Review
This Review is part of a thematic series on Exosomes in Cardiovascular Disease, which includes the following articles:
Role of Exosomes in Myocardial Remodeling
Exosomes: Nanoparticles Involved in Cardioprotection?
Exosomes and Cardiac Repair After Myocardial Infarction
Microvesicles as Cell–Cell Messengers in Cardiovascular Diseases
Ali J. Marian, Editor
Role of Exosomes in Myocardial Remodeling
Anders Waldenström, Gunnar Ronquist
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Abstract: Exosomes are nanovesicles released from cells through exocytosis and are known to be mediators of
proximal as well as distant cell-to-cell signaling. They are surrounded by a classical bilayered membrane with an
exceptionally high cholesterol/phospholipid ratio. Exosomes were first described in 1977, then named prostasomes,
and in 1987 the name exosome was coined. Exosomes contain surface proteins, some of which can act as labels in
order to find their target cells. Exosomes also contain messages in the form of proteins and nucleic acids (RNA and
DNA) that are transferable to target cells. Little is known and written about cardiac exosomes, although Gupta
and Knowlton described exosomes containing HSP60 in 2007. It is now known that exosomes from cardiomyocytes
can transfect other cells and that the metabolic milieu of the parental cell decides the quality of exosomes released
such that they induce differential gene expression in transfected cells. Future clinical use of exosomes in diagnosis,
monitoring disease progress, and treatment is promising. (Circ Res. 2014;114:315-324.)
Key Words: biogenesis
■
myocytes, cardiac
C
ardiac vascular disease (CVD) continues to be the most
common cause of death, being 47% of all deaths according to European Cardiovascular Disease Statistics.1 In most
Western countries, the CVD death rates are declining possibly because of progress in treatment strategies, such as valvular surgery, coronary bypass surgery, balloon dilatation of
coronary vessels, β-blockers in acute myocardial infarction, as
well as preventive initiatives, with smoking cessation being the
most effective one. Earlier (particularly surgical) treatments
have aimed to restore normal anatomy, whereas future therapies increasingly will be relying on cellular, subcellular, and
molecular myocardial actions. A major step forward in molecular genetics in clinical cardiology took place with the finding
by the Seidman group in 1989 that hypertrophic cardiomyopathy, in part, is due to a mutation in the myosin heavy chain
gene.2 Since then, the ambition has been to look for a possible
genetic basis in hitherto unexplained cardiac diseases as well
as to explore new means of diagnosis and treatment based on
■
nucleic acids
■
transfection
modification of gene expression, microRNA (miR), and protein handling of the cell (synthesis, folding/chaperones, degradation by the ubiquitine–proteasome pathway). It has been
reported that misfolding of proteins can lead to proteotoxicity,
which is an important pathophysiologic mechanism in many
heart diseases.3 Exosomes are vesicles released from cells via
exocytosis, and myocardial exosomes may play a major role
in many of the abovementioned processes. Although knowledge about specific myocardial exosome function and importance is limited, the subject is extensively studied at present.
Analogous functions of cardiac exosomes are now looked for
based on knowledge about exosomes from other cell systems.
The first description of exosomes (prostasomes) was in
epithelial glandular tissue.4–6 Glandular epithelial structures
as well as unicellular systems were those initially thought to
release exosomes.7–9 The first identification of exosomes in
­cancer-derived cells was made in prostate cancer cell lines
PC3, Du145, and LnCaP grown in monolayer.10 This initial
Original received March 27, 2013; revision received May 6, 2013; accepted May 6, 2013. In November 2013, the average time from submission to first
decision for all original research papers submitted to Circulation Research was 14.6 days.
From the Department of Cardiology, Heart Centre, and Department of Public Health and Clinical Medicine, Umeå University, Umeå, Sweden (A.W.);
and Department of Medical Sciences, Clinical Chemistry, University Hospital of Uppsala, Uppsala University, Uppsala, Sweden (G.R.).
The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.
114.300584/-/DC1.
Correspondence to Anders Waldenström, MD, Department of Cardiology, Heart Centre, and Department of Public Health and Clinical Medicine, Umeå
University, 90185 Umeå, Sweden. E-mail [email protected]
© 2014 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/CIRCRESAHA.114.300584
315
316 Circulation Research January 17, 2014
Nonstandard Abbreviations and Acronyms
CVD
miR
MMP
MVB
TGF
cardiovascular disease
microRNA
matrix metalloproteinase
multivesicular body
transforming growth factor
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finding was followed by investigations on exosomes of other
cancer cells.11–14 Accordingly, subsequent investigations proved
that most cells were able to produce exosomes.15–21 Gupta and
Knowlton22 reported in 2007 that cardiomyocytes grown in
vitro could release exosomes and that they contained HSP60.
Moreover, extracellular HSP60, when not in exosomes, may
be apoptosis-inducing to the surrounding cardiac myocytes because of a probable activation of Toll-like receptor 4.23 Still, it is
suggested that exosomes may be involved in mediating messages to proximal and distant cells for normal events such as cardiac development/growth, normal function, as well as pathology.
Plasma Membrane
Cardiac exosomes are derived from plasma membrane elements, and recognition of some properties of plasma membranes is important to understand extracellular vesicle
formation (including exosomes) and behavior. About 100 years
ago, Overton24 asserted that the barrier function of the plasma membrane could best be understood if it were attributed
to lipids. Gorter and Grendel25 suggested a lipid ­double-layer
structure and the idea was further explored by Davson and
Danielli.26 They reasoned that the amphipathic nature of the
phospholipid molecules and their conspicuous place as membrane components made them a dominant feature of the membrane. According to the model, the hydrocarbon sidechains
were directed inwardly toward the center of the membrane, and
the polar groups outwardly together with a layer of proteins
made up the surfaces. In 1960, Robertson27 demonstrated the
basic similarity of images of several membranes of a cell (ie,
plasma membrane, nuclear membrane, and inner mitochondrial membrane) obtained by electron microscopy (unit membrane), in that many membranes of a cell are fundamentally
similar in structure, consisting of a bilayer, 7 to 9 nm thick.
The fluid model is another expression for the lipid bilayer
model, with emphasis on the probable mobility of protein components in the lipid matrix. Singer and Nicolson28 introduced
the concept of membrane surface proteins and transversing proteins, thus allowing some of the membrane proteins to move
laterally on the cell surface. This was described as the fluid
mosaic membrane. The phospholipid bilayer was no longer
considered a continuum because of the presence of transversing
proteins. Simons and Ikonen29 launched the raft hypothesis in
1997 describing a heterogeneous organization of the membrane
(including lipid composition) with functional lipid raft microdomains, described in more detail elsewhere in this review.
History of Exosomes
In 1977, we described the extracellular presence of vesicles
surrounded by a bilayer membrane in prostatic and seminal
fluids.4–6 Electron microscopy examinations of acinar cells
from surgically removed specimens of human prostatic tissue revealed that the extracellular vesicles, which we called
prostasomes, corresponded to intracellular vesicles inside another larger vesicle, a so-called storage vesicle, equivalent to
a multivesicular body (MVB)/multivesicular endosome of late
endosomal origin.30 We also measured the diameter of prostasomes in 3 different locations: an intracellular location within
storage vesicles, extracellularly in the acinar lumen, as well
as (extracellularly) in isolated and purified prostasomes from
prostatic and seminal fluids. We found similar mean values
of ≈150 nm for all 3 locations. Moreover, the ultrastructure
of the vesicles (prostasomes) was suggestive because, while
inside the storage vesicles within the acinar cell, they could
be released after fusion with the surrounding membrane of the
storage vesicle and the plasma membrane of the acinar cell
into the extracellular space, that is, exocytosis, into the glandular duct.30 Vesicles released in this way, thus, formally fulfill
the criterion to be termed exosomes. Functional and beneficial
effects of prostasomes on sperm cells were recognized early.31
Concomitantly, it was recognized that other cells could also be
in receipt of these beneficial properties.32
Electron microscopic studies by Pan et al7 in 1985 on maturing reticulocytes disclosed an intracellular sac filled with
small membrane-enclosed structures nearly uniform in size.
When these sacs fused with the plasma membrane, the vesicular contents were released,7 again an example of exocytosis.
A similar finding was reported in 1984 by Harding et al8 for
reticulocytes of another species. The Johnstone group9 interpreted this process to be something like reverse endocytosis,
with internal vesicular contents released in contrast to external molecules internalized in membrane-bound structures,
and in 1987, they named the extruded structures exosomes.
The functionality of exosomes in case of reticulocytes was interpreted to represent shedding of some membrane proteins,
which were known to diminish during the final stage of development to mature erythrocytes.33
In 1996, Raposo et al34 demonstrated in B-lymphocytes that
both the limiting membrane and the internal vesicles contain
major histocompatibility complex class II, and the released
exosomes were perceived to have a role in antigen presentation in vivo. Subsequently, it was realized that many cell
types release exosomes, including hematopoietic cells, B- and
T-lymphocytes, dendritic cells, mast cells, platelets, intestinal
epithelial cells, astrocytes, neurons, and tumor cells.15–21 In addition to exosomes, cells can also shed other types of extracellular membrane vesicles, namely, microvesicles, microparticles,
and apoptotic bodies,35,36 after various biological stimuli, including induction of programmed cell death. Exosomes (especially those harvested from growth media of cells grown in
vitro) have generally undergone a filtration step in their purification, and they, therefore, represent a population of membrane
vesicles rather homogeneous in size (40–100 nm in diameter).
Accordingly, regarding size, exosomes can be categorized into
those with a narrow size range (because of filtration in the preparatory procedure) and those not filtered, with a broader size
range (eg, prostasomes). In contrast, microvesicles, microparticles, and apoptotic bodies represent heterogeneous populations
of extracellular vesicles (100 to >1000 nm) that are the result
of a direct budding from the plasma membrane.
Waldenström and Ronquist Exosomes and Myocardial Remodeling 317
Extracellular vesicles, including exosomes, have potent proinflammatory effects; they can affect the function of endothelium
and promote coagulation. Therefore, they may play a role in the
pathogenesis of CVDs. Hence, cancer cell– and endothelial cell–
derived exosomes stimulate endothelial cells by receptor activation and transfer of exosomal effectors leading to the progression
of angiogenesis in vitro and in vivo.37–39 It should be kept in
mind, however, that exosomes derived from mesenchymal stromal cells have been claimed to exert an a­ nti-inflammatory effect
on lung vessels. In this way, hypoxic pulmonary hypertension is
inhibited via the suppression of macrophage action.40 This reasoning is relevant in revascularization of ischemic myocardium
and justifies new approaches in trying to master exosome production and movement in interstitial fluid and peripheral blood.
Exosome Biogenesis
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Better understanding of the biogenesis of exosomes has led to
more insight during the past decades into their function.41–44
Exosomes are a subgroup of extracellular vesicles generally
ranging in size from 40 to 200 nm, which are released extracellularly through exocytosis.30,33,34 Interactions between cells,
which are mediated by exosomes, lead to a much more complex
event compared with those mediated by a single ligand with a
single receptor. Most cells are capable of releasing exosomes.
The plasma membrane of mammalian cells is organized heterogeneously, that is, there is no bilayer continuum. Instead, the
bilayer is interrupted by so-called transversing proteins, which
extend across the biological membrane. The plasma membrane
also contains specific microdomains known as detergent-resistant membrane domains, lipid rafts, or caveolae.45–48 More than
20 years ago, we observed an extraordinary composition of the
membrane of prostasomes.49 The prostasomal membrane contains a high amount of saturated phospholipid acyl chains and
sphingomyelin, which in turn results in an affinity for cholesterol. Hence, the cholesterol/phospholipid ratio was strikingly
high (≈2, contrasting to most other biological membranes with
a corresponding ratio of ≈0.8), and electron spin resonance
studies revealed that lipids in the prostasome membrane were
highly ordered.49 The implications of these findings were not
clear at first but were explained some years later when Simons
and Ikonen29 formulated the raft hypothesis. According to this
hypothesis, sphingolipid–cholesterol microdomains are involved in numerous cellular functions, from membrane trafficking and cell morphogenesis to cell signaling. These rafts
recruit a specific set of proteins and exclude others. The exact
mechanisms of selection are not known. Endocytic vesicles
arise at the lipid raft domain of the plasma membrane through
clathrin- or nonclathrin-mediated endocytosis, leading to the
intracellular formation of early endosomes. These early endosomes are subjected to a maturation process that includes an
interaction with the Golgi complex to become late endosomes.
The bilayer membrane surrounding late endosomes can in
turn display invaginations, giving rise to intraluminal vesicles
completing the formation of MVBs/multivesicular endosomes
(or, in case of prostasome nomenclature, the so-called storage
vesicles),30,50,51 rendering the membrane surrounding the intraluminal vesicles a right-side-out position in relation to the plasma membrane (Figure 1).52 The components of the endosomal
sorting complex required for transport pathway are critical for
the formation of MVBs (Figure 2). However, the relationship
between the endosomal sorting complex required for transport
pathway and the secretion of exosomes remains unclear. The
fusion of MVBs with the plasma membrane results in the release of their cargo, the exosomes, to the extracellular space.
This process completes the exocytosis event.
The protein composition of exosomes produced in vitro
has been studied in various ways, including Western blotting,53,54 flow cytometry of exosome-coated beads,55 and mass
spectrometry.56–58 These studies have demonstrated that exosomes from different cellular origins share some common
characteristics. Such characteristics are the lipid bilayer with
exceptionally high cholesterol/phospholipid ratio, size, density, and a basic collection of lipid and protein composition.
Among these proteins are those derived from the cytoplasm
or some that are membrane-bound, such as tubulin, actin,
actin-binding proteins, annexins and Rab proteins, and some
glycolytic enzymes. Others represent those responsible for
signal transduction, such as protein kinases and heterotrimeric
G-proteins.59–64 Many exosomes typically contain major histocompatibility complex class I and class II molecules65,66 and
heat shock proteins62,67 that are involved in antigen binding
and presentation. However, the protein family most characteristically associated with exosomes would seem to be the
tetraspanin and integrin proteins (targeting and cell adhesion),
including CD9, CD63, CD81, and CD8268–70 (Figure 3).
Although differential centrifugations, including preparative
ultracentrifugation, do not discriminate between exosomes and
other small vesicles, or large protein aggregates, exosomes float
on sucrose gradients, and their densities range from 1.13 g/mL
(B-cell–derived exosomes) up to 1.19 g/mL (epithelial cell–derived exosomes). Accordingly, contaminating protein material
can be separated from exosomes by floatation on sucrose gradient.67 Other modes of enrichment of prostasomes/exosomes
include size exclusion chromatography14,30,31 filtration,56 immunoaffinity purification,71 and specific isolation kits.72
The sorting behavior of lipids in MVBs/storage vesicles is
not known. It has been claimed that it might be determined
by the nature of their hydrophobic tails.73 We have noted the
unusual distribution of phospholipids in prostasomes with
sphingomyelin being predominant and with relatively high
amounts of phosphatidylethanolamine and phosphatidylserine as well as with lysophospholipids, whereas phosphatidylcholine and especially phosphatidylinositol were found at
consistently low levels.49 This prostasome/exosome-specific
phospholipid pattern was subsequently confirmed by others.74,75 Exosomal lipids might be biologically active,57 and
we reported in 1993 the presence of prostaglandin E in prostasomes.76 Exosomal prostaglandins have been shown to be
able to trigger ­prostaglandin-dependent intracellular signaling pathways within the target cells.77 The local enrichment
of prostaglandins in prostasomes/exosomes may favor a more
efficient biological activity as compared with what can be
achieved with prostaglandins in soluble form.78 This reasoning also holds true for other soluble molecules.
Exosome–Target Cell Interaction
Intercellular communication is essential for multicellular organisms to maintain vital functions. Direct cell-to-cell contact
318 Circulation Research January 17, 2014
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Figure 1. Biogenesis of exosomes. Exosomes are generated in the late endosomal compartment and carry recycled proteins from coated
pits/lipid rafts in the cellular membrane, proteins directly sorted to the MVBs from RER and GC, mRNA, microRNA, and DNA. Note that
the generation of exosomes by inward budding of the limiting membrane of MVB ensures that the membrane-bound proteins preserve
the same orientation and folding on the exosomal membrane as those on the plasma membrane. The exosome-filled MVBs are either
fused with the plasma membrane to release exosomes or sent to lysosomes for degradation. Exosomes are different from ectosomes and
apoptotic bodies because the latter extracellular vesicles are the result of a direct budding process of the plasma membrane. GC indicates
Golgi complex; MV, microvesicle internalized from the cellular membrane, early endosomes; MVB, multivesicular body; and RER, rough
endoplasmic reticulum. The role of placental exosomes in reproduction. (Illustration credit: Ben Smith). Reprinted from Mincheva-Nilsson
and Baranov52 with permission of the publisher. Copyright © 2010 John Wiley & Sons A/S. Authorization for this adaptation has been
obtained both from the owner of the copyright in the original work and from the owner of copyright in the translation or adaptation.
or transfer of secreted molecules can accomplish this communication. A third mode of contact between cells is the release of
extracellular vesicles such as exosomes, with interaction and
uptake by another cell. Using free-zone electrophoresis, we
found that both prostasomes and spermatozoa (presumed target
cells) had a net negative surface charge (prostasomes less negative than spermatozoa), favoring repulsive forces. Nevertheless,
spermatozoa and prostasomes interacted strongly with each
other, and the interaction site most probably had a hydrophobic character.79 This type of interaction enables prostasomes to
act in close vicinity to spermatozoa. Not only prostasomes, but
also other exosomes will certainly display net negative surface
charges that facilitate the solubility and integrity of exosomes
in body fluids such as blood plasma. The transfer of a message
to distant cells could occur by 3 possible mechanisms: by direct
contact between the exosomal membrane and the plasma membrane of the target cell, by fusion of the 2 membranes, or by target cell internalization of the exosome (Figure 4).80,81 By these
means, a parental cell is able to communicate with a target cell
in its vicinity or at a distance through an amplification process.
Cardiac Remodeling and Exosome Function
Modern therapy of acute myocardial infarction has resulted in
early increased survival, although heart failure due to loss of
contracting myocardium is still a problem in survivors.82 The
remaining myocardium has to adapt to meet the work requirement performed earlier by a larger myocardial mass.
Many different stress factors can lead to heart failure. Most
often, such stressors lead to events in the myocardium, including certain adaptive measures. When these adaptations are insufficient, a state of maladaptation occurs. Obvious adaptation
is hypertrophy (increased wall thickness and decreased wall
stress) concomitantly with change in cellular metabolism and
contractility to increase cost-effectiveness (switch to the fetal gene program).83 The stress may be pressure-induced (hypertension, aortic stenosis), volume-induced/increased blood
flow (valve insufficiency), or induced by the loss of contractile
myocardium (myocardial infarction or dilated cardiomyopathy). The heart strives for the maintenance of adequate cardiac
output, that is, through increased sympathetic tone, which in
the short term is adaptive, but in the long term is deleterious.
The adaptive measures are orchestrated by different signal
substances, such as chemokines, growth factors, miRs, many
of which are mediated via exosomes from cardiac cells.
In 1998, Fire et al84 described short, ≈19 to 23 nucleotides,
noncoding ribonucleic acid molecules (miRs) that play important gene regulatory roles. They bind to complementary
sequences on target mRNA, causing translational repression
or target degradation and gene silencing.85 Several cellular
processes such as proliferation, differentiation, and apoptosis
Waldenström and Ronquist Exosomes and Myocardial Remodeling 319
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Figure 2. Electron microscopy picture of a multivesicular
body (MVB) containing intraluminal vesicles/exosomes. Note
the surrounding double-layered membrane and intraluminal
vesicles/exosomes of different sizes and densities. Image
reprinted with permission from Dr Jastrow’s electron microscopic
atlas (www.drjastrow.de).
are regulated by miRs.86 Recent studies revealed that miRs
are aberrantly expressed in the cardiovascular system under
some pathological conditions. Hence, complex changes in
miRs occur during prolonged CVDs, such as cardiac hypertrophy.87–89 Myocardial miRs are downregulated in the early
stage of hypertrophy. miR-21 reaches 8 times upregulation
by day 14 of induction of experimental cardiac hypertrophy
and regulates apoptotic mechanisms. Also, miR-23 has a similar pattern of expression and function.90,91 miR-21 and miR26a also regulate the expression of matrix metalloproteinase
(MMP)-2, known to be important in extracellular matrix remodeling during hypertrophy.92,93 miR-26 and miR-133a/b are
highly expressed in muscle and heart, but are only expressed
in late-stage hypertrophy. miR-133 regulates the IP3 channel
receptor gene, leading to prohypertrophic calcium signaling.
miR-499 is embedded in the myosin heavy chain gene and is
important in its regulation during hypertrophy.94 miR-30b-5p
is downregulated in cardiac hypertrophy and regulates ­Ca2+/
calmodulin-dependent protein kinase A. Such complex changes in miRs discussed for long-standing CVDs are also valid
during acute events such as myocardial infarction.95,96
Signaling between endothelial cells, endothelial progenitor
cells, and stromal cells is crucial for the establishment and
maintenance of vascular integrity and involves exosomes,
among other signaling pathways. van Balkom et al97 showed
that miR-214 (a miR that controls endothelial cell function
and angiogenesis) plays a dominant role in exosome-mediated
signaling between endothelial cells. Endothelial cell–derived
exosomes turned out to stimulate migration and angiogenesis
in target cells, whereas exosomes from miR-214–depleted
endothelial cells failed to stimulate these processes. Results
from another research group98 revealed that atheroprotective
stimuli induced communication between endothelial cells and
smooth muscle cells through a miR- and extracellular vesicle–mediated mechanism, suggesting a promising strategy to
combat atherosclerosis. Another possible antiatherosclerotic
exosome-mediated mechanism is already commented upon.40
Although subjected to debate, a recent investigation established that the majority of miRs in blood plasma (and
saliva) are enclosed in exosomes.99 Few years ago, Ji et al100
applied an analogous experimental approach to identify circulating miRs that might accurately reflect myocardial injury in vivo. Accordingly, miRs apparently circulate in an
­exosome-shielded form in body fluids such as blood plasma,
and this established a basis for the idea that circulating miRs
could serve as a new generation of biomarkers for CVDs.101–104
With the rapid expansion in stem cell research (including
cardiosphere-derived stem cells), hope arose that stem cells
could be seeded in myocardial cell–deficient tissue. Despite
great efforts with seeding of different types of stem cells, this
has not led to the expected positive result. Moreover, it was
observed that despite a measurable increase in cardiac function, this could not be attributed to an abundance of stem cell
invasion alone. Another explanation was that a paracrine function of stem cells and other cells could induce remodeling and
cell protection, as well as improved function and facilitate cell
transformation to cardiomyocytes.105–108 Recent findings suggest that a surprisingly high number of different miRs can independently trigger cardiomyocyte mitosis in the border zone
of an infarction, emphasizing the superiority of exosomal
miRs over stem cell implantation in restitution of infarcted
myocardium. This opens up for a new treatment strategy replacing stem cell implantation.109
The paracrine functional capacity of cardiac exosomes
has been shown indirectly by our group as well as by others.
Cardiac cells can release exosomes that contain heat shock
proteins, among others, and nucleic acids.22,81,110 The population of such cardiomyocyte-derived exosomes is not homogeneous. They differ in size from 40 to 300 nm, and some are
electron-lucent, and others electron-dense.81 When characterized by surface proteins, 80% contain flotillin-1 and 30% are
positive for caveolin-3. Moreover, exosomes released from
cultured HL-1 murine cardiomyocytes contained 1595 different mRNAs, of which 1520 also were detected in cardiomyocytes and 423 could be directly connected to a biological
network.81,111 Accordingly, 35 genes coding for proteins in the
small and large ribosomal subunit and additional 8 genes could
be connected to a network. Finally, 33 genes coded for proteins
in the mitochondria. These exosomes were internalized by fibroblasts when cocultivated, and it could be demonstrated that
exosomes were forwarded to the nucleus where both exosomal
DNA and RNA colocalized. Exosomes were functional in that
they induced a gene response of the transfected cells, giving
rise to 333 differentially expressed genes (175 upregulations
and 158 downregulations) compared with controls. Moreover,
343 different chromosomal DNA sequences were identified.
The question arises whether these unequivocal effects are applicable to other cardiomyocyte cell lines. This relevant question cannot be answered until proper comparisons are made.
It is notable that the milieu of the parental cell may influence the quality of exosomes released. Thus, when HL-1 cells
320 Circulation Research January 17, 2014
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were cultured with different growth factors, namely, transforming growth factor (TGF)-β2 or platelet-derived growth factor
BB, individual responses were induced concerning the quality of exosomes released. Exosomes were isolated from each
group of the treated cardiomyocytes and were cocultivated
with fibroblasts. In the 3 groups of transfected fibroblasts (with
exosomes derived from cells treated with TGF-β2, or plateletderived growth factor BB, or controls), a common pool of 235
transcripts was found in all 3 groups where 14% were ribosomal and 5% were connected to the energy supply system.
Apart from this, there were 138 transcripts unique for controls.
Cardiomyocyte-derived exosomes contain a basic stock of transcripts common for exosomes derived from controls as well as
growth factor–stimulated myocytes. The products are involved
in intracellular transport, mitogen-activated protein kinase signaling pathways, and the nucleus. TGF-β2–derived exosomes
induced 201 platelet-derived growth factor BB 74–specific
transcripts, apart from the 138 in untreated controls.110 It may
be concluded that cardiomyocyte-derived exosomes carry a
basic package of transcripts and growth factor stimulators of
cardiomyocytes, resulting in the alteration of the transcriptional content in a specific way. Thus, the conditions under
which the parental cell maintains life is decisive for the quality
of the exosomal message sent by the cell. It is of fundamental
interest to know that cardiomyocytes can send messages with
specific content according to the need or will of the sending
cell. Examples of this are outlined below when the myocardium
adapts to the external stimuli (such as different forms of stress),
inducing hypertrophy and changes in contractility. Such adaptation to increased workload (valvular disease, hypertension,
loss of myocardium/infarction) can generally be described as
remodeling.
Literature on cardiac exosomes is limited, but certain assumptions can be made based on the knowledge about exosomes
from other tissues. Remodeling of the heart involves interplay
mainly between cardiac cells, myocytes, fibroblasts, endothelial
cells, smooth muscle cells, and extracellular matrix (and inflammatory cells). In particular, myocyte–fibroblast interaction is of
importance.112 This interplay enables myocardial cells to grow
or proliferate or be substituted by stem cells/fibroblasts transformed into cardiomyocytes. This includes angioneogenesis for
regeneration of scarred myocardium or poorly perfused myocardium.109 This complicated and only partly known interplay
is to some extent governed by signals delivered by exosomes.
In 2008, a series of important articles from Utrecht demonstrated that a mesenchymal stem cell–conditioned medium,
when given intravenously just before reperfusion after ischemia, induced limited infarct size in both pigs and mice.113
Injection of isolated exosomes into a tail vein before reperfusion of coronary ligated mice resulted in limitation of myocardial infarct size.114 This article also suggests an explanation for
the paracrine effect of mesenchymal stem cell implantation on
tissue repair. Moreover, it was shown that exosomes derived
from cardiomyocyte progenitor cells stimulate the migration of
Figure 3. Cartoon of an exosome. The exosomal contents comprise mRNA, DNA, microRNAs, proteins (ie, enzymes, growth factors,
and cytokines), and transmembrane proteins of different kinds, including tetraspanins, annexin, and intracellular cell adhesion molecule.
For further information, see text. Reprinted from Hu et al124 with permission of the publisher. Copyright © 2012, Frontiers in Genetics.
Waldenström and Ronquist Exosomes and Myocardial Remodeling 321
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Figure 4. Confocal image of a transfected fibroblast with
exosomes derived from cardiomyocytes stained with acridin
orange. Arrow shows DNA in the nucleus of fibroblasts. When
switching to red wavelength, RNA was also envisaged (not shown
in picture). Reprinted from Waldenström et al81 with permission of
the publisher. Copyright © 2012, Waldenström et al.
endothelial cells. Human cardiomyocyte progenitor cells were
cultivated, and exosomes were isolated from the medium. A
major factor for endothelial cell migration was the exosomal
contents of MMPs. A membrane-bound MMP activator is also
found in these exosomes, which induces MMP and vascular
endothelial growth factor release from neighboring cells.115
Progenitor cell–derived medium, including exosomes, modulates cell differentiation, proliferation, and survival of cardiac
progenitor cells, cardiomyocytes, and fibroblasts.116–119 It was
reported recently that when the mechanism of protection was
further studied in this mouse ischemia reperfusion model,
ventricular dilatation was prevented and myocardial function
improved. It was suggested that exosomes activated the survival pathways and restored ATP depletion. These exosomes
also carry CD73 (5′-nucleotidase) that can produce extracellular adenosine, a well-known mediator of protection in ischemic preconditioning.120–122 Finally, the inflammatory reaction
to ischemia was shown to be reduced.123 It is still not known
which exosomal factors are most important for the effects described above, but this body of results so far supports the interpretation that certain growth factors and cytokines, such as
basic fibroblast growth factor, tumor necrosis factor-α, ­TGF-β,
epidermal growth factor, heat shock proteins (HSP20, 60, and
70), and miRs known to be carried by exosomes, are, at least in
part, responsible for the response.110,124,125–128
Ischemic preconditioning was first described by Murry
et al129 in 1986. Several factors have been shown to function
in this phenomenon, including adenosine, ATP-dependent K+
channels, mitochondrial transition pores, and some other factors.130–132 It was recently found that this complex system is
also governed by factors that are known to be e­ xosome-borne,
much the same way as in ischemia/reperfusion injury
described above. They are likely to be important especially in
the late phase of preconditioning, as first described by Bolli.133
Similarly, miRs that are involved in ischemic injury and preconditioning and known to be carried by exosomes include
miR-1 (post-transcriptional regulation of HSP60 and 70),
miR-21 (control of cell survival and expression of MMPs),
miR-29 (regulates p53 and induces apoptosis), miR-133a
(regulates hypertrophy and apoptosis), and miR-499 (inhibits
apoptosis).134–138 MMPs (regulated by miRs) induce degradation of scar tissue and substitution by invading transformed
cells.115 Dying or apoptotic cells convey emergency signals,
and rescue packages are sent back from stem cells, which to a
certain extent are mediated via exosomes.124
Exosomes with their narrow spectrum of molecules (nucleic acids, proteins, and lipids) may well be mediators of all
these processes, thereby modulating cellular responses and
signal transduction.57 It should be kept in mind that protein
and RNA sorting into exosomes is highly regulated,139 and this
allows cells to produce tailormade exosomes with different
functional characteristics, in turn governed by the nature of
signals triggering their production and release. After release,
exosomes are targeted specifically to conjugated and distant
cells as well. Accordingly, exosome release and cellular uptake are tightly controlled, and if we are able to govern this
release and understand the mechanisms involved, we might be
able to control the process.
Future Aspects
The era of clinical use of exosomes is just beginning. With the
increased rate of publications, it can be expected that the field
of use will also develop into unexpected areas.
Exosomes in peripheral blood most probably reflect the status of the whole organ of exosomal origin and can, therefore,
be expected to be used for:
•
•
•
•
•
•
Diagnosis, for example, acute myocardial infarction
Screening, for example, silent ischemia and hypertrophic
cardiomyopathy
Prognosis, for example, myocardial infarction
Monitoring disease progression; exosome number and quality would reflect progression
Monitoring treatment efficacy, for example, normalization
of exosome number and quality
Treatment, for example, as vehicle for gene therapy
Because the status of the parental cell decides the quality of
exosomes released, it is assumed that exosomes can be used to
diagnose organ disease. The methods for diagnosis and prognostication of prostate cancer are under development.140 This
method will have advantages over biopsies because exosomes
reflect the whole part of sick tissue, whereas biopsies might
miss the diseased part. The quality and quantity of exosomes
might tell severity of the disease as, for example, in cancer but
also in congestive heart failure and hypertrophic cardiomyopathy depending on which miR, mRNA, and DNA are found.110
Moreover, the treatment effect could be monitored by these
means. Many monogenic diseases do not start at a given time,
for example, the onset of hypertrophic cardiomyopathy ranges
from childhood to adulthood. The monitoring of the quality of
circulating exosomes might give a hint of when to start therapy
322 Circulation Research January 17, 2014
like β-blockers or implantable cardioverter defibrillator, and
they will also be excellent for screening purposes. Finally,
exosomes loaded with therapeutic contents (DNA, RNA, and
miRs) will probably be possible to generate in the future.
Acknowledgments
Professors Michael Haney and Joe Hayes are greatly appreciated for
their careful reading and linguistic improvement of the article.
Sources of Funding
The Swedish Heart and Lung Foundation.
Disclosures
None.
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Role of Exosomes in Myocardial Remodeling
Anders Waldenström and Gunnar Ronquist
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Circ Res. 2014;114:315-324
doi: 10.1161/CIRCRESAHA.114.300584
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