Extracellular vesicles: new players in regulating vascular barrier function.

Extracellular vesicles (EVs) have attracted rising interests in the cardiovascular field not only because they serve as serological markers for circulatory disorders but also because they participate in important physiological responses to stress and inflammation. In the circulation, these membranous vesicles are mainly derived from blood or vascular cells, and they carry cargos with distinct molecular signatures reflecting the origin and activation state of parent cells that produce them, thus providing a powerful tool for diagnosis and prognosis of pathological conditions. Functionally, circulating EVs mediate tissue-tissue communication by transporting bioactive cargos to local and distant sites, where they directly interact with target cells to alter their function. Recent evidence points to the critical contributions of EVs to the pathogenesis of vascular endothelial barrier dysfunction during inflammatory response to injury or infection. In this review, we provide a brief summary of the current knowledge on EV biology and advanced techniques in EV isolation and characterization. This is followed by a discussion focusing on the role and mechanisms of EVs in regulating blood-endothelium interactions and vascular permeability during inflammation. We conclude with a translational perspective on the diagnostic and therapeutic potential of EVs in vascular injury or infectious diseases, such as COVID-19.

INTRODUCTION

Extracellular vesicles (EVs) were originally considered as inert debris released by cells to dispose of metabolic wastes (61, 154). One of the seminal discoveries in the history of EV research was made in 1967, when Peter Wolf (154) identified platelet-derived vesicles as “platelet dust” having coagulative properties. The term “exosomes” was used for the first time by Trams et al. (139) in 1981 to describe EVs released from cell cultures. Other notable studies in the 1980s and 1990s by Johnstone et al. characterized EV production from maturing reticulocytes (58, 60) and described functional changes in the plasma membrane due to shedding of redundant proteins in EVs (59). Rapidly evolving research in the past few decades has revealed that EVs are in fact a nonclassical means of intercellular communication, along with the classical means of cell-cell contact and secretion of soluble molecules (96). EVs have gained significant interests in the scientific community as novel contributors to physiological and pathological processes, as they carry specialized cargos capable of altering a variety of molecular reactions (47). Unlike secreted solutes, EV cargos contain proteins and nucleic acids that are enclosed within a lipid bilayer membrane that can be easily transported in the circulation without losing biological activity. The lipophilic nature of these tiny vesicles makes them highly interactive with recipient cells as they can be easily fused with cell membranes and endocytosed (129). Hence, circulating EVs are currently recognized as not only biomarkers but also regulators in cardiovascular diseases.

Despite a recent surge in EV research, a lot of questions about EV biology and their function in circulatory homeostasis or disease remain unanswered (85). The endothelial cells (ECs) lining the luminal surface of blood vessels provide a semipermeable barrier that restricts plasma leakage and immune cell infiltration into surrounding tissues. The integrity of this barrier is an important determinant of circulatory homeostasis, and endothelial hyperpermeability is a hallmark of inflammatory response to injury or infection. EVs have recently been shown to regulate vascular inflammation and permeability response. In this review, we attempt to summarize the current knowledge of EV biology, followed by discussing the roles of circulating EVs in regulating vascular inflammation and permeability.

EV BIOGENESIS AND TARGETING

EVs are a heterogeneous collection of membranous vesicles produced by a variety of cells via complex intracellular pathways involving different subcellular compartments (Fig. 1). The International Society of Extracellular Vesicles and Extracellular RNA Communication Consortium have classified them into three groups: 1) exosomes, small vesicles with diameters ≤100–150 nm that are formed inside multivesicular bodies (MVBs), which can be pelleted at centrifugation speed of 100,000 g (119); 2) microvesicles (MVs), medium-size vesicles of plasma membrane origin ranging in diameters up to 1,000 nm, which sediment at centrifugation speed of 20,000 g (89); and 3) apoptotic bodies, large vesicles with diameters >1,000 nm that can be collected at centrifugation speed of around 2,000 g (89).

Fig. 1.

A: generation of extracellular vesicles (exosomes, microvesicles, and apoptotic bodies) from donor cells. Exosomes are produced as a result of a fusion of a specialized subset of multivesicular bodies (MVBs) in the endosomal pathway to the plasma membrane. Microvesicles are formed by outward budding of the plasma membrane. Apoptotic bodies are formed by outward blebbing of the plasma membrane of apoptotic cells. B: different mechanisms of uptake of extracellular vesicles into the target cell. C: cargo composition of extracellular vesicles comprising lipids, proteins, and nucleic acids. CM, ceramide; MVB, multivesicular body; PS, phosphatidylserine; RER, rough endoplasmic reticulum; SER, smooth endoplasmic reticulum; SM, sphingomyelin. Some images of cells or organelles were obtained from Smart-Servier Medical Art (https://smart.servier.com).

Two complex mechanisms have been identified in the generation of exosomes. The first pathway is dependent on the endosomal sorting complex required for transport (ESCRT) machinery, which consists of four subunits (ESCRT-0, ESCRT-I, -II, and -III) that recruit ubiquitinated proteins to the early endosome and act in concert with other associated proteins (e.g., VPS4, VTA1, ALIX) in the assembly of exosome cargo to intraluminal vesicles (ILVs). Invagination of late endosomal membranes results in the formation of ILVs within larger endosomal multivesicular bodies (30). Once fusion of MVBs to the plasma membrane occurs, ILVs are released into the extracellular space as exosomes (96). Exosome biogenesis also involves the syndecan-syntenin-ALIX pathway and proteins such as heparanase, syndecans, heparan sulfate, ADP-ribosylation factor 6, phospholipase D2, and syntenin (136). The second pathway in exosome biogenesis is ESCRT independent and mediated by sphingomyelinase-dependent ceramide production (136). Exosome release is facilitated by multiple proteins, such as Rab GTPases, diacylglycerol kinase-α, and SNARE proteins (30, 136).

Microvesicles (MVs) are formed via blebbing or budding of the plasma membrane. This process is less well characterized compared with exosomes; rather, it is considered a sequela of loss of membrane asymmetry as a result of cell activation or injury. The outer leaflet of plasma membrane is enriched in lipids, including phosphatidylcholine and sphingomyelin, whereas the inner leaflet contains phosphatidylserine (PS) and phosphatidylethanolamine (29). This asymmetry is maintained by coordinated activity of membrane phospholipid transporter, flippase (inward translocation of PS), floppase (outward translocation of PS), and scramblase (bidirectional transporter that shuttles phospholipids along concentration gradient and Ca2+ influx) (29). Cell activation or injury causes increased Ca2+ influx, enhanced activity of floppases, and diminished activity of flippases, leading to PS outward exposure. This reaction is coupled with activation of caspases and/or calpains. The membrane budding process is facilitated by cytoskeletal remodeling, which involves RhoA, ADP-ribosylation factors 1 and 6, extracellular signal-regulated kinase, and myosin light chain kinase (MLCK) (79, 142). There is also a report that sphingomyelinase converts sphingomyelin to ceramide, which in turn regulates membrane curvature and MV release (12). MVs expressing PS on their surface are identified by positive annexin V labeling. PS+ MVs are known to be procoagulant, as they provide a negatively charged surface for prothrombinase and tenase complexes to bind, thereby promoting coagulation (104). In addition to the PS+ MVs, vesicles that do not expose PS on their surface exist. The mechanism behind the generation of PS− MVs is unclear. A study suggests their formation is via smaller endosomal vesicle binding to plasma membrane or via cleavage of cytoskeleton components during basal conditions, as opposed to activated states that produce PS+ MVs (104). In addition to annexin V, a recent study in cancer cell-culture models described annexin 1 and annexin 2 as novel MV markers that were exclusively present in MVs and not in exosomes (57). However, it must be noted that characterization of EVs is heavily dependent on methods of EV isolation and purification to reliably separate vesicular from nonvesicular fractions and soluble proteins in biofluids.

Apoptotic bodies are larger vesicles released by apoptotic cells via membrane blebbing, with or without cytoskeletal spikes (23). They are generally distinguished from other MVs based on size. Further details are discussed in other reviews (41, 135).

EVs have been detected in various biofluids, including plasma, urine, bile, saliva, bronchoalveolar lavage, ascitic fluid, lymph, and postoperative drainage fluid (67, 125). A variety of cells have been identified as parent cells capable of generating EVs, including platelets, leukocytes, endothelial cells, epithelial cells, hepatocytes, brain cells, and cancer cells. Most of them also serve as recipients or effectors of EV targeting. Once docked onto the recipient cell surface, EVs are internalized via membrane fusion or endocytosis; the latter occurs via clathrin-mediated endocytosis, caveolin-mediated endocytosis, lipid raft-mediated endocytosis, macropinocytosis, or phagocytosis (1, 90) (Fig. 1). The level and mechanism of uptake vary depending on the recipient cell type, state of activation, and presence of receptors. For example, PS+ EVs target endothelial cells via developmental endothelial locus-1, or the tyrosine kinase receptor AXL that recognizes Gas6 on EV surface (35, 49). Heparan sulfate proteoglycans on the recipient cell surface also participate in EV uptake and internalization (11). The circulation kinetics and biodistribution profile of EVs in vivo depend on the route of administration, cell type of origin, and surface protein composition (152). A study shows that EVs labeled with the infrared lipophilic dye DiR, when intravenously injected in mice at a concentration of 1 × 1010 particles/g body wt, are mainly distributed in the liver, spleen, gastrointestinal (GI) tract, and lungs 24 h after injection (152).

EV CARGO COMPOSITION

EV cargos are enriched in nucleic acids, proteins, and lipids (Fig. 1). Studies related to EV cargo packaging under different conditions are reported in public databases, including ExoCarta (91), Vesiclepedia (63), and EVpedia (63).

The nucleic acid contents of EVs include DNA (chromosomal and mitochondrial), mRNAs, miRNAs, short noncoding RNAs, tRNA fragments, piwi-interacting RNA, vault RNA, and Y RNA (1). The RNAs are enclosed in the lipid membrane rendering them resistant to RNAse degradation. The RNAs also exist in combination with ribonucleoproteins such as Argonaute 2 (AGO2), or high-/low-density lipoproteins (142). The existence of miRNAs in EVs indicates that EVs may influence gene expression in distal tissues via their cargo miRNA.

The protein composition of EVs has been studied using proteomic approaches. EV proteins are mainly derived from endosomes, plasma membranes, and cytosolic components. In contrast, those from the nucleus, endoplasmic reticulum, and Golgi apparatus are mostly absent (30). Some proteins are cell specific as shown in Table 1; others are commonly detectable irrespective of cell origin. For example, exosomes contain tetraspanins (CD9, CD63, CD81, CD37), heat shock proteins (Hsp 70, 90), tumor susceptibility gene 101 protein, major histocompatibility complex II, flotillin, and ALG-2-interacting protein X (ALIX) (1). Among them, CD9 is one of the most abundant exosomal proteins (65). Larger EVs are enriched with cytosolic components (enzymes, histones, ribosomal proteins, and proteasomes), transmembrane molecules (integrins, receptor kinases, and G proteins), and cytoskeletal elements (actin, cofilin, and tubulin) (30).

Table 1.

Cell-specific EV markers

Cell of origin	Markers
Platelet	CD41+, CD61+, CD42b+, P-selectin+
Endothelial cell	CD144+, CD106+, E-selectin+, CD31+/CD41-, CD144+/CD105+
Neutrophil	CD66b+, CD11b+, L-selectin+, CD15+ (human), Ly6g+ (mouse)
Monocyte	CD14+, CD16+, Ly6c+ (mouse)
Lymphocyte	CD3+, CD4/8+ for T cells, CD19+ for B cells
Red blood cell	CD235a+ (human), TER-119+ (mouse)

EV, extracellular vesicle.

Although the lipid profile of EVs has not been fully established, current known lipids include sphingomyelin, phosphatidylserine, cholesterol, diglycerides, saturated fatty acids, glycerophospholipids, ganglioside GM3, ceramides, and their derivatives (30, 76).

TECHNIQUES IN EV ISOLATION AND CHARACTERIZATION

Studying EVs has been challenging, owing to their small size and underdeveloped standardization protocols. Currently, methods of EV isolation include ultracentrifugation, density gradient centrifugation, sequential filtration, immunoaffinity isolation, size exclusion chromatography, polymer-based precipitation, and microfluidics (Table 2) (69). The most commonly used method is differential centrifugation by which different-sized EVs are collected under different centrifugation speeds: apoptotic bodies (2,000–2,500 g), MVs (10,000–20,000 g), and exosomes (100,000–120,000 g), respectively. This method allows processing of larger sample volumes but has limitations in separation of different types of EVs, as sedimented EVs could be contaminated with protein aggregates. EV purity can be enhanced via ultracentrifugation on a sucrose or iodixanol gradient as well as by size exclusion chromatography (SEC), each of which reduces soluble protein contamination (87). Both these techniques have their challenges; ultracentrifugation at speeds ∼100,000 g for prolonged times could damage EVs, whereas SEC that involves passing EVs through a column with nanosized pores could cause sample clogging or damage (87). Moreover, the latter is time-consuming and does not allow large sample volumes. Commercially available isolation kits use precipitation methods for fast isolation; however, their purity and specificity are often debated. In contrast, isolation by immunoaffinity method, i.e., EV capture by antibody-coated beads, produces high-quality EVs, but the yield is generally low because EVs that do not express the particular marker are missed during immunocapture (75).

Table 2.

Common methods of EV isolation and characterization

	Advantages	Disadvantages	Ref.
Methods of isolation
Ultracentrifugation,differential centrifugation	Large sample volumes, cost effective	Non-EV impurities, specialized equipment, physical damage to EVs	(32)
Density gradient ultracentrifugation	Pure EVs with minimal contamination from soluble protein and nonvesicular fractions	Labor-intensive and complex procedure, particles with same sedimentation rate cannot be separated	(69, 102)
Ultrafiltration	Large sample volumes, concurrent processing of multiple samples	Filter plugging, impurities, deformation of vesicles	(80)
Size exclusion chromatography	Pure EVs, separation of EV fraction from soluble proteins	Low sample volumes, labor-intensive, specialized equipment and complexity, column plugging	(75, 101)
Polymer-based precipitation	Quick and simple procedure	Impurities, additional chemicals	(69)
Immunocapture	Purity and high selectivity	Selective subsets of EVs only, high cost of antibodies, EV damage after elution	(32)
Microfluidics	Rapidness, purity, efficiency	Specialized equipment and complexity, low sample capacity	(158)
Methods of isolation
Nanoparticle tracking analysis (NTA); tunable resistive pulse sensing (TRPS)	Assessment of size distribution and particle concentration, fluorescent labeling for NTA	Cell debris, impurities can be mistaken for EVs, specialized equipment and training, high cost	(156)
Microscopy (EM, confocal, atomic force, super-resolution)	Direct visualization and study of uptake in cells, size distribution and morphology assessment, immunogold/fluorescent labeling	Dye aggregates can be mistaken for EVs, specialized equipment and training, high cost	(82)
Flow cytometry (conventional, nano-FACS, imaging flow cytometry)	Multicolor fluorescent labeling, estimation of particle concentration with counting beads, high resolution with nano-FACS, and imaging FCM	Smaller EVs (<200 nm) are missed with conventional flow cytometers, specialized equipment and training	(87)
Molecular studies (Western blot, qPCR, ELISAs, mass spectrometry, arrays)	Bulk EV cargo assessment	Cannot differentiate between different subsets of EVs	(32)

EM, electron microscopy; EV, extracellular vesicle; FCM, flow cytometry; qPCR, quantitative polymerase chain reaction.

EV characterization approaches include nanoparticle tracking analysis (NTA), tunable resistive pulse sensing (TRPS), microscopy, flow cytometry, and molecular studies (Table 2) (156). NTA is a commonly used technique to quantitate EV numbers with a size resolution threshold of ∼30 nm. Particles are visualized by scattering of light and based on Brownian motion; the average particle size is then calculated by the Stokes–Einstein equation, according to which a particle’s size is inversely proportional to its diffusion capacity (126). TRPS is another particle analysis technique combining the Coulter principle with size-tunable pores and uses information obtained from the resistive pulse signal to determine particle numbers, size, and surface charge (70). Further confirmation of EV numbers, size, and morphology can be obtained by transmission electron microscopy (TEM), cryo-EM, and atomic force microscopy. Interaction of EVs with target cells can be determined by confocal microscopy (156). In addition, super-resolution microscopy, such as stimulated emission depletion (STED) microscopy, uses technology that bypasses the diffraction limit of light microscopy and thus is able to image small structures 20–40 nm in size; it also enables dynamic monitoring of EV interaction with recipient cells (109). As EVs are membranous and rich in nucleic acids, lipophilic dyes (DiR, PKH26, Di-8-Anepps) and nucleic acid dyes (Sytox RNA) have been used to stain EVs. A caveat is that dye aggregates can form in the EV size range, confounding the reading. Other dyes used for EV labeling include amine reactive fluorescent label and 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE); these dyes show less aggregates than lipid or nucleic acid dyes but have more background noise, and they require repeated washes, causing reduced EV yield (103).

EVs can also be quantitatively characterized by multicolor flow cytometry using antibodies specific to EV markers, sizing reference polystyrene beads, and EV counting beads (87). Conventional flow cytometry has a limitation in detecting particles smaller than 200 nm; the problem is minimized with the new generation of high-resolution flow cytometers (nano-FACS) or imaging flow cytometers equipped to quantitate EVs as small as 20–40 nm (87, 115). In addition, quantitative PCR, Western blotting, ELISAs, and mass spectrometry are commonly used to quantitatively measure specific EV components.

ENDOTHELIAL BARRIER STRUCTURE AND FUNCTION

Plasma fluid and proteins move across the endothelial barrier via the paracellular (major) and/or transcellular pathway (Fig. 2). The paracellular pathway is controlled by cell-cell junctions, including the adherens junction (AJ) formed by homophilic dimers of vascular-endothelial (VE)-cadherin linked to actin through catenins (α, β, γ, and p120), as well as tight junction (TJ) composed of occludin, claudins, and zonula occludens (ZO-1,2,3) that connect to the actin cytoskeleton (50, 167). Proteins associated with junction barrier also include junctional adhesion molecules (JAMs), platelet endothelial cell adhesion molecule-1, CD99, and nectins (143). The transcellular pathway is controlled by caveolae, vesiculo-vacuolar organelles (VVOs), and transmembrane molecules like gp60 that mediate transcytosis of albumin across the endothelium (Fig. 2) (111). Caveolin-1 is a major caveolar protein that plays a dual role in barrier regulation. On the one hand, it stabilizes caveolae and facilitates transcytosis; on the other hand, it inhibits endothelial nitric oxide (NO) synthase (eNOS), thereby reducing NO-induced paracellular permeability (121).

Fig. 2.

The paracellular and transcellular transport pathways for endothelial permeability. The paracellular pathway is maintained by interendothelial junctions comprising adherens junctions (AJs) and tight junctions (TJs), which are anchored to the actin cytoskeleton. The transcellular pathway comprises caveolae containing caveolin-1 (Cav-1) and vesiculo-vacoular organelles (VVOs). JAMs, junctional adhesion molecules. Some images of cells or organelles were obtained from Smart-Servier Medical Art (https://smart.servier.com).

During infection or acute inflammation, the junction proteins undergo posttranslational modifications or conformational changes, disrupting barrier integrity and enhancing permeability. Among multiple forms of posttranslational modifications, phosphorylation is best characterized; for example, VE-cadherin/catenin phosphorylation by protein kinases (c-Src, PKC) leads to AJ disassociation (36). Likewise, phosphorylation of caveolar proteins has been shown to increase EC transcellular permeability (36, 71). In addition, cytoskeleton modifications, for example, myosin light chain phosphorylation catalyzed by MLCK, trigger actomyosin contraction and intercellular gap formation (122). Recently, other forms of posttranslational modifications, including eNOS-mediated nitrosylation (48) and palmitoyltransferase-mediated palmitoylation (15), have been shown to regulate endothelial permeability. In particular, our laboratory has reported a novel function of DHHC-containing palmitoyl acyltransferases (PATs) in mediating endothelial inflammation and barrier disruption during inflammation (15).

Vascular permeability is also indirectly modulated by leukocyte-EC interactions. Neutrophil rolling and adhesion on microvascular endothelium increase permeability via intracellular cell adhesion molecule-1 (ICAM-1)-mediated signaling (130). Although constitutive PKC predominantly regulates ICAM-1-dependent permeability under physiological conditions, inducible Src activity plays a major role in ICAM-1 signaling during inflammation (130). Interestingly, many of these signaling pathways have been linked to EVs (25).

EV FUNCTION IN VASCULAR PERMEABILITY DURING INFLAMMATION

Circulating EVs are mainly derived from megakaryocytes and platelets, red blood cells, endothelial cells, and leukocytes, of which platelet EVs form the majority under normal conditions. EV production and cargo composition are altered during inflammation (117). For example, vascular inflammation is accompanied by increased ICAM-1+, VCAM-1+, and E-selectin+, and decreased endothelial protein C receptor (EPCR)+ EVs (25). In sepsis, plasma levels of total EVs and subsets derived from platelets, leukocytes, and endothelium are increased (117, 128, 153). In chronic vascular diseases like atherosclerosis, increased numbers of EVs derived from platelet, endothelium, and leukocyte origin are detected in the plasma (37). More importantly, EVs exert a detrimental role in promoting inflammation and microvascular permeability. Table 3 lists the EV cargo proteins and RNAs that participate in the regulation vascular inflammation and permeability. The role of EVs as pro-inflammatory mediators is further supported by the finding that blocking exosome generation with GW4869 inhibited cytokine production and attenuated systemic inflammatory injury in septic mice (42). In diabetic mice, plasma EVs laden with IgG are able to activate the classical complement pathway and promote microvascular leakage in the retina (53, 54). Similarly, patients with metabolic syndrome have a higher level of circulating EVs; when injected to normal mice, these EVs cause impaired endothelium-dependent vasorelaxation and decreased eNOS expression (3). Hence, EVs are thought to play a self-perpetuating role in augmenting inflammation in both acute and chronic systemic inflammatory diseases.

Table 3.

EV cargo regulating inflammation and endothelial junction permeability

Origin	EV Cargo	Known Function	Ref.
Platelets	IL-1b	Increase permeability by activating inflammasome pathway in ECs	(51)
	CCL5; P-selectin; sCD40L	Leukocyte recruitment and activation	(9, 92)
Endothelial cells	c-Src;	Tyrosine phosphorylation of EC junction and cytoskeletal proteins to increase permeability
	ICAM-1; VCAM-1	Increase leukocyte-EC adhesion	(25)
Neutrophils	Annexin 1;	Improve EC permeability	(33)
	MPO; PAF	Oxidative damage, inflammation, and thrombosis	(113, 149)
Monocytes	IL-1b; tissue factor	Increase EC permeability and thrombosis	(118)
Microglia	IL-1b; miR-155	Increase neuroinflammation	(73)
	Nef	Increase BBB permeability by decreasing ZO-1	(118)
Red blood cells	miR-451a-Ago2 (RBC infected with malaria parasite)	Increase EC permeability by downregulating caveolin-1 and activating transcription factor 2	(84)
Cancer cells	miR-25-3p (colon cancer)	Downregulate TJ proteins, alter VEGFR2 expression	(160)
	miR103a (hepatoma)	Decrease VE-cadherin, p120 catenin, and ZO-1 expression in EC junctions	(43)
	miR-181c (brain metastatic breast cancer)	Disrupt actin cytoskeleton and increase EC permeability	(138)
	Semaphorin 3A; VEGF-A (glioblastoma)	Increase brain vascular permeability	(140, 141)

EC, endothelial cell; EV, extracellular vesicle; ICAM-1, intracellular cell adhesion molecule-1; MPO, myeloperoxidase; PAF, platelet activating factor; RBC, red blood cell; TJ, tight junction; VEGF-A, vascular endothelial growth factor A.

Although many studies demonstrate a detrimental role of EVs in vascular inflammation, controversy exists. There is evidence suggesting that septic EVs protect vascular reactivity, and EV-leukocyte conjugates negatively correlate with organ dysfunction and mortality in sepsis (106, 127). Nevertheless, EVs not only serve as markers of cell stress or activation but also actively participate in pathophysiological responses to stress (Fig. 3). In the following section, we provide a detailed discussion about circulating EVs of different cell origins, centering on their roles in regulating vascular inflammation and permeability.

Fig. 3.

Inflammation alters the circulating extracellular vesicle (EV) profile that interacts with the vasculature to cause endothelial injury. Inflammation-associated EVs increase thrombogenicity, immune cell activation, adhesion, and transmigration through the endothelium. EVs also cause direct injury to the endothelium by increasing expression of pro-inflammatory adhesion molecules, modulating adherens junction (AJ), tight junction (TJ), caveolar, and cytoskeletal proteins through their protein and miRNA cargos and disturbing nitric oxide homeostasis. Some images of cells or organelles were obtained from Smart-Servier Medical Art (https://smart.servier.com).

PLATELET-DERIVED EVs

Platelet EVs show both pro-inflammatory and anti-inflammatory effects via interactions with endothelial cells and leukocytes. They carry cytokines like interleukin 1 beta (IL-1b), IL-6, and tumor necrosis factor alpha (TNFa), which stimulate neutrophil adhesion to endothelium and transendothelial migration (19, 83, 155). In particular, P-selectin and soluble CD40 ligand (sCD40L) carried by platelet EVs bind to P-selectin glycoprotein ligand-1 and CD40 expressed on leukocyte surface, respectively, causing leukocyte activation (9). Platelet EVs increase monocyte-EC binding by upregulating CD11a/b and CD14 on monocytes (14) and delivering c-c-motif chemokine ligand 5 (CCL5) to ECs (92). They also prolong neutrophils’ effect on tissue injury by reducing neutrophil apoptosis (20). With respect to their roles in vascular function, IL-1b-rich platelet EVs increase vascular permeability by activating EC inflammasome pathway (51). Recent mechanistic investigation reveals their effects to cause aortic endothelial injury and increase glomerular endothelial permeability by damaging glycocalyx, decreasing nitric oxide production, and increasing reactive oxygen species (ROS) production, via a mechanism dependent on mammalian target of rapamycin complex 1 (mTORC1) (145). In contrast to these pro-inflammatory actions, however, platelet EVs have been shown to exert anti-inflammatory effects, as they decrease c-c-motif chemokine ligand 4, TNFa, and colony-stimulating factor-1 release from macrophages (74), reduce TNF-a and IL-8 secretion from plasmacytoid dendritic cells (24), and inhibit IL-17 and interferon gamma production by regulatory T cells (24). In addition, they attenuate thrombin-induced endothelial barrier dysfunction in vitro and vascular endothelial growth factor (VEGF)-induced microvascular hyperpermeability in vivo (100).

ENDOTHELIAL-DERIVED EVs

Different inflammatory stimuli promote the production of endothelial-derived EVs (EC-EVs) with different compositions (25). In vitro, EC-EVs can be induced by TNFa, IL-1b, thrombin, lipopolysaccharide (LPS), C-reactive protein, and plasminogen activator-1 (52). In addition, laminar shear stress, high glucose conditions, and hypoxia are strong inducers of EC-EV generation (52). Several studies have demonstrated that EC-EVs bind leukocytes and contribute to their activities in inflammatory disorders (52, 125). Our recent study (25) shows that EC-EVs upregulate CD11b expression on neutrophil surface and induce formation of neutrophil extracellular traps (NETs). As citrullinated histone H3 (cit-H3) comprises the major protein content of NETs, increased circulating level of cit-H3 is associated with vascular injury (95). We have identified its direct effects to cause endothelial paracellular leakage (94).

In addition to activating leukocytes, EC-EVs can directly target the vascular endothelium. For example, endothelial cell monolayers exposed to EC-EVs, in the absence of leukocytes, display adherens junction discontinuity coupled with paracellular hyperpermeability (25). At the molecular level, EC-EVs promote phosphorylation of myosin light chain and VE-cadherin, which triggers a cytoskeleton contractile response characterized by cortactin translocation from cell periphery to center and actin stress fiber formation. Further mechanistic studies revealed that these effects involved c-Src signaling transferred by EV cargo, as c-Src-depleted EVs failed to cause barrier disruption (25). In line with this finding, EC-EVs have recently been shown to regulate blood-brain barrier (BBB) permeability (99, 123). In neuroinflammatory diseases (e.g., multiple sclerosis), brain-derived EC-EVs are increased, which bear markers that are specific to brain ECs (112). EVs derived from LPS-stimulated brain microvascular EC (BMEC) promote angiogenesis and/or vascular leakage by upregulating VEGF expression in pericytes (157). EC-EVs also contribute to the pathogenesis of cerebral malaria complicated by BBB injury (31).

LEUKOCYTE-DERIVED EVs

All subtypes of white blood cells can produce EVs, with the neutrophil being the main source especially during acute inflammation. Neutrophil-derived EVs are capable of targeting endothelial cells and increasing their production of IL-6, monocyte chemoattractant protein 1, and tissue factor (TF) (97, 98). They also cause oxidative injury in ECs by transferring cargo containing myeloperoxidase (113). Their effects to reduce TJ protein expression and to increase junction permeability have been observed in human cerebral ECs (6). In response to endotoxin stimulation, neutrophils release EVs that are rich in platelet-activating factor, which is known to promote inflammation and coagulation (149).

On the other hand, some studies show that neutrophil EVs exhibit beneficial effects. For example, extravasating neutrophils deposited CD18+ EVs that are barrier protective (81). Neutrophil EVs carry anti-inflammatory molecules, such as annexin 1 which is capable of reducing neutrophil-EC adhesion in vitro and neutrophil infiltration in vivo (33). Neutrophil EVs also reduce the release of pro-inflammatory cytokines and enhance the release of anti-inflammatory transforming growth beta 1 from macrophages (45) and natural killer cells (114).

Monocyte-derived EVs are known to induce endothelial inflammation in many ways. EVs produced from THP-1 (monocytic cell line) or primary monocytes bind to ECs, causing ERK1/2 phosphorylation and increasing the expression of ICAM-1, VCAM-1, and E-selectin in ECs via the nuclear factor-κB (NF-κB) pathway (147), or they transfer cargo carrying IL-1b and components of the inflammasome pathway. Blocking NLR family pyrin domain-containing 3 (NLRP3) in THP1 cells, or IL-1b receptor on ECs, mitigated the pro-inflammatory effects of monocytic EVs (147). Additionally, monocyte EVs increased nitrative stress via enhanced production of peroxynitrites (88). They also promote thrombogenicity by upregulating tissue factor, exposing phosphatidylserine, and downregulating anticoagulant tissue factor pathway inhibitor and thrombomodulin in ECs (5). This is evident in patients with meningococcal sepsis who display TF+ monocytic EVs coupled with severe disseminated intravascular coagulation (108). In vitro treatment of monocytes with LPS or interferon alpha produced EVs with unique miRNA signatures, which interacted with human brain microvascular ECs to increase inflammatory signaling and monocyte chemotaxis (34). Despite their well-recognized pro-inflammatory nature, however, monocytic EVs do not seem to be a potent barrier-damaging factor in human brain EC monolayers (150).

Compared with neutrophils and monocytes, lymphocyte EVs are relatively less well studied in the context of vascular inflammation and permeability. It is plausible that T cell-derived EVs play a crucial role in the chronic stage of inflammation, autoimmune disease, and cancer, based on the studies showing their effects to decrease nitric oxide (NO) production and increase ROS production in ECs; both contribute to impaired vasorelaxation responses (86, 105).

Microglia, the tissue-resident macrophages in the central nervous system, release EVs that regulate the blood-brain barrier and cerebral microcirculation in various neuroinflammatory diseases. Extracellular ATP, a common damage-associated molecular pattern (DAMP) associated with sterile inflammation and tissue injury, is known to increase the release of microglia EVs that are rich in IL-1b and GAPDH (18, 133). Microglia-derived EVs also play a complex role in the pathogenesis of neuroinflammation after traumatic brain injury (TBI). Although they are generally known as promoters of neuroinflammation after TBI (73), a study shows that microglia EVs containing miR-124-3p inhibit neuronal inflammation and promote neurite growth (56).

During infectious conditions, microglia release pro-inflammatory EVs in response to bacterial and viral pathogens. In particular, LPS stimulates the production of microglia EVs that are rich in IL-1b and miR-155 conferring pro-inflammatory effects (73). Microglia transfected with the HIV auxiliary protein Nef, release EVs that can disrupt the BBB by downregulating an important TJ protein, ZO-1. Nef+ microglia EVs also promote neuroinflammation by increasing production of proinflammatory cytokines like IL-6, IL-8, and IL-12 (118). EVs isolated from plasma of LPS-injected mice, when stereotactically introduced to the brain of healthy mice, are preferentially internalized by microglia as opposed to other neuronal cells (163). Thus, microglia play an important role in brain-blood crosstalk during inflammation through EVs.

RBC-DERIVED EVs

Red blood cells are a significant source of circulating EVs under physiological conditions, and their levels are increased in malaria (13), hematological disorders, systemic inflammation, and after prolonged blood storage (4, 8, 77, 117). RBCs infected with the malaria parasite Plasmodium falciparum show increased production of EVs, which carry functional miR-451a-Ago2 complexes capable of increasing endothelial permeability by downregulating caveolin-1 and activating transcription factor 2 (84). RBC-EVs also carry heme and iron that can cause oxidative damage to phospholipids on the cell membrane (21) and scavenge nitric oxide, resulting in impaired vascular function (38). RBC-EVs from stored blood are procoagulant in vitro, as they are rich in phosphatidylserine and can induce tissue factor upregulation and activate platelets (7). RBC-EVs have also been implicated in neutrophil priming and CD11b upregulation for enhanced adhesiveness to vascular endothelium (16, 22).

TUMOR-DERIVED EVs

EVs released by cancer cells circulate in the blood; they increase endothelial permeability and promote angiogenesis, thus facilitating tumor progression. A recent study indicates that metastatic tumor-derived EVs increase EC activity and permeability through exosomal miRNAs (120). Another study shows that colon cancer cells produce EVs that can increase vascular permeability by altering the expression of vascular endothelial growth factor receptor 2 (VEGFR-2) and tight junction proteins ZO-1, occludin, and claudin-5, through exosomal miR-25-3p (160). Likewise, hepatoma cells secrete EVs containing miR103a that increases vascular permeability by downregulating VE-cadherin, p120 catenin, and ZO-1 expression in EC junctions (43). Moreover, glioblastoma-secreted EVs containing protein semaphorin 3A increase brain vascular permeability, an effect attenuated by blocking semaphorin 3A or by inhibiting its receptor neuropilin-1 (140). These EVs also carry VEGF-A that increases the permeability of brain ECs (141). In addition, brain metastatic cancer cells produce EVs containing miR-181c, which is shown to disrupt actin cytoskeletal dynamics leading to increased BBB permeability (138).

CLINICAL AND TRANSLATIONAL PERSPECTIVE

Biomarker role of EVs.

Distinct EV signatures have been identified as potential biomarkers for cardiovascular diseases (28). For example, increased cystatin C, serpin F2, and CD14 protein levels in EVs are associated with an elevated risk for adverse vascular events and mortality in patients with atherothrombotic disease (64). In patients with diabetes, reduced miRNA-126-3p and miRNA-26 in circulating EVs correlate with prevalence of cardiovascular complications (110). Moreover, increased blood level of CD31+/annexin V+ EC-EVs is considered an independent predictor of adverse cardiovascular outcomes in subjects with stable coronary artery disease (124, 151). In patients with ultrasound evidence of subclinical atherosclerosis, increased CD11a+ leukocytic EVs are detected in their blood (26). Although the diagnostic potential of these EV markers is quite promising, more in-depth and large-scale analyses are required to establish their validity and clinical utility.

Therapeutic potential of EVs.

EVs are highly biocompatible because of low toxicity and immunogenicity. They are stable in the circulation and can easily be taken up by tissues, making them excellent therapeutic candidates for multiple diseases (131). Currently, engineering stem cell EVs represents an attractive field. With respect to the treatment of diseases associated with vascular barrier dysfunction, mesenteric stem cell EVs (MSC-EVs) have shown promise in attenuating pulmonary vascular permeability and lung injury in hemorrhagic shock (116). In a model of ex vivo human lung perfusion, MSC-EVs improved alveolar fluid clearance, decreased lung edema, and improved respiratory as well as hemodynamic function (46). Likewise, EVs from endothelial progenitor cells (EPCs) carrying miR-126-5p/3p increased the survival rate in mice after septic challenge and suppressed lung vascular leakage by increasing expression of TJ proteins (164). In addition, engineered EVs are being designed as part of drug delivery system or vectors to deliver specific miRNAs, peptides, antibodies, or synthetic compounds (132). These techniques involve multiple procedures using viral (adenoviral/retroviral) or nonviral (incubation, lipofection, sonication, electroporation) approaches (132).

The use of engineered EVs as nanotherapeutics for combating systemic inflammatory diseases is still at its stages of infancy. Several approaches have been proposed to modify EV surface and cargo for improved targeting of inflamed tissues. For example, integrins αLβ2 and α4β1 expressed on leukocyte EVs can sense and traffic toward ICAM-1 and VCAM-1 on inflamed endothelium (159). This property of integrin-bearing EVs can be used to deliver EV-based therapeutics. In addition to native proteins, other synthetic approaches to engineer EV surface and cargo by adding antibodies, peptides, RNAi, small molecules, and aptamers have been used to achieve tissue-specific targeting. For example, Tian et al. (137) used click chemistry-based modification of EVs to decorate EV surface with αvβ3 integrin-specific RGD peptides for targeting the ischemic brain in a stroke model. For ischemic heart diseases, myocardium-targeting peptides introduced on EV surface by fusion with lamp2 protein have been used (66, 148). ICAM-1 and VCAM-1 binding synthetic peptides are available; however, when conjugated to EVs, their ability and specificity to target inflamed tissues have yet to be confirmed (107, 162).

EV-based RNAi therapeutics, such as EVs loaded with miR-155 inhibitors, miR-15a mimics, or Myd88 siRNA, have shown promise as anti-inflammatory therapies to reduce LPS-induced acute lung injury (161). In a mouse study, EVs with designer mRNAs were delivered through implantation of engineered cells in the brain, which were shown to successfully reduce neuroinflammation (68).

Other EV nanotherapeutics have used the anti-inflammatory mechanisms intrinsic to cells. Some notable studies include injecting EVs loaded with IκBα, an inhibitor of transcription factor NFκB, which helped reduce mortality and inflammation in septic mice (27). Similarly, EVs loaded with catalase, an anti-oxidant enzyme, reduced oxidative stress, improved neuronal activity and attenuated neuroinflammation in a mouse model of Parkinson’s disease (166). In addition, neutrophil-derived EVs loaded with resolvin D2, and stem cell EVs loaded with curcumin, reduced inflammation and restored the BBB in ischemia-reperfusion-induced brain injury models (39, 62). In a recent study, decoy EVs expressing cytokine receptors lacking the signaling domain were generated and proved effective in neutralizing pro-inflammatory cytokines in an in vitro model of inflammation (40). Drugs like dexamethasone and piceatannol have been synthesized into macrophage/neutrophil-derived EVs, which successfully reduced endothelial inflammation in the kidney and lungs (44, 134). It must be noted that most of these approaches have been tested at the preclinical stages and their clinical utility, specificity, and safety need further validation (131).

Potential role of EVs in the pathogenesis and treatment of COVID-19.

Our understanding of COVID-19 pathophysiology has been rapidly evolving. A current view emphasizes its vasculitis nature, characterized by microcirculatory dysfunction, barrier leakage, and coagulopathy (2). Severe cases present with cytokine storm and advance to acute respiratory distress syndrome (ARDS) culminating in multiple organ failure (55, 144). EVs likely participate in the pathophysiological response of pulmonary and systemic circulation to viral infection. Previous studies show that severe acute respiratory syndrome-related coronavirus (SARS-Cov), responsible for the SARS outbreak in 2003 and sharing a high degree of amino acid homology with the novel SARS-Cov, enters host cells through endocytosis and is processed in the endosomal pathway (146). Given that exosome biogenesis involves the endosomal pathway, we speculate that EVs released by infected host cells might carry viral proteins or nucleic acids as part of their cargo (Fig. 4). This is supported by the fact that many other viruses, such as paramyxoviruses, hepatitis C virus, rhabdoviruses, filoviruses, herpes viruses, and hepatitis B virus, use the ESCRT pathway to promote their release (93). These viral components assembled in EVs can act as pathogen-associated molecular patterns (PAMPs) which interact with pattern recognition receptors (PRRs), such as Toll-like receptors, in host cells, thereby activating downstream inflammatory processes (Fig. 4).

Fig. 4.

A: severe acute respiratory syndrome-related coronavirus 2 (SARS-Cov-2)-infected cells can release extracellular vesicles (EVs) expressing viral antigens that may play a role in inflammation and act as candidates for cell-free vaccines. B: potential role of mesenchymal stem cell-derived EVs (MSC-EVs) in mitigating inflammatory injury of the cardiovascular and the respiratory system in COVID-19. ACE-2, angiotensin-converting enzyme 2. Some images of cells or organelles were obtained from Smart-Servier Medical Art (https://smart.servier.com).

Developing effective vaccines is crucial to combating COVID-19. As a natural means of cell-to-cell and tissue-to-tissue communication, EVs provide an efficient vehicle for presentation and distribution of antigens through the circulation for them to reach distal organs. The presence of adhesion molecules and integrins on exosome surface ensures specific binding and uptake by host cells. Exosomal vaccines containing the S protein of SARS coronaviruses have been shown to induce high levels of neutralizing antibodies (72). We believe that similarly designed exosomes can be used as a viable cell-free vaccine platform for SARS-Cov-2 (Fig. 4).

Mesenchymal stem cells have been extensively used in regenerative medicine to attenuate acute lung injury and facilitate tissue healing or recovery (78). Currently, there are many ongoing clinical trials worldwide testing the effect of MSC therapies against ARDS in patients with COVID-19 (ClinicalTrials.gov Identifier: NCT04288102, NCT03042143, NCT04355728, NCT04333368). However, as cell-based therapies are associated with an increased risk of tumor development, immune rejection, and microvessel obstruction, MSC-EVs may become a preferred therapeutic option because of lower allogenic immune rejection, easier preservation, and higher stability compared with MSCs. Currently, MSC-EVs have gained significant interest as therapeutics in cardiovascular regenerative medicine and have shown promise in mitigating ischemic heart injury and LPS-induced acute lung injury in preclinical models (10, 165). Given the commonality of cardiovascular and pulmonary responses to respiratory infection, MSC-EVs might represent a novel therapeutic strategy to mitigate COVID-19-associated organ injury (Fig. 4).

Future perspectives.

The future of EV research lies in the translational studies that test and validate their clinical applications in disease diagnosis as biomarkers, as well as in disease treatment as therapeutics or drug vectors. In the past few years, there has been tremendous progress in this field, evidenced by the surge in clinical trials involving exosome-based interventions, although many roadblocks need to be overcome before such EV-based approaches can be widely used in clinical settings. For example, there must be consensus in standardizing protocols for EV isolation and purification from human samples or preclinical models. Within this context, combining two different techniques of EV isolation (e.g., ultrafiltration or ultracentrifugation followed by size exclusion chromatography) to increase efficiency and purity is gaining popularity (17, 101). Another unmet challenge lies in EV quality and quantity with respect to large-scale production of reproducible batches of clinical grade EVs for therapeutics. In addition, the route of EV administration to achieve a consistent and desired therapeutic outcome needs to be optimized. Nevertheless, the future of applied EV research looks very promising.

Conclusion.

EV production from blood cells and/or endothelium is increased during acute and chronic cardiovascular diseases, and they carry distinct molecular signatures and serve as unique biomarkers for disease diagnosis. Functionally, circulating EVs induce vascular inflammation by promoting thrombosis, immune cell activation, leukocyte/platelet adhesion, and transmigration across the endothelium. They also directly impair vascular barrier function by altering endothelial junction and cytoskeleton dynamics via transfer of cargo containing permeability signaling molecules. Although many studies suggest EVs as inflammatory mediators, there is ample evidence supporting their anti-inflammatory and barrier-protective function, especially in the processes associated with inflammation resolution and tissue repair. The pleiotropic effects of EVs depend on the physiological state of their parent cells, cargo profile, and pathological conditions that promote their release. Further molecular insights into EV biogenesis, cargo loading mechanisms, and functional consequences of EV-targeting tissues will help us to better understand their enigmatic role in the regulation of various cardiovascular diseases. Moreover, as promising biomarkers and vaccine delivery vehicles, EVs and their engineered products may significantly contribute to the development of novel diagnostics and therapeutics for vascular injury in inflammatory or infectious diseases.

GRANTS

This work was supported by National Heart, Lung, and Blood Institute Grants R35 HL150732 and GM097270 (to S. Y. Yuan) and HL120954 (to M. H. Wu) and Department of Veterans Affairs Grants 101BX000799 and IK6BX004210 (to M. H. Wu).

DISCLOSURES

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

AUTHOR CONTRIBUTIONS

M.H.W. and S.Y.Y. conceived and designed research; V.C. prepared figures; V.C. drafted manuscript; X.Y., Y.M., M.H.W., and S.Y.Y. edited and revised manuscript; V.C., X.Y., Y.M., M.H.W., and S.Y.Y. approved final version of manuscript.