Extracellular vesicles: Potential impact on cardiovascular diseases

Abstract

Extracellular vesicles (EVs) have received considerable attention in biological and clinical research due to their ability to mediate cell-to-cell communication. Based on their size and secretory origin, EVs are categorized as exosomes, microvesicles, and apoptotic bodies. Increasing number of studies highlight the contribution of EVs in the regulation of a wide range of normal cellular physiological processes, including waste scavenging, cellular stress reduction, intercellular communication, immune regulation, and cellular homeostasis modulation. Altered circulating EV level, expression pattern, or content in plasma of patients with cardiovascular disease (CVD) may serve as diagnostic and prognostic biomarkers in diverse cardiovascular pathologies. Due to their inherent characteristics and physiological functions, EVs, in turn, have become potential candidates as therapeutic agents. In this review, we discuss the evolving understanding of the role of EVs in CVD, summarize the current knowledge of EV-mediated regulatory mechanisms, and highlight potential strategies for the diagnosis and therapy of CVD. We also attempt to look into the future that may advance our understanding of the role of EVs in the pathogenesis of CVD and provide novel insights into the field of translational medicine.

Introduction

Cardiovascular diseases (CVD), including coronary heart disease, heart failure, stroke, congenital heart disease, rhythm disorders, and hypertension, remain the leading causes of morbidity and mortality around the world [1]. In the United States, the prevalence of CVD in adults 20 years of age and older is 48.0% overall (121.5 million in 2016) (NHANES 2013–2016) [2]. The prevalence of CVD increases with age in both males and females. In 2016, approximately 161,438 (19.2%) Americans who were < 65 years old died of CVD; 306,638 (36.5%) of the deaths attributable to CVD occurred before the age of 75 years [2]. In 2016, the Chinese Cardiovascular Disease Report estimated that 290 million (21%) Chinese had CVD, the leading cause of mortality (61%) [3]. Globally, CVD produces immense health and economic burden [4], [5], [6]. In the United States, the cost of informal caregiving for patients with CVD is projected to increase from $616 billion in 2015 to $1.2 trillion in 2035 [7]. Thus, increasing the efficacy in the prevention and treatment of CVD has a huge potential in improving global health outcome and decreasing financial burden [8], [9].

Cell-to-cell communication, a key process in multicellular organisms, is required to guarantee proper coordination among the different cell types within tissues and thus, fundamental for the functioning of biological systems. There are multiple modes of intercellular communication, including soluble factors, tunneling nanotubes, and extracellular vesicles (EVs), which allow the transfer of surface molecules or cytoplasmic components from one cell to another [10], [11], [12]. Among them, EVs have received a lot of attention in biological and clinical research due to their ability to mediate cell-cell crosstalk. EVs originate from diverse subcellular compartments of most eukaryotic cells and are released into the extracellular space in normal and disease states [13], [14], [15]. EVs are categorized as exosomes, microvesicles, and apoptotic bodies, according to their abundance, size, and composition. They contain different materials, which include many functional molecules, such as microRNAs (miRNAs), messenger RNAs (mRNAs), long non-coding RNAs (lncRNAs), proteins, DNA fragments, and lipids [12], [13], [14]. Those components can be delivered from the cell of origin to a recipient cell, whether the recipient cell is in the vicinity or distant from the cell of origin. The transferred molecules are potentially capable of eliciting changes in function and gene expression in the recipient cell. Numerous studies have shown that EVs may serve as biomarkers for the diagnosis and treatment of diseases, including cardiovascular diseases [16], [17], [18]. In this review, we discuss the evolving understanding of the role of EVs in CVD, summarize the current knowledge of EV-mediated regulatory mechanisms, and highlight potential strategies for the diagnosis and treatment of CVD, and also attempt a look into the future. These may advance our understanding of the role of EVs in CVD and provide novel insights into the field of translational medicine.

EVs were first described in 1967; this subcellular fraction, identified by electron microscopy, consists of small vesicles, with a diameter between 20 and 50 nm, and termed “platelet dust” from activated platelets [19]. EV is an umbrella term for all types of vesicles released from prokaryotic and eukaryotic cells. Currently, there is an ongoing debate on the classification of EVs with some investigators having preference to classify the EVs according to their source, such as endothelial cell-derived EVs, vascular smooth muscle cell-derived EVs, cardiac fibroblast-derived EVs, macrophage-derived EVs, etc. [20], [21], [22]. However, this manner of classification for EVs does not reflect the character of a particular EV. Moreover, there is also some controversy on the nomenclature and sizes of the different types of vesicles. Therefore, in 2014 and updated in 2018, the International Society for Extracellular Vesicles (ISEV) provided some standards in the classification of EVs, based on various morphological, biochemical, and biogenic parameters [23], [24]. However, until now, there is no consensus on the nomenclature of EVs that define the cellular origin or classification of EVs once they have been secreted or shed from the cell of origin [25]. For this reason, the term “extracellular vesicle” is suggested by the ISEV as a generic name for all secreted vesicles, and also as a keyword in publications [26]. Recent reviews have categorized EVs into three groups [27], [28], [29]: exosomes, microvesicles, and apoptotic bodies.

Exosomes are a homogenous population of EVs derived from the endocytic compartment. These cell-derived nanovesicles, ranging in size between 30 and 100 nm in diameter, are surrounded by a lipid bilayer [30], [31]. Usually, exosomes are able to float on a sucrose gradient at a density of 1.13–1.19 g/mL. Exosomes typically display a cup-like shape when observed by transmission electron microscopy, but this may be an artifact [32]. Their biogenesis is initiated by the inward budding of multi-vesicular endosomes. The transmembrane proteins are endocytosed and trafficked to early endosomes, which invaginate to generate intraluminal vesicles and form multivesicular bodies (MVBs). Then, the MVBs can either fuse with the plasma membrane and the intraluminal vehicles are released as exosomes into the extracellular space (Fig. 1); the intraluminal vesicles can also fuse with lysosomes for eventual degradation [33], [34].

Exosomes are released from cells via a constitutive or inducible mechanism, depending on the cell type of origin [35]. In general, exosomes are wrapped by a phospholipid bilayer, enriched with sphingomyelin, ceramide, and cholesterol [36], [37]. The biochemical composition of exosomes depends on the cell source. Cells release subpopulations of exosomes with different sizes, compositions, and molecular and biological properties [38]. The mechanisms proposed for their release, include Rab GTPases (Rab11/35, Rab27), tetraspanins, and SNAREs (soluble N-ethylmaleimide-sensitive attachment protein receptors) complex [39], [40], [41]. Tetraspanins, including CD9, CD63, CD81, CD82, and CD151, exist in released exosomes and accumulate in plasma membrane endosomes and microdomains [42], [43]. In the plasma membrane, tetraspanins form tetraspanin-enriched microdomains (TEMs). TEMs are involved in many biological process, including exosome biogenesis, exosome internalization, selection of exosome cargo, and antigen presentation [44], [45]. In addition to tetraspanins and lipids, there are other components, such as cargos, in EVs, including sorting proteins (e.g., Alix, clathrin, TSG101), related to MVB biogenesis, membrane proteins (e.g., Rab GTPase and annexins) regulating exosome docking and membrane fusion, cytoskeleton proteins (e.g., actin, tubulin), inflammatory cytokines (e.g., IL-10, transforming growth factor-β [TGF-β]), metabolic enzymes (e.g., pyruvate kinase, α-enolase), as well as different RNA species (e.g., mRNA, miRNA, lncRNA), and DNAs [12], [13], [14], [46], [47]. Via the above cargos, exosomes can exert their vast and varied physiological functions, through either direct interaction with surrounding cells, where they are generated, or transported to distant recipient cells by their release into the circulatory fluid system.

Microvesicles, also known as microparticles, ectosomes, and membrane particles, represent a more heterogeneous population than exosomes. They are a class of EVs typically ranging in size between 100 and 1000 nm in diameter [48], [49]. Although exosomes are generally smaller than microvesicles, their sizes may overlap. Microvesicles float on sucrose gradient at a density of 1.04–1.07 g/mL. They have been characterized predominantly, as products of platelets, red blood cells, and endothelial cells.

In addition to the size and density differences, microvesicles differ from exosomes by their mechanisms of release and biogenesis. Microvesicles are derived from activated or apoptotic cells through outward budding and fission of membrane vesicles from the plasma membrane [50], [51]. In response to stimuli, outward blebbing may be dependent on various enzymes and mitochondrial or calcium signaling. Microvesicle shedding shares a similar process with virus budding. Moreover, microvesicles may be assembled selectively in the lipid-rich microdomains of the membrane, including lipid rafts or caveolae [52], [53]. In mammals, microvesicles are released from almost all cell types, including blood cells (e.g., platelets, erythrocytes, leukocytes), vascular smooth muscle cells (VSMCs), and endothelial cells [54], [55], [56]. The release can be observed within a few seconds after stimulation [41].

Microvesicles reflect the nature and the activation state of the parent cell and are identified by the expression of phosphatidylserine on their surfaces, which is indicative of their release from apoptotic or activated cells. The preferential binding of annexin to phosphatidylserine can be used to detect the exposed phosphatidylserine on the surface of apoptotic cells and phosphatidylserine-positive microvesicles subclass [57], [58]. However, not all microvesicles express phosphatidylserine, and therefore, certain microvesicle populations fail to bind annexin V [59], suggesting that some microvesicles might be formed by other undetermined mechanisms. Microvesicles have markers different from those of exosomes, e.g., tryptophanyl-TRNA synthase 1 and C1q [41]. It should be noted that there are some similarities between exosomes and microvesicles. For example, both contain adhesion molecules, membrane receptors, tissue factors, cytoskeletal proteins, chemokines, various enzymes and cytokines, as well as DNAs and RNAs (mRNA, microRNA). Microvesicles can carry nuclear proteins that originate from apoptotic cells [60], [61].

Apoptotic bodies, generally ranging in size from 1 to 5 μM in diameter (approximately the size of platelets), are produced from the plasma membrane as blebs when cells undergo apoptosis. Apoptotic bodies are closed structures and are larger than exosomes and microvesicles. They float on a sucrose gradient at a density between 1.16 and 1.28 g/mL, overlapping with the density of exosomes. Apoptotic membrane blebbing is a late stage of programmed cell death that is controlled by caspase-mediated cleavage, and subsequent activation of Rho-associated protein kinases [62], [63]. They are characterized by the presence of externalized phosphatidylserine and permeable membrane. Apoptotic bodies contain several intracellular fragments and cellular organelles, including histones, DNA fragments, degraded proteins, nuclear fractions, coding RNAs, noncoding RNAs, and DNAs, similar to those inside microvesicles [62], [63], [64], [65].

Apoptotic bodies may provide an easier system for cellular clearance, since they are smaller than cells and are, therefore, easier to phagocytose [66]. Apoptotic bodies have been suggested to act by “dispatching suicide notes” on the surrounding environment. Indeed, apoptotic body membranes show increased permeabilization by releasing proteins into the microenvironment during the early stages of apoptosis. In turn, the surrounding cells, which lose their membrane integrity, affect apoptotic cells during secondary necrosis [67]. However, the exact function of apoptotic bodies is still unclear. Moreover, little is known about their molecular composition. Therefore, the role of apoptotic bodies in the pathophysiology of disease is not covered in this review.

As stated above, because of the difficulties in the isolation and detection of EVs, their classification cannot be rigorously determined in most settings at this time. The ISEV has provided some standards to classify EVs, according to various morphological, biochemical, and biogenic parameters. EVs continue to be classified into exosomes, microvesicles, and apoptotic bodies [27], [28], [29]. With the improvement of technologies, more criteria for the classification for EVs could be added, such as refractive index, potential energy, and chemical composition [68], [69].

The isolation and purification of EVs have attracted attention because of their potential to aid in the diagnosis and treatment of a broad range of disorders. Today, diverse biochemical and biophysical properties of EVs are used in their isolation, including buoyant density, size, shape, charge, and surface composition [24], [70], [71], [72]. The most widely used method for EV isolation is differential centrifugation, originally developed by Johnstone et al. for the separation of EVs in reticulocyte tissue culture fluid and subsequently optimized by Théry et al. [32], [73]. Differential centrifugation can separate vesicle particles based on their size and density by sequentially increasing the centrifugal force to pellet cells and debris (< 1500 g), large EVs (10,000–20,000 g), and small EVs (100,000–200,000 g) [74]. This method is widely used for various biological samples and is considered the standard for isolating EVs.

In addition to differential centrifugation, other methods have been developed for the isolation of EVs. These include density gradient centrifugation, size-exclusion chromatography, ultrafiltration, immunocapture, and various precipitation-based methodologies, using different reagents [24], [75]. In theory, EVs may be isolated based solely on their physicochemical properties, because they are larger than protein fractions but smaller than whole cells, more dense than the lipid fractions, and have a defined density range [76]. However, the above methods have not reached the ultimate goal of having homogeneous EV subpopulations and precise study of their targets. All of these approaches have their respective limitations, which should be taken into consideration; technical standards are not yet fully established [77]. Some researchers have proposed that the choice of a specific isolation method may depend not only on the type of sample (for example, proteinuric urine or non-proteinuric urine) but also on the type of downstream analyses used for “omics” characterization (e.g., transcriptomics or proteomics) [28]. Furthermore, currently, most researchers perform one or more other methods after the main steps, such as washing in EV-free buffer, ultrafiltration, and further purification by density gradient [78]. Thus, currently, there is no ideal method to isolate relatively pure samples that only contain EVs. Further improvements of the above or new methods to isolate EVs are needed.