Molecular characterisation of plasma membrane-derived vesicles
Antwi-Baffour Journal of Biomedical Science
Molecular characterisation of plasma membrane-derived vesicles
Samuel S. Antwi-Baffour 0
0 Department of Medical Laboratory Sciences, School of Biomedical and Allied Health Sciences, College of Health Sciences, University of Ghana , P. O. Box KB 143,Korle-Bu, Accra , Ghana
Plasma membrane-derived vesicles (PMVs) are released into circulation in response to normal and stress/pathogenic conditions. They are of tremendous significance for the prediction, diagnosis, and observation of the therapeutic success of many diseases. Knowledge of their molecular characteristics and therefore functional properties would contribute to a better understanding of the pathological mechanisms leading to various diseases in which their levels are raised. The review aims at outlining and discussing the molecular characteristics of PMVs in order to bring to the fore some aspects/characteristics of PMVs that will assist the scientific community to properly understand the role of PMVs in various physiological and pathological processes. The review covers PMVs characterisation and discusses how distinct they are from exosomes and endosomes. Also, methods of PMVs analysis, importance of proper PMV level estimation/characterisation, PMVs and their constituents as well as their therapeutic significance are discussed. The review concludes by drawing attention to the importance of further study into the functions of the characteristics discussed which will lead to understanding the general role of PMVs both in health and in disease states.
PMVs; Flow-cytometry; Characterisation; Proteins; Membrane; Microparticles
Plasma membrane-derived vesicles (PMVs), also referred
to as microparticles (MPs), microvesicles (MVs) or more
rarely ectosomes, are sub-membrane fragments shed
from the plasma membrane of cells during cell growth,
activation, proliferation, senescence, apoptosis and when
stimulated . Many cell types including leukocytes,
platelets and endothelial cells release these small
membrane fragments. PMVs therefore originate from cells
which are surrounded by plasma membranes .
The presence of basal levels of PMVs is common in
healthy individuals and an increase in their release
although a controlled event, is a hallmark of cellular
alteration . Therefore, pharmacological modulation of
circulating PMV concentrations could become a major
therapeutic target in the future . Various publications
have discussed PMVs in relation to various disease in
which their levels are raised . This suggest the
importance of PMVs as a key role player in various cell
processes rather than just inert bi-products of cellular
activation and therefore in disease pathogenesis .
PMVs shed from both normal and cancerous cells may
serve as a means of intercellular communication as they
carry proteins, lipids and nucleic acids derived from the
host cell [6, 7]. Their isolation and analysis from blood
samples has the potential to provide information about
the state and progression of malignancy in terms of cancer
[6, 7]. PMVs are also likely to prove of great clinical
importance as biomarkers for a variety of disease states in
other words of potential diagnostic value as well as a
therapeutic tool . However, a standardized protocol for
isolation has not yet been agreed upon . It is often
unclear what the content of the isolates are and whether the
isolated PMVs, were present in vivo or whether they were
created during the isolation procedure . Vesicular
structures, which are sized from 1 μm down to 50 nm, are
present in isolates of many body fluids. Isolates of PMVs
sometimes contain different populations that differ in size
and shape, which indicates that methods of isolation and
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determination of the number of PMVs in the peripheral
blood need to be elaborated and improved upon [9, 10].
A difficulty in the study of PMVs has been comparing
results between laboratories. Since PMVs exist as
heterogeneous species and express several different cell surface
markers, which can vary according to the cell of origin
and depending on the stimulus that caused their release,
it is unlikely that any single marker will efficiently label
all PMVs . Thus, the need for precise and
reproducible quantification is necessary if PMVs are to be a
reliable marker for diagnosis. To date, the measurement
and detection of levels of PMVs in various conditions
have not translated into therapeutic or diagnostic
strategies in the management of disease conditions in which
they have been shown to be relevant [9, 10]. However
they have helped to shift the understanding of the
pathophysiological mechanisms of several diseases such as
sickle cell and thrombotic thrombocytopenic purpura
. The detection of chronically elevated levels of
circulating PMVs in patients with sickle cell disease provides
an insight into the chronic endothelial attack that
characterises this condition, and may provide an important
tool in measuring the protective effects of therapeutic
interventions in an early and non-invasive manner .
Exosomes, endosomes and PMVs are distinct
Exosomes were described in the early 1980s as
5’-nucleotidase activity-containing vesicles and to range in
size from 30 to 90 nm (Fig. 1). In contrast to PMVs,
exosomes are preformed membrane vesicles, which are stored
in cellular compartments named multivesicular bodies
(MVB), and secreted when the MVBs fuse with the cell
membrane . Many eukaryotic cells release vesicles like
exosomes spontaneously or under appropriate
stimulation. They were shown to be involved in the removal of
the transferrin receptor from maturing reticulocytes by
‘invagination’ blebbing. These blebs (called endosomes)
pinch off very small (30–90 nm) ‘intraluminal’ vesicles
[12, 13]. These endosomes containing ‘intraluminal
vesicles’ are stored in the MVB and once the ‘intraluminal’
vesicles become secreted, i.e. after membrane fusion of
MVB and the surrounding plasma membrane - they are
called exosomes .
Endosomes are therefore membrane-bound vesicles,
formed via a complex family of processes collectively
known as endocytosis, and found in the cytoplasm of
virtually every animal cell. The basic mechanism of
endocytosis is the reverse of what occurs during
exocytosis or cellular secretion. It involves the invagination of
a cell’s plasma membrane to surround macromolecules
or other matter diffusing through the extracellular fluid
. The encircled foreign material is then brought into
the cell, and following a pinching-off of the membrane
(termed budding), is released into the cytoplasm in a
sac-like vesicle .
PMVs and exosomes/endosomes are morphologically
distinct vesicle populations . A recent study revealed
that protease-containing PMVs shed from tumor-cell
lines appear to be rather heterogeneous in size and
shape as opposed to exosomes, which are a more
uniform population of vesicles . The same study also
showed that microvesicles sediment at lower speeds
relative to exosomes, which pellet at 100,000 g. Also,
exosomes from red blood cells contain the transferrin
Fig. 1 Schematic presentation of modes of emission of PMVs vs. Exosomes. This figure shows the formation of an endosome by invagination. By
inward blebbing of the endosomal membrane, intraluminal vesicles are formed. Endosomes containing intraluminal vesicles are called multivesicular
bodies (MVBs). Cells release the contents of their MVB when the membrane of the MVB fuses with the plasma membrane and in contrast
to exosomes, PMVs are formed by major structural rearrangements of the cytoskeleton and are ‘budded’ off from the outer cell membrane
receptor found in reticulocytes, which is absent in
mature erythrocytes and are therefore not seen in PMVs.
Again, dendritic cell-derived exosomes express MHC I,
MHC II, and costimulatory molecules and have been
proven to be able to induce and enhance
antigenspecific T cell responses in vivo. Exosomes can also be
released into urine by the kidneys, and their detection
might serve as a diagnostic tool. In fact, urinary
exosomes may be useful as treatment response markers in
prostate cancer. Furthermore, exosomes are remarkably
more stable in bodily fluids than PMVs, strengthening
their case as good reservoirs for disease biomarkers.
Methods of PMV analysis
Although several methods for analysis of PMVs have
been reported, conventional Flow-cytometry and ELISA
are the most widely adopted . Florescence Activated
Cell sorting (FACS) (flow cytometry method) is the
commonly used method for analysing the number, size, and
properties of PMVs [17, 18]. With flow cytometry, it is
possible to determine the number of events (forward
scattering) and the density of the events (side scattering)
allowing quantification of the PMVs identified in each
case . However, because of the sizes, detection is
limited by ‘noise’ in the flow cytometer as conventional
FACS instruments are mostly designed to measure cells,
which are 10-to 100-fold greater in diameter than PMVs
[17, 18]. Antibodies to cell surface molecules allow the
identification of specific PMV subpopulations as in the
case of FACS analysis of cells, but because of the size of
PMVs, the amount of any surface marker is drastically
reduced in comparison to intact cells, thereby limiting
detection . In addition to proteins, the presence of
phosphatidylserine on the outer leaflet of the PMV plasma
membrane allows binding of annexin V, which is also used
to identify and enumerate the PMVs via FACS [17, 18].
Some investigators identify PMVs with a forward angle
light scatter smaller than the 1-1.5 μm latex beads most
instruments use as an internal standard whilst others
also employ a lower size limit of 100 nm and any
particles detected by the flow cytometer under this size are
not considered true PMVs leading to underestimation
. Using the upper size (1.5um) limit criteria could
also lead to an underestimation of true PMV numbers as
some investigators consider PMVs to be up to 2 μm in
size [17, 18]. As different flow cytometers are used to
analyse PMVs, it may be possible that the flow cytometer
technique and the way they are used in terms of
interpreting results could have a greater part to play in PMV
analysis. For example, laser alignments can differ and
different instruments capture PMVs in different ways.
Bench top flow cytometers such as FACSCalibur have a
fixed laser in constant alignment needing no human
adjustment whereas FACSVantage SE uses removable
lasers, which need manual adjustment .
For capture, FACSCalibur systems use a device
referred to as a catcher tube to sort PMVs. The catcher
moves in and out of the sample stream to collect a
population of desired PMVs, with a maximum rate of
300 per second [17–19]. The laser alignment and stream
velocity are fixed, making the time it takes for PMVs to
travel from the laser to the catcher tube constant.
Conversely, the FACSVantage SE isolates a cell of interest by
vibrating the stream. The sample stream vibrates and
separates the sample into drops, with the distance
between drops being fixed. When the sheath velocity and
the vibration speed are constant, the pattern of drop
formation is fixed, allowing the distance between drops to
be calculated followed by PMV isolation . Even
though no known studies appear to have used
FACSVantage SE to analyse PMVs, highlighting such differences
alerts investigators prior to undertaking experiments
that flow technology and its incorrect implementation
could lead to erroneous results.
An alternative method for identifying PMVs involves
binding assays in a solid-phase or microtitre plate format
. In this approach, antibodies to cell surface
molecules can capture particles for subsequent detection by
another antibody or a functional assay . While such
assays can assess the total amount of PMV-related
material in a specimen, they cannot provide information on
the number or size of particles [20, 22].
PMVs analysis can be impacted not only by type of
assay, but also by the manner in which sample (blood) is
collected and processed, including sampling site, needle
diameter, centrifugation, re-suspension and washing of the
isolated PMV pellet [18, 19]. Also it is important to ensure
that platelets are removed, since they interfere with PMV
detection, giving false positive signals . At present,
PMV analysis is constrained by lack of standardized PMV
Importance of proper PMV level estimation/
PMVs constitute a dynamic circulating storage pool
by themselves, able to induce vascular responses to
proapoptotic, inflammatory, or thrombotic stimuli .
Therefore, the clinical background or outcome associated
with elevated PMV levels or accelerated clearance should
be taken into close consideration in deciphering their
multiple effects . For instance, misleading
quantification or phenotypes could result from accelerated
degradation by secretory phospholipase A2, interactions with the
vascular wall, or trapping in cell–cell aggregates or within
the thrombus .
An additional complexity recently observed was that
circulating PMVs may bear antigens from different
cellular origin, pointing to multiple transcellular
PMVsmediated exchanges . Antigens specifically expressed
during cell activation could prove useful in identifying
the various pathways of PMV release and discriminating
underlying pathologies and associated damages . The
circulating levels of PMVs may have a direct
pathophysiological role in the development of several diseases
and thus quantification/characterisation of PMVs may
serve as an early diagnostic screening tool for those
diseases . PMVs are implicated in cardiovascular diseases
as their composition and microRNA content are specific
signatures of cellular activation and injury . Also, the
potential of PMVs in transferring biological information
to neighbouring cells have given them an active role in
inflammatory diseases, including atherosclerosis and
angiogenesis. For example, they promote joint
inflammation in rheumatoid arthritis by transporting
proinflammatory cytokine interleukin-1 . PMVs have again,
been implicated in the process of remarkable anti-tumor
reversal effect in cancer, tumor immune suppression,
metastasis, tumor-stroma interactions and angiogenesis
along with having a primary role in tissue regeneration
. Their levels have also been seen to be raised in
thalassaemia and sickle cell diseases [11, 30].
Although increased PMVs, compared to healthy
controls, have been associated with increased severity of
diseases such as β-thallasaemia and Sickle Cell [11, 19],
there is only evidence to support a circumstantial
association at this stage as many studies are only quantitative
and does not look much into the specific role of PMVs
in disease states and their contribution to pathogenesis
of the diseases [11, 30]. Hopefully, more experiments
that focus on the function of PMVs in disease states will
emerge in the future to give concrete evidence of the
role PMVs play in diseases. A recent study on
erythrocyte PMVs and other circulating PMV subtypes in sickle
cell disease found that they did not detect Tissue Factor
+ (TF+) PMVs, monocyte (CD14+) PMVs (MPMVs) or
endothelial cell (CD 144+, CD146+, CD62E+) PMVs
(EPMVs) in their samples . Contrastingly, another
group did not just detect TF+ PMVs, EPMVs and
MPMVs, but concluded that the observed increase gave
weight to the hypothesis that sickle cell disease is an
inflammatory state with endothelial cell and monocyte
activation along with abnormal vessel wall activity .
Unsurprisingly, both groups used different methods for
PMV isolation , but as seen in Table 1, PMVs
generally carry proteins (surface markers) characteristic of
their parent cell.
PMVs and their constituents
Owing to the plasticity of the lateral organization of the
plasma membrane into raft domains, known to segregate
particular proteins and lipid species, a given stimulus
Table 1 A table showing the cell of origin of PMVs and their
characteristic surface markers
can be expected to elicit the release of PMVs . These
PMVs may carry microRNA, mRNA, numerous
membrane proteins, bioactive lipids and cytoplasmic
constituents, characteristic of their parental cells which are
implicated in a variety of fundamental processes .
PMVs also carry the majority of non-conventionally
secreted proteins released into culture supernatants, and
potentially carry them within the PMVs themselves .
This help in the export of proteins lacking a signal
peptide as an alternative to conventional protein
export. Amongst these, epimorphin, fibroblast growth factor
1 and 2 (FGF-1 and FGF-2), macrophage migration
inhibitory factor (MIF) and galectin 3 (Gal-3) are all transported
to the plasma membrane via the adenosine triphosphate
cassette transport channel (ABCA1) needed for the release
of PMVs or by exocytosis of exosomes .
The potential of PMVs to transmit proteins between
cells play an important part in intercellular
communication and the maintenance of homeostasis under
physiological conditions, or initiation of deleterious processes in
the case of excess or when carrying pathogenic
constituents . Most recently PMVs derived from embryonic
stem cells (ESC) were found to carry Wnt-3, which is
involved in haematopoietic differentiation and such PMVs
were shown to reprogramme haematopoietic progenitor
cells . The ability of cells stimulated by sublytic
complement to release PMVs has been demonstrated by work
showing that these PMVs carry surface molecules such as
TGF-β1 with which they in turn cause promonocytic/
monocytic cells to differentiate along the monocyte/
macrophage differentiation lineage (Table 2) . PMVs
can therefore be considered a disseminated storage pool
of bioactive effectors, the nature and proportion of the
latter accounting for duality, more particularly evidenced in
vascular disease, inflammation, and immunity .
Phenotypical differences in PMVs from normal vs.
PMVs in cancer patients were first documented in 1978,
when they were identified in cultures of spleen nodules
and lymph nodes of a male patient with Hodgkin disease
. The amount of PMVs shed by tumor cells has been
shown to correlate with their invasiveness both in vitro
Table 2 A table with selected surface protein markers that
enrich PMVs from circulation and the diseases they code for
EGFR, EGFRvIII, PDPN, IDH1
CD63 and caveolin 1
and in vivo and there are phenotypical differences in
PMVs isolated from normal as against cancer patients
[38, 39]. Isolated PMVs from cancer patients tend to
have a distinct molecular profile from that isolated from
normal controls: in addition to conventional PMV
markers (LAMP1 or MHC-I), they contain
membraneassociated transforming growth factor-β1, FasL, MICA/
MICB and myeloid blasts markers CD34, CD33 and
CD117. These PMVs have the ability to decrease natural
killer cell cytotoxicity and down-regulate the expression
of NKG2-D in normal natural killer cells. In a recent
study by Miroslaw J. Szczepanski et al, the PMVs
fractions obtained from sera of AML patients had
significantly greater protein content (75 ± 12 μg protein/mL)
than those isolated from sera of normal controls (1.2 ±
0.4 mg protein/mL) . Generally, PMVs derived from
human cancer cells have received a good deal of
attention because of their ability to participate in the
horizontal transfer of signalling proteins between cancer cells
and contribute to their invasive activity.
Therapeutic significance of PMVs
Taking into consideration the ability of PMVs to
transmit information between cells through proteins, lipids
and nucleic acids (DNA, mRNA, microRNA) transfer
and on their ability to create a communication network
between cells, it is plausible that they could serve as
tools with veritable therapeutic potential . In this
context, ligands carried by PMVs can directly interact
with receptors in target cells and induce signal
transduction. In addition, membranes of PMVs can fuse with the
plasma membrane of target cells, leading to the transfer
of membrane components and delivery of PMV
cytoplasmic content . This can result in the activation or
inhibition of intracellular pathways of target cells or in
the modification of their phenotype . Through their
action, PMVs have a great effect on angiogenesis. This is
because they can induce the production and transfer
of proangiogenic or antiangiogenic factors, modify
endothelial cell function concerning adhesion,
migration, or proliferation - the 3 key steps in the
formation of new vessels (angiogenesis) and subsequently
promote cancer progression as well as the
pathogenesis of other diseases [44, 45]. Cancer progression is
dependent on abnormal angiogenesis, in particular,
when exacerbated neovascularization forms new blood
vessels that supply adequate nutrients, oxygen, and
growth factors to facilitate the growth of the tumor
and metastasis development . Tumor angiogenesis
therefore results from an imbalance of proangiogenic
and antiangiogenic factors released from tumor cells
and circulated by PMVs .
Tumour cells can produce PMVs constitutively
without any apparent need for stimulus but vesiculation
can be increased by stress, including exposure to
chemotherapeutic drugs and heat . Cancer patients
not only exhibit tumour -derived PMVs but also high
levels of platelet-derived PMVs and the hypercoagulation
typically observed with malignancy can be attributed in
part to these procoagulant PMVs . It has also been
postulated that tumour cells can evade apoptosis by
releasing PMVs that contain caspase-3, thereby preventing
its intracellular accumulation . Inhibition of this
release has been shown to result in caspase-3 accumulation
and subsequent apoptosis . Again, metastatic cancer
cells can degrade the extracellular matrix (ECM) through
the transfer of surface proteases such as matrix
metalloproteinases (MMPs), urokinase-type plasminogen
activator (uPA) and cathepsins via PMVs . Tumour cells
have been shown to release PMVs rich in MMPs and uPA,
which degrade the ECM, allowing for cell invasion .
There is also the PMVs assisted dissemination of
cancer multidrug resistance, by which drug sensitive
cells can acquire the resistance phenotype via
longrange communication [53, 54]. Multi-drug resistance
can also come from the expulsion of therapeutic
drugs from tumor cells through PMVs release . In
a demonstration, tumor cells treated with doxyrubicin
accumulated and released the drug in shed PMVs,
implying PMV shedding as a drug-efflux mechanism involved in
drug resistance . By virtue of their ability to harness
select bioactive molecules and propagate the horizontal
transfer of these cargoes, shed PMVs can have an
enormous impact on tumor growth, survival and spread .
PMVs have also been implicated in the pathogenesis of
thrombosis, diabetes, inflammation, atherosclerosis and
vascular cell proliferation. From evidences, as outlined
above, it implies that PMVs are important mediators of
cell communication and vital components of the tumor
microenvironment niche as well as in the pathogenesis of
Since knowing the characteristics of PMVs are so
important, there have been various studies undertaken in
this regard in trying to come out with a set of standards
by which they can be measured, such as the appropriate
centrifuge speed, sample removal and storage, flow
cytometry (types and mode of operation) amongst others
to offset the various pre-analytical variables known. It is
also important to note that flow technology and its
incorrect implementation could lead to result variability as
well and therefore requires standardisation. This review
discussed typical characteristics of PMVs and their
therapeutic significance which could be a bench mark
for PMVs analysis as we move to the era of using PMVs
in diagnosing diseases and as a therapeutic tool. It is
important that further studies are carried out to ascertain
the functions of the characteristics discussed which will
lead to understanding the general role of PMVs both in
health and disease states.
ABCA1: ATP-binding cassette transporter A1; AML: Acute myelocytic leukaemia;
CD: Cluster of differentiation; DNA: Deoxyribonucleic acid; ECM: Extracellular
matrix; ELISA: Enzyme linked immunosorbent assay; ESC: Embryonic stem cell;
FACS: Fluorescence activated cell sorting; FasL: Fas ligand; FGF: fibroblast
growth factor; Gal-3: Galectin - 3; LAMP-1: Lysosomal-associated membrane
protein 1; MHC: Major histocompatibility complex; MICA: MHC class I
polypeptide-related sequence A; MICB: MHC class I polypeptide-related
sequence B; MIF: Macrophage migration inhibitory factor; MMPs: Matrix
metalloproteinases; MPs: Microparticles; MVB: Multivesicular bodies;
MVs: Microvesicles; NKG2-D: Natural Killer group 2 - D; PMVs: Plasma
Membrane-derived Vesicles; RNA: Ribonucleic acid; TF: Tissue Factor;
TGF – β1: Tissue Growth Factor – beta 1; uPA: Urokinase-type
I am grateful to Prof. Patrick F. Ayeh-Kumi the Dean of the School of Biomedical
and Allied Health Sciences (SBAHS), College of Health Sciences, University of
Ghana for his assistance in the development and drafting of the manuscript. I
will also like to acknowledge the following persons for their help in putting the
manuscript together: Dr. I.A Bello, Dr. Charles Brown and Mr. David Nana Adjei
all of SBAHS.
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