Extracellular Vesicles and Inflammatory Diseases: Comparison
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Inflammation is the defense mechanism of the immune system against harmful stimuli such as pathogens, toxic compounds, damaged cells, radiation etc. and characterized by tissue redness, swelling, heat generation, pain, and loss of tissue functions. Inflammation is essential in the recruitment of immune cells at the site of infection, which not only aids in the elimination of the cause, but also initiates the healing process. However, prolonged inflammation often brings about several chronic inflammatory disorders, hence, a balance between the pro- and anti-inflammatory responses is essential in order to eliminate the cause while producing least damage to the host. Growing body of evidence indicates that extracellular vesicles (EVs) play a major role in cell-cell communication via the transfer of bioactive molecules in the form of proteins, lipids, DNA, RNAs, miRNAs etc. between the cells.

  • extracellular vesicles
  • classification
  • inflammation
  • inflammatory diseases

1. Introduction

The ability of cells to communicate with each other holds an important step in the differentiation and development of multicellular organisms. Numerous mechanisms govern how cells interact with each other, such as cellular secreted molecules, direct interaction between the adjacent cells through the cell-adhesion molecules, and the formation of cytoplasmic bridges or nanotubules [1]. However, a growing body of evidence identifies a unique mechanism by which cells convey signals between one another, the release of extracellular vesicles (EVs) [2][3][4]. EVs are membrane-enclosed nano-sized bodies, shown to be released from almost every cell type [5][6]. As EVs are derived from cells, they often carry cellular components such as proteins, lipids, and genetic materials in the form of DNA, RNA, microRNA (miRNA), etc. [7], and upon transferring these bioactive molecules, EVs generally modulate the function of the target recipient cells [8][9][10]. A wide variety of non-coding RNAs (ncRNAs) including miRNAs regulate the fundamental cellular processes which can be therapeutically targeted in the context of cancer [11][12]. The uptake mechanisms of EVs by the recipient cells include the direct fusion of EVs with the plasma membrane or endocytosis [4][8][13]. EVs are readily detected in every biological fluids including blood, urine, saliva, synovial fluid, sputum, breast milk, bronchoalveolar lavage fluid (BALF), and cerebrospinal fluid (CSF) and even in interstitial spaces between the cells [6][14][15][16][17][18]. Based on the biogenesis, content, size, and function, EVs are extensively categorized into three major groups, microvesicles, exosomes, and apoptotic bodies (Figure 1) [5][6].
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Figure 1. Biogenesis and uptake of EVs. EVs are composed of microvesicles (MVs), exosomes, and apoptotic bodies. MVs are produced by outward budding of the plasma membrane, whereas exosomes are generated by endocytic mechanism. Invagination of the early endosomal membrane produces the exosomes inside the endocytic vesicles which mature into multivesicular bodies (MVBs). MVBs are eventually fused directly with the plasma membrane to release the exosomes outside the cells. Sometimes, MVBs also fuse with the autophagosomes to form amphisomes. Amphisomes, in turn, fuse with the plasma membrane to release their content including the exosomes outside the cells. Apoptotic bodies, on the other hand, are generated during the contraction of the cells, leading to the dissociation of plasma membrane from the cytoskeleton. The induction of apoptosis often results in the fragmentation of DNA which is incorporated into the apoptotic bodies. Both MVs and exosomes, which carry cargoes in the form of RNA, miRNA, proteins, etc., are readily taken up by the recipient cells via either direct fusion with the plasma membrane or endocytosis. In the case of endocytosis, inside the recipient cells, the EVs are further fused with the membrane of endocytic vesicles, thereby releasing the cargoes into the recipient cells’ cytosol. In contrast, direct fusion of EVs with the target cells’ plasma membrane results in the release of the EVs’ cargoes in the cytosol of the recipient cells.
Microvesicles. Microvesicles (MVs) or microparticles (MPs) or ectosomes are recognized as plasma membrane ‘buds’ of the cells [7][19]. The crosstalk among cytoskeletal components such as actin and microtubules, molecular motor proteins such as kinesin and myosin, fusion machineries such as soluble N-ethylmaleimide-sensitive factor activating protein receptor (SNARE), and tethering factors essentially regulates the formation and release of MVs from the cells [2][3][20][21][22][23]. The size of MVs is believed to range from 100 nm to 1 µm in diameter [5][6][14]. MVs, because of being generated by plasma membrane outward budding, are shown to carry cytosolic and plasma membrane-associated proteins such as tetraspanins, which often serve as a universal marker for the MVs, regardless of the cells’ origin [24]. Moreover, cytoskeletal proteins such as heat shock proteins, integrins, and proteins associated with posttranslational modifications including glycosylation, phosphorylation, etc., are, sometimes, found to be enriched in MVs [25].
Exosomes. Exosomes, the smaller EV class having a diameter of 30–150 nm [26], are generated by the endocytic mechanism [25]. Typically, invagination of the early endosomal membrane produces these exosomes which are matured into multivesicular bodies (MVBs) [25]. MVBs are eventually fused with the plasma membrane, thereby releasing the exosomes outside the cells [25]. Exosomes’ biogenesis often requires the active involvement of endosomal sorting complexes required for the transport (ESCRT) pathway [25]; therefore, ESCRT pathway-associated molecules including TSG101, Alix, HSP90β, and HSC70 are shown to be present in the exosomes [27][28], which are also used as exosomal markers. However, ESCRT-independent exosomal biogenesis also occurs, which is reported to be associated with sphingolipid ceramide [29].
Recently, a unique exosomal release mechanism has been identified which involves the autophagic pathway. Autophagy is the process of eliminating non-functional and futile components of the cells depending on lysosomal mechanisms [30]. The sequestration of a cytoplasmic portion by a membranous organelle, called a phagophore, generates autophagosomes, which in turn fuse with the MVBs to produce the amphisomes [31][32]. Amphisomes are often found to be enriched with endosomes as well as autophagosome markers, LC3 and CD63, respectively. Moreover, cytosolic DNA and nucleosomes are also present in the amphisomes. Amphisomes are either fused with the plasma membrane, resulting in the release of amphisomal content including the exosomes outside the cell, a phenomenon called ‘exophagy’, or their fusion with the lysosomes leads to the degradation of the amphisomal components by lysosomal enzymes.
Apoptotic bodies. In contrast to MVs and exosomes, apoptotic bodies are larger in size, ~50 nm to 5 µm in diameter [33]. These are released from the apoptotic cells via the separation of the plasma membrane from the cytoskeleton due to immense hydrostatic pressure, generated during the cell contraction [34]. Apoptotic bodies are often found to contain cell organelles, nuclear chromatin, and a few glycosylated proteins; therefore, mitochondrial proteins, such as HSP60, Golgi, and endoplasmic reticular proteins, such as GRP78, and nuclear histones appear to be markers for apoptotic bodies [33][35][36][37].
EV isolation procedures: A comparative analysis. The present section briefly discusses different techniques of EVs isolation in a comparative approach. Currently, the widely accepted procedures for the isolation of EVs include centrifugation, precipitation, size exclusion, affinity purification, and micro-/nano-fluidics or chips [38].
Centrifugation. This is the most commonly used method for isolating EVs by several research groups, principally based on the particle size, density, shape, and viscosity of the medium. This is further classified into differential ultracentrifugation, density-gradient centrifugation, and rate-zonal centrifugation [39][40]. (1) Differential centrifugation separates the EVs based on the size, shape, and density [39][41]. The influencing factors in this method include temperature, sample dilution, and duration of centrifugation [42][43]. Although the procedure is easy, has average yield, and needs no additional steps for the preparation of samples, it is time-consuming, laborious [39][44][45][46], and incapable of differentiating between different EVs types [47]. In addition, protein contaminants are the major issue in this EV isolation procedure [46]. (2) In contrast to differential centrifugation, density-gradient centrifugation employs a preconstructed density-gradient medium such as sucrose and iodixanol for the isolation of EVs [39][48]. This method has the advantage of separating EVs from the contaminating proteins [39], and different types of EVs can be separated according to their density [49]. However, average yield and the need for longer isolation time are the two major pitfalls of this approach [50][51]. (3) Rate-zonal centrifugation, on the other hand, utilizes the combined principle of density-gradient and sedimentation in which the sample is loaded on top of the tube, and following centrifugation, EVs with higher density are shown to pass through the dense layer as compared to lighter EVs [38]. The additional advantages of this technique over the other centrifugation procedures are that EVs with same density but different size can be separated [52] and the high yield recovery of the EVs [39].
Precipitation. This method employs the use of a water-excluding compound, such as Polyethylene glycol (PEG), which is mixed with the EV sample, followed by centrifugation or filtration. PEG dries up the sample, leading to the precipitation of the other molecules [53][54][55]. Although this method is easy and applicable for both small and larger volume of samples, more often it results in the co-precipitation of the non-EV components. Therefore, precipitation is always combined with other techniques to improve the quality and selectivity [39][54][56][57].
Size exclusion. This procedure explores the different size distributions of EVs for their isolation. Size exclusion techniques include ultrafiltration, sequential filtration, isolation kits, field-flow fractionation, size-exclusion chromatography, and hydrostatic filtration dialysis. (1) In ultrafiltration, the EVs samples pass through different pore-sized membrane filters, leading to the separation of the EVs based on their size and molecular weight [39][54]. Despite the fast and inexpensive separation of the EVs [39][56], this method has several disadvantages. Often, the EVs become entrapped in the membrane [39][56]. Moreover, poor efficiency and EVs’ deformation due to membrane pressure further lead to the lower efficiency of the process [39][56][58][59]. (2) Sequential filtration, a semi-automated technique, is basically a system composed of multiple filters of different sizes. When an EV sample is loaded, the larger particles are trapped in the filters, and the smaller ones pass through. Although this technique is fast [58], it often results in membrane plugging and hence low yield [58][60][61]. (3) Recently, isolation kits have been developed which also separate EVs based on their size. For example, Exomir Kits are composed of two membranes: the upper one is of a higher pore size (200 nm), whereas the bottom one has a lower pore size (20 nm) [39]. Another isolation kit, ExoTIC, contains multiple filters, and the EV samples, when applied to it, are separated according to their size. These kits often produce high yield EVs [62]. (4) In field-flow fractionation, the EV samples are loaded into a chamber in which a crossflow is generated. The larger particles, due to the cross-flow, are positioned on the chamber wall, whereas the smaller particles are eluted first [39][63]. This technique is fast, is efficient, provides higher recovery, and facilitates the isolation of EVs from a very small sample volume [64]. (5) Size-exclusion chromatography allows the elution of larger particles from the column followed by the release of smaller particles through the pores [39][58][65][66]. This not only obtains the biological integrity of the EVs but also offers no damage of sample pre-treatment [58][65]. (6) Hydrostatic filtration dialysis employs hydrostatic pressure for the isolation of EVs. It is a tube-based technique in which the small particles are diffused through the membrane, whereas the larger ones are retained in the tube [39][67].
Affinity purification. Affinity purification of EVs involves antibody-mediated purification of the EVs against surface antigens [39]. In this technique, the purity of the EVs is shown to be the highest [39]; however, at the same time, poor yield limits the efficiency of the method [39][57]. Also, the availability of antibodies against unique antigens on the EVs further adds to the difficulties of affinity purification [38]. However, combinational techniques, in association with affinity purification, are found to be quite effective [68].
Micro/nano fluidics or chips. Biochemical features such as electrophoretic, acoustic, and electromagnetic properties of the EVs are often explored to develop micro-/nano-chips for the isolation of EVs [39][54]. For example, the development of micro-chips is based on the size, immunoaffinity, and density of the EVs [38]. Nanowires, viscoelastic flow, and nano-sized deterministic lateral displacement (nano-DLD) are the other techniques that fall into this category. The nanowires’ principle is very similar to size-exclusion chromatography, which contain silicon micropores [38]. The elastic lift forces of different sized EVs vary in a viscoelastic medium, which is utilized in EV isolation by the viscoelastic flow [69][70]. On the other hand, nano-DLD utilizes the pillar-array-based microfluidic mechanism for the isolation and analysis of the EVs [69]. The acoustic separation method employs the ultrasonic radiation, in which the EVs are exposed, for the separation of the EVs. Based on their size, the frequency of the waves is controlled to separate the EVs. The larger particles, influenced by the heavier waves, move to the pressure node at a faster rate [39][71]. This often leads to the yield of highly purified EVs [72].
Heterogeneity in EV preparations: Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV2018). In the past three decades, the advancement of EV research has also increased the complexity of EV characterization. Depending on the cells of origin, biogenetic mechanisms, and various physiological and pathological functions, different research groups apply different terminology for the EVs, such as exosomes, microparticles, microvesicles, ectosomes, apoptotic bodies, oncosomes, and many others. However, the disparity in size within different methods of EV preparation often turns out to be the primary limitation for EV characterization. In this regard, the International Society for Extracellular Vesicles (ISEV) proposed a guideline for the isolation and characterization of EVs, termed as ‘Minimal Information for Studies of Extracellular Vesicles’ (MISEV), in 2014 which was further updated in 2018 [73]. A worldwide ISEV survey from 2015 [74] indicates that the differential ultracentrifugation was the most frequently used technique for separating and concentrating EVs over the other conventional methods, such as density-gradient centrifugation, precipitation, filtration, size-exclusion chromatography, affinity purification, etc., with moderate purity and recovery. However, for better specificity and recovery, several other techniques were further used which are mentioned in MISEV2018 [73]. These include tangential flow filtration and variations thereon, asymmetric flow field-flow fractionation, field-flow fractionation, field-free viscoelastic flow, variations on size exclusion chromatography (SEC), acoustics, alternating current electrophoretics, ion exchange chromatography, fluorescence-activated sorting, microfiltration, DLD arrays, novel precipitation/combination techniques, novel immunoisolation or other affinity isolation technologies, hydrostatic filtration dialysis, a wide variety of microfluidics devices which combine one or more principles, as mentioned above, and high-throughput/high-pressure methods including fast protein liquid chromatography/high performance liquid chromatography (FPLC/HPLC) involving some chromatography techniques [73].
Selectivity of EVs in the uptake by target cells. There is mixed evidence which indicates the movement of EVs towards specific target cells. Although EVs are shown to be non-selectively taken up by a wide variety of recipient cells [75], at times, the release of specific morphogens by the target recipient cells may guide the EVs towards them [76]. However, an interesting study by Sharif et al. demonstrates that Wharton’s jelly-mesenchymal stem cell (WJ-MSC)-derived EVs specifically deliver miR-124 to glioblastoma multiforme (GBM), resulting in the down-regulation of GBM migration while increasing its chemosensitivity [77]. This indicates the possibility of a ligand–receptor interaction in the specific uptake of EVs by the target recipient cells. In this context, the role of EVs’ membrane proteins, lipids, and glycans becomes indispensable.
EVs’ membrane proteins. EVs’ membrane proteins play a major role in their uptake by specific target cells. Tetraspanins (CD63, CD9, CD82, and CD81), the abundantly expressed molecules on the surface of EVs [78], in association with other adhesion molecules such as intercellular adhesion molecule (ICAM) [79] essentially mediate the docking and uptake of EVs by the recipient cells upon interacting with cellular integrins and other adhesion molecules [78]. Hoshino et al. further demonstrate that α6β4- and α6β1-integrin + EVs are associated with lung metastasis, whereas αvβ5-integrin + EVs are involved in liver metastasis, and targeting the EVs’ integrins not only interferes with the EVs’ uptake but also decreases the EV-associated metastasis [80].
Lipids of EVs’ membrane. EVs are enriched with a negatively charged phospholipid, phosphatidylserine (PS), which is indirectly identified by the growth arrest-specific protein 6, Gas6, leading to the activation of Mer receptor tyrosine kinase (MERTK) on the surface of macrophages, thereby facilitating the EVs’ uptake and associated anti-inflammatory response [81].
EVs’ membrane glycans. In most cases, glycans are abundantly found on the surface of the EVs, and targeting glycans, more specifically proteoglycans, is believed to reduce EVs’ uptake by interfering with the glycans–lectin interaction [82]. Moreover, mannose-containing glycoproteins are glycan structures that are often found on the EVs’ membrane whose inhibition significantly down-regulates the uptake of the EVs by ovarian cancer cells [36].

2. EVs in Various Diseases

The abundance and heterogeneity of different cargoes entrapped within EVs often turn out to be important biomarkers in various pathophysiological conditions. For example, the level of pro-coagulant tissue factor (TF) expression is shown to be well-elevated on the plasma EVs of Gram-negative sepsis-induced urinary tract infection (UTI) patients, which often contributes significantly to the hyper-coagulative responses [83]. In contrast, EVs derived from activated platelets are believed to confer anti-coagulative effects [84]. In the case of atherosclerosis, the plaque-derived EVs transport ICAM-1 to the endothelial cells depending on the PS, thereby leading to the recruitment of inflammatory cells to promote atherosclerotic plaque progression [85]. Moreover, in acute kidney injury (AKI), fetuin-A and AQP1 + EVs may be used as diagnostic biomarkers. The level of fetuin-A is significantly up-regulated in the urinary EVs, whereas EVs’ AQP1 expression is shown to be down-regulated in AKI [86]. Furthermore, ten signature miRNA molecules (miR-199a-5p, miR-143-3p, miR-4532, miR-193b-3p, miR-199b-3p, miR-199a-3p, miR-629-5p, miR-25-3p, miR-4745-3p, and miR-6087) are found to be up-regulated, whereas another ten miRNAs (miR-23b-3p, miR-10a-5p, miR-141-3p, miR-98-5p, miR-382-5p, miR-200a-3p, miR-200c-3p, miR-483-5p, miR-483-3p, and miR-3911) are significantly down-regulated in the human follicular fluid (HFF)-derived EVs of polycystic ovary syndrome (PCOS) patients, which can serve as PCOS biomarkers [87]. The cerebrospinal fluid (CSF) of patients with Parkinson’s disease (PD) is shown to be enriched with α-synuclein + EVs which facilitate the aggregation of α-synuclein in healthy cells, leading to the progression of PD [88]. Circulating EVs from the differentiating myoblasts actively participate in the enhancement of muscle regeneration during congenital myopathies, and thus the elevated level of circulating EVs could be considered as the biomarker for congenital myopathy progression [89]. The composition of microbial EVs in the feces, blood, and urine of patients with gastrointestinal tract disease is significantly altered as compared to healthy individuals, and hence these EVs have the potential of being recognized as a diagnostic biomarker for microbial infection [90]. Numerous studies have mentioned the important contributions of EVs in the progression of cancer. For example, breast cancer cell-derived EVs transfer miR-125b to the normal fibroblasts in the tumor microenvironment (TME) rendering their transformation into cancer-associated fibroblasts (CAFs) [91]. Moreover, the population of triple-negative breast cancer (TNBC) cell-secreted EVs is shown to be significantly increased in the presence of FVIIa, which imparts epithelial to mesenchymal transition (EMT) to the EMT-negative cells via miR-221 transfer, leading to the progression of TNBC [9]. Again, the level of miR-144 is shown to be well-elevated in the EVs derived from nasopharyngeal carcinoma (NPC) which is readily transferred to the endothelial cells following EVs uptake, thereby resulting in the enhanced migration, invasion, and angiogenesis of the endothelial cells [92]. In the majority of instances, EVs have been associated with the modulation of inflammatory responses in different ways and are often considered an important regulator in various inflammation-associated diseases.
The role of EVs in inflammatory diseases. Inflammation, the defense mechanism of the immune system against harmful stimuli [93] such as radiation [94], toxic compounds [95], damaged cells [96][97][98][99], and most importantly pathogens [100], is characterized by tissue redness, swelling, heat, pain, loss of tissue functions, and recruitment of the immune cells at the site of infection [101][102][103], the results of which help eliminating the harmful cause and initiate the healing process [104][105][106]. However, just as ‘too much of anything is bad’, prolonged inflammation often gives rise to several chronic disorders [107][108][109][110]. Therefore, a balance between pro- and anti-inflammatory responses is a prerequisite in the removal of injurious stimuli with minimal damage to the host. Inflammation is often shown to play a pivotal role in various pathophysiological anomalies such as neurological disorders, cardiovascular diseases, respiratory syndrome, defects in the digestive and integumentary systems, disease associated with musculoskeletal, urinary, and reproductive systems, and endocrine as well as lymphatic disorders. Moreover, it has been established that inflammation and blood coagulation are intrinsically related: the activation of one process often leads to the activation of the other [111][112][113]. The latter part of the review focuses on how EVs influence the inflammatory responses in various coagulation-associated disorders.
Evs in neuroinflammatory diseases. Emerging evidence implicates the active involvement of Evs in various neuroinflammatory diseases. For example, the concentration of plasma Evs is shown to be significantly up regulated in the central nervous system (CNS). Autoimmune disease, multiple sclerosis (MS) [114], and EVs of endothelial as well as platelet origin from the plasma of MS patients have been revealed to induce blood–brain barrier (BBB) permeability, leading to the transmigration of myeloid- and T-cells into the CNS, thereby contributing to the neuropathology in MS [115][116][117]. Moreover, EVs in the plasma and CSF of patients suffering from neurodegenerative disorders such as Alzheimer’s disease (AD), PD, etc., are enriched with neurotoxic molecules including β-amyloid (Aβ), α-synuclein, and tau, whose origin are believed to be microglia and neuronal cells, and the uptake of toxic molecule-laden EVs to the local and distant neurons contributes to the neuronal loss, the characteristic feature of neurodegenerative disorders [118][119][120][121]. Another neurodegenerative disease, Creutzfeldt–Jakob disease (CJD), is caused by the misfolded and transmissible form of the prion protein (PrP) PrPSc. PrPSc is readily detected in the plasma EVs of CJD patients [122], and the selective packaging of PrPSc into the neuronal EVs often contributes to the EV-associated pathogenetic spread of CJD [123]. EVs often contribute to CNS infection. For example, JC polyomavirus (JCPyV), the causative agent of progressive multifocal leukoencephalopathy (PML), is shown to be transferred via serum EVs between glial cells and is highly infectious and leads to the pathogenesis of PML [124]. Furthermore, Plasmodium-infected red blood cells and other host cells have been demonstrated to release a significant amount of EVs in circulation [125] which contribute to the pathogenesis of cerebral malaria (CM), the most severe form of malaria, and targeting EV biogenesis has proven to be highly effective against CM in an animal model system [126]. In contrast to the above, EVs have also proved to be beneficial in a few instances.
In stroke, MSC-derived EVs have been reported to perturb the microglial differentiation of pro-inflammatory M1 phenotypes, thereby prohibiting neuroinflammation and brain injury following middle cerebral artery occlusion (MCAO) in rats [127]. Again, during spinal cord injury (SCI), infiltrating macrophages release NADPH oxidase 2 (NOX2)-loaded EVs which are readily taken up by the injured neuronal axons, and inside the neurons, NOX2 inactivates PTEN, thereby stimulating the PI3K-AKT pathway to regenerate neuronal outgrowth [128]. In addition, microglial EVs are shown to be enriched with miR-124-3p in conditions such as traumatic brain injury (TBI), which not only inhibits neuronal inflammation but also induces neurite outgrowth via PDE4B-targeted down-regulation of the mTOR signaling pathway [129]. In the majority of the above-mentioned studies, differential centrifugation techniques have been employed to isolate the EVs, which often reduces the purity, always leaving behind the possibilities of protein contaminants’ presence in the EV preparation which could affect the inflammatory responses of the EVs. However, Asai et al. [118] and Robertson et al. [122] used ultracentrifugation followed by density-gradient centrifugation for isolating the EVs, which improves the purity of the EVs markedly. In addition to this, Guo et al. utilized ExoQuick-TC PLUS followed by ultracentrifugation for EVs isolation which also yields highly purified EVs [121]. Figure 2 illustrates how EVs contribute to the progression of different neuroinflammatory diseases via different mechanisms.
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Figure 2. The role of EVs in different forms of neuroinflammatory diseases. EVs from multiple sclerosis (MS) patients induce BBB permeability, leading to the transmigration of T-cells and myeloid cells into the CNS, contributing to MS neuropathology. In the case of Alzheimer’s disease (AD) and Parkinson’s disease (PD), microglial EVs, enriched with neurotoxic molecules, are incorporated into the neurons, leading to neuronal loss. EVs, loaded with infectious PrPSc, are released from infected neurons and are readily incorporated into healthy neurons, leading to pathogenic spread of Creutzfeldt–Jakob disease (CJD). In progressive multifocal leukoencephalopathy (PML), JCPyV-laden EVs are transferred between the glial cells which contribute to the pathogenesis of PML. Plasmodium-infected RBC-derived EVs are also known for increasing the pathogenesis of cerebral malaria (CM). In stroke, MSCs-EVs perturb microglial neuroinflammatory responses and the subsequent brain injury. Again, in spinal cord injury (SCI), EVs from infiltrating macrophages transport NOX2 to the neuronal cells which leads to the regeneration of neuronal outgrowth. Similarly, microglial EVs carried miR-124-3p, which not only prohibits neuronal inflammation but also induces neurite growth in the context of traumatic brain injury (TBI).
EVs and cardiovascular inflammatory responses. Inflammation plays a key role in the pathogenesis of various cardiovascular diseases such as atherosclerosis, myocardial infarction and ischemic heart disease, heart failure, aneurysms, etc.
A growing body of evidence highlights the active participation of EVs in these inflammation-associated cardiovascular anomalies. For example, during initial atherogenic stages, EVs from atherogenic plaque, circulating monocytes, and neutrophils induce the endothelial expression of ICAM-1. This facilitates leukocyte recruitment, adhesion, and trans-endothelial migration, mostly via the activation of pro-inflammatory signaling pathways [85][130][131]. This is followed by the plaque maturation stages, wherein EVs from platelets and adipose cells play a pivotal role by enhancing the formation of foam cells depending on the pro-inflammatory signaling. Platelet-derived EVs trigger the macrophages’ phagocytosis of oxidized LDL (ox-LDL) [132]. Adipose cell-derived EVs, on the other hand, perturb the cholesterol efflux of macrophages [133]. Both the platelet- and adipose cell-derived EVs are shown to stimulate the formation of foam cells. In the final stage, atherosclerotic plaque progression essentially requires calcification, and EVs from pro-inflammatory macrophages are shown to induce microcalcification both in human and murine systems [134][135]. EVs also play a pivotal role in inflammation-associated myocardial infarction (MI) and ischemic heart disease. For example, EVs in the myocardium, originating from cardiomyocytes and endothelial cells, trigger the secretion of pro-inflammatory cytokines and chemokines from infiltrating monocytes. These pro-inflammatory molecules contribute to the pathogenesis of MI and ischemic heart disease [136]. On the other hand, EVs’ miR-155 is reported to be transferred from activated macrophages to cardiac fibroblasts. This leads to the inhibition of fibroblast proliferation and triggers the inflammatory responses, thereby contributing to the cardiac rupture [137]. EVs are also demonstrated to be involved in heart failure (HF)-associated inflammatory responses. For example, cardiac fibroblasts are well known for releasing miR-27a*- and miR-21*-laden EVs, capable enough of promoting cardiac hypertrophy [138][139]. On the other hand, cardiac hypertrophy is also driven by cardiomyocytes which promote fibroblast proliferation via the release of miR-217-laden EVs [140]. Moreover, the role of EVs in aneurysm is widely documented. For example, neutrophil EVs, in the intraluminal thrombus of aortic aneurysms, are known for carrying ADAM10 and ADAM17 which, due to their proteolytic activities, cause the degradation of aortic walls [141]. Additionally, ficolin-3 + platelet-derived EVs are well elevated in the plasma of aortic aneurysms patients which contribute to the progression of aneurysms [142]. In the above-mentioned studies, the authors used either differential centrifugation or ultracentrifugation for EVs’ isolation, which reduces EVs’ purity and hence could influence the inflammatory behavior of the EVs. Figure 3 briefly summarizes the role of EVs in the progression of different cardiovascular inflammatory diseases.
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Figure 3. The role of EVs in cardiovascular inflammatory diseases. EVs are shown to play important roles in the progression of various cardiovascular inflammatory diseases including atherosclerosis, myocardial infarction (MI) and ischemic heart disease, heart failure (HF), and aneurysm. In atherosclerosis, during initial atherogenic stages, monocyte-, neutrophil-, and plaque-derived EVs interact with the endothelium, leading to transendothelial migration of leukocytes. During plaque maturation stages, platelet- and adipose cell-derived EVs convert macrophages into foam cells. Furthermore, during plaque progression stages, inflammatory macrophage derived EVs promote calcification of the plaque. In MI and ischemic heart disease, endothelial cell- and cardiomyocyte-derived EVs trigger macrophage pro-inflammatory responses. On the other hand, macrophage-EVs induce cardiac fibroblasts’ pro-inflammatory responses. In the case of HF, cardiomyocyte derived EVs promote the proliferation of cardiac fibroblasts, thereby contributing to cardiac hypertrophy. Moreover, EVs generated from cardiac fibroblasts also trigger cardiac hypertrophy. In aortic aneurysms, neutrophil-derived EVs cause degradation of aortic walls whereas platelet-derived EVs contribute to the progression of aneurysms.
EVs in respiratory inflammatory diseases. EVs often influence inflammation-associated respiratory diseases, such as acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease (COPD), pulmonary hypertension (PH), idiopathic lung fibrosis (ILF), asthma, etc. In ALI and ARDS, EVs are released into the BALF upon infection (LPS or Gram-negative bacteria) or sterile stimuli (acid aspiration or oxidative stress) from alveolar macrophages or alveolar type-I epithelial cells, respectively. These EVs trigger the release of pro-inflammatory cytokines and mediators from naïve alveolar macrophages, leading to the development of lung inflammation [143]. In the case of COPD, bronchial epithelial cell-derived EVs are shown to be enriched with miR-210. These miR-210-laden EVs are associated with autophagy functions and myofibroblasts differentiation, the dysregulation of which leads to the pathogenesis of COPD [144]. Furthermore, in PH, more specifically pulmonary arterial hypertension (PAH), miR-143-laden EVs from pulmonary arterial smooth muscle cells (PASMCs) promote migration and angiogenesis of pulmonary arterial endothelial cells (PAECs) [145]. These contribute to the pathogenesis of PH. BALF-EVs of ILF patients have an abundance of WNT5A, believed to originate from the lung fibroblasts, and are shown to promote fibroblast proliferation and the pathology of ILF [146]. In asthma, plasma EV-associated miR-145 plays a crucial role in epithelial and smooth muscle cell functions [147] related to inflammation. The inhibition of miR-145 is often observed during asthma which is accompanied by low eosinophilic inflammation, Th2 cytokine production, airway hyperresponsiveness, and hypersecretion of mucous, characteristic features of asthma-induced bronchial stress [148]. The use of ultracentrifugation in EV isolation, in the above-mentioned studies, limits the purity of the EVs except for Martin-Medina et al. who used highly purified EVs, isolated by ExoQuick followed by ultracentrifugation, in their study [146]. Figure 4 briefly demonstrates how EVs play their part in various respiratory inflammatory syndromes.
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Figure 4. The role of EVs in various respiratory inflammatory diseases. EVs’ role is well-established in various inflammation-associated respiratory diseases, such as acute lung injury (ALI) or acute respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease (COPD), pulmonary hypertension (PH), idiopathic lung fibrosis (ILF), and asthma. In ALI/ARDS, infected alveolar macrophage- and oxidative stress-induced alveolar type-I epithelial cell-derived EVs trigger pro-inflammatory cytokines’ release from naïve alveolar macrophages. In COPD, bronchial epithelial cell secreted EVs promote myofibroblast differentiation and influence autophagy functions. In PH, PASMC-released EVs promote migration and angiogenesis of PAECs. ILF-infected lung fibroblast-derived EVs promote proliferation of normal lung fibroblasts. EVs from the plasma of asthma patients influence the functions of lung epithelial and smooth muscle cells.
EVs in inflammatory diseases of the digestive system. A growing body of evidence indicates that EVs also play important roles in the inflammatory diseases of the digestive system, such as necrotizing enterocolitis (NEC) and inflammatory bowel disease (IBD). Numerous studies have demonstrated the active involvement of EVs in influencing NEC and IBD; however, the present review highlights a few of them. NEC is considered to be one of the catastrophic diseases of newborns with mortality rates of ~20–30% [149]. EVs from stem cells often show protective responses against NEC, indicating the therapeutic potential of the stem cell-derived EVs in NEC. Pisano et al., in a recent study, demonstrated that pre-treatment of intestinal epithelial cells (IEC) with bone marrow (BM)-derived EVs, which are abundant in the breast milk, rescues IEC against hypoxia/reoxygenation (H/R)-triggered inhibition of proliferation and induction of apoptosis in a rat model [149]. Furthermore, amniotic fluid stem cell (AFSM)-derived EVs are shown to promote epithelial proliferation and anti-inflammation, leading to the regeneration of normal intestinal epithelium, ultimately contributing to the intestinal recovery following NEC [150]. IBD, the other inflammatory disease of the gastrointestinal tract, is caused by the dysbiosis of the intestinal microenvironment, currently affecting more than 3.5 million people worldwide [151]. IECs, under physiological conditions, produce TGF-β1-laden EVs which induce regulatory T-cells (Treg) and immunosuppressive dendritic cells, thereby decreasing the severity of IBD [152]. Moreover, mast cell (MC)-derived EVs transfer miR-223 to the IECs, hence targeting IECs’ Claudin 8 (CLDN8), resulting in the loss of intestinal epithelial tight junctions which leads to increased intestinal epithelial permeability, the characteristic feature of IBD [153]. IBD-induced injury to the epithelial barrier triggers the release of annexin A1 (ANXA1) + EVs from the IECs, which is associated with the activation of the wound repair process [154]. Unlike others, Li et al. [150] and Jiang et al. [152] used the ExoQuick kit for the isolation of EVs in their studies, which not only improves the yield as compared to conventional ultracentrifugation but also consumes less time. However, ExoQuick-purified EVs without subsequent centrifugation steps may result in a high degree of lipoprotein contamination.
The role of EVs in integumentary inflammatory diseases. EVs are sometimes shown to be involved in the inflammatory responses of various integumentary diseases such as systemic lupus erythematosus (SLE), psoriasis, atopic dermatitis (AD), etc. In SLE, the number of circulating EVs is found to be well-elevated, and those EVs target the endothelial cells leading to the secretion of pro-inflammatory cytokines, induction of endothelial apoptosis, and enhancement of vascular permeability, ultimately contributing to secondary tissue leukocyte infiltration [155]. In psoriasis, interferon α (IFN-α)-induced mast cell-derived EVs transfer cytoplasmic phospholipase A2 (PLA2) to nearby CD1a-expressing cells, thereby generating neo lipid antigens and their recognition by CD1a-reactive T-cells to induce the release of IL-22 and IL-17A, ultimately leading to skin inflammation [156]. Furthermore, in AD patients, Staphylococcus aureus-derived EVs (SEVs) trigger dermal microvascular endothelial cells (DMECs) to induce the expression of E-selectin, ICAM-1, VCAM-1, and IL-6 release via TLR4-NF-ĸB signaling, thereby promoting leukocytes’ adhesion to the endothelium and their subsequent transmigration to promote AD progression [157]. In the above-mentioned studies, the isolation of EVs was carried out through differential or ultracentrifugation. However, it is important to note that these methods leave behind the possibility of soluble protein contaminants, which can have a significant impact on the inflammatory responses under investigation.
EVs’ role in musculoskeletal inflammatory diseases. An increasing body of evidence indicates that EVs also play a crucial role in inflammatory responses associated with musculoskeletal diseases, which include osteoporosis (OP), osteoarthritis (OA), etc. For example, oxidative stress and aging result in the elevated expression of miR-183-5p in the EVs isolated from bone marrow interstitial fluid (BMIF). miR-183-5p is shown to arrive from aged bone marrow stromal cells (aBMSCs) and is capable of targeting heme oxygenase-1 (Hmox1) in young BMSCs (yBMSCs), thereby not only inhibiting the proliferation and osteogenic differentiation of yBMSCs but also promoting yBMSCs senescence, the characteristic features of OP [158]. In OA, EVs from IL-1β-stimulated synovial fibroblasts (SFBs) are observed to induce MMP-13 and ADAMTS-5, whereas inhibiting COL2A1 and ACAN expression in articular chondrocytes contributes to the pathogenesis of OA [159]. Unlike others, Kato et al. [159] used both ultracentrifugation and ExoQuick for the isolation of EVs in their studies. As stated before, the use of ExoQuick without subsequent ultracentrifugation improves the yield significantly but leaves behind the possibility of lipoprotein contamination. Figure 5 briefly illustrates the role of EVs in different inflammation-associated diseases of the digestive system, integumentary system, and musculoskeletal system.
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Figure 5. The role of EVs in various inflammatory diseases associated with the digestive system, integumentary system, and musculoskeletal system. (1) The role of EVs in digestive inflammatory diseases. In necrotizing enterocolitis (NEC) (sky dotted arrows), BM-EVs promote IEC proliferation and anti-apoptosis. Moreover, amniotic fluid stem cell derived EVs promote the proliferation and anti-inflammation of IEC. In inflammatory bowel disease (IBD) (red dotted arrows), IEC-EVs promote the induction of Treg and immunosuppressive dendritic cells, as well as wound repair. MC-derived EVs increase IEC permeability. (2) EVs’ roles in integumentary inflammatory diseases. In systemic lupus erythematosus (SLE) (violet dotted arrows), plasma EVs promote endothelial apoptosis, permeability, and release of pro-inflammatory cytokines, leading to leukocytes transmigration. In psoriasis (green dotted arrows), MC-EVs containing PLA2 are taken up by CD1a-expressing cells which present a lipid antigen (red dot) to the CD1a-reactive T-cell, leading to the release of pro-inflammatory cytokines IL22 and IL17A. In atopic dermatitis (AD) (blue dotted arrows), SEVs trigger the expression of E-selectin, VCAM-1, and ICAM-1 and the release of IL-6, thereby promoting vascular permeability to induce leukocytes transendothelial migration. (3) EVs in musculoskeletal diseases. In osteoporosis (OP) (pink dotted arrows), aBMSC-derived EVs inhibit proliferation and differentiation, while promoting senescence of yBMSC. In the case of osteoarthritis (OA) (orange dotted arrows), SFB-EVs induce pathogenicity to articular chondrocytes.

The role of EVs in urinary inflammatory diseases. EVs also play critical roles in the progression of several urinary inflammatory diseases. For example, the level of plasma or urine-derived EVs is often used as a predictive biomarker for the progression of acute kidney injury (AKI) [160]. Guan et al. showed that hypoxia or ischemia-reperfusion (I/R)-induced injured tubular epithelial cells (TECs) release significant amount of miR-150-laden EVs which develop profibrotic manifestations to renal fibroblasts. Moreover, the expression of urinary EVs’ chemokine (C-C motif) ligand 2 (CCL2) mRNA is shown to be significantly higher in IgA nephropathy (IgAN) patients as compared to other glomerulopathy controls, which is correlated with the tubular interstitial inflammation and C3 deposition, reflecting renal injury and impaired renal functions [161]. Again, as in the majority of cases, using ultracentrifugation to isolate EVs frequently results in a drop in EVs purity.

EVs’ role in inflammatory diseases of the reproductive system. In the uterine microenvironment (UME), EVs play a crucial role in maternal-embryo interaction by promoting implantation defects which often lead to several pregnancy-related disorders. Maternal immune macrophages-derived EVs are shown to be endocytosed by placental trophoblasts, resulting in the release of pro-inflammatory cytokines, thereby contributing to the maternal inflammatory responses to protect the fetus [162]. On the other hand, placental trophoblast-derived EVs are loaded with chromosome 19 miRNA cluster (C19MC) which attenuate autophagy-mediated virus replication in non-placental cells, thereby protecting from the embryo from viral infections [163]. Delorme-Axford [163], unlike others, employed ultracentrifugation followed by density-gradient centrifugation in their EVs preparation which is shown to yield highly purified EVs.

The role of EVs in inflammatory diseases of the endocrine system. A few studies indicate the active participation of the EVs in inflammatory responses of the endocrine system. For example, EVs, derived from obese adipose tissues and plasma show a significantly lower expression of miR-141-3p, which is associated with glucose intolerance and insulin resistance [164][165]. EVs, released into the serum from brown adipocytes contain significant level of miR-99b, which target FGF21 in the liver, thereby contributing to metabolic dysfunctions such as glucose intolerance in obesity [166]. Adipose tissue macrophages (ATMs)-EVs are shown to be over-expressed with miR-155 under obese conditions, which target PPARγ in adipocytes, myocytes, and primary hepatocytes, leading to glucose intolerance and insulin resistance [167]. As with most cases, the use of ultracentrifugation to isolate EVs in mentioned studies raises questions about the presence of protein contaminants in the EV suspension.

EVs of the lymphatic system in inflammatory diseases. EVs of the lymphatic system often influence various inflammation-associated diseases. For example, the concentration of EVs, derived from the lymph, is shown to be well-elevated in atherosclerotic conditions as compared to healthy controls, which is believed to contribute to lymphatic dysfunction and associated-inflammatory disease progression [168]. Pronounced inflammation-induced vascular leakage promotes the egress of platelet-derived EVs into the lymphatic system, which is shown to contribute to the pathogenesis of rheumatoid arthritis (RA) [169]. Figure 6 demonstrates the role of EVs in inflammation-related diseases of the urinary system, reproductive system, endocrine system, and lymphatic system.

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Figure 6. The role of EVs in various inflammatory diseases, associated with the urinary, reproductive, endocrine, and lymphatic system. (1) EVs and urinary inflammatory diseases. In acute kidney injury (AKI), I/R-induced TEC-derived miR-150-laden EVs promote profibrotic manifestations to renal fibroblasts. In IgA nephropathy (IgAN), CCL2 mRNA-loaded EVs promote inflammation-induced renal injury and impaired renal functions. (2) EVs’ roles in reproductive inflammatory diseases. In pregnancy-related diseases, maternal macrophage-derived EVs trigger inflammatory responses in the placental trophoblast. On the other hand, EVs from placental trophoblast prevent virus replication of non-placental cells, thereby protecting the embryo from viral infections. (3) EVs and endocrine inflammatory responses. In obesity, adipose tissue-derived EVs influence glucose intolerance and insulin resistance. Again, brown adipocytes-derived EVs promote glucose intolerance after migrating to the liver tissues. Furthermore, ATM-EVs target adipocytes, myocytes, and primary hepatocytes leading to glucose intolerance and insulin resistance. (4) EVs of the lymphatic system influencing various inflammatory diseases. In atherosclerosis, EVs’ level in the lymph is significantly increased, contributing to inflammation, and associated disease progression. On the other hand, in rheumatoid arthritis (RA), inflammation-induced vascular leakage renders the transmigration of platelet-EVs into the lymph, thereby contributing to the pathogenesis of RA.

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