non-EV fraction should be performed to assess the specificity of EVs.

Cardiovascular disease (CVD) is the most prevalent cause of morbidity and mortality worldwide even with state-of-the-art therapies [7]. Recently, EV-based treatment has been proven to be a promising CVD intervention. Compared to traditional cell-based treatment, EV-based therapy can enhance clinical efficiency and improve long-term survival of patients [8]. EVs can be derived from multiple cell types like cardiomyocytes, smooth muscle cells, macrophages, monocytes, and fibroblasts, and carry a variety of contents such as lipids, nucleic acids, and proteins [2, 3, 9, 10]. By delivering these biologically active molecular cargos into recipient cells, EVs can regulate many physiological and pathological processes, including modulation of angiogenesis [11], cardiomyocyte hypertrophy [12], apoptosis/survival [13], and cardiac fibrosis [14]. These physiological functions of EVs can be harnessed as therapeutic strategies. Also, due to their presence in body fluids, such as blood and urine, EVs have also been used as potential biomarkers of CVD. The primary focus of this review will be the contributions of EVs in regulating local and systemic inflammation in CVD.

2. Sources of EVs

EVs have been shown to be generated from almost all cell types, including stem cells [10, 15], immune cells, and the cells of the central nervous systems [16–18]. Additionally, cancer cells secrete copious amounts of EVs in vitro and in vivo [19, 20]. Depending on the sources, EVs transduce biological signals between cells and regulate a diverse range of intercellular communication. For example, EVs from mesenchymal stem cells (MSC) could promote tissue regeneration after myocardial infarction (MI) [21, 22], and EVs originated from tumor antigen-presenting dendritic cells have been implicated in cancer immunotherapy [23]. In the brain, neuron-derived EVs can facilitate a wide range of neurobiological functions like synaptic plasticity [24] and glutamatergic activity [25], indicating their potential role for future therapies.

Interestingly, some EVs have been proven to be detrimental while under other circumstances, they can be beneficial. For instance, endothelial EVs can reduce vasorelaxation by decreasing nitric oxide production in recipient cells. This is found to be modulated via a reduction in phosphorylation and activation of endothelial nitric oxide synthase [26], regional oxidative stress [27], or an enhanced NADPH oxidase activity [28], leading to impaired vascular relaxation abilities. In contrast, other studies reported that endothelial-derived EVs promote blood vessel regeneration and vascular protection [29]. Endothelial EVs containing caspase-3 secrete the cargo, which reduces intracellular levels of this proapoptotic protein [29]. Additionally, annexin I/phosphatidylserine receptor-dependent EVs can protect endothelial cells (ECs) from apoptosis [30]. Thus, the sources of EVs determine their diverse functions and downstream signaling pathways.

3. Isolation and characterization of EV subtypes

To accurately investigate the characteristics of EVs, it is essential to isolate the EVs correctly. Several methods have been used to get highly pure EVs from different samples. Detailed protocols of EV isolation have been reviewed previously [1]. Here, we briefly discuss the commonly used EV isolation procedures. Ultracentrifugation is one of the most widely used approaches and suitable to isolate EVs from large sample volumes. However, this method is time-consuming, and probably not suitable for rare clinical samples due to their limited volumes [31]. Compared to the ultracentrifugation method, density-gradient centrifugation is quicker and removes contaminants with sucrose solution, which ensures higher separation efficiency and generates purer EVs, especially exosomes. This method also ensures the integrity of EV structure and function [31]. Ultrafiltration is another fast and efficient method of EV isolation, but this approach fails to remove protein contaminants [32]. Although it requires higher sample volumes and has complex steps of EV enrichment using magnetic beads, immuno-affinity purification can produce higher quantity and quality EVs [33]. The selection of EV separation procedure is determined by sample types and experiments.

After isolation, the next step is to characterize EVs based on their size, morphology, density, and porosity. Several techniques have been used to assess these parameters, including nanoparticle tracking analysis, flow cytometry and transmission electron microscopy. For nanoparticle tracking analysis, the process of sample preparation and detection is quick and straightforward. This technique can detect particles with diameters as low as 30 nm. However, the technology is expensive and the correct sample dilution remains controversial [34]. Flow cytometry is the most frequently used technique for EV analysis. It allows analysis of different physical and chemical characteristics via measuring the size and structure of EVs. However, flow cytometry is inefficient for directly detecting smaller particles like exosomes, and flow cytometers with high sensitivity are expensive [35]. Transmission electron microscopy is widely used to define not only the structure and morphology of EVs but also the size of various biological cargos. However, this technique is expensive and involves multiple steps, which may change the morphology of EVs [3]. Generally, at least two methods should be utilized to characterize EVs isolated from samples.

4. Mechanisms of EV secretion

The secretion of EVs occurs in two distinct ways: direct outward budding of MVs and inward budding of exosomes. Apoptotic bodies are secreted by dying cells and contain undamaged organelles, chromatin, and some glycosylated proteins [1]. During apoptosis, cells tend to contract themselves to increase hydrostatic pressure, and then release various contents by separating the plasma membrane from the cytoskeleton [36]. In this review, we primarily focus on MVs and exosomes. Once generated, MVs can directly pinch off from the cell surface, while exosomes rely on the endocytic system and require additional steps, including MVB transport and their fusion with the membrane before being released into the surrounding microenvironment. Thus, EV secretion depends on multiple steps namely EV generation, transportation, fusion to the plasma membrane and release into the extracellular space.

EV generation:

As a relatively heterogeneous group, MVs are normally formed through an outward budding of the cell membrane. This process is mediated by membrane lipid microdomains and regulatory factors like ADP-ribosylation factor (ARF) 6 [37]. In contrast, exosomes represent a population of EVs containing homogeneous cargoes, and the generation of these EVs begins with early endosome formation. This relies on a procedure called endocytosis. The cell membrane buds inward and catches bioactive molecules simultaneously. These molecules are dissociated into smaller particles that transfer from the plasma membrane into endosome lumen, generating vesicles. During this process, the late endosomes have multivesicular appearance and therefore they are also named MVBs. These MVBs can either fuse with lysosomes or with the plasma membrane. Considering that exosomes carry cytokines and other regulatory factors, fusion with lysosomes will result in degradation and recycling of their nucleotide, protein, lipid and other contents. Instead, fusion with the plasma membrane can initiate both autocrine and paracrine signaling, allowing the intercellular communication. Thus, the balance between secretion and degradation is essential for exosome generation. The separating machineries of exosomes are largely dependent on endosomal sorting complexes required for transport (ESCRT) or ceramide [38, 39]. Particularly, the ESCRT machinery, along with its adornment proteins, such as tumor susceptibility gene 101 protein and ALG-2-interacting protein X (ALIX), is crucial to exosome formation and MVBs transportation [40, 41]. The syndecan-syntenin-ALIX pathway, for example, has been shown to regulate exosomes secretion [40]. Conversely, ubiquitylation of MHC class II targets MVBs to lysosomal degradation [42], which interferes with exosome generation.

EV transportation:

The transportation of MVBs is another key step of exosome secretion. The ESCRT machinery and accessory proteins, such as ALIX, are able to combine exosome cargoes and incorporate them into intraluminal vesicles [40]. Once matured, they are transported along microtubules to the plasma membrane. This process is regulated by various RAS- related protein (RAB) GTPases. For instance, RAB27A and RAB27B, along with their associated factors synaptotagmin-like protein 4 and exophilin 5, have been found to be crucial to MVB transport and docking to the plasma membrane by regulating sub-membrane actin cytoskeleton rearrangement, and exosomes secretion [43, 44].

EVs fusion and release:

Based on the distinguishing mode of biogenesis, EVs can be released from cells through multiple ways. The release of MVs is directly triggered once the cargoes fuse with the plasma membrane. This process depends on actin and myosin, and is ATP-dependent [45]. The stimulation of small GTP-binding proteins, like ARF1 and ARF6, contributes to the phosphorylation of myosin light chain and actomyosin contraction, which promotes the particles to pinch off from the cell membranes of cancer cells [37, 46]. Moreover, in HeLa cells, CDC42 has been reported as another mediator of actin dynamics [47]. The docking and fusion of MVBs with the plasma membrane have been reported to be partially modulated by synaptotagmin family members and soluble N-ethylmaleimide sensitive factor attachment proteins [48].

5. EV contents and their roles in CVD and inflammation

EVs contain micro RNAs (miRNAs) [49–51], which can affect the functions of inflammatory leukocytes and secretion of cytokines. In table 2, we present miRNAs with prognostic properties in CVD. Besides miRNAs, EVs contain a myriad of molecular components such as proteins, nucleic acids, and lipids [52, 53]. All of these cargos are important in precipitating and resolving inflammation.

Table 2:

miRNAs with prognostic properties in CVD

Disease	Marker	Effect	References
Cardiac hypertrophy and HF	miR-21-3p	Deletion or inhibition can diminish the development of cardiac hypertrophy in murine model	[91]
	miR-215p	Increased levels promote heart repair and augment cardiomyocyte survival	[92]
	miR-132	Regulates adverse cardiac remodeling in HF	[128]
	miR-425, miR-744	Suppresses HF pathogenesis and cardiac remodeling	[143]
MI	miR-15	Promotes cardiac remodeling	[124, 125]
	miR-34a, miR-192, miR-194	Heightened levels can be used as prognostic biomarkers	[94]
	miR-92a-3p	Modulates angiogenesis in recipient cells via thrombospondin 1	[179]
	miR-143, miR-222	Regulate endothelial proliferation and differentiation	[109]
	miR-126, miR-199	High levels linked with decreased major adverse cardiovascular events in patients with stable CAD	[89]
	miR-208, miR-499	Highly elevated after MI	[77]
PAH	miR-143	Increased in PAH	[109, 118]
PH	miR-210	Increased in PH	[119]
	miR-342-5p, miR-181	Can reverse pulmonary vascular remodeling	[131,141]
Cardiomyopathy	miR-92b	Elevated in HF	[85, 180]
Atherosclerosis	miR-92a	Increased in atherosclerosis	[85, 120]
	miR-155	Reduces inflammatory processes via macrophages towards M2 polarization	[131, 142]
	miR-342-5p	Anti-atherosclerotic effect on ECs	[131,141]
CAD	miR-126, miR-199a	Lower levels reflect on the lower survival rates in CAD patients	[86, 88, 94–96, 181]

Due to the different generation and transportation pathways, some cargos are specific for a particular class of EVs and can be used as distinguishing markers. As mentioned previously, ESCRT along with their accessory proteins like ALIX and tumor susceptibility gene 101 protein are involved in the production and secretion of exosomes and are expected to be detected within these particles regardless of different parental cells [54]. This group of proteins is considered as exosomal markers.

The EVs are surrounded by a bilayer membrane enriched in different lipids similar to the cell of origin [55, 56]. Exosomal membrane is enriched in lipids such as sphingomyelin, ceramides phosphatidylserine, glycosphingolipids, and cholesterol [39, 56–59], which provide the stability required for their cargo transportation over short and long distances within the extracellular space and body fluids [59, 60]. Unlike exosomes, MVB membrane is enriched in not only cholesterol and sphingolipids but also glycosylphosphatidylinositol-anchored proteins, MHC II, αB-crystallin and flotillin-1. Proteolipid proteins are abundant in oligodendroglia precursor cell derived EVs and support the membrane [39, 56]. The membrane lipid bilayer of EVs also contains integrins, cell adhesion molecules and proteins from the tetraspanin family such as CD9, CD81, and CD63 [32, 59]. These proteins are called EV-associated proteins that participate in EV biogenesis, cell recognition and binding [32, 59].

Phosphatidylserine and annexin V, which are found on the surface of MVs, are pivotal to cell cycle signaling leading to apoptosis, cell adhesion and EV uptake by target cells [61, 62]. Additionally, exosomes tend to have high levels of glycoproteins while MVs contain proteins with higher levels of post-translational modifications such as phosphorylation and glycosylation [63]. This property could be used as an alternative way to distinguish these particles based on their contents rather than sizes.

We can also find plasma proteins inside EVs. For example, various proteins were identified in EVs isolated from the serum of patients with acute coronary syndrome (ACS) [64]. Cystatin C, complement factor C5a, and polygenic immunoglobulin receptor were significantly elevated in ACS compared to the control group. EVs from patients with either ST-elevation MI or stable coronary artery disease (CAD) [65] contained proteins involved in the inflammatory response post cardiac injury. Of note, some of these proteins are also involved in the thrombogenesis pathway, underscoring the contribution of EVs in the atherothrombotic incidents leading to MI [65].

EV contents can also be used as biomarkers for CVD. There are correlations between EV protein concentrations and secondary vascular events or mortality in patients with vascular diseases [66]. SerpinF2, SerpinG1, CD14, and cystatin C were found in EVs as potential prognostic markers. The plasma levels of these proteins have linear correlations with the risk of new vascular events and mortality [66, 67]. EVs also harbor cytokines, such as IL-1β and matrix metalloproteinases [68], which are important in cardiac remodeling after MI. Congruently, the Canakinumab Anti-inflammatory Thrombosis Outcome Study (CANTOS) showed that treatment with an IL-1β inhibitor curtailed the incidence of serious CVD cases in patients post MI [69]. Studies have shown left ventricular (LV) dilation in transgenic mice with cardiac-specific overexpression of tumor necrosis factor (TNF) due to elevated levels of matrix metalloproteinases [68, 70, 71].

It was reported that EVs contain cytokines like, IL-2, IL-4, IL-10, IL-12, IL-15, IL-16, IL-18, IL-21, IL-22, IL-33, eotaxin, IFN-γ-inducible protein-10, inducible T cell alpha chemoattractant, M-CSF, MIG, macrophage inflammatory protein-3 α, TGF-β, and TNF-α [72]. IL-17, IL-2, IL-12p70, IL-4, C-X-C motif chemokine 11, IL-21, IL-33, IL-22, IFN-γ, TGF-β, and TNF-α were detected in EVs derived from T-cells and monocytes [73]. Interestingly, different stimuli can alter the relative amounts of cytokines secreted as soluble form and incorporated in EVs [72, 73]. IL-1β and TNF-α were found in EVs produced by stimulated T-cells [72], while monocyte chemoattractant protein-1 was identified in apoptotic bodies [74]. Furthermore, EVs isolated from the blood of STEMI patients contained different cytokines and chemokines such as IL-1β, IL-6, IL-21, eotaxin, IL-22, IL-33, MIP-1α, IL-8, IL-16, IP-10 [75], suggesting that these cytokines can be therapeutically targeted to reduce MI pathogenesis.

6. EVs induce inflammation in cardiovascular disease

Reports also showed associations between the levels of circulating miRNAs and specific disease conditions[51, 76] including heart failure (HF) [12, 51, 77, 78]. The degree of tissue damage can be assessed by the presence of systemic miRNAs, which can be used as a biomarker for different cardiac diseases [51]. Additionally, exosomes have been used as biomarkers and therapies for diseases [79–81]. For example, increased exosome levels are characteristics of atherosclerosis, cardiomyopathy, and MI. Furthermore, exosomes carry proteins and RNAs, particularly miRNAs, and can be used as a drug delivery vehicle [81]. In Figure 1, we depict some of the prognostic and diagnostic miRNAs in CVD. In table 3, we summarize the regulation of cardiovascular inflammation by EVs originating from multiple cell types.

Figure 1: Schematic overview of miRNAs that can play as prognostic and diagnostic biomarkers in cardiovascular disease.

The levels of certain miRNA, lipids and membrane proteins change in CVD. Plasma miRNA concentrations could be used for prognostic and diagnostic purposes.

Table 3:

The role of hematopoietic cell derived EVs in the regulation of cardiovascular inflammation

Cells	Contents/Pathway	Cardiovascular disease	Relative role
Monocytes/Macrophages	KBTBD7/p38/ NF-κB	Cardiac hypertrophy and HF	Promote pro-inflammatory response	[82]
	miR-21/TLR8/ TNF-α, IL-6	Cardiac hypertrophy and HF	Contribute to antimicrobial activities	[83]
	cytochrome coxidase I	CAD	Indicates high risk of cardiovascular events	[99]
	miR-192	MI	Elicits an anti-inflammatory phenotype	[105]
	miR-92a/TLR	DCM	Enhances innate inflammatory response	[121]
Cardiomyocytes	Heat shock protein 90, IL-6/the transducer and activator of transcription-3 pathway	Cardiac hypertrophy and HF	Promote inflammation and disease pathogenesis	[90]
	miR-143 and miR-222	MI	Regulate endothelial proliferation and differentiation	[109]
Endothelial cells	thrombospondin 1/TGF-β	atherosclerosis	Serve as immunomodulatory agents	[92]
Cardiac fibroblast	miR-21-3p	Cardiac hypertrophy and HF	Mediates cardiomyocyte hypertrophy	[12]

6.1. Inflammation mediated by EVs in cardiac hypertrophy and failure

EVs have been reported to modulate the signal transduction between cardiomyocytes and fibroblasts and can be used as biomarkers for diagnosis and prognosis of cardiac hypertrophy and HF [13]. Several studies have pointed out the essential role of EV-derived miRNAs and proteins in mediating inflammation during the progression of HF [13]. Kelch repeat and BTB domain containing 7 (KBTBD7) promotes damage-associated molecular pattern-triggered pro-inflammatory response in macrophage via p38 and nuclear factor κB (NF-κB) [82]. It was observed that miR-21 can be a potential therapeutic target due to its ability to target KBTBD7 and attenuate maladaptive response to inflammation [82]. Exosomal-miR-21 increases the expression of Toll-like receptor (TLR) 8 in macrophages and triggers the generation of proinflammatory cytokines such as tumor necrosis factor alpha and interleukin (IL)-6 [83]. This miRNA binds to multiple genes, suppresses their functions, and contributes to the antimicrobial activities [84]. miR-21-5p-loaded EVs promote cardiac repair by activating angiogenesis and increasing cardiomyocyte survival via the phosphatase and tensin homologue/protein kinase B pathway [13]. Moreover, cardiac fibroblast-derived EVs enriched in miR-21-3p act as a paracrine signal to mediate cardiomyocyte hypertrophy. It can target sorbin and SH3 domains containing protein 2 to induce hypertrophy. The inhibition of miR-21-3p has been shown to diminish the development of cardiac hypertrophy in angiotensin II-infused mice [12]. Another miRNA that has been reported as a biomarker for HF is miR-192-5p, which is p53-responsive [85, 86] and activates M1 macrophages [87]. Elevation of miR-192 expression has been found to be associated with hypertrophic cardiomyopathy [88, 89]. EVs released from hypertrophied cardiomyocytes harbor proteins like heat shock protein 90 and IL-6, which target the transducer and activator of transcription-3 pathway, essential in inflammation progression and disease pathogenesis [90].

6.2. EVs in regulating atherosclerosis and CAD-associated inflammation

EVs have been found to play vital roles in regulating the inflammatory processes in various cell types, including ECs, smooth muscle cells (SMCs), platelets, and immune cells. This is pivotal in the progression of atherosclerosis and angiogenesis [91]. It has been reported that among patients with stable CAD, ECs and platelets are two main sources of EVs, and the miRNAs they contain are causally associated with disease development. For example, under atherosclerotic conditions, miR-92a-3p can be packaged into endothelial EVs and modulate angiogenesis in recipient cells via activating thrombospondin 1 [92], which can promote the expression of transforming growth factor (TGF)-β and serve as an immunomodulatory agent [93]. Furthermore, the high levels of miR-126 and miR-199a in EVs, rather than in plasma, are associated with major adverse cardiovascular events [94] and better survival, respectively, compared to those with lower levels of these miRNA [95, 96]. Additionally, miR-126 propagates anti-inflammatory effects by suppressing the expression of vascular cell adhesion molecule 1 and reducing leukocytes adherence to ECs [97]. miR-126-5p encourages cell proliferation by inhibiting delta-like 1 homolog [98], indicating the potential role of miR-126 in vascular inflammation. miR-199a exhibits anti-inflammatory properties via targeting IKKβ, which leads to down regulation of NF-κB. Low mitochondrial-encoded cytochrome c oxidase I expression in monocyte-derived EVs can be used to identify a new patient population with high risk of cardiovascular events [99], linking monocyte-specific EVs with prognosis of CAD patients.

6.3. EVs control inflammation after MI and ischemia-associated heart remodeling

Ischemic CVD can promote cardiomyocyte death and secretion of cell contents such as danger-associated molecular patterns, which ignite cytokine secretion. This activates platelets that arbitrate leukocyte migration and their extravasation into tissue [100, 101], and mobilizes neutrophils into the myocardium. This, in turn, damages ECs and release chemokines, cytokines and proteases [100, 102]. Local cell death is also followed by monocytes recruitment [100, 103, 104]. In Figure 2, we depict the contribution of miRNA cargoes in inflammation.

Figure 2: The contributions of EV cargoes in inflammation in CVD.

EVs contain miRNA, proteins and lipids. ECs secrete miR-92-3p that induce angiogenesis on neighboring ECs via thrombospondin 1 which also increase proinflammatory signals [92]. miR-126 suppresses VCAM-1-VLA-4 binding, which reduces leukocytes adherence to ECs, resulting in lower leukocyte influx [89]. EC-derived EVs contain mir-199a which suppresses monocytes recruitment and inflammation via inhibition of the NF-κB signaling pathway (which includes TLR, IL-1R, MyD88, IRAK1/4, RAF6, TRAF6, IRAK1, IKKβ, IKKα, IKKγ, IkB, P65, P50, NF-κB) [99]. This could be a potential treatment to reduce inflammation after a CVD event [155]. miR-21-3p and miR-21-5p target cardiomyocytes and promote hypertrophy by inducing cell growth via inhibiting the PTEN signaling (which includes phosphoinositide (4,5) P2, phosphoinositide (3,4,5) P3, PDK1, AKT, and mTOR). Fibroblast cells release EVs that harbor miR-21-3p, miR-21-5p and exo-miR-21 [13]. Exo-miR-21 promotes inflammation via stimulating the NF-κB signaling in macrophages [83]. Thrombospondin activates CD36, which activates CD47 and Fas protein that encourage apoptosis and promotes secretion of damage-associated molecular pattern. Cardiomyocytes secrete miR-143 and miR-222 that are engulfed by ECs [109]. These two miRNAs regulate cell proliferation and differentiation and are associated with inflammation and MI pathogenesis. Platelet-derived extracellular vesicles that contain IL-1β and caspase-1 activates the inflammasome pathway in neutrophils [156]. IL-1β neutralization is a potential therapy to dampen inflammation in cardiovascular disease [69]. This figure was created with www.biorender.com.

The levels of miR-192, miR-194, and miR-34a in the plasma are linked to inflammation and can serve as prognostic biomarkers in MI patients. EV miR-192 has been shown to elicit an anti-inflammatory phenotype in macrophage [105]. miR-194 increases TLR4 expression in THP-1 cells, a human monocytic cell line, in presence of palmitic acid [106]. miR-34 is involved in the regulation of apoptosis [107], which has been shown to initiate inflammatory cascade after a sterile tissue injury. Specifically, the levels of these miRNAs can predict the development of HF and ventricular remodeling within one year after MI onset. These three miRNAs are found to be modulated by p53 and then packaged into CD63⁺ EVs, decreasing viability of recipient cells [89]. Additionally, foam cell-derived EVs can enhance SMCs migration and adhesion via stimulation of extracellular signal-regulated protein kinase and protein kinase B [108]. Furthermore, under the local ischemic, hypoxic, and inflammatory conditions after MI, cardiomyocytes can secrete EVs containing miRNA, such as miR-143 and miR-222, to regulate endothelial proliferation and differentiation [109]. Therefore, a complex EV-miRNAs-protein network has been reported to be associated with inflammation and MI pathogenesis. Considering the fundamental role that inflammation plays after MI [109–115], several potential interventions targeting EVs can be utilized for the improvement of current traditional therapies.

6.4. EVs in exacerbating inflammation and lung remodeling in pulmonary arterial hypertension (PAH)

EVs generated in PAH have the potential to stimulate transcription and translation of numerous proangiogenic proteins and their receptors in human pulmonary ECs (hPAECs) [116]. EVs augment the mRNA expression of vascular endothelial growth factor (VEGF)-A, VEGF receptor, placental growth factor, and fibroblast growth factor-2 in hPAECs [116]. Although the proteins carried by EVs have not been fully characterized, EVs have been linked to inflammation in PAH.

miRNA cargoes in EVs mediate the cell-cell cross talk between ECs and SMCs in PAH [117]. It has been demonstrated that PAEC migration and angiogenesis are induced by miR-143 carried by exosomes. Elevation in miR-143 levels can be used as a potential biomarker in PH. Furthermore, genetic and pharmacological inhibition of miR-143 in mice can prevent the progress of hypoxia-induced PH [118]. We have shown that miR-210 carried by hematopoietic cells targets pulmonary ECs and promotes vascular remodeling in the lungs [119]. We also observed that influx of blood Ly6C^high and Ly6C^low monocytes into the lungs in response to hypoxia is miR-210-mediated.

6.5. Association of EVs and inflammation in diabatic cardiomyopathy (DCM)

Studies on patients with DCM presented different subtypes of miR-92 as biomarkers for the disease [85, 120]. miR-92a-1 and miR-92a-2 assemble miR-92a, which has altered expression in CVD [85]. Both miR-92a-1 and miR-92a-2 have similar basic sequences, but they are located in different loci. In miR-92a-deficient mice, uncontrolled cell proliferation has been observed due to increased expression of mitogen-activated protein kinase 4, which activates c-Jun N-terminal kinase/stress-activated protein kinase [121]. Furthermore, decreasing miR-92a levels in macrophages upregulates TLR expression that leads to enhanced innate inflammatory responses [121]. Increased expression of miR-92b is observed in chronic HF patients with reduced ejection fraction [85, 120, 122]. Additionally, the levels of exosomal-miR-92b-5p, a subgroup of miR-92b, are elevated in patients with DCM [85].

7. Potential therapies

Future EV-based therapies should be directed to reduce inflammation and encourage cardiac regeneration. As we discussed above, EVs can have both inflammatory and anti-inflammatory roles, which are determined by EV origin, their content, and disease condition. For example, in hypoxic conditions, exosomes from bone marrow mesenchymal stem cells post MI contain miR-125b-5p that mitigates ischemic cardiac insult by suppressing cardiomyocyte apoptosis [123]. In contrast, exosomes originating from mature dendritic cells elevate endothelial inflammation and atherosclerosis via TNF-α- mediated NF- kB pathway [123].

7.1. Preclinical & clinical studies targeting inflammation and implication of EV-specific therapies in CVD

EV contents can directly or indirectly perpetuate inflammation after an acute injury such as MI. miRNA and proteins present in EVs can be targeted as therapies. Although miRNA treatment is still in its infancy, it is already being tested in clinical trials. In this section, we will talk about recent studies and their implications on EV therapies in CVD.

miR-15, which is present in EVs in high amounts after MI, was inhibited to reduce cardiac remodeling post MI [124, 125]. Targeting miR-92a, which is abundantly present in EVs, prevented intima formation due to vascular damage in atherosclerosis [126]. Furthermore, CCR2 could be blocked using pharmacological or genetic approaches to reduce inflammatory monocyte recruitment in atherosclerotic plaques and myocardial infarcts [68]. miR-208a inhibition in rats reduced the levels of miR-208b and miR-499, which are associated with the initiation and progression of CVD pathogenesis [77]. Additionally, targeting miR-29b decreased cardiac fibrosis by preventing TGF-β/Smad3 signaling [127]. Thus, inhibition of this miRNA could be a novel mechanism to reduce cardiac fibrosis in patients with HF [68, 124, 127]. Moreover, CD132L was used as an anti-miRNA treatment against miR-132 to mitigate adverse cardiac remodeling in HF patients [128].

As discussed above, the Canakinumab treatment, an IL-1β neutralizing antibody, diminished the occurrence of secondary cardiovascular events in patients with MI [69, 124, 129], and Pentoxifylline, a TNF inhibitor, was tested in clinical trials to reduce LV dilation [68]. The results from the CANTOS trial suggest that HF patients with an inflammatory phenotype can be identified and treated to reduce systemic inflammation and prevent future cardiovascular events [68]. For example, HF patients with high C-reactive protein, IL-1 β or TNF-α can benefit from an anti-inflammatory treatment.

7.2. Potential clinical therapy

EV contents are able to target neighboring and distant cells. miRNAs present in EVs bind to the genes of target cells based on partial sequence complementarity. For example, in PAH patients, treatment with miR-181a-5p and miR-324-5p reversed pulmonary vascular remodeling [130, 131]. Exosomal miRNA-143 derived from pulmonary artery smooth muscle cells-initiated ECs migration and angiogenesis and induced apoptosis in donor cells [79, 131–140]. miR-342-5p present in adipose derived MSCs reduced atherosclerotic inflammation [131, 141]. Furthermore, miR-155 could be therapeutically used to reduce inflammatory processes in atherosclerosis via polarization of macrophages to M2 cells [131, 142]. In HF, miR-425 and miR744 inhibition activated cardiac fibroblasts and was associated with the increased production of collagen type 1 and α-SMA, leading to cardiac remodeling [143]. Thus, miRNA present in EVs could be targeted to reduce cardiovascular pathogenesis. Different cell lines can secrete EVs containing various cargos [144], which can be used for drug delivery [145, 146]. In general, MSCs are one of the main sources of EVs, especially exosomes, which act as immunomodulatory agents [144, 146]. Additionally, cancer cell lines can be used to produce EVs [145, 146].

7.3. Bioengineered EVs for CVD therapy

EVs have unique properties that help them to survive in the extracellular space, evade biological barriers and carry their content to recipient cells [147]. Despite these properties, the major limitation of EV therapy is their low numbers. EVs can be bioengineered to not only have better stability [147]. This can also improve protein targeting of recipient cells and thereby increase their numbers [147]. Specific cargos can be incorporated in EVs using novel techniques depending on their sizes. For example, since dendritic cells can secrete as a response to inflammation, smaller EVs, which contain ADAM10, syntenin-1, annexin XI, and EHD4 or mitofilin and actinin-4, we can bioengineer these proteins and incorporate them in small or large EVs [148, 149]. In another study, to suppress immune response in macrophages, we can bioengineer EVs containing miRNAs present in EVs originated from MSCs. These miRNAs suppress macrophage-mediated immune response via modification of the NF-kB pathway and downstream reduction of TLR signaling [148, 150]. Additionally, the lipid bilayer could be modified to increase targeting specificity of EVs. For example, CD47 could be expressed on EVs to augment the delivery of the EVs to targeted tissues [151, 152].

Clinical uses of bioengineered EVs are still limited by the lack of large-scale production. Bioengineered EVs can be categorized as semi-synthetic and fully synthetic. Semi-synthetic EVs can be generated by transfecting cell lines with plasmid encoding cargoes of interest [145, 153]. In contrast, fully synthetic EVs are formed from cell fragments from a variety of cells [145, 153]. Also, these EVs can be modified to express different membrane markers such as tetraspanin and lipid rafts [145, 154].

8. Conclusions

EVs have clinical potential to be used as prognostic, diagnostic, therapeutic, and in drug delivery approaches. They engage in physiological and pathological processes in various types of diseases. EVs are released in stress and physiological conditions as a response to alterations in the microenvironment. EVs carry membrane receptors, nucleic acids, lipids, and proteins, which initiate and prolong an inflammatory response in cardiovascular disease and can be therapeutically targeted to ameliorate disease pathogenesis. However, a comprehensive knowledge in biosynthesis, elimination, secretion and cellular signaling of EVs will be required for these purposes. EVs elicit both pro- and anti-inflammatory effects, which are determined by many factors such as EV origin, disease condition, etc. To leverage the beneficial anti-inflammatory properties of EVs, we will need to choose right donor cells and determine the appropriate time after a cardiac injury. This is still an evolving and enigmatic field, and future mechanistic studies will shed light on the determinants of EV functions.

Abbreviations

ACS

acute coronary syndrome

ARF

ADP-ribosylation factor

ALIX

ALG-2-interacting protein X

CANTOS

Canakinumab Anti-inflammatory Thrombosis Outcome Study

CVD

cardiovascular disease

CAD

coronary artery disease

DCM

diabetic cardiomyopathy

ECs

endothelial cells

ESCRT

endosomal sorting complex required for transport

ES

extracellular space

EVs

extracellular vesicles

HF

heart failure

hPAECs

human pulmonary endothelial cells

IL

interleukin

LV

left ventricular

MSCs

mesenchymal stem cells

MVs

micro-vesicles

micro-RNA

miRNA

MVBs

multivesicular bodies

MI

myocardial infarction

PH

pulmonary hypertension

PAH

pulmonary arterial hypertension

RAB

RAS-related protein

SMCs

smooth muscle cells

TGF-β

transforming growth factor TGF-β

TNF

tumor necrosis factor

VEGF

vascular endothelial growth factor