Exosomes and Microvesicles

Dildar Savcı1, Gamze Işık1, Saliha Hızlıok1, Oytun Erbaş1

1ERBAS Institute of Experimental Medicine, Illinois, USA & Gebze, Türkiye

Keywords: Exosomes, extracellular vesicles, stem cell, mesenchymal stem cell, microvesicle.

Abstract

Extracellular vesicles (EVs) are released by all cells and have a variety of physiological functions. Microvesicles (MV), exosomes, oncosomes, and apoptotic bodies, are the four forms of EVs. Exosomes are spherical EV with an endocytic double-layered lipid membrane. As for their identification, it's based on isolation methods, cell type, and cell surface markers. Moreover, their functions differ according to the cell from which they are obtained. They are involved in the immune system, neural communication, drug delivery, and many other processes. Another type of EV is MVs, which are a diverse collection of vesicles formed by the outward budding of the plasma membrane. Differentiating these two types of EVs is mostly based on the process of biogenesis. MVs play a variety of pathological and physiological roles. MVs were hypothesized to have a role in the immunomodulatory function of mesenchymal stem cells (MSCs). Following that, it was discovered that MVs secreted by damaged cells send precise signals to MSCs. Given their physiological contact with the target cell, MVs are regarded to be therapeutic targets in cancer treatment. This chapter provides information about exosomes and microvesicles, which are extracellular vesicles.

Introduction

Extracellular vesicles (EVs) are secreted by practically all cell types as a continuation of membrane structures and are responsible for many vital physiological functions. Exosomes, a form of EVs, were first observed in the 1980s. These nano-sized spherical vesicles secreted by cells contain a wide variety of lipids and proteins as well as nucleic acids in their composition and can fulfill a broad range of functions depending on the source from which they are obtained.[1-9]

Waste molecule removal, intercellular contact, cargo package transportation, immune system, reproduction and development, neural communication, and cell proliferation are some of the processes these organelles can participate in.[2,4]

Microvesicles (MVs), another type of EVs, are formed directly by the budding of the cell membrane. Although MVs resemble exosomes in many ways, these vesicles have different properties and functions that distinguish them, including size distribution and biogenesis mechanism.[10-13] Moreover, having the ability to transport various bioactive molecules, MVs are involved in many pathological and physiological events as well as intercellular interactions.[14-17] Different extracellular factors, in addition to cytoskeletal components, molecular motors, and numerous signaling molecules, influence the formation of microvesicles.[13,18]

Mesenchymal stem cells (MSCs), which can differentiate into several lineages and play a key role in the physiological system in addition to their ability to self-renew, can also release various chemicals that influence the immune system. It is considered that EV secretion may influence MSCs’ numerous physiological effects.[19-25] The involvement of EVs in crucial events and processes including inflammation, fibrosis, and cancer, implies that they may play a role in disease development. Furthermore, stem cell-derived EVs have been shown to be beneficial in numerous treatment processes.[26,27]

EXOSOMES

Exosomes are nano-sized endocytic EV with a diameter of 30-100 nm, float at a density of 1.13-1.19 gmL-1 in a sucrose gradient solution, and are attached to a double-layered lipid membrane.[2-6] They are classified based on the cell type from whence they were derived, isolation techniques, size, and cell surface indicators.[2,5]

Exosomes are composed of a variety of molecules including nucleic acids like DNA and RNAs such as microRNA (miRNA), long non-coding RNA (lncRNA), non-coding RNA (ncRNA), circular RNA (circRNA), ribosomal RNA (rRNA), small nucleolar RNA (snoRNA), messenger RNA (mRNA), piwi-interacting RNA (piRNA) and more. various lipids such as cholesterol, ceramide, sphingomyelin, phosphatidylinositol, phosphatidylserine, phosphatidylcholine, phosphatidylethanolamine, and glycosphingolipid, along with a range of proteins like transmembrane protein, heat shock proteins (HSP), cytoskeletal proteins such as actin, cophilin, and tubulin, glycoproteins, there are also several molecules and complexes necessary for certain intra- and extracellular activities, including endosomal sorting complex required for transport (ESCRT) components, include ALG-2-interacting protein X (Alix) and tumor susceptibility gene TSG-101 as well as proteins involved in membrane trafficking and fusion such as GTPases, annexin, Rab, dynamin, and syntaxin, other molecules essential for the immune system like, cytokines, Tumor necrosis factor-alpha, Transforming growth factor (TGF)-beta, and TNF-associated apoptosisinducing ligand (TRAIL), Major histocompatibility complex (MHC) class I and II, cluster of differentiation (CD) molecules including CD9, CD37, CD53, CD63, CD81, and CD82, signaling receptors Fas ligand (FasL), TNF receptor and Transferrin receptor.[28-30] Together with different types of biomolecules involved in transcription and protein syntheses such as Histone1,2,3 and Ubiquitin.[7-8] In exosomes, tetraspanins, antigen-presenting molecules, glycoproteins, and adhesion molecules are located among the transmembrane proteins, while HSPs, cytoskeletal proteins, components of the ESCRT mechanism, proteins responsible for membrane transport and fusion, cytokines, and developmental factors are located in the lumen.[8]

Those biomolecules cover a vast variety of activities, for instance, integrins, lactadherin, and intracellular adhesion molecule-1 are involved in cell-cell communication, Tsg101, Aliks, Rab proteins, involved in multivesicular body biogenesis, lysosomal-associated membrane proteins 1/2, CD13 and PG regulator-like protein, on the other hand, are essential for membrane transport.[31] Moreover, there are molecules involved in transcription and protein synthesis, proteins responsible for signaling, aspartate aminotransferase, aldehyde reductase, ATPase, and similar metabolic enzymes, antiapoptotic proteins like Aliks, Thioredoxin, peroxidase; death receptors FasL, TNF-associated apoptosis-inducing ligand also, transferrin receptor which is involved in iron transport.[6,31]

In the same context, Leukotrienes (LTs) like LTA4, LTB4, LTC4, and related LTA4 Hydrolase and LTC4 synthase enzymes are associated with triggering polymorphonuclear leukocyte migration in exosome functioning, cyclooxygenase isoenzymes (COX-1 and -2) are engaged in immunosuppression, and prostaglandin 2 (PGE2), as well as PGE synthase enzyme, are involved in inflammation.[32-35] Additionally, phosphatidic acid, phospholipase (PLD) 2, and diglyceride kinase increase exosome production; arachidonic acid, lysophosphatidylcholine, calcium-dependent PLA2, and calcium-independent PLA2 participate in the formation of membrane curvature and PLA- II, and V in PGE biosynthesis; and while ceramides are involved in the sorting of cargo into multivesicular bodies (MVBs), Bis (monoacylglycerol) phosphate is involved in the formation of them, phosphatidylserine (PS) determines exosome fate and sphingomyelins are known to be associated with the triggering of calcium influx.[6]

Exosome Biogenesis

Structurally, exosomes consist of early endosomes, which are intracellular, small bodies formed by the inward budding of the plasma membrane due to the drawing of intracellular fluid. Early endosomes enter the maturation process, where late endosomes are formed.[36] Intraluminal vesicles (ILVs) form as late endosomal membranes invaginate, and as ILVs accumulate, MVBs rich in ILVs form.[8,37]

The newly formed MVBs either go through hydrolyzation and fragmentation by fusion with lysosomes; or get transported to the plasma membrane via the cytoskeleton and microtubular network, where they undergo ion-dependent fusion and are released as exosomes into the extracellular space.[2,8,38]

The ESCRT is a biogenesis and secretion process. This mechanism is made up of four complexes: ESCRT-0, ESCRT-I, ESCRT-II, and ESCRT-III, as well as vacuolar protein Sorting-associated protein 4A (VPS4A), Tsg101, and ALIX.[7] The MVB pathway is initiated by ESCRT-0 conveying ubiquitinated cargo proteins via their lipid domains. Following that, ESCRT-I and II drive the inward budding and induce stabilization, and ESCRT-III detaches from the MVB membrane via VPS4A, completing its role in membrane remodeling.[6,7,38] Tsg101 ensures that ESCRT-1 is active in the process by connecting with ESCRT-0, whereas ALIX is a protein implicated in ILV production by binding ESCRT-III subunits.[39]

Independent of the ESCRT mechanisms involved in cargo loading, endosomal sorting pathways, exosome release, and exosome biogenesis, there is a different pathway called tetraspanin, in which exosome-rich transmembrane proteins and lipids are involved.[37,38]

Tetraspanin-rich microdomains (TEMs) are involved in the classification of membrane-bound receptors and signaling proteins and tetraspanin CD81 participates in the sorting of intracellular components of TEMs and target receptors towards exosomes and in exosome biogenesis; meanwhile, CD63 is found to help in the loading of exosomal proteins, with Epstein-Barr virus being an example, additionally, EVs are reduced as a result of CD63 knockout, indicating that CD63’s involvement in exosome biogenesis; Furthermore, tetraspanins are valuable exosome biomarkers.[40,41]

Another mechanism independent of ESCRTs that plays an important role in secretion is the sphingolipid (ceramide Cer) method where ceramides are produced by sphingomyelinase or neutral sphingomyelinase which are involved in the budding of intracellular vesicles into MVBs, and inhibition of neutral sphingomyelinases results in a decrease in exosome secretion.[38,42-45]

Aside from that, miRNAs are considered another way of sorting cargo, given exosomes are enriched with them. Thanks to a specific motif contained in miRNA, A2B1 ribonucleoprotein interacts with miRNAs and plays a role in their loading. The KRAS gene is also known to be involved in this process. Likewise, it is thought that mRNAs can be separated into vesicles due to the enrichment of fragments at their 3’ ends.[37]

More importantly, it has been proposed that the Trans-Golgi network, Rab-GTPase, Rab family proteins, p53 activation, microtubules, microfilaments, SNARE complex, Na+/Ca2+ channels, H+ pumps, pH level, calcium ionophores, statins, heparanase, molecular motors, and tethering factors are all involved in the release of exosomes.[2,7,37,38]

As for the exosomes’ entry to the target cells, there are three different ways. The first is direct interaction, where the ligands in exosomes bind to the receptor of the target cell.[46] The second is fusion with the plasma membrane, where the exosomes fuse with the membrane of the target cell via adhesion molecules.[47] Another method is internalization, which is divided into five subdivisions. In this method, exosomes are introduced into target cells by clathrin-mediated endocytosis, lipid rafts-mediated endocytosis, caveolin-mediated endocytosis, phagocytosis of the exosome in the target cell, and finally, macropinocytosis, in which macropinosomes are formed by inducing invagination of the plasma membrane using actin-driven lamellipodia.[48]

Exosome Functions and Relationship with Stem Cells

Exosomes are linked to various functions depending on the source of the cell from which they are obtained.[9] Including removal of unnecessary molecules, intercellular communication and transport, immune system, reproduction and development, neural communication, cell proliferation-homeostasis maturation, and drug delivery, besides functioning as vaccines and biomarkers.[2,4]

B cells and dendritic cells-derived exosomes provide co-stimulatory proteins to activate T cells together with tumor antigens, resulting in anti-tumoral and immunostimulatory effects. These effects are observed in melanoma, prostate, lymphoma, and kidney cancer patients.[49]

Additionally, the epithelial cell adhesion molecule (EpCAM) and CD24 found in tumor-derived exosomal (TD-exosomes) miRNAs act as an essential diagnostic for the early identification of ovarian cancer and detection of TD-exosomes in general.[50] likewise, exosomal miRNAs in saliva serve as a biomarker of aging and as a marker for diagnosing various central nervous system diseases, cancer types, and cardiovascular diseases. Moreover, the ones found in amniotic fluid serve as a biomarker of fetal sex.[51,52]

As for their role in the immune system, they participate in a range of functions including acting as an immune activator, and immune suppressor, taking part in antigen-presenting, immune tolerance, immunoregulation, as well as establishing communication between mast cells, dendritic cells, natural killer cells (NKs), and antigen-presenting cells (APCs).[5] It was demonstrated that exosomes released in vivo from rodent mast cells promote dendritic cell maturation and present antigens to T lymphocytes, the ones released from immunosuppressive dendritic cells suppress autoimmunity, and finally, exosomes derived from bone marrow-derived dendritic cells showed its ability to inhibit the development of inflammatory bowel disease.[53-56]

An excellent example of exosome involvement in immunosuppression is helping neoplastic lesions to form and grow in vivo, leading to a decreased proliferation and cytotoxicity of NKs and T cells, as well as decreased numbers of APCs.[5] These effects are accomplished through autocrine signaling depending on the type of cell line from which the exosomes originate. For instance: exosomes from pancreatic cancer cells can inhibit the growth of some neoplastic lesions by inducing apoptosis via the Notch signaling pathway, whereas exosomes from breast cancer and gastric cancer promote neoplastic lesions.[55] They are also implicated in developing a matrix for neoplastic cell adhesion, aiding their movement to the sentinel lymph node, promoting angiogenesis, and establishing an environment that encourages metastases by suppressing the immune system.[5,57]

Moreover, the significance of exosomes in the nervous system’s signaling between sensory and motor neurons, interneurons, and glial cells, as well as their slowing influence on the hypothalamus’s endocrine function concerning aging, added to that, the exacerbation of some diseases such as Alzheimer (AD), Huntington, and Parkinson’s due to the release of amyloid beta peptide, misfolded proteins, and prions by exosomes, suggests the role the exosomes play in the pathogenesis of neurodegenerative diseases.[4,9,58]

As for the process of homeostasis and cell maturation, exosomes reduce intracellular stress by clearing toxic materials and selectively removing unwanted cargo in the maturation process of reticulocytes to erythrocytes, maintain homeostasis by secretion of exosomes, however, when secretion is suppressed, the innate immune response is exacerbated, causing reactive oxygen species (ROS)-dependent DNA damage responses and inducing apoptosis.[59]

Interestingly, exosomes contribute to reproductive and developmental processes such as sex cell maturation, cell behavior, horizontal transfer of genetic material, protection of the fetus by reducing immune cell functions during pregnancy, organ development, protection of the placenta against infections with exosomal miRNAs and resident immunity in the genital area, fertilization, and embryo implantation. Likewise, exosomes in breast milk, sometimes called tolerosomes, influence immunological tolerance modulation and development.[60-63] In addition, TD-exosomes can be employed as immunotherapeutic vaccines against cancer with modifications of various antigen-presenting proteins due to their effects on the immune system.[2,4] In that context, exosomes derived from intestinal epithelial cells induce immunotolerance in an antigen-specific manner during pregnancy, whereas exosomes derived from pancreatic carcinoma cells induce tolerance by stimulating migration in NKs, exosomes derived from breast milk are involved in immune tolerance regulation.[63,64] Moreover, breast tumor-derived exosomes have a role in the regulation of the immune system. The FasL on the exosomes’ surface demonstrates immunosuppressive effects by inducing apoptosis.[2] In contrast, Some tumor-derived antigen-containing exosomes suppress tumor growth by triggering antitumoral T-cell responses. And on top of that, exosomes generated from MSCs have immunomodulatory and cytoprotective properties and can even be used as therapeutic agents.[65,66] Several studies proposed that exosomal miRNAs increase anticancer activity in mammary carcinoma and glioma and that dendritic cell-derived exosomes modified with rabies virus glycoprotein have therapeutic effects.[67-69]

Exosomes released by human retinal pigment epithelial cells (ARPE-19) in response to oxidative stress were shown to promote apoptosis and inflammatory responses.[9,70]

Furthermore, exosomes can act as intermediaries in drug delivery due to their biodistribution and biocompatibility properties, their ability to cross the blood-brain barrier, and their immunogenicity.[9] For instance, RVG exosomes are used to reduce the loss of dopaminergic neurons in the treatment of parkinsonism, while cisplatin-loaded M1 exosomes are given as chemotherapeutics in ovarian cancer.[71,72]

Exosomes originating from immature dendritic cells also play a role in targeted drug delivery due to their low immunogenicity and toxicity. superparamagnetic iron oxide nanoparticles (SPION)-equipped exosomes, significantly inhibit the growth of tumor cells by inducing the apoptotic pathway.[73] It was found that quercetin-loaded exosomes potentiate brain targeting of the drug in AD and are involved in the amelioration of cognitive dysfunctions.[9] Further, increasing the solubility of resveratrol in primary microglia-derived exosomes in spinal cord injuries has the potential to boost targeted drug delivery and improve neuronal function.

Additionally, catalase-loaded exosomes showed neuroprotective effects in a mouse model of Parkinson’s disease, and treatment with gemcitabine-loaded autologous exosomes resulted in tumor suppression in tumor-bearing mice.[74,75]

As for stem cell-derived exosomes effects, human adipose tissue-derived MSCs (hADSCs) and human bone marrow-derived MSCs (hBM-MSCs) exosomes were found to be beneficial in AD, with the former being more effective due to the noticeable reduction in amyloid beta they cause.[76] Meanwhile, human menstrual blood MSCs (MenSCs) and hBM-MSCs-exosomes help neurite outgrowth in cortical and sensory neurons unlike exosomes derived from the human chorionic plate MSCs and human umbilical cord MSCs (hUC-MSCs) in neurodegenerative diseases. In comparison with exosomes obtained from human synovial membrane-derived MSCs (SMSCs), exosomes from induced pluripotent stem cells (iPSCs)-derived MSCs have more significant therapeutic effects with chondrocyte migration and proliferation in osteoarthritis disease.[77,78]

The anti-inflammatory properties of MSC exosomes are mediated by macrophage polarization from the pro-inflammatory phenotype M1 to the anti-inflammatory M2 macrophages. Exosomes derived from human jaw BM-MSCs and BM-MSCs, accelerate wound healing and decrease bronchopulmonary dysplasia, and exosomes obtained from MenSCs reduce inflammation and repair wounds in diabetic rats. They are both examples of M2 macrophage polarization.[79] Furthermore, it was reported that hUC-MSCs-exosomes’ immunosuppressive capabilities alleviate inflammation, help in diabetic cutaneous conditions, enhance wound healing and burn inflammations, and exosomes from mouse BM-MSCs (mBM-MSCs) regress osteosclerosis, and decrease atherosclerotic plaques, myocardial ischemia-reperfusion damage, and infarct size.[80-82]

Exosomes derived from hBM-MSCs are therapeutically effective by inducing M2 macrophages in intestinal bowel disease in mice and are also useful in improving motor skills in a mouse model of multiple sclerosis.[83] On top of that, exosomes derived from ADSCs depending on the species from which they are derived can demonstrate various effects including, showing a decrease in cardiac damage, fibrosis, and apoptosis when derived from rats; increasing the expression of M2 macrophages when derived from humans; increasing the polarization of M2 macrophages in obese mice when derived from mice; and are effective in the treatment of autoimmune type 1 diabetes mellitus.[84,85] Exosomes from hBM-MSCs and hADSCs not only help reduce skin photoaging ensure skin flap longevity and reduce inflammation respectively, but also they are useful in reducing the pathological symptoms of atopic dermatitis, meanwhile, exosomes from rat BM-MSCs (rBM-MSCs) help improve regeneration with a decrease in histopathological findings, blood urea nitrogen, creatine levels, oxidative stress, apoptosis, and inflammation in kidney injury.[86-89] Human placental MSCs exosomes are reported to be useful in reducing tissue fibrosis and inflammation in Duchenne muscular dystrophy.[90] In neonatal mice, models of bronchopulmonary dysplasia, exosomes from hUC-MSCs exhibit improvements in lung, heart, and brain pathologies, and in post-stroke neurodegeneration, hBM-MSCs-exosomes can demonstrate neuroprotection, induction of neurogenesis, and angiogenesis. In addition to that, in diabetic peripheral neuropathy, exosomes derived from mBM-MSCs are capable of increasing the nerves’ conduction velocity, intraepidermal nerve fibers’ density, myelinization, and axon diameter, as well as preventing disease progression in intervertebral disc degeneration.[91,92]

Along with that, inhibition of cardiac fibrosis, reduced inflammation, improvement in cardiac function in myocardial infarction, as well as induced survival and injury healing with decreased apoptosis and inflammation in acute lung injuries, were reported as impacts of rBM-MSCs-exosomes.[93,94] In graft versus host disease (GVHD), exosomes derived from hUC-MSCs and hBM-MSCs modulate immune cells and prevent acute GVHD. While, in status epilepticus, they reduce inflammation in the hippocampus, decrease glutamatergic and gamma-aminobutyric acid (GABA) loss, and improve learning and memory disorders.[95,96] With similar effects, exosomes derived from rBM-MSCs and hADSCs enhance spatial learning and motor behavior in traumatic brain injury, reduce neural inflammation, increase cognitive performance, increase white matter connectivity, improve autistic-like behaviors, and improve social behaviors in Autism spectrum disorder.[77,97] Besides, exosomes derived from human Wharton’s jelly MSCs, also known as UC-MSCs, help reduce neuroinflammation and neuron-specific cell death in perinatal brain injury.[98]

Exosomes inhibition of the M1 phenotype in macrophages, and promotion of M2 polarization, along with the suppression of macrophages migration, lead to suppression of proliferation, activation, migration, and cytotoxicity of NKs; dendritic cells maturation and activation suppression, increasing in Treg cells; inhibition of proliferation, differentiation, and immunoglobulin secretion in B cells, in addition to suppression in T cells proliferation and activation.[99]

With that mechanism, MSC-exosomes prevent inflammation in the central nervous system and relieve neurobehavioral symptoms in multiple sclerosis. When they target the spinal cord, they reduce inflammation by the inflammatory inactivation of NALP3 autoimmune damage is prevented by inducing Treg cells with anti-inflammatory factors when T and B lymphocytes are targeted. Further, when the inflammation of islet cells is prevented, it leads to an increase in plasma insulin levels; likewise, they promote the recovery of cognitive impairment by repairing neurons and astrocytes.[100-105] Similarly, when exosomes target suppressor cells of myeloid origin in Sjögren’s Syndrome, the disease’s progression is slowed by raising levels of reactive oxygen species and nitric oxide, which is accomplished by activating the Janus Kinase 2/ Signal Transducer and Activator of Transcription 3 (JAK2/STAT3) signaling pathway, and when salivary gland epithelial cells are targeted, serum autoantibody levels are suppressed by preventing APC activation.[106]

Using exosome targeting abilities, RNA loading is becoming a popular application in drug delivery. Nowadays, BM-MSCs-exosomes can be applied for various tumors treatments like loading miRNA for glioma inactivation, reducing breast cancer activation via miRNA-379, playing a neuroprotective role via miRNA-133b, promoting angiogenesis via miRNA-132, and repairing spinal cord injury via miRNA-29b, while, hUC-MSC-exosomes are used for the inhibition of pancreatic ductal adenocarcinoma via miRNA-145-5p, treatment of aging-related vascular dysfunction via miRNA-675, treatment of diabetes via miRNA-21. Additionally, dental pulp MSCs suppress the proliferation of breast cancer cells via miRNA-34a, and SMSCs are involved in the treatment of osteoarthritis via miRNA-155-5p.[107-113]

In a similar mechanism, BM-MSC-exosomes are loaded into Norcantharidin to treat hepatocellular carcinoma and into Doxorubicin to treat osteosarcoma; hUC-MSCs-exosomes into Paclitaxel to target breast cancer; and MSC-exosomes are loaded into Honokiol to treat some cancer cell lines.[114]

Aside from that, they are loaded into proteins. Exosomes obtained from MB-MSC are used in the treatment of cystic fibrosis by loading zinc finger protein and for anti-tumoral purposes with TRAIL proteins; while exosomes obtained from hUC-MSCs increase angiogenesis with Angiopoietin-2 protein and play a role in the progression of cardiac regeneration through Akt protein.[115,116]

MICROVESICLES

Microvesicles are a diverse set of membrane-enclosed vesicles that are discharged into the extracellular space as a result of the plasma membrane being compressed by budding outward. Their diameters range from 100 to 1000 nm.[10,11] Surface markings distinctive to MVs are absent. They can, however, express the cell surface markers from which they are produced.[14] DNA, mRNA, miRNA, and proteins are examples of bioactive molecules that can be transported by MVs. They act as a conduit for communication between cells.[12,14,17] As a result, they can play a role in a variety of physiological and pathological processes, including cell apoptosis, autophagy, tumor development, and metastasis.[14-16]

Microvesicle Biogenesis

One of the main points in the classification and naming of EVs is their biogenesis mechanism. It has been shown that the biogenesis mechanism of MVs is different from the biogenesis mechanism of other EVs such as exosomes, in which intracellular events are effective in their formation, and apoptotic bodies, which are formed indiscriminately by bubbling of almost any surface.

The biogenesis mechanism of EVs is one of the key factors in their classification and nomenclature. Further, it has been demonstrated that the biogenesis method of MVs differs from that of other EVs such as exosomes, which are effectively generated by intracellular events, and apoptotic bodies, which are created randomly by outward budding of virtually any surface. In contrast to exosomes, which are formed by the continuation of intracellular events, MV biogenesis involves budding and compression directly outward from the plasma membrane. In this process, the vertical transport of molecular cargo across the plasma membrane and the use of contractile mechanisms for vesicle compression can be observed.[117-119]

Another point that distinguishes MVs from the better-characterized exosomes is the size difference arising from their formation mechanism.

Since MVs are formed by directly bulging from the cell membrane, they are larger than exosomes and can be found in a wider range of diameters.[11-13] However, the biogenesis mechanism is primarily taken into account in the separation of the two types of EVs due to their common size range.[18,120]

Biofluids such as urine, blood, and cerebrospinal fluid can also be a source for EVs, and the sources from which they are obtained may play a major role in their naming. However, the fact that some cells such as endothelial cells, platelets, and cancer cells can release exosomes as well as MVs into the circulation makes it difficult to distinguish between them both. Nevertheless, EVs can also be named variously in the literature as epididymosomes, argosomes, exosome-like vesicles, microvesicles, archaeosomes, promininosomes, dexosomes, exosomes, nanovesicles, apoptotic bodies, and oncosomes depending on the isolation source or isolation method.[11,120-122]

Depending on their cell of origin, MVs, which may have a plasma membrane, endosome, cytosol, or other cellular protein contents and molecular loads, are released directly into the extracellular space and shed from the surface of the cells. Microvesicles, which mostly contain proteins clustered on the surface of the plasma membrane, may also contain some marker proteins and various proteins with translational modifications and therefore are thought to be used as biomarkers.[11-13]

Regulation of microvesicle formation is a process involving various cell molecules including cytoskeletal components such as actin and microtubules and molecular motors such as myosins. Here, Ras homology family GTPases (Rho) play a critical role in actin reorganization, and thus, MV secretion.[13,18,27,123].

At the same time, studies are showing that the redistribution of heterogeneous phospholipids in the cell, and mainly the translocation of PS to the outer leaflet leads to the formation of MVs.[11,120]

Regulation of the process of microvesicle release, however, is achieved by a signaling cascade that starts with PLD activation in the cell. Here, as a result of the activity of ADP-ribosylation factor 6, one of the GTP-binding proteins, extracellular signal-regulated kinase is recruited to the plasma membrane and phosphorylation of myosin light chain and MV release occurs with its increased activity.[120,124] In addition, RhoA/Rho-associated kinase signaling is thought to support MV release via a different pathway.[11,18,27]

The number of MVs formed varies depending on the physiologic state of the released and retrieved cells as well as their microenvironment. More importantly, temperature and extracellular calcium concentration are thought to be effective in the formation of these vesicles.[13,18] Microvesicles content can also be analyzed to understand the mechanisms involved in the release of MVs loaded with various proteins and nucleic acids.[11,124]

While it was initially thought that MVs, like most EVs, were involved in the removal of waste material from the cell, it was revealed that these vesicles, like exosomes, have an important role in intercellular interaction.[11,125] With this discovery, interest in MVs has increased and the processes of packaging and delivering active cargoes containing various proteins, nucleic acids, and lipids to the target cell have begun to be investigated. Additionally, it is thought that glycan-binding proteins on the surface of MVs may be an effective factor, especially in the transportation of MVs to target cells and regulation of the function of the recipient cell.[13,126]

Relationship of Microvesicles with Stem Cells

Stromal cells, also known as MSCs, were initially identified only in the bone marrow.[127] Nevertheless, to date, they have so far been isolated from a wide variety of other tissues, including umbilical cord blood, Wharton’s jelly, and placental, adipose, and lung tissue.[128,129]

The perspective on the nature and function of MSCs has undergone several paradigm shifts over time. They play multiple roles in the physiological system as well as their ability to differentiate into multiple lineages and contribute to organized cell replacement therapy.[19] They not only have the ability to self-renew by undergoing multiple cell divisions but also exhibit anti-inflammatory and immunosuppressive properties by directly interacting with several immune cells.[20,21] Moreover, MSCs have provided short-term therapeutic benefits to numerous diseases.[130,131] To reconcile these different findings, it has long been known that MSCs produce growth factors and cytokines, many of which modulate the immune system. However, despite this knowledge, most of these proteins lack signal peptides. The fact that they are packaged in membrane-bound vesicles with mRNAs and miRNAs partly explains how MSCs can exert multiple effects on the physiological system.[19,22-26]

Bioactive molecules secreted by MSCs can function as paracrine or endocrine mediators that modulate immune responses, interact with neighboring cells, and promote self-repair.[129]

According to several recent researches, MSCs’ capacity to release MVs may play a role in mediating some of their immunomodulatory function.[132] Therefore, MVs released by injured cells may provide specific signals to stem cells that enable their differentiation. They may also represent a mechanism involved in physiologic tissue repair.[133] Stem cell-derived MVs can cause changes in the phenotype of tissue cells that regulate regeneration and cell differentiation.[134] It has been shown that MVs isolated from MSC culture supernatants may perfectly replicate the inhibitory effects of MSCs on CpG-induced B cell proliferation and differentiation in the co-culture system of peripheral blood mononuclear cells in a dose-dependent way. Allogeneic and syngeneic T lymphocyte proliferation has been shown in certain investigations to be effectively inhibited by MVs derived from BM-MSCs. Additionally, it was shown that these microparticles can cause activated T-cells to undergo apoptosis. Additionally, spleen cells grown with MSC-MVs showed enhanced production of TGF-1 and IL-10. These findings imply that tolerogenic signaling may be induced by MSC-MVs.[132]

Numerous studies have demonstrated that EVs play a key role in numerous crucial cellular processes and events, including fibrosis, cancer, and inflammation. Considering different pathological situations, EVs may also effectively contribute to the onset of illness.[135,136]

Some of the EVs released by tumor cells influence their microenvironment, promote immune suppression, and promote the proliferation and spread of cancer cells.[136-141] The fact that EVs promote the spread of cancer while simultaneously offering hope for a cure for the disease is particularly intriguing.[142-144]

The potential of tumor cells to produce MVs, like many other cells, raises the possibility that MVs play a significant role in the development, progression, and dissemination of cancer.[135,137,145-147] The thought that MVs might be exploited as potential therapeutic targets in the treatment of cancer boosts interest in them given their capacity to package and securely deliver a variety of cargo, including nucleic acids, to the target cell as well as their function in intercellular communication.[148-152] In particular, MSCs can be treated with chemotherapeutics and the treatment process can be shortened with drug-carrying EVs released by them. [153,154]

Stem cells have a significant role in cancer in addition to microvesicles. It has been found that MVs are effective in protecting cancer stem cells, which are known to have a significant impact on the development of tumors in liver cancer, one of the malignant malignancies. Additionally, it is believed that cell-to-cell pathways created by EVs safeguard the stem cell lineages of ESCs.[136,137]

Even though there have been several studies on cancer, there is still a great demand for investigation into metastasis and other topics. The importance of gaining MVs is rising at this stage. Patients’ serum may simply be used to isolate MVs, and urine and plasma can also be used in this way.[137,155,156] A liquid biopsy of cancerous tissue can also yield MVs, but this approach is still relatively new.[153,157,158]

In conclusion, the molecular makeup of exosomes and MVs and the kind of stem cell or tissue they originate from the impact their function. The capacity of EV to biodistribution, pass the blood-brain barrier and reflect the molecular characteristics of the cells from which they arise has drawn attention in pharmacology and translational medicine. Stem cells are prominent in drug delivery by loading drugs through EVs and thus play a role in the treatment of different types of cancer; at the same time, EVs have been noted to cause positive clinical effects in the treatment of neurodegenerative diseases, antitumoral effects, regeneration and repair processes by loading miRNA and protein types. As biomarkers, EVs are used in the early detection and diagnosis of diseases and even the gender determination of the fetus. As a result, it is thought that the therapeutic effects of EVs in cancer will also lead to vaccine studies. Although EV-stem cell studies offer many promising developments, their negative effects such as creating a microenvironment supporting metastasis, promoting lesions, inducing apoptosis, and immune suppressor behaviors should also be considered.

Cite this article as: Savcı D, Işık G, Hızlıok S, Erbaş O. Exosomes and Microvesicles. JEB Med Sci 2024;5(1):19-32.

Conflict of Interest

The authors declared no conflicts of interest with respect to the authorship and/or publication of this article.

Financial Disclosure

The authors received no financial support for the research and/or authorship of this article.

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