Recent Advances in Extracellular Vesicles and their involvements in Vasculitis

Abstract

Systemic vasculitis is a heterogeneous group of multisystem autoimmune disorders characterized by inflammation of blood vessels. Although many progresses in diagnosis and immunotherapies have been achieved over the past decades, there are still many unanswered questions about vasculitis from pathological understanding to more advanced therapeutics. Extracellular vesicles (EVs) are double-layer phospholipid membrane vesicles harboring various cargoes. EVs can be classified into exosomes, microvesicles (MVs), and apoptotic bodies depending on their size and origin of cellular compartment. EVs can be released by almost all cell types and may be involved in physical and pathological processes including, inflammation and autoimmune responses. In systemic vasculitis, EVs may have pathogenic involvement in inflammation, autoimmune responses, thrombosis, endothelium injury, angiogenesis and intimal hyperplasia. EV-associated redox reaction may also be involved in vasculitis pathogenesis by inducing inflammation, endothelial injury and thrombosis. Additionally, EVs may serve as specific biomarkers for diagnosis or monitoring of disease activity and therapeutic efficacy, i.e. AAV-associated renal involvement. In this review, we have discussed the recent advances of EVs, especially their roles in pathogenesis and clinical involvements in vasculitis.

1. Introduction

Systemic vasculitis is a heterogeneous group of multisystem autoimmune disorders, including Kawasaki disease (KD), antineutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV), Takayasu’s arteritis (TA), Behçet’s disease (BD), Polyarteritis nodosa (PAN), Giant cell arteritis (GCA), etc. [1, 2]. Vasculitis is characterized by inflammation in particular-sized blood vessels and commonly accompanied by thrombosis, blood vessel stenosis/occlusion, aneurysm formation or hemorrhage [3]. Although progress has been achieved for earlier diagnosis and the new immunotherapies in the past few decades, patients still experience premature mortality, relapse, comorbidity and poor quality of life [4]. Therefore, better understanding of etiology and pathogenic mechanisms are still needed in order to find better diagnostic biomarkers and establish more advanced therapeutic strategies.

Extracellular vesicles (EVs) are membrane vesicles released by almost all cell types when they undergo cell activation or programmed cell death [5–7]. Many EVs are in relatively low levels under physiological conditions, while may be elevated in various pathological conditions or diseases, such as inflammation [8], thrombosis [9], cardio-metabolic diseases [10], autoimmune disease [11], sepsis [12] as well as cancer [13]. Brogan et al. reported that children with active systemic vasculitis have significant higher levels of plasma platelet EVs and endothelial EVs compared to those in inactive group and healthy controls [14]. The levels of these EVs are closely associated with disease activity [14]. In our early publication, EVs have been shown to mediate intracellular communication, and participate in vasculitis associated pathological processes [11]. In this review, we mainly focus on the most recent progress of EV generation and the potential roles of EVs in the pathogenic involvements and clinical applications in systemic vasculitis.

2. Recent progress in extracellular vesicle research

EVs are cell-derived vesicles that are enclosed by a membrane lipid bilayer. EVs can be released from almost all cell types, such as platelets, erythrocytes, endothelial cells (ECs), monocytes, neutrophils, lymphocytes, mesenchymal stem cells, and malignant cells when they undergo cell activation or several types of programmed cell death [5–7, 11, 15, 16]. By carrying various bioactive molecules, i.e. proteins, lipids, DNA, messenger RNA, long noncoding RNA, microRNA, and circular RNA [7, 17], EVs can exert various biological functions. According to their size and different cellular compartment biogenesis, EVs can be categorized into three subtypes, including exosomes (30–100 nm in diameter), microvesicles (MVs) or microparticles (MPs) (100–1000 nm in diameter), and apoptotic bodies (1000–5000 nm in diameter) [18–20]. EVs can be found in almost all types of body fluid (e.g. blood, urine, milk, semen, synovial fluid, broncho-alveolar lavage fluid, malignant ascites), feces, and solid organ/tissues [19, 21–24]. Here, we briefly summarize the recent progress in EV generation and EV-associated isolation and detection techniques.

2.1. Extracellular vesicles and programmed cell death

It has long been known that EVs can be released during apoptosis [25]. Recent studies indicate that EVs can also be generated during other types of programmed cell death including necroptosis, pyroptosis and NETosis, in addition to apoptosis. Pyroptosis is an inflammatory programmed cell death mediated by caspase-1-dependent canonical inflammasome pathway and non-caspase-1-dependent (caspase-11 in mice, caspase-4/5 in human) pathways [26–29]. Recent study showed that a heterogeneous population of EVs packaged with Gasdermin D (GSDMD), caspase-1and Fas-associated death domain can be released by pyroptotic monocytes [30, 31]. Caspase-1 activation, potassium efflux, and concomitant calcium influx may directly regulate EV secretion and cargo loading [32]. Furthermore, EVs with both cleaved GSDMD and active caspase 1 can induce vascular endothelial injury [30].

Necroptosis is another form of regulated cell death that generally manifests with morphological features of necrosis. This type of cell death is caspase-independent and mediated by the receptor-interacting serine-threonine kinase 1 (RIPK1)- and RIPK3-mixed lineage kinase domain-like (MLKL) signaling axis [33]. The necroptotic cells can release EVs into extracellular space prior their loss of cell viability or membrane integrity [6, 34–36]. Though mechanisms of necroptotic EV biogenesis are still not fully understood, RIPK3-dependent-MLKL phosphorylation and conformational change are indispensable [6]. Compared with apoptotic bodies, these EVs are smaller and have less DNA content. Besides, they contain phosphorylated MLKL, endosomal sorting complex required for transport III members, mitochondria and other proteins [6, 34–37]. Normally, during apoptosis, Ca2⁺-dependent translocation of phosphatidylserine (PS) to the outer leaflet of the plasma membrane occurs [38], while the necroptotic PS externalization occurs independently of Ca2⁺ influx, and may be a direct, local, and proportional consequence of MLKL-mediated [33, 39, 40]. However, PS externalization on the necroptotic EVs mediates their recognition and phagocytosis by phagocytes, and involves in the inflammatory response [34, 36].

Neutrophils are the most abundant circulating leukocytes [41]. NETosis, a unique type of neutrophil cell death, is characterized by nuclear envelope rupture, nuclear chromatin externalization and formation of neutrophil extracellular traps (NETs) [42, 43]. Recent publications indicate that NETotic neutrophils can also release membrane EVs during an early stage of NETosis when cytoskeletal rearrangement takes place [44]. Though mechanisms are still not fully understood, a recent work demonstrates that calcium depletion at high glucose concentrations reduce the activity of peptidylarginine deiminase 4 (PAD4) and promote the vesicular NET release [45]. Interestingly, a recent work demonstrates that neutrophils can bind and uptake EVs from the plasma of AAV patients, resulting in NET formation [46]. Therefore, generation of EVs and NETs may be related in certain conditions.

2.2. Extracellular vesicles and redox biology

Oxidation-reduction (redox) reaction is essential in physiological homeostasis and disorders. Redox signaling is also associated with generation and bio-function of EVs. EV formation can be induced by oxidative stress in erythrocytes [47]. In addition, inhibition of NADPH oxidase and nitric oxide synthase-2 attenuates production of EVs from neutrophils [48]. Generally, main steps of EV biogenesis are as follow: formation of clustered microdomains with cargoes, membrane budding, and fission either at the plasma membrane or at the limiting membrane of the multivesicular endosomes [49]. Redox signaling may be involved in different process during EV generation. Calcium influx is a major inducer of EV release, as elevated cytoplasmic calcium enhances fusion of MVBs with plasma membrane and also promotes plasma membrane blebbing and EV formation [50, 51]. Several calcium channels are redox sensitive [52, 53]. Thom and colleagues have shown that reactive oxygen species (ROS) can induce thiol oxidation of inositol-1,3,5-triphosphate receptors, and that triggers calcium release from the endoplasmic reticulum to the cytoplasm, thus consequently resulting in EV release [54]. Additionally, other bio-molecules with redox-sensitive groups (like thiols on cellular surface, actin cytoskeleton, phospholipid flippases, thiol-rich fusion proteins) are potential targets for regulation of EV biogenesis [55].

Redox reactions may also result in the changes of contents in EVs. Alternatively, cells under oxidative stress can remove oxidized proteins through release of EVs, which can be transferred to neighbor or distant cells, thereby triggering an intercellular oxidative stress response between cells [56]. Similarly, oxidative stress increases the abundance of membrane complement regulatory proteins in EVs [57]. In addition, anti-oxidants may affect the composition of EVs. As a reducing reagent, N-acetylcysteine amide has been shown to inhibit the release of EV-associated of TF and PS from cells exposed to oxidative status, thus decreasing the pro-coagulant properties of EVs [55]. Therefore, altered EV concentrations and composition are associated with redox biology, especially under oxidative stress. Additional research is required to identify the specific redox-dependent mechanisms that are involved in regulation of EV generation, and the biofunction, as these may be potential targets for pharmaceutical modulation of EV signaling.

EVs also serve as massage trafficker to modulate redox reactions. A recent proteomic analysis has reported a list of antioxidant activities and related molecules in EVs derived from endothelial cells [58]. Furthermore, these endothelial-derived EVs contain the enzymatic machinery necessary to synthesize NADPH using blood metabolites to feed different biosynthetic pathways [59]. Additionally, nuclear factor erythroid 2-related factor 2 (Nrf2)-targeting miRNAs or other potential components can be incorporated into EVs, thus contributing to redox regulation through the Nrf2/Antioxidant signaling pathway [60]. EVs and ROS have been reported to be closely interacted with each other [61]. On the one hand, EVs can directly modulate intracellular or extracellular contents of ROS, as they carry anti-oxidant/pro-oxidant machinery that may either produce or scavenge ROS [61]. On the other hand, after interacting with their target cells (indirect effect or horizontal transfer), EVs can also increase or reduce ROS metabolism in target cells by transferring enzymatic components, signaling molecules, and/or regulating gene expression that are involved in regulation of redox responses [61].

Redox and inflammatory changes occur during vascular inflammation and endothelial injury [62, 63]. As major free radicals, ROS and reactive nitrogen species (RNS) are involved in vascular diseases [64]. In vasculitis, imbalance of pro-oxidants/anti-oxidants system was observed. For example, enhanced production of ROS through NADPH oxidase in neutrophils is associated with thrombosis in BD patients [65]. In the following section (section 3.), we have discussed pathological involvements of EV-associated redox signaling in vasculitis.

2.3. Isolation and detection of extracellular vesicles

Growing interests in EV research have led to a focus on the development of more advanced techniques in EV isolation, to obtain EVs with high quality for further analysis and applications. EVs are heterogeneous, there are no “one-size-fits-all” standardized optimal methods available until now [7, 66]. Based on the isolation principle, namely size-, charge-, and affinity-based techniques, modern isolation and separation techniques in the field of EV research can be subdivided into three groups [66]. Thereinto, separation methods like ultracentrifugation or density gradients are still commonly used, while the method of size exclusion chromatography has also been gradually used [67]. General criteria for efficient isolation and purification techniques include being capable of isolating EVs with satisfactory quality and quantity, as well as being fast, automatic, simple, and suitable for further applications.

In the context of EV detection, flow cytometry is the most commonly used technique according to their physical characteristics and surface markers based on fluorescence and scattering signals [7]. Conventional flow cytometry can only detect EVs larger than 200 nm [23]. However, according to differential fluorescence intensity, high-resolution flow cytometer can detect EVs with a diameter of 100nm [68, 69]. Other techniques including electron microscopy, fluorescence-based antibody array system, nanoparticle tracking analysis, confocal fluorescent microscopy, enzyme-linked immunosorbent assay (ELISA), immunoblotting as well as electrophoresis, have been used by our [7, 16, 70, 71] and other groups [72–74]. More importantly, in vivo identification of apoptotic and extracellular vesicle-bound live cells using image-based deep learning has been reported [75]. The authors also identified cell subsets binding with naturally occurring EVs and EV-associated decoration in vivo [75]. This breakthrough favors clarifying the function of EVs binding with different cell types in various tissues under normal and pathological conditions. Furthermore, new technologies such as single extracellular vesicle detection [76], label-free surface-enhanced raman spectroscopy [77], highly efficient magnetic labelling and magnetic resonance imaging (MRI) tracking [78], have also been introduced to the field. These new techniques alone with classical methods together will contribute to better understanding of EVs and their involvement in physiology and pathophysiology of various human diseases, including vasculitis.

In addition to the above discussed basic research progress in EV generation, and technical progress in EV detection, recent studies have also demonstrated new understandings and new roles of EVs in human pathophysiology. Very interestingly, recent publications have reported that EVs can transfer intergenerational information of metabolic disease risk [27, 79] or even transmit intergenerational stress of the paternal environmental experience [80] from parents to their offspring in an epigenetic fashion. These emerging functions of EVs challenge the classical concepts of genetic transmission across generations [27, 79, 80]. Importantly, the new understanding in EVs expands over the classical mechanisms of cellular interaction [81], and these knowledge may change our way of thinking about human pathophysiology in the subcellular scale [81]. In the following sections of this review article, we will focus on the progress and the most recent information about pathophysiological involvement of EVs in vasculitis.

3. Pathological involvement of extracellular vesicles in vasculitis

EVs are known to participate in the pathological processes of vasculitis through different mechanisms [11]. Based on the current understanding of the vasculitis pathogenesis, we have summarized the potential pathologic involvement of EVs in vasculitis (figure 1.).

Fig. 1. Pathological involvements of EVs in vasculitis.

A. EVs and inflammation in vasculitis: EVs are shown to have proinflammatory effect by interacting with recipient cells and inducing the release of proinflammatory mediators, and directly transporting proinflammatory mediators as well. B.EVs and autoimmunity in vasculitis: EVs participate in autoimmune procedures of vasculitis, including autoantigen loading and presenting, ICs formation and deposition and impaired EV-ICs clearance, NETosis and DAMPs associated innate immunity. C.EVs and Thrombosis in vasculitis: EVs exert prothrombotic ability in vasculitis mainly due to the surface expression of TF and PS. Besides, EVs could participate in three basic prerequisites of thrombosis of Virchow’s triad. D. EVs and vascular endothelial injury in vasculitis: Endothelial cell- (EC-), neutrophil- and monocyte-derived EVs could induce vascular endothelial injury. E. EVs and angiogenesis in vasculitis: EVs could contribute to all steps of angiogenesis, including enzymatic degradation of the vessel’s basement membrane, ECs proliferate and migrate to form vascular tube as well as the stabilization and maturation of the newly formed vascular structures by recruiting pericytes. F. EVs and intimal hyperplasia in vasculitis: As vascular smooth muscle cells (VSMCs) are central to intimal hyperplasia, EVs involve in phenotypic changes of VSMCs through transferring of bioactive cargoes, including HMGB1, IL-1β and miRNAs.

Abbreviations: ECs, endothelial cells; C, complement; Casp-1, caspase-1; MPO, myeloperoxidase; MMPs, matrix metalloproteinases; PR3, proteinase 3; MHC, Major Histocompatibility Complex; HMGB1, High Mobility Group protein B1; TCR, T cell receptor; ICs, immune complexes; PS, phosphatidylserine; TF, tissue factor; VSMCs, vascular smooth muscle cells; PDGF, platelet derived growth factor; VEGF, vascular endothelial growth factor; ROS, reactive oxygen species; GSDMD, Gasdermin D; MVs, microvesicles.

3.1. Extracellular vesicles and inflammation in vasculitis

Systemic vasculitis is an autoimmune rheumatic disease that are characterized by vessel inflammation [82]. EVs are shown to have proinflammatory effects by interacting with immune and nonimmune cells, and inducing the release of proinflammatory mediators/cytokines, and by directly transporting proinflammatory mediators [83–86]. For instance, our recent studies demonstrated that EVs can induce the release of TNF-α from cultured peripheral blood mononuclear cells [87, 88], while TNF-α is a pivotal cytokine in the pathogenesis of vasculitis. EVs released by neutrophils can contribute to inflammatory response in AAV by promoting the release of IL-6 and IL-8 and the expression of intercellular adhesion molecule-1 (ICAM-1) by ECs [89]. EVs released by anti-PR3-IgG-activated neutrophils can promote the release of pro-inflammatory cytokines in ECs in vitro [90]. Interestingly, the levels of these pro-inflammatory cytokines released by ECs are also elevated in blood of patients with GPA [90]. Therefore, one may speculate that EVs from neutrophils in GPA might induce the release of pro-inflammatory cytokines from ECs of vasculature, contributing to pathogenesis in patients with GPA. Moreover, B1 receptor-positive endothelial EVs in patients with vasculitis may contribute to the chronic inflammation by inducing neutrophil chemotaxis [91].

On the other hand, EVs can exert proinflammatory effects through transporting cellular receptors, proteins and miRNA, etc. Leukocyte-derived EVs can transfer kinin B1-receptors to glomerular ECs and promote kinin-associated inflammation in vasculitis [92]. Additionally, our previous study indicates that high mobility group box-1 (HMGB1) can be loaded into EVs and released from activated monocytes [71]. As a representative damage-associated molecular patterns (DAMPs), HMGB1-positive EVs may participate in the pathogenesis of inflammatory and autoimmune diseases [71]. Interestingly, EV-associated oxidized proteins can be recognized as DAMP and induce inflammation [93]. Xie and colleagues identified differentially expressed protein profile in serum exosomes of patients with coronary artery aneurysm (CAA) caused by KD compared with healthy controls [94]. Further analysis have shown that many of these differentially expressed proteins (like complement system, immunoglobulin, leucine-rich alpha-2-glycoprotein) are associated with immune responses and inflammation [94]. Recent advances also show that EVs contain differential miRNAs that are related to inflammation. Wang et. al. detected a differential exosomal miRNA profile in KD patients with CAA as compared to healthy controls [95]. Differential exosomal miRNA target genes are mainly involved in IL-6 signaling pathways, indicating that exosomal microRNAs are associated with the pathogenesis and development of CAA through inflammatory mechanisms in KD [95].

More interestingly, as mentioned above, EVs can be released by pyroptotic monocytes [96]. These EVs with GSDMD and caspases-1 could induce vascular endothelial injury [30]. Recent study showed that pyroptosis plays an important role in KD. In KD patients, serum levels of pyroptosis-related proteins are significantly increased [97]. Moreover, THP-1 cells treated with KD sera could induce NLRP3 (NACHT, LRR, and PYD domains-containing protein 3)-dependent endothelial pyroptosis through HMGB1/ RAGE(receptor for advanced glycation end-products)/cathespin B signaling pathways [97]. Thus, EVs, that are derived from pyroptotic cells, might be generated during inflammation and are involved in the pathologic process in vasculitis.

In contrast, EVs may also have anti-inflammatory effects depending on the environmental conditions. Kolonics et al. showed that EVs released from resting neutrophils, but not activated or apoptotic neutrophils, exert anti-inflammatory action by inhibiting production of reactive oxygen species (ROS) and releasing anti-inflammatory cytokines from neutrophils [98]. Additionally, inflammatory stimulation of mesenchymal stromal cells (MSCs) results in the release of EVs with altered protein and nucleic acid composition, along with enhanced anti-inflammatory properties [99]. These observations represent a new paradigm for generation of anti-vasculitis EVs for potential treatment of vasculitis.

Taken together, EVs may contribute to a autoimmune inflammation and proinflammatory milieu in vasculitis either indirectly by triggering recipient cells to release proinflammatory mediators, or directly by EV-associated proinflammatory cellular receptors, proteins, miRNA, etc. Depending on environment, certain EVs may exert anti-inflammatory effect as well. One specific kind of cell-derived EVs may exert opposite effects towards inflammation under different metabolic status. Further studies, particularly in vivo analyses, are needed to verify these delicate interactions.

3.2. Extracellular vesicles and autoimmunity in vasculitis

Elevated levels of circulating EVs have been described in various autoimmune diseases, including autoimmune vasculitis [7, 100, 101]. Our previous publication has systematically reviewed the role of EVs in autoimmune diseases [7, 102]. During EV generation, certain cargoes from nucleus, cytoplasm and cell membrane can be clustered as autoantigens in EVs [102]. Stimulated EVs from GPA patients can produce ROS and carry dsDNA [46]. Moreover, EVs are known to be able to express peptide-MHC complexes, presenting autoantigens and activating immune cell responses [100]. Significant infiltration of neutrophils in/around blood vessels is a representative histopathological finding of vasculitis. In addition, neutrophil-derived EVs also contribute to vasculitis as bioactive autoantigen carriers. As autoantigens, neutrophil proteinase-3 (PR3) or myeloperoxidase (MPO) can induce the production of corresponding autoantibodies and promote the pathogenesis of AAV. Both PR3-positive EVs and MPO-positive EVs have been reported [103, 104], suggesting their potential role to serve as auto-antigens and their contributions to autoimmune responses in AAV. On the other hand, these neutrophil-derived MPO-positive EVs could induce MPO-mediated damage of vascular endothelium [105]. Similarly, the level of MPO-positive EVs expressing both C3a and C5a is significantly higher in AAV patients with renal involvement [106].

Furthermore, neutrophil NETs can also associate with a wide range of granular enzymes, such as MPO [107, 108]. NETs can serve as autoantigens, activating complement system, or acting as DAMPs and inflammasome activators [107]. Recent works have shown significant increase of NETs formation in vasculitis [109–112]. As we discussed, neutrophils undergoing NETosis can also release EVs [44]. Thus, both NETotic EVs and NETs in extracellular milieu may contribute to pathogenesis of vasculitis. Besides, NETs can also serve as a source of extracellular HMGB1 [113]. Our published work indicated that HMGB1 could be associated with EVs [71]. Other study also showed HMGB1 plays an important role in AAV [114–116], Henoch-Schonlein Purpura [117, 118], KD [119, 120] and BD [121]. Thus, both EV- or NET-associated HMGB1 in the extracellular milieu may participate in pathological processes by activating HMGB1 associated innate immunity in vasculitis.

In autoimmune disease, especially systemic lupus erythematosus (SLE), circulating or tissue resident EVs have been reported to form immune complexes (ICs) and lead to autoimmune responses [122–124]. ICs also present in patients with vasculitis and can be found in the serum of children with KD [125], in the kidneys of AAV patients [126], as well as in vessel walls of leukocytoclastic vasculitis [127]. On the one hand, EVs with ICs could activate monocytes, T and B cells in vitro, and stimulate the expression of pro-inflammatory mediators, therefore amplifying the autoimmune responses and tissue damage [123, 128, 129]. Furthermore, EV-associated ICs may hinder phagocytes to recognize and clear these EVs. By carrying CD47, EVs might also inhibit phagocytosis through CD47-signal regulatory protein α (SIRPα) signaling [130]. Thus, proinflammatory EVs and cell debris may accumulate when phagocytosis is dampened, and leading to excessive activation of autoimmune responses. Since phagocytes from AAV patients have impaired function [131], EVs-mediated interruption of phagocyte capacity may contribute to the pathogenesis of vasculitis.

In summary, during EV generation, autoantigens could be clustered in EVs, especially the cargoes of PR3 and MPO during EV generation and/or NET release from neutrophils. Following autoantigen presentation, the corresponding production of autoantibodies, formation and deposition ICs in vasculature induce subsequent autoimmune responses in vasculitis. Besides, attenuated phagocytosis leads to the accumulation of these detrimental EVs, contributing propagation of autoimmune responses in this autoimmune vascular disease.

3.3. Extracellular vesicles and thrombosis in vasculitis

Vascular thrombosis is a common phenomenon in vasculitis and prompts tissue injury. It has long been known that exposure of procoagulant PS and tissue factor (TF) on EVs make them highly potent in their pro-thrombotic functions. Shifting of PS from the inner leaflet to the cell membrane can favor membrane budding and formation of EVs [49]. PS externalization is also associated with the prothrombotic activity of EVs by electrostatic interaction between negatively charged PS and positively charged carboxyglutamic acid in the clotting proteins [132]. To date, PS positive EVs have been reported to be released from cell surface of almost all cell types including platelet, monocytes, lymphocytes, or ECs [133, 134].

TF is an initiator of the extrinsic coagulation cascade. Previous results of our and others have demonstrated that TF can be carried on EVs derived from monocytes [9, 135–137]. TF activity is enhanced by PS and phosphatidylethanolamine (PE) on the membrane surface of their parental cells [138]. Importantly, procoagulant activity of EVs can also be enhanced when PS and TF are co-existed [132] as well as after oxidation of the thiol group of the TF molecules [139]. Besides, monocyte-derived EVs can also increase TF expression on ECs, and decrease the levels of anticoagulant tissue factor pathway inhibitor and thrombomodulin [140]. On the other hand, TF-bearing EVs can also be released from ECs after stimulation by TNF-α [141]. EVs released by TNF-α-activated human ECs can promote vascular inflammation and monocyte migration [142]. Positive feedback mechanisms are present between monocytes, monocyte-derived TF⁺ EVs, ECs, and EC-derived TF⁺ EVs, thus propagating prothrombotic conditions in vasculitis. Further investigations are needed to explore their interactions and mechanisms in pathogenesis in vasculitis.

Prothrombotic ability is the first documented pathological feature of membrane EVs that are released from platelets [143], and EVs are associated with thrombogenesis in systemic vasculitis [144]. BD patients have significantly higher levels of CD62P⁺ platelet EVs as compared to healthy controls [145]. Another recent study showed the increased levels of procoagulant EVs in BD patients [146]. Furthermore, BD patients with a history of thrombosis have the increased levels of total and TF positive EVs, but have a lower proportion of EVs with tissue factor pathway inhibitor, as compared to those without a history of thrombosis [147]. Moreover, TF can be expressed on EVs and NETs released from neutrophils in AAV. ANCA can induce the release of TF-expressing EVs or NETs in C5a-primed neutrophils [148]. TF plays a pivotal role in EV-dependent thrombin generation as implicated by antibody neutralization studies [149]. In clinical settings, Mendoza et al. demonstrated that elevated EV-TF activity in AAV remission is a strong indicator of venous thromboembolism (VTE) independent of renal function, suggesting that EV-TF activity might be a useful biomarker for identifying patients that are prone to develop VTE [150]. Therefore, EVs exert prothrombotic ability in vasculitis mainly due to their surface expression of TF and PS, and may serve as a potential biomarker of VTE in vasculitis patients.

According to Virchow’s triad, vascular endothelial injury, blood flow disturbance, and blood hypercoagulability are three basic prerequisites for thrombosis [151]. Firstly, as discussed above, monocyte- and neutrophil-derived EVs with TF, cleaved GSDMD, active caspase 1 and MPO could mediate vascular endothelial damage in vasculitis. Endothelial injury could also be caused by pro-inflammatory properties of EVs. Secondly, blood vessel stenosis/occlusion and aneurysm formation are common manifestations of vasculitis. Elevated pro-thrombotic EVs with TF and PS can initiate coagulation system, leading to activation of coagulant factors and platelets. These contributors can affect blood flow disturbance. Thirdly, elevated coagulant factors and inflammation-associated hypercoagulability are also involved in blood hypercoagulability. Collectively, EVs could be involved in thrombosis through different mechanisms.

3.4. Extracellular vesicles and vascular endothelial injury in vasculitis

Endothelial activation and injury, characterized by increased adhesion molecule expression in ECs and prothrombotic endothelial phenotype, are central to the pathogenesis of vasculitis [152]. EC-derived EVs are biomarkers of endothelial activation/ dysfunction and for monitoring disease activity in systemic vasculitis [153–157]. Circulating EC-derived EVs can impair endothelium-dependent vasorelaxation and reduce nitric oxide (NO) production and bioavailability [158]. In contrast, endothelial EVs can transfer miR-10a to monocytic cells and inhibit inflammatory signaling [159]. This suggests that altered endothelial EV release may lead to vascular injury under the complex and delicate regulation network.

In addition, EVs, derived from other cell types, also contribute to vascular injury. EVs, derived from PR3-ANCA stimulated neutrophils, can bind and activate endothelium via induction of ROS and thrombin generation [89]. Neutrophil EVs expressing active MPO can induce endothelial injury in vasculitis through activation of MPO-hydrogen peroxide-chloride pathway [105]. By co-presence of acute phase reactant pentraxin-3 (PTX3) and HMGB1, MPO-positive EVs may participate in the pathogenesis of AAV by promoting endothelial activation and vascular inflammation [104]. EVs from apoptotic T cells can increase the level of inducible NO-synthase and cyclooxygenase-2 in blood vessel and promote vascular dysfunction [160].

Furthermore, EVs can also induce endothelium injury through kallikrein-kinin and complement systems. Excessive complement deposition on the endothelium is reported to promote endothelial injury, and the complement-bearing endothelial EVs can be found in vasculitis patients [161]. Endothelial complement fixation rapidly induces neutrophil-endothelial adhesion, which is an early event in neutrophil recruitment into acute inflammatory lesions [162]. In AAV patients, ANCA-stimulated neutrophils exhibit greater ability to activate the alternative complement pathway as compared to the primed neutrophils from healthy controls [163]. AAV patients also have significantly elevated expression level of C3a and C5a on MPO-positive EVs [106]. Therefore, interaction between neutrophils and complement system may propagate vasculitis. Since blocking the kallikrein-kinin system can reduce endothelial complement activation [161], the effect may be due to different mechanisms.

Since EVs can be released during pyroptosis and NETosis as mentioned above, pyroptosis- and NETosis-mediated EVs may also induce endothelium injury. However, few studies have been conducted in these contexts. Recent report revealed that EVs-contained both cleaved GSDMD and active caspase 1, two markers of pyroptosis, could induce vascular endothelial injury [30]. In addition, ANCA-induced NETs can provoke endothelial injury [164]. NET-related endothelial dysfunction or damages are dependent on their associated nuclear histones from their parental cells [165, 166]. These studies indicate contributions of pyroptosis and NETosis, as well as their released EVs and/or NETs to endothelial injury in vasculitis. More investigations are needed to explore the role of EVs in vasculitis in the context of pyroptosis and NETosis.

All in all, EVs (especially derived from ECs, neutrophil and monocyte) can contribute to vascular endothelial injury in vasculitis, through complement activation, ROS production, or extracellular nuclear histone-associated injury. Besides, endothelial hypoxia caused by EV-associated thrombosis and vessel stenosis/occlusion also contribute to vascular endothelial injury. Furthermore, neuropeptides might also participate in EV-induced endothelial injury. In human dermal microvascular endothelial cells, neuropeptide, especially substance P, can increase expression of VCAM-1 which is associated with inflammation and endothelium activation [167]. Since EV-associated neuropeptides have not been reported, further studies on neuropeptides and EVs, as well as their pathological roles in vasculitis are still needed.

3.5. Extracellular vesicles and angiogenesis in vasculitis

Angiogenesis is the formation of new capillaries from pre-existing vasculature, which is an important pathological change in vasculitis [168]. In vasculitis, angiogenesis may represent a compensatory response to the hypoxic environment which leads to stenosis or occlusion of blood vessel [169]. Moreover, newly-formed vessels can further promote inflammation by expressing adhesion molecules, colony-stimulating factors and chemokines [169]. Angiogenesis is a process regulated by the balance between stimulatory and inhibitory signals. Altered levels of angiogenic and anti-angiogenic factors have been described in vasculitis patients [168, 170–172]. Interestingly, EVs derived from differential cell types can deliver either pro- or anti-angiogenic signals to ECs in vasculitis [173].

Vasculogenesis is the differentiation of angioblasts into ECs and formation of vascular labyrinth followed by angiogenesis which is the further expansion of the vascular network [174]. Enzymatic degradation of the basement membrane in vasculature is the first step of sprouting angiogenesis [173]. Matrix metalloproteinases (MMPs)-containing EVs can cause extracellular matrix remodeling that paves the way for endothelium invasion and revascularization [175]. Furthermore, EVs, that are taken up by recipient cells, can induce further production of MMPs and their release via EVs [176]. Secondly, ECs proliferate and migrate to form vascular tube [173]. In this step, EVs are also involved. Activated platelet-derived EVs can promote the proliferation and migration of vascular ECs by inducing overexpression of miR-126 and angiogenic factors, including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), or transforming growth factor-β1 (TGF-β1) [177]. Thirdly, pericytes are required for the stabilization and maturation of the newly formed blood vessel [173]. Platelet-derived growth factor (PDGF)-B and PDGF receptor-beta play critical roles in recruitment of vascular pericytes [178]. Given that platelet-derived EVs can trigger angiogenesis by EV-associated cytokines (e.g. VEGF, bFGF, and PDGF) [179], platelet-derived EVs may have the potential to recruit vascular pericytes. Thus, EVs could contribute to all steps of angiogenesis through different ways.

In contrast, EVs may also exert anti-angiogenic effects. Lymphocyte-derived EVs can inhibit angiogenesis by increasing oxidative stress and inhibit VEGF-associated signaling [180]. Oxidized low-density lipoprotein (ox-LDL)-stimulated human monocyte-derived macrophages release EVs that can inhibit the proliferation, migration, and angiogenesis of human coronary artery vascular ECs in vitro [181]. Although the role of EVs in vascular formation is well documented, further investigations to study the specific involvement of endogenous EVs in the process may help us to develop EV-related therapeutic strategy.

3.6. Extracellular vesicles and intimal hyperplasia in vasculitis

Intimal hyperplasia is an important pathological process in vasculitis. In response to vascular injury and inflammation, proliferation, migration and decreased expression of smooth muscle cell markers can occur in vascular smooth muscle cells (VSMCs), resulting in intimal hyperplasia [182, 183]. In this pathological process, various cell types, like dendritic cells (DCs), CD4 T cells and macrophages, recruit and produce cytokines, MMPs, as well as growth factors, contributing to intimal hyperplasia [184, 185].

As VSMCs is crucial to intimal hyperplasia, EVs are involved in phenotypic changes of VSMCs through transferring of bioactive cargoes, including HMGB1, IL-1β and miRNAs. HMGB1 have been reported to be critical regulator of intimal hyperplasia after arterial injury [186, 187]. HMGB1 increases IL-1β production in VSMCs via NLRP3 inflammasome, thus prompting VSMC inflammation [188]. In turn, IL-1β enhances VSMC proliferation and migration via P2Y2 receptor-mediated RAGE expression and HMGB1 release [189]. So HMGB1 and IL-1β can regulate VSMC inflammation, proliferation and migration, which are essential to intimal hyperplasia. Our previous publication indicated that nuclear HMGB1 could be externalized and released with EVs from macrophages [71]. EVs with co-expression of MPO and HMGB1 are also associated with AAV disease activity [104]. IL-1β containing EVs have also been detected [190, 191]. These indicate the potential role of EVs in intimal hyperplasia through transporting HMGB1 and IL-1β to VSMCs. Additionally, transfer of miRNAs by EVs also participate in phenotypic changes of VSMCs. It has been reported that transfer of exosomal miR-21–3p from nicotine-stimulated macrophages to VSMCs can enhance migration and proliferation of VSMCs [192]. Besides, transfer and direct overexpression of miR-221–3p from adipose tissue-secreted EVs dramatically enhance VSMCs proliferation and migration [193]. In contrast, EVs may also inhibit migration and proliferation of VSMCs, indicating a potential therapeutic role in vasculitis. Significant inhibitory effects of adipose MSCs-EVs on VSMC proliferation and migration in vitro have been reported to limit intimal hyperplasia [194]. Furthermore, exosomes derived from human umbilical cord MSCs have also been reported to inhibit intimal hyperplasia in a rat model [195].

Collectively, since VSMCs are central to intimal hyperplasia, EVs are involved in phenotypic changes of VSMCs through transferring of bioactive cargoes, including HMGB1, IL-1β and miRNAs. While EVs can either promote or attenuate intimal hyperplasia. One of the major challenges, that the expanding field of EV research faces, is the highly heterogenous of vesicle subgroups and the complexity of the characteristics, compositions and biological functions of EVs.

4. Clinical involvement of extracellular vesicles in systemic vasculitis

As discussed above, EVs may play pivotal role in the pathogenesis of systemic vasculitis mainly through their effects in inflammation, pro-coagulation, autoimmune processes, endothelial injury, and intimal hyperplasia. In clinical aspect, EVs can also be important to serve as biomarkers for diagnosis, to predict treatment efficacy for disease activity monitoring, and to be a potential therapeutic target in systemic vasculitis. Here, we have summarized the published works regarding the involvement of EVs in vasculitis in Table 1.

Table 1.

Summary of the published works about EVs in systemic vasculitis

EV subtype	Cellular origin	Markers	Associated specific component	Samples	Techniques	Study findings	Ref.	Year
MVs	Platelets; ECs	PS, CD42a, P-selectin, E-selectin, CD105, ICAM-1, VCAM-1	NA	Platelet-poor plasma	Flow cytometry	Increased levels of circulating endothelial and platelet MVs were found in children with active vasculitis. The level of endothelial MVs correlated with disease activity of vasculitis.	[14]	2004
EVs	NA	CD63, CD81	NA	Platelet-poor plasma	Flow cytometry, nanoparticle tracking analysis	Leukotriene B4 and 5-oxo-eicosatetraenoic acid from EVs of GPA patients can stimulate primed neutrophils.	[46]	2020
MVs	Neutrophils	PS, MPO, PR3, CD18, CD11b, CD66b	NA	Supernatant of cultured neutrophils, platelet-poor plasma	Flow cytometry	ANCAs can stimulate primed neutrophils to release MVs that activate ECs and promote the generation of thrombin.	[89]	2012
EVs	Neutrophils	NA	NA	NA	Flow cytometry, nanoparticle tracking, miRNA screening	EVs released by activated neutrophils can promote the release of pro-inflammatory cytokines in ECs corresponding with the regulatory network of miRNAs/mRNAs comprising both EVs miRNAs and ECs transcripts.	[90]	2021
MVs	ECs	CD105, CD144	B1-receptors	Platelet-free plasma	Flow cytometry	B1 receptor-positive endothelial MVs in patients with vasculitis may contribute to chronic inflammation by inducing neutrophil chemotaxis.	[91]	2017
MVs	Neutrophils, monocytes	CD45, CD66	B1-receptors	Plasma, renal biopsies	Flow cytometry, TEM	Patients with vasculitis have higher level of plasma leukocyte derived MVs bearing B1-receptors that can dock on glomerular endothelial cells and induce inflammation.	[92]	2017
Exosomes	NA	CD63, HSP90a, Flotillin	tetranectin , Retinol-binding protein 4, leucine-rich alpha-2-glycoprotein 1, apolipoprotein A-IV	Serum	ExoQuick precipitation, TEM, Western blot	Proteomic profile of serum exosomes from children with CAA caused by KD was established.	[94]	2019
Exosomes	NA	NA	let-7i-3p	Serum	ExoQuick precipitation	Exosomal miRNA (let-7i-3p) is a potential biomarker for CAA patients caused by KD.	[95]	2019
MVs	NA	NA	MPO, PTX3, HMGB1	Platelet-poor plasma	Flow cytometry	Concentration of MPO-positive MVs is increased in plasma from AAV patients. PTX3 and HMGB1 expressed on MPO-positive MVs were associated with disease activity.	[104]	2020
MVs	NA	NA	MPO, C3a, C5a	Platelet-poor plasma	Flow cytometry	The levels C3a and C5a expressed on MPO-positive MVs was associated with renal involvement and disease activity in AAV patients.	[106]	2020
MVs	Platelets, ECs, neutrophils	PS, CD62E, CD11, CD41	TF	Platelet-poor plasma	Flow cytometry	Enhanced MV-mediated thrombin generation is associated with thrombotic disease in systemic vasculitis.	[144]	2011
MVs	Platelets	CD42a, CD62P	NA	Plasma	Flow cytometry	BD patients had significantly higher level of CD62P+ platelet MVs compared with healthy controls.	[145]	2011
MVs	NA	PS	NA	Platelet-free plasma	ELISA	The level of procoagulant MVs increased in BD patients.	[146]	2016
MVs	NA	PS	TF	Platelet-poor plasma	Flow cytometry	BD patients with a history of thrombosis had increased total and TF-positive MV numbers, but had a lower proportion of issue factor pathway inhibitor positive MVs, compared to those without a history of thrombosis.	[147]	2016
MVs	Neutrophils	PS	TF	Supernatant of cultured neutrophils	Flow cytometry	C5a primed neutrophils activated by ANCAs release TF expressing MVs and NETs, which subsequently activate the coagulation system and generate thrombin.	[148]	2015
MVs	Neutrophils	PS, CD66b	TF	Serum	Flow cytometry	Expression of TF in NETs and neutrophil derived MVs proposes a novel mechanism for the induction of thrombosis and inflammation in active AAV.	[149]	2014
MVs	NA	NA	TF	Platelet free plasma	Centrifugation	Elevated TF activity of MVs and increased anti-Plg in remission are strong indicators of VTE independent of renal function in AAV patients.	[150]	2019
MVs	ECs, leukocytes, platelets	PS, CD62E, CD105, CD42a, CD62P, CD45, CD11b	NA	Platelet-poor plasma	Flow cytometry	Endothelial MVs are elevated in active adult AAV. Endothelial MV level correlate with disease activity and might serve as a marker of endothelial activation and damage.	[153]	2008
MVs	ECs	CD144, CD62E, CD105	NA	Platelet-poor plasma	Flow cytometry	The increased levels of endothelial MVs have significant correlation with decreased values of flow-mediated dilation, both of which may reflect endothelial dysfunction in child KD.	[155]	2014
MVs	Platelets, leukocytes, ECs	PS, vWF, CD105, CD62e, CD45, CD11b, CD42a, CD62p	NA	Platelet-poor plasma	Flow cytometry	MVs in this CSS patient were markedly elevated at initial presentation and rapidly decreased during follow-up and closely paralleled the course of the Birmingham vasculitis activity score	[156]	2008
MVs	ECs	PS, CD144, CD62E	NA	Platelet-poor plasma	Flow cytometry	Elevated levels of circulating endothelial cells , endothelial MVs, endothelial progenitor cells , VEGF, and angiopoietin 2 occurs during active vasculitis, and both circulating endothelial cells and endothelial MVs can be used to monitor disease activity in children with vasculitis.	[157]	2010
MVs	ECs	CD144, CD105	C3, C9; CD46, CD55	Plasma before/after perfusion over primary glomerular endothelial cells	Flow cytometry	Plasma of vasculitis patients had significantly more C3- and C9-positive endothelial MVs and can induced the release of significantly more complement-positive MVs from glomerular endothelial cells. Blockade of the kallikrein-kinin system can reduces endothelial complement activation and thereby the inflammatory response on the endothelium.	[161]	2019
MVs	Neutrophils	PS, CD66	NA	Supernatant of cultured neutrophils;	Flow cytometry	Neutrophils from AAV patients have a greater ability to activate the alternative complement pathway in vitro compared with those from healthy controls. AND this increased ability could be caused by increased release of MVs.	[163]	2019
MVs	Platelets, ECs, leukocytes	CD41a, CD31, CD45, CD62P	Bioactive proteins	Platelet-free plasma	Flow cytometry	Increased levels of platelet derived MVs may be actively involved in enhancing an acute inflammatory state in AAV.	[198]	2019
MVs	Neutrophils, platelets	CD41a, CD66b	NA	Plasma, supernatant of cultured neutrophils	Centrifugation	Patients with vasculitis have increased level of peripheral MVs derived from neutrophils and platelets compared from healthy controls.	[199]	2006
MVs	ECs	CD144	miR-145–5p, miR-320a	Serum	Flow cytometry	MicroRNA-145–5p and microRNA-320a encapsulated in endothelial MVs might be involved in inflammatory cytokine regulation and the pathogenesis of vasculitis in acute KD.	[203]	2018
MVs	ECs	PS, CD105, E-selectin, ICAM-1, VCAM-1, CD144, CD31	NA	Platelet-poor plasma	Flow cytometry	The level of CD105 positive endothelial MVs in the KD group, particularly in the CAA present group, were higher versus healthy control.	[204]	2015
MVs	Platelets, Erythrocytes, neutrophils, monocytes, T cells, B cells, ECs	CD42, CD235, CD66b, CD14, CD3, CD19, CD144	NA	Platelet-poor plasma	Flow cytometry	The number of KD MVs, mainly derived from ECs and T cells, is significantly increased. MVs may develop from endothelial damage and cell activation.	[205]	2011
MVs	ECs	CD31, CD146	NA	Platelet-poor plasma	Flow cytometry	Elevated level of endothelial MVs, a biomarker of endothelial cells damage, concomitant with increased levels of TNF-a and IL-6, is seen in patients with KD.	[206]	2013
Exosomes	NA	CD9, CD81, flotillin	Differential proteins	Platelet-poor plasma	Electron microscopy, Western blot, 2Delectrophoresis/MALDI-TOF mass spectrometry	Differential proteome profile was found in patients with CAD caused by KD compared with healthy controls, and the majority of the proteins are involved in the inflammation and coagulation cascades.	[207]	2016
Exosomes	NA	TSG101, CD9, CD81	miR-1246, miR-4436b-5p, miR-197–3p, miR-671–5p	Serum	ExoQuick precipitation, Western blot, real-time quantitive PCR, microarray	Serum exosomal miRNAs can be candidate diagnostic biomarkers for Kawasaki disease.	[208]	2017
Exosomes	NA	NA	miR-328, miR-575, miR-134, miR-671–5p	Serum	Microarray	Serum exosomal miR-328, miR-575, miR-134 and miR-671–5p may act as potential biomarkers for the diagnosis of KD and the prediction of outcomes for the IVIG therapy by influencing the expression of inflammatory genes.	[209]	2018
Exosomes	NA	CD9, flotillin	Differential proteins	Serum	Electron microscopy, Western blot, 2Delectrophoresis/MALDI-TOF mass spectrometry	Differential proteomic profile of serum exosomes of patients with Kawasaki disease before and after intravenous immunoglobulin therapy was recognized.	[210]	2017
MVs	Platelets	NA	NA	Platelet-poor plasma	Centrifugation, ELISA	Platelet derived MVs can be used as an index to show the degree of platelet activation and the inflammation in children with KD.	[211]	2019
MVs	Platelets	glycoprotein Ib	NA	Platelet-poor plasma	ELISA	Platelet derived MV level before aspirin treatment was significantly higher in acute-phase KD patients than in control group.	[212]	2014
MVs	NA	PS	NA	Platelet-poor plasma	ELISA	Platelet-derived MVs might be used as biomarker of antiplatelet therapy in KD.	[213]	2017
MVs	ECs, T cells, B cells, monocytes	PS, CD144, CD3, CD19, CD14	NA	Platelet-free plasma	Flow cytometry	Concentrations of endothelial MVs are correlated with inflammation in Takayasu arteritis and may be useful markers to assess disease activity.	[214]	2019
MVs	Platelets	CD62	NA	Whole blood	Flow cytometry	BD patients with previous thrombosis had a higher percentage of circulating CD62-positive platelets and a higher number of circulating microaggregates than those without thrombosis. However, no significant differences in the number of platelet derived MVs per 5,000 platelets was detected between the two groups.	[222]	2007
MVs	ECs, platelets, leukocytes, neutrophils, monocytes	PS, CD105, CD144, CD41, CD18, CD16b, CD14	NA	Platelet-poor plasma	Flow cytometry	MV counts and platelet reactivity correlated well with disease activity in GPA. Furthermore, MVs were found to activate vascular endothelial cells and platelets in vitro.	[223]	2015
MVs	ECs	CD144, CD146	NA	Platelet-free plasma	Flow cytometry	Circulating endothelial MVs could be used as a surrogate marker for subclinical inflammation in Henoch-Schönlein purpura.	[224]	2011

4.1. Extracellular vesicles and renal vasculitis

Systemic vasculitis with renal involvement is common and may become kidney failure in severe cases. Many studies showed EVs play important roles in the pathogenesis of vasculitis-associated renal failure. We have recently reviewed the roles of EVs in pathophysiology and clinical implications in lupus nephritis [102]. AAV mainly affects systemic small vessels and is characterized by the presence of ANCAs in the circulation [196]. Renal involvement is present in the majority of AAV patients (almost 100% in MPA, 70–80% in GPA, 25% in EGPA), and their renal function is closely correlated with survival and the risk of end-stage renal disease in patients [197]. Since there are very few studies about EVs in renal vasculitis that affects large and medium vessels, here, we mainly focused on the studies in AAV (with illustration in Fig. 2).

Fig. 2. Pathological involvements of EVs in renal AAV.

Neutrophil -derived EVs and NETotic EVs could express the ANCA autoantigens (MPO, PR3), TF, kinin B1-receptors or extracellular histones. These EVs are involved in the pathogenesis of renal AAV through promoting autoimmunity, inflammation, thrombosis, endothelial injury and tissue damage. Kinin-receptors antagonists might be used to alleviate vasculitis.

Abbreviations: ANCA, Antineutrophil cytoplasmic autoantibody; AAV, ANCA-associated vasculitis; C, Complement; ECs, Endothelial Cells; EVs, Extracellular Vesicles; MPO, Myeloperoxidase; PAD4, Peptidylarginine deiminase 4; PR3, Proteinase 3; TF, Tissue Factor.

EVs can be detected in the circulation and may be associated with disease severity of renal injury in AAV patients [198]. Leukocyte- and endothelial-derived EVs are important, although many EVs are of platelet origin in patients with ANCA disease [14, 89, 199]. Pauci-immune necrotizing and crescentic glomerulonephritis (GN) is the typical glomerular pathological feature in AAV patient with renal involvement [200]. The most prominent infiltrating cell types in pauci-immune necrotizing GN are neutrophilic granulocytes [201]. ANCA can induce generation of EVs from neutrophils [89, 148]. These EVs expressed a variety of bioactive molecules, including PR3, MPO, and TF, thus promoting autoimmune inflammation of the vessel wall and hypercoagulability in these patients [89, 148]. Additionally, NETs are formed during neutrophil NETosis and are closely associated with EVs [44, 46], as we discussed above. NETs are involved in ANCA-mediated vascular injury and the production of ANCAs, therefore, involved in the pathogenesis of AAV [196]. In renal biopsy of early focal phase ANCA-associated polyangiitis patients, NET-associated components, citrullinated histone, MPO and PAD4 concurrently deposited around fibrinoid necrosis in necrotizing GN, and along an interlobular arterial wall [202]. However, it is not known if EVs from NETotic neutrophils also contain histones, MPO, and PAD4 which can mediate renal AAV. Besides, neutrophil-derived EVs expressing kinin B1-receptors can dock on and transfer kinin B1-receptors to glomerular ECs in kidney biopsies from patients with vasculitis [92]. This may result in a sustained inflammatory response. Furthermore, kinin-receptor antagonists could inhibit the release of complement-positive endothelial EVs and reduce glomerular C3 deposition [161], indicating the role of interrupting transfer of kinin-receptors from neutrophil-EVs to endothelial-EVs in amelioration of vasculitis.

Therefore, by expressing ANCA autoantigens, TF, or kinin B1-receptors, neutrophil-derived EVs are involved in pathogenesis of renal AAV through promoting autoimmune inflammation and hypercoagulation. Moreover, kinin-receptors antagonists might be used for amelioration of vasculitis. In addition, EVs from NETotic neutrophil may also be involved in renal AAV, but further investigation is need.

4.2. Extracellular vesicles in vasculitis diagnosis and disease severity evaluation

Over the past decades, a number of studies have demonstrated the roles of EVs in diagnosis and disease evaluation of vasculitis. In KD, the levels of EC-derived EVs were increased [203–206], particularly in patients with CAA [204]. Differential proteome profile has been found in KD patients with coronary artery dilatation (CAD) as compared with healthy controls, and majority of the EV-associated proteins are involved in inflammation and coagulation cascades [207]. Recently, Wang and colleagues reported that serum exosomal microRNA let-7i-3p could distinguish KD complicated CAA patients from healthy children, as well as virus-infected patients [95]. Furthermore, exosomal microRNAs (miR-1246, miR-4436b-5p, miR-197–3p and miR-671–5p) are differentially expressed between KD patients, other febrile patients and healthy controls, indicating a role as candidate diagnostic biomarkers for KD [208]. Zhang et al also found the differential expression of exosomal miRNA between the healthy children and KD patients, or between KD patients and KD patients following IVIG therapy [209], indicating the role of serum exosomal miRNA as potential biomarkers for diagnosis of KD, and the predictive value for outcomes of the IVIG therapy. Similarly, differential exosomal proteomic profiles have been found in patients with KD before and after intravenous immunoglobulin therapy [210]. Besides, significant higher levels of platelet-derived EVs have been reported in KD children [211], while intravenous immunoglobulin (IVIG) significantly decreased the levels of platelet-derived EVs [205, 212, 213]. Moreover, levels of platelet-derived EVs were significantly higher in IVIG-resistant patients than those in IVIG-effective patients before the IVIG treatment [211].

As for TA patients, Cheng and colleagues showed that the concentrations of EC-derived EVs, but not EVs from T cells, B cells or monocytes, increased significantly in TA patients and are associated positively with disease activity [214]. Moreover, the concentrations of EC-derived EVs increased during disease relapse in four TA patients [214]. However, the authors pointed that since the concentrations of EC-derived EVs also increased in patients with other vasculitis, the use of EC-derived EVs as a diagnostic marker in TA might be limited [214].

Similarly, the levels of EC-derived EVs are also increased in adult patients with active AAV and correlated with disease activity [153]. MPO-positive EVs expressing PTX3 and HMGB1 are significantly higher in AAV patients, and are correlated with disease activity in these patients [104], indicating that PTX3 and HMGB1 expressing MPO-positive EVs might be used as promising biomarkers to monitor disease activity in AAV patients [104]. In the study from Miao et al, the levels of platelet-derived EVs are significantly correlated with disease activity, inflammation, and renal damage in AAV patients [198]. Additionally, compared to patients in remission, differentially expressed proteins in platelet-derived EVs from patients with active disease are associated with chemotaxis, cell adhesion, growth and apoptosis [198]. Overall, EVs from different cellular origins as well as their cargoes could be used as candidate biomarkers in diagnosis and disease activity evaluation for vasculitis including KD, TA and AAV.

4.3. Extracellular vesicles and clinical treatment of vasculitis

Aside from being promising biomarkers, EVs can also be used as novel vehicles containing endogenously and exogenously packaged molecules that exert therapeutic role. EVs harbor many features and molecules from their parental cells. Therefore, they generally have low immunogenicity and toxicity with increased circulating time, and can protect RNA or DNA from hydrolysis by nucleases in blood [8, 215]. Therefore, using drug-loaded EVs as therapeutic vehicles has been used as promising strategy for drug delivery.

One approach is to load exogenous cargoes inside EVs to target endothelium. During vascular inflammation, ICAM-1, which binds to integrin β2 on neutrophil membrane, is upregulated in endothelium through NF-κB signaling pathway [216]. Inspired by this interaction, nanovesicles expressing integrin β2 have been generated [217]. Administration of neutrophil nanovesicles loaded with TPCA-1 (a NF-κB inhibitor) markedly mitigated mouse inflamed vasculature [217]. Moreover, rabies virus glycoprotein-decorated EVs with HMGB1-siRNA have also been reported [218]. Therefore, one may expect to administrate neutrophil-derived EVs loaded with HMGB1-siRNA or specific drugs for inhibition of HMGB1 activation and its associated innate immunity as potential therapeutics.

Another approach is to equip endogenous EVs with specific cargoes. MicroRNA-100 transferred by MSC-derived EVs inhibits in vitro angiogenesis through down-regulation of VEGF [219]. The miRNA-126–3p from human umbilical cord MSC enhances endothelial function in an EV-associated manner in vitro and attenuates intimal hyperplasia in vivo [220]. Interestingly, engineered decoy EVs are generated from genetically modified donor cells which express cytokine receptors without the signaling domain [221]. Expression of cytokine receptors on surface of EVs can decoy the pro-inflammatory cytokines (like TNF-α and IL-6) specifically and significantly attenuate inflammation in vitro and in vivo [221]. Therefore, engineered EVs have been shown great potential to be the next generation biotherapeutics.

5. Conclusions

In summary, we have summarized the latest progress in EV generation and EV-associated isolation and detection techniques in this review. In addition to apoptosis, EVs could also be generated from necroptotic, pyroptotic and NETotic cells. We have also summarized the association between EVs and redox biology. The article also summarized the current literature and provide our interpretation based on our experiences in EV research for over a decade. In the article, we summarized EVs secreted by various cell types possess important biological properties in the pathogenesis of systemic vasculitis, including inflammation, autoimmune responses, thrombosis, endothelium injury, angiogenesis and intimal hyperplasia. Thereinto, redox metabolism involves in many aspects. As for clinical implications, EVs may be used as biomarkers for diagnosis and disease activity monitoring. Importantly, EVs play an important role in AAV associated renal involvement. EVs may also reflect the therapeutic responses of immune therapy in patients with vasculitis, thus might predict the prognosis. Moreover, EVs could also be used as novel vehicles for therapeutic purpose. Further studies including animal experiments will allow us to investigate the specific involvement of endogenous EVs in vasculitis and are necessary to determine when and how EVs can be protective or pathogenic.

Abbreviations:

AAV

ANCA-associated vasculitis

ANCA

antineutrophil cytoplasmic autoantibody

BD

Behçet’s disease

bFGF

basic fibroblast growth factor

CAA

coronary artery aneurysm

CSS

Churg-Strauss Syndrome

DAMPs

damage-associated molecular patterns

DCs

dendritic cells

ECs

endothelial cells

EGPA

eosinophilic granulomatosis with polyangiitis

ELISA

enzyme-linked immunosorbent assay

EVs

extracellular vesicles

GCA

giant cell arteritis

GN

glomerulonephritis

GPA

granulomatosis with polyangiitis

GSDMD

Gasdermin D

HMGB1

high mobility group box-1

ICs

immune complexes

ICAM-1

intercellular adhesion molecule 1

IL

interleukin

IVIG

intravenous immunoglobulin

KD

Kawasaki disease

MHC

major histocompatibility complex

MLKL

mixed lineage kinase domain-like

MMPs

matrix metalloproteinases

MPs

microparticles

MPO

myeloperoxidase

MPA

microscopic polyangiitis

MRI

magnetic resonance imaging

MSCs

mesenchymal stromal cells

MVs

microvesicles

NA

not available

NETs

neutrophil extracellular traps

NF-κB

nuclear factor-kappaB

NLRP3

NACHT, LRR, and PYD domains-containing protein 3

NO

nitric oxide

Nrf2

Nuclear factor erythroid 2-related factor 2

ox-LDL

oxidized low-density lipoprotein

PAD4

peptidylarginine deiminase 4

PAN

polyarteritis nodosa

PDGF

platelet derived growth factor

PE

phosphatidylethanolamine

PR3

proteinase-3

PS

phosphatidylserine

RIPK

receptor-interacting serine-threonine kinase

ROS

reactive oxygen species

SIRPα

signal regulatory protein α

SLE

systemic lupus erythematosus

TA

Takayasu’s arteritis

TEM

transmission electron microscopy

TF

tissue factor

TGF-β1

transforming growth factor-β1

TNF-α

tumor necrosis factor-α

VCAM-1

vascular cell adhesion molecule 1

VEGF

vascular endothelial growth factor

VSMCs

vascular smooth muscle cells

VTE

venous thromboembolism

vWF

von Willebrand factor

WG

Wegener’s granulomatosis