Extracellular vesicles in circulation

Extracellular vesicles in circulation
Extracellular vesicles (EVs) are membrane limited vesicles, originating from cells and located in the
extracellular space(1). The term extracellular vesicles comprises exosomes, microvesicles (MV) and
apoptopic bodies (AB) and is chosen as a step towards standardization of terminology, as currently
many more exotic terms are in use, such as oncosomes, minisomes, microparticles nanovesicles etc.
The differences between exosomes, MVs and ABs lie in their size and origin. Exosomes are
membrane vesicles with a diameter of 40–100 nm that are secreted by many cell types into the
extracellular milieu. Before secretion, they are present as the internal vesicles of an endosomal
compartment, the multivesicular body (MVB) and are released upon exocytic fusion of this organelle
with the plasma membrane(2). MVs are shed from the plasma membrane and have a diameter of
100-1000 nm(3) and ABs have a diameter of >1 µm and are formed by large blebs which occur during
programmed cell death(4).
Figure 1: Schematic representation of extracellular vesicles by Abcam®. Exosomes, exosome-like vesicle, microvesicles and
membrane particles are membrane limited vesicles, originating from the cell and located in the extracellular space. Despite
different origins inside the cell, they are all EVs and are able to act as carriers of information between cells.
EVs is a synonym of membrane vesicles proposed by Thery et al, but EVs emphasizes the
extracellular nature of the vesicles and this denomination is easier to distinguish from intracellular
membrane vesicles(5). The emergence and growth of this bourgeoning research field is characterized
by the recent formation of the International Society of Extracellular Vesicles (ISEV).
Secretion of EVs differs between vesicle type and depends on the cell type. For example epsteinBarrvirus (EBV)-transformed B cell lines, primary dendritic cell (DC) cultures and tumor cells show
constitutive secretion of EVs(6)., while other cells, like presynaptic nerve terminals(7) and
erythrocytes(8) need a stimulus, like an increase in internal Ca2+, to stimulate exosome secretion.
Another trigger for EVs is induced stress, such as hypoxia and shear stress, which often is regulated
by the P53 protein(9). These factors do not only activate a cell to secrete EVs, but can also influence
the molecular content of the vesicles.
So why are these EVs so interesting? Recent studies indicate that these vesicles are used for
communication between cells(10). This communication includes transmission of surface-membrane
lipids, proteins and horizontal transfer of proteins and RNAs between cells. Transfer of these
biomolecules can have profound effects on the activity of the acceptor cell. For example a number of
studies have suggested a functional role for transfer of vesicles in the proliferation of tumors (11).
Summarizing, functional transfer of EVs is important for the general homeostasis of cell populations
in physiological conditions, but can also be a factor that aggravates pathological conditions(12).
Since it is shown that EVs can act as carriers of RNA and proteins, it is very interesting to investigate
their potential as drug delivery carriers to improve the efficacy of drugs. This technique could be
used to enhance currently available pharmaceuticals, but can also be used to deliver potential new
drugs, which are currently not suitable because of their fast clearance rate. Besides their potential as
drug carriers, EVs can play an important role in diagnostic applications. It appears that every cell type
has its own unique ‘fingerprint’ of protein and RNA distribution in their EVs and that this fingerprint
can change depending on the (pathological) milieu. By screening the blood for elevated or altered
levels of EVs and determining their origin it is possible to diagnose diseases in an early stage(13).
Furthermore an interesting subject is to disrupt communication by EVs in order to alter disease
outcome (14). These three topics are the drivers of this upcoming research field and in this review a
brief summary of the current research and opportunities will be given of EVs. Although EVs are found
in all body fluids(15), this review will focus on EVs in circulation, because it is the most prevalent fluid
in the body and has the most therapeutical relevance.
Vesicles in circulation
In the bloodstream EVs from four different cell types mainly are present, namely red blood cells,
endothelial cells (ECs) leukocytes, and platelets.
Type of EVs
Platelet(17, 18)
237 × 106/L
Specific Markers
Apoptopic blebs
Glycoprotein IIb/IIIa
64 × 106/L
Apoptopic blebs
E-selectin and Vascular
Endothelial Cadherin
46 × 106/L
Apoptopic blebs
Phosphatase Receptor
(21, 22)
28 × 106/L
Glycophorin A
Table 1: Overview of the cell lines and their vesicles which are mainly present in the bloodstream.
HSP 70
cd 63
Effects attributed to these EVs are activation of the plasma coagulation system and modulation of
the vascular tone. Furthermore several studies suggest prothrombotic, pro-inflammatory and proangiogenic activities(23).
Already in 1967, Wolf showed that EVs from platelets, named platelet dust, had the same effect as
whole activated platelets(24). Platelet EVs (PEVs) are well known for their procoagulant activity,
which is increased by the presence of anionic phospholipids, particularly phosphatidylserine (PS) and
tissue factor (TF)(25). PS is a phospholipid which normally resides at the inner layer of the cell
membrane. It is capable to form an electrostatic interaction between positively ɣ-carboxyglutamic
acid (GLA) domains in clotting proteins like coagulation factor VIIa (26).TF is a transmembrane
glycoprotein which serves as a receptor and catalytic cofactor for coagulation factor VIIa and this
complex is the major trigger of the coagulation protease cascade(27). TF also plays an important role
in tumor survival and progression. It is shed into circulation on tumor derived EVs and promotes
intravasation and orchestrates thrombin-, platelet- and fibrin-dependent tumor progression(28).
PEVs have also been shown to contribute to vascular contractions, by metabolizing arachidonic acid
to tritiated TXB2. Furthermore a study showed that arachidonic acid is able to transfer from ECs to
PEVs. Without PEVs, no TXB2 was formed and this is likely because cyclooxygenase is needed, which
is present in the EVs(29).
Endothelial cells
Besides PEVs, lots of research is focused on endothelial-derived EVs (EEVs) and especially their
angiogenic properties. Angiogenesis plays an important role in cancer(30) and many other diseases, it
is a tightly regulated process that involves endothelial cell survival, proliferation, migration,
differentiation and morphological changes, such as tube formation. EEVs have been shown to
influence vascular network formation of human umbilical vein endothelial cells (HUVECs). Effects
included decreases in total capillary length, number of meshes and the amount of branching points
and an increase in mesh area(31). EEVs also affect vascular tone by modulating the NO and PGI2
pathway (32). Some effects of EEVs seem to be dose-dependent as is shown on HUVEC
invasiveness(33) and on plasminogen activation(34), in which the latter showed a saturable effect,
where high dosages lead to effect inhibition. A clear review about EEVs and their role is written by
Leroyer et al(35).
Vascular tone is also affected by EVs originating from lymphocytes(36). Besides influencing
endothelial cells, EVs from DCs have potent immunoregulatory properties. EVs from immature DCs
have shown immunosuppressive therapeutic effects(37), while EVs from mature DCs (DEVs) can be
used as immunostimulating vaccines(38). After capturing antigens, DCs incorporate MHC-antigenic
peptide complexes in DEVs. After migration to regional lymph nodes, DEVs can present the antigenpeptide complexes to immature DCs, which thereby acquire the ability to stimulate CD4+ and CD8+ T
As is shown in table 1 erythrocytes mostly lack the formation of exosomes and ABs. Their
predecessor, reticulocytes, do secrete exosomes, since these EVs are part of the remodeling process,
that accounts for the volume shrink, which occurs during erythrocyte maturation(39). MVs are shed
from erythrocytes and have, like PEVs, procoagulant activity because of the presence of PS. Besides
being procoagulant, it is hypothesized that these EVs are able to transfer their PS to healthy
nucleated cells which leads to ‘falsely marked’ apoptotic cells(40). Furthermore it is hypothesized
that erythrocyte EVs may not only serve to remove modified hemoglobin, but may also help to
eliminate premature removal molecules from otherwise functional erythrocytes(41). Besides vesicle
secretion intended for clearance, studies have shown miRNA in erythrocytes(42). Given the general
view of erythrocytes lacking protein translation this could imply miRNA regulation through
erythrocyte EVs.
RNA’s in EVs
One of the most intriguing aspects of EVs is their ability to functionally transfer miRNAs and mRNA.
The first evidence of encapsulation of mRNAs into EVs was reported by Valadi et al(43) who stated
that mast cell exosomes containing mRNA and miRNA from mouse, are transferred to both human
and other murine cell lines. After this transferral, new murine proteins were found in the recipient
cells, demonstrating that exosomal mRNA can be passed into other cells. Thus, it was established
that the message delivered on from the donor cells to the neighboring cells via exosomes, does not
simply mirror the transcriptional status of the donor cell.
Another study identified and defined the profile of exosome-encapsulated miRNAs circulating in the
plasma of healthy individuals, where they found that 37 miRNAs were expressed at significantly
different levels between plasma EVs and peripheral blood mononuclear cells, and that the majority
of these miRNAs were predicted to regulate cellular differentiation of blood cells and metabolic
pathways(44). A third study showed that EVs from human bone marrow-derived mesenchymal stem
cells and stem cell residing in the liver contain selective patterns of expression of miRNAs, which
could be transferred to target cells, where they down regulated their targets(45). A table with
examples of miRNAs involved in cell-to-cell communication is compiled by Redis et al(46).
Endothelial cells to vascular
Rice plant cells to small
intestinal epithelial cells to LDLRAP1
human hepatoma cells
Monocyte leukemia cells to
HMEC-1 cells
Tumor-associated macrophages
to breast cancer cells (SKBR3,
EBV-positive nasopharyngeal
carcinoma cells to endothelial
cells (HUVECs)
Activation of tissue repair and
Decreased levels of LDL (lowdensity lipoprotein)
Promote cell migration
The Mef/βRegulation of invasiveness of
breast cancer cells
CXCL11 and Repression
immunostimulatory genes
Table 2: Examples of miRNAs involved in cell-to-cell communication(46).
MiRNAs are known to regulate the expression of hundreds of target mRNAs, thereby modulating a
large number of processes, some of which are described in the table (e.g. angiogenesis, apoptosis
and tumor growth). These and other miRNAs might be used to knock down or over express certain
proteins in target cells to achieve therapeutic effects, for example by over expressing miR-15a/16-1,
to inhibit leukemia cell growth(52).
Binding of EVs to their target cells
EVs are able to bind and fuse to their target cells. Binding of monocyte EVs to activated platelets
occurs through a molecular bridge. After activation, platelets translocate a vascular cell adhesion
molecule, P-selectin, to the plasma membrane. P-selectin is a member of the selectin family of
adhesion molecules and recognizes a lineage-specific carbohydrate as well as a protein component.
P-selectin glycoprotein ligand-1 (PSGL-1) is P-selectins dominant ligand in vivo(53) and this ligand is
localized on EVs. Besides a docking function, P-selectin has also been shown to have a signaling
function, which up regulates TF expression(54).
Molecules present on blood-borne exosomes are lactadherin, CD11a, CD54, PS and tetraspanins CD9
and CD81. These molecules can bind to αv/β3 integrin, CD11a and CD54 on DCs, followed by
internalization of the EVs(55). After internalization, exosomes are sorted into recycling endosomes
and then through late endosomes/lysosomes. This leads to irreversible maturation, Antigen
presentation and ends with apoptotic cell death(56). Immature DCs are better capable to endocytose
vesicles than mature DCs. Besides DCs, also specialized splenic macrophages and kupffer cells are
able to bind blood-borne exosomes.
Target modification
Besides natural occurring ligands, studies have shown it is possible to induce targeting by modifying
the EVs. A recently developed technique is to fuse targeting peptides to the extra-exosomal N
terminus of a protein found abundantly in exosomal membranes(57). In this study they fused three
different peptides to murine lysosome-associated membrane protein 2b (Lamp2b), which is a type 1
integral membrane protein with a large luminal domain which is heavily glycosylated(58). The
peptides used were a FLAG epitope, rabies viral glycoprotein (RVG) peptide and a muscle-specific
peptide (MSP).
The first peptide fused to Lamp2b was FLAG. This is a small peptide consisting of 8 nucleotides which
can be targeted with commercial available antibodies(59). This peptide was used to determine the
topology of Lamp2b and a pulldown assay showed the epitope is localized to the external exosomal
surface, but FLA is not used as a targeting peptide. The second peptide, The 29- amino-acid RVG
peptide, specifically binds to the acetylcholine receptor expressed by neuronal cells and provides a
non invasive approach for the delivery across the blood-brain barrier(60) and the third peptide, MSP,
is a heptapeptide (ASSLNIA) which is identified by in vivo phage display and which has an increased
binding affinity for skeletal and cardiac muscle(61). Besides (ASSLNIA), even more specific MSPs have
been found, which can target for example specifically laryngeal muscle surface receptors(62).
Other targeting peptides are being investigated as well. Many viruses are specialized in binding and
transducing information to host cells. Proteins which are responsible for this effect can be
incorporated in vesicles and this is called pseudotyping. An often used protein is the spike
glycoprotein of the vesicular stomatitis virus (VSV-G). Over expression of VSV-G in human cells
induces the release of fusogenic vesicles named gesicles. These gesicles have a number of
similarities, like the sites of budding(63), the molecular content and the targeting signals(64), with
EVs. Gesicle formation is , at its core, a form of EV biogenesis(65).
VSV-G is able to attach to a broad range of host cells(66) and these gesicles incorporate proteins
from their producer cells and are able to deliver them to recipient cells. This protein delivery
technique has been used to transfer membrane, cytoplasmic and nuclear proteins in precise amounts
(67). Nuclear proteins were efficient in activating transcription, but for increased performances a
fusion to the H-RAS c terminus was needed, since this motif is farnelysed in vivo and redirects
proteins to the cell membrane.
Another study which used over expression of VSV-G, tried to include a second targeting peptide,
namely biotin acceptorpeptide- transmembrane domain (BAP-TM). This genetically engineered
receptor allows binding to biotinylated ligands via a streptavidin bridge(68). After incubation with
streptavidin-conjugated magnetic beads, it is possible to attract exosomes with a magnetic field,
which can be used for specific targeting(69). These and other techniques could be used to target
drug loaded vesicles to their destinations, which will lead to great improvements to their
Only recently have we learned that EVs are more than waste and cell debris. EVs are continuously
present in the bloodstream, where they provide communication between cells and help to maintain
homeostasis. Besides maintaining homeostasis they also play an important role in pathological
states and the development of cancer. Here we discussed the most important and prevalent EVs in
circulation. Evs from platelets, ECs, leukocytes and erythrocytes are always present and this should
be taken in mind when developing experimental, diagnostic or therapeutic tests. Several important
effects of EVs originating from these cell types are highlighted and their RNA content is discussed.
Furthermore, target binding is elucidated and target modification is discussed. By fusing targeting
proteins or pseudotyping, the binding of EVs to their target cells can be improved. These techniques
can further improve the delivery of miRNAs/mRNAs or therapeutic proteins. All in all this upcoming
research field is very promising in understanding the cancer microenvironment and can give rise to
many breakthroughs in the field of diagnostics, gene silencing and drug delivery.
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