Membrane vesicles mediate pro-angiogenic activity of equine

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The Veterinary Journal
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / t v j l
Membrane vesicles mediate pro-angiogenic activity of equine
adipose-derived mesenchymal stromal cells
Luisa Pascucci a,*, Giulio Alessandri b, Cecilia Dall’Aglio a, Francesca Mercati a,
Paola Coliolo a, Cinzia Bazzucchi a, Sara Dante a, Stefano Petrini c, Giovanni Curina c,
Piero Ceccarelli a
a
Department of Veterinary Medicine, University of Perugia, Via San Costanzo, 4, 06126 Perugia, Italy
IRCCS Foundation, Neurological Institute ‘C. Besta’, Cerebrovascular Diseases Unit, Via Celoria 11, 20133 Milan, Italy
c Experimental Zooprophylactic Institute of Umbria and Marche, Immunology Unit, Via G.Salvemini 1, 06126 Perugia, Italy
b
A R T I C L E
I N F O
Article history:
Accepted 21 August 2014
Keywords:
Angiogenesis
Horse
Mesenchymal stromal cells
Membrane vesicles
Microvesicles
A B S T R A C T
Multipotent mesenchymal stromal cells (MSCs) have attracted a great deal of interest, due to several distinctive features, including the ability to migrate to damaged tissue and to participate in tissue regeneration.
There is increasing evidence that membrane vesicles (MVs), comprising exosomes and shedding vesicles,
represent a key component, responsible for many of the paracrine effects of MSCs. The aim of the present
study was to establish whether equine adipose-derived MSCs (E-AdMSCs) produce MVs that are capable
of influencing angiogenesis, a key step in tissue regeneration.
A morphological study was performed using MSC monolayers, prepared for transmission and scanning electron microscopy and on ultracentrifuged MSC supernatants, to identify production of MVs. The
ability of MVs to influence angiogenesis was evaluated by means of the rat aortic ring and scratch assays.
The results demonstrated that MVs, constitutively produced by E-AdMSCs, are involved in intercellular
communication with endothelial cells, stimulating angiogenesis. Although many questions remain regarding their formation, delivery, content and mechanism of action, the present study supports the concept
that MVs released by MSCs have the potential to be exploited as a therapeutic tool for regenerative
medicine.
© 2014 Elsevier Ltd. All rights reserved.
Introduction
Use of multipotent mesenchymal stromal cells (MSCs) for regenerative medicine has been proposed in humans and veterinary
species (Fortier and Travis, 2011; Sykova and Forostyak, 2013). The
beneficial effect of MSCs in enhancing tissue regeneration was initially thought to result from their proliferation and differentiation
into tissue-specific mature cells (Kopen et al., 1999; Mezey et al.,
2000; Caplan and Bruder, 2001). However, the observation that only
a small number of transplanted MSCs survive and integrate into host
damaged tissues has highlighted the possibility that alternative
mechanisms might exist. In particular, evidence of extensive interaction with the surrounding microenvironment has led researchers
to focus more on MSCs as activating agents of regenerative pathways, rather than simply replacing damaged cells (Iso et al., 2007;
Horwitz and Prather, 2009; Prockop, 2009; Vrijsen et al., 2010; Baglio
et al., 2012).
* Corresponding author. Tel.: +39 75 585 7632.
E-mail address: luisa.pascucci@unipg.it (L. Pascucci).
MSCs produce a plethora of trophic factors, cytokines and signalling molecules, able to influence neoangiogenesis, fibroblast
proliferation, inhibition of apoptosis and even recruitment of resident stem cells (Caplan and Dennis, 2006; Caplan, 2007; Gnecchi
et al., 2008; Horwitz and Prather, 2009; Boomsma and Geenen, 2012),
thereby creating optimal environmental conditions for tissue regeneration via a paracrine mechanism. This complex interaction
between MSCs and the tissue microenvironment might involve
soluble factors as well as production of membrane vesicles (MVs),
containing molecules such as short peptides, proteins, lipids, and
various forms of RNAs (Gyorgy et al., 2011).
The term ‘exosome’ is used to describe a specific subtype of MV,
derived from multivesicular bodies (MBs). These represent a type
of ‘late endosome’, containing luminal nanovesicles (30–100 nm),
formed by the inward budding of the outer endosomal membrane
that are released by fusion of MBs with the plasma membrane
(exosomes). The term ‘shedding vesicles’ refers to another type of
vesicle that is released into the extracellular environment by direct
budding of the plasma membrane. The size of shedding vesicles has
not been clearly defined, but it is estimated that their diameter
ranges between 50 and 1000 nm (Gyorgy et al., 2011).
http://dx.doi.org/10.1016/j.tvjl.2014.08.021
1090-0233/© 2014 Elsevier Ltd. All rights reserved.
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MVs can be constitutively produced or induced and may act as
vehicles of bi-directional information exchange between cells
(Camussi et al., 2010). Through their membrane envelope, they are
able to reach, interact, and eventually fuse with recipient cells, modulating a variety of physiological and pathological processes (Raposo
and Stoorvogel, 2013). MVs derived from MSCs (MSC-MVs) have been
isolated, visualised, and their function evaluated in several studies,
but their mechanism of action remains relatively unclear (Gatti et al.,
2011; Bruno et al., 2013; Kim et al., 2013). Several studies have
shown that MSC-MVs can have a similar therapeutic efficacy to donor
cells, when used for tissue repair and even in anti-cancer therapy,
suggesting that development of innovative ‘MV-based therapy’ might
reduce the difficulties and risks associated with whole cell transplantation (Tetta et al., 2011; Baglio et al., 2012).
In veterinary medicine, equine MSCs have been particularly
studied for their beneficial effects on tendon regeneration in sport
horses (Godwin et al., 2012). Here, a complex set of mechanisms
are likely to be involved, with stimulation of angiogenesis a major
contributory factor (Caplan and Dennis, 2006). The aim of the present
study was to investigate the capacity of MVs, derived from horse
adipose tissue MSCs (E-AdMSC-MVs), to influence angiogenesis.
Materials and methods
MSC culture and MV isolation
Adipose tissue samples were obtained under general anaesthesia, with informed owner consent, from subcutaneous fat of four donor horses, 1–3 years of
age, undergoing abdominal surgery. The tissue sampling procedure was approved
by the University of Perugia Ethics and Welfare Committee (Protocol number 2012034; approved 12 September 2012).
Samples were washed with sterile phosphate buffered saline (PBS), supplemented with 200 U/mL penicillin, 200 mg/mL streptomycin, and 12.5 mg/mL
amphotericin B (Sigma–Aldrich). Tissues were then finely minced and digested with
0.075% collagenase type I (Worthington Biochemical) at 37 °C for 45 min and centrifuged at 600 g for 10 min, to obtain a pellet containing the stromal vascular cell
fraction. Cells were incubated at 37 °C with 5% CO2 for 72 h in tissue culture flasks
with complete basal medium, consisting of Dulbecco’s modified Eagle Medium
(DMEM; Gibco), 10% fetal bovine serum (FBS; Gibco), 100 U/mL penicillin and 100 mg/
mL streptomycin. The adherent cells were expanded until they achieved 80%
confluence. Cells were recovered using 0.5 mM EDTA/0.05% trypsin (Gibco) and passaged at a density of 5–10,000 cells/cm 2 (passage one). The E-AdMSCs were
maintained in complete basal medium until passage three, at which time they were
prepared for MV isolation and for observation by electron microscopy.
E-AdMSCs were differentiated along adipogenic, osteogenic or chondrogenic lineages, using specific inductive media, as described previously (Zuk et al., 2002) (see
Appendix: Supplementary Fig. S1). Additionally, cells at passage three were evaluated for their expression of CD90 and CD44, two of the main MSC markers in
veterinary species (Pascucci et al., 2011) (see Appendix: Supplementary Fig. S2).
Cells at passage three (~20 × 106) from each donor horse were used for recovery of MVs. Cells were incubated for 72 h in FBS-free DMEM, supplemented with
0.5% bovine serum albumin (BSA). The culture supernatant was centrifuged at 2000 g
for 20 min to remove debris and dead cells. The cell-free supernatant was then centrifuged at 100,000 g for 60 min at 4 °C (Beckman-Coulter ultracentrifuge XL100K), washed in 0.1 M PBS, pH 7.3, and subjected to a second ultracentrifugation
step under the same conditions. The MV pellet was finally suspended in 1 mL PBS.
In order to quantify and compare the MVs recovered from the four MSC samples, a
Bradford assay (BioRad) for protein determination was performed. As a control, an
aliquot of FBS-free DMEM with 0.5% BSA was subjected to the same procedure described for conditioned medium. The ‘mock pellet’ was resuspended in PBS and
employed in the formulation of control media used in functional assays, to exclude
the possible involvement of trace amounts of BSA in the endothelial cell response.
Electron microscopy of cell monolayers and MVs
For transmission electron microscopy (TEM) of cell monolayers, cells were fixed
with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (CB), pH 7.3, for 2 h at room temperature, detached from the well by means of a cell scraper, and centrifuged at 600 g
for 10 min to remove the fixative. Pellets were subsequently washed twice in CB,
post-fixed in 2% osmium tetroxide, dehydrated in a graded series of ethanol up to
absolute, pre-infiltrated, and embedded in Epoxy Embedding Medium (Sigma–
Aldrich). Ultrathin sections (90 nm) were mounted on 200-mesh copper grids then
stained with uranyl acetate and lead citrate. For MVs, approximately 20 μL of each
MV suspension, were initially placed on Parafilm. A Formvar-coated copper grid (Electron Microscopy Sciences) was gently placed on the top of each drop for about 60 min
in a humidified chamber. Grids were then washed in 0.1 M CB, pH 7.3 and finally
fixed with 2.5% glutaraldehyde (Fluka) in CB. After washing in CB, MVs were contrasted with 2% uranyl acetate then air dried. The observation was performed using
a Philips EM208 transmission electron microscope equipped with a digital camera
(CUME – University Centre of Electron Microscopy).
For scanning electron microscopy (SEM), cells were grown on glass coverslips,
fixed with 2.5% glutaraldehyde in 0.1 M CB, pH 7.3, for 2 h at room temperature and
dehydrated, in a graded series of ethanol up to absolute. Coverslips were placed on
metal stubs and coated with gold to a thickness of 15 nm. MVs suspended in PBS
were allowed to adhere to Formvar-coated copper grids and fixed with the same
procedure as described for TEM. The preparations were attached to metal stubs and
coated with gold to a thickness of 15 nm. SEM was performed using a Philips XL30
scanning electron microscope.
Isolation of equine vascular endothelial cells (E-VECs)
The femoral veins of adult horses, euthanased on clinical grounds unrelated to
the study, were removed within 30 min post mortem, with informed owner consent.
Subjects with clinical or pathological evidence of vascular disease or endotoxaemia
were excluded from the study.
The vessels were rinsed with sterile PBS, pH 7.4, supplemented with 200 U/mL
penicillin and 200 mg/mL streptomycin. One end of each vessel was closed with a
bowel clamp and 0.1% type I collagenase in PBS was introduced inside the lumen.
The open end of the vessel was clamped shut with another sterile bowel clamp and
the vessel was incubated at 37 °C for 30 min. After a gentle massage of the vein, the
endothelial cell-collagenase suspension was poured off and diluted in a 1:4 ratio with
complete ENDOGRO medium (Millipore). The vessel lumen was washed twice with
PBS, supplemented with penicillin/streptomycin and the combined cell suspension centrifuged at 500 g for 10 min.
The supernatant was discarded and cells were suspended in 20 mL complete
ENDOGRO medium, seeded in six-well plates coated with collagen (BD Biosciences), and incubated at 37 °C, with 5% CO2. E-VECs were subcultured once a week
at a 1:3 ratio and were not used for experiments beyond passage four. Due to the
absence of antibodies that are specific for equine endothelium, the endothelial cell
origin of our cultures was evaluated by morphological examination, whereby endothelial cells form a typical ‘cobblestone’ monolayer of polygonal cells (Jaffe et al.,
1973). Additionally, we evaluated the capacity of E-VECs to form cord structures when
seeded on a tridimensional matrix (Matrigel, Sigma–Aldrich) as previously described (Kleinman and Martin, 2005). This is considered a functional feature of
endothelial cells and is not retained by fibroblasts (Ponce, 2009).
Scratch assay
A scratch assay was used to assess the angiogenic potential of MSC-MVs. Since
protein content, as assessed by the Bradford test, was similar in all MV preparations, the same arbitrary amount of MV suspension was used. Silicone culture inserts
(Ibidi) for cell migration assays were gently transferred to collagen-coated 48-well
plates. E-VECs (2000 cells at passage three) suspended in 100 μL complete ENDOGRO
medium, were seeded into each chamber of the Ibidi insert and incubated at 37 °C,
5% CO2. After attachment, the cells were allowed to reach confluence, then the insert
was gently removed using sterile tweezers and the wells filled with 200 μL MV suspension diluted 1:10 in basal ENDOGRO medium without growth factors or serum.
ENDOGRO basal medium was used as negative control. Plates were incubated at 37 °C
and observed at 24 and 48 h. The test was conducted in triplicate for each MV sample.
E-VECs migrating into the intervening space were counted at 20 × magnification in
five random fields. Mean ± SD values are shown and P-values calculated using the
Student’s t test, with P < 0.05 taken to represent statistical significance.
Rat aortic ring assay
The rat aortic ring assay was carried out as previously described (Aplin et al.,
2008) to evaluate the angiogenic potential of E-AdMSC-MVs. The dorsal aorta was
excised from 6-week old Sprague-Dawley rats (Charles River Laboratories) and dissected into 1 mm thick rings. Each aortic ring was placed inside a well of a fourwell plate with 40 μL collagen solution prepared as previously described (Aplin et al.,
2008). The plates were incubated at 37 °C for 30 min to obtain collagen jellification.
The wells were filled with 500 μL control medium, composed of 1:1 DMEM and EBM
(Lonza) or with 500 μL E-AdMSC-derived MVs diluted 1:1 in EBM. In both cases, FBS
was omitted from the medium. The plates were incubated at 37 °C, 5% CO2 for 7 days.
Formation of vascular structures was assayed daily using a Zeiss inverted light microscope and the total number of tubular structures was counted using a 10 × objective.
Results
Preparations obtained by ultracentrifugation of E-AdMSC supernatants revealed the presence of a variety of MVs. By TEM, these
appeared to be mainly round in shape and ranged in size from 30
to 200 nm. MVs were observed to be present as isolated vesicles
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or aggregated in clusters, showing a peripheral electron-dense rim
enclosing a homogeneous electron-lucent to moderately electrondense content (Fig. 1A). Observation of cell monolayers by electron
microscopy, allowed capture of different stages of MV formation and
release, allowing identification of distinguishing features of shedding vesicles (Fig. 1B) and exosomes (Fig. 1C). Using SEM, isolated
(Fig. 1D) or in situ MVs (Fig. 1E) revealed the same characteristics,
in terms of morphology and dimensions.
In the scratch assay, E-VEC migration was significantly promoted after incubation with MVs, with the gap almost filled within
48 h (Fig. 2), and the number of cells migrating into the space significantly greater in the MV-treated samples than in controls (Fig. 3).
In the treated samples, cells facing the wound edge exhibited a
typical polarised and migrating morphology (see Appendix:
Supplementary Fig. S3).
In the rat aortic ring assay, exposure to MVs significantly increased neovessel formation from aortic rings. Sprouting of
microvessels initiated as early as 36 h after addition of MVs and was
maximal after 7 days of culture (Figs. 4 and 5). As previously demonstrated for this angiogenesis assay, where both pericytes and
endothelial cell migration are typically stimulated (Nicosia and
Ottinetti, 1990; Aplin et al., 2010), the newly formed microvessels
sprouting from rings treated with E-AdMSC-MVs revealed complete structural organisation, suggesting that E-AdMSC-MVs probably
transport more than one angiogenic molecule. Neovessel sprouting was minimal or absent after incubation of aortic rings with
control medium.
Discussion
Fig. 1. (A) Electron micrograph showing MVs isolated by ultracentrifugation from
E-AdMSC supernatants. MVs are round in shape and measure between 30 and 200 nm.
TEM, scale bar = 200 nm. (B) Image shows an electron-lucent vesicle shedding from
the cell surface. TEM, scale bar = 200 nm. (C) Image shows a maturing multivesicular
body measuring around 700 nm and containing less than 100 nm large intraluminal exosomes. TEM, scale bar = 500 nm. (D) MVs isolated by ultracentrifugation
from E-AdMSC supernatant and observed by SEM. Scale bar = 5 μm. (E) Image shows
a great number of MVs (arrows) surrounding and emerging from an E-AdMSC (asterisk). SEM, scale bar = 2 μm.
Over recent years, some of the biological effects of MVs as mediators of intercellular communication have been revealed (Gyorgy
et al., 2011). By virtue of their ability to transfer proteins, lipids, and
various forms of RNAs, they can modulate a variety of physiological and pathological processes in a paracrine and endocrine fashion
(Gyorgy et al., 2011). Considering the current interest in the biological properties of equine MSCs and their increasing use in
regenerative medicine, we aimed to establish whether E-AdMSCs
produced MVs that are able to influence angiogenesis, a key pathway
of tissue regeneration (Lamalice et al., 2007). By means of electron microscopy, we demonstrated that E-AdMSC-derived MVs, used
in functional assays, contained a heterogeneous mixture of exosomes
and shedding vesicles. Previous studies have indicated the difficulty in isolating a pure exosomal population (Barile et al., 2012; van
der Pol et al., 2012). Examination of cell monolayers by electron microscopy, allowed us to clearly distinguish the different pathways
of shedding vesicle and exosome formation, demonstrating that both
fractions were produced by E-AdMSCs.
We hypothesised that horizontal transfer of membrane components, such as MVs, could be a possible mechanism of intercellular
communication between E-AdMSCs and vascular endothelial cells.
The results of the study suggest that MVs might promote
neovascularisation by stimulating migration and proliferation of endothelial cells, as well as by inducing complete microvascular
formation, as evidenced in the aortic ring assay. These data strongly
suggest a capacity for MVs to interact, not only with endothelial cells,
but also with cells of the vessel wall (i.e. pericytes).
Previous studies have reported that MVs can induce angiogenesis by two mechanisms; MV-mediated transfer of cytokines or
growth factors that directly stimulate angiogenesis (Kim et al., 2012),
or MV-mediated transfer of RNAs that induce changes in the
secretome of target cells, either increasing production of
proangiogenic factors or decreasing production of anti-angiogenic
factors (Deregibus et al., 2007; Sahoo et al., 2011; Li et al., 2013).
In both cases, MVs operate as a trans-cellular delivery system that,
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Fig. 2. Results of scratch assay. In untreated samples, endothelial cells display a very scant migratory ability from time 0 (A) to 24 h (B) and 48 h (C). In contrast, E-EVEC
migration is promoted after incubation with MVs from time 0 (D) to 24 h (E), such that the gap is almost completely filled by 48 (F). In A and D, the dotted lines indicate the
margin of the scratch.
by virtue of its biological cargo, is capable of eliciting a specific response, or reprogramming target cell function.
It has been shown by Sahoo et al. (2011) that the angiogenic
effects of exosomes, derived from human CD34+ bone marrow stem
cells, on vascular endothelial cells were probably due to the presence of microRNAs 126 and 130a. Additionally, bone marrow MSCderived exosomes have been shown to favour tumour growth and
angiogenesis in a mouse xenograft model of gastric carcinoma,
through increased expression of vascular endothelial growth factor
(VEGF) (Zhu et al., 2012). Other studies have demonstrated the ability
of MVs, or of isolated exosomes, to influence angiogenesis in both
in vitro and in vivo experimental models, by virtue of their RNA
content (Deregibus et al., 2007; Li et al., 2013). In some studies, MVs
appeared even more potent than the originating stem cells themselves, possibly because of a delay in their degradation in culture
or at the site of injury (Collino et al., 2010; Martinez and
Andriantsitohaina, 2011; Shai and Varon, 2011).
Few studies have investigated the protein content of MSCderived MVs. Work by Kim et al. (2012) identified more than 700
proteins in bone marrow MSC-derived MVs, most possessing tissue
regenerative properties. According to the ExoCarta database1, over
4000 different proteins have been identified in exosomes derived
from different cell sources, thus suggesting a role for MV-mediated
transfer of cytokines and growth factors in cell–cell interactions.
1
See: www.exocarta.org.
Fig. 3. Scratch assay analysis. E-VEC migration into the intervening space was quantified by counting the cells at different time intervals at 20 × magnification in five
random fields. Data are shown as means + SD of four MV specimens, with each migration test performed in triplicate for each specimen. *P < 0.01 vs. control (CTRL).
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Fig. 4. Rat aortic ring assay. (A) Neovessel sprouting after incubation of vessel rings with control medium. Light microscopy, 4 × magnification. (B) Sprouting of microvessels
after incubation with MVs. Light microscopy, 4 × magnification. (C) Outgrowth of new microvessels from rings stimulated with E-AdMSC-MVs. Light microscopy, 20 × magnification.
The results of the present study demonstrate that E-AdMSCMVs are involved in modulation of different stages of the angiogenic
process, both on isolated equine endothelial cells and in a rat model
of blood vessel growth. Although the precise mechanisms by which
MVs induce angiogenesis remain to be resolved, they might be able
to influence endothelial cell function both via their effects on the
transcriptome and secretome and via cytokines or growth factors.
The different types of response induced by MVs in target cells
is probably determined by the contribution of each fraction (shedding vesicles and exosomes) and of each mechanism (RNAs or
protein-mediated) that, in turn, are influenced by the microenvironment. This suggests that MVs may represent a flexible system
and have the capacity to be manipulated to allow different responses to be elicited, depending upon the environmental conditions.
Conclusions
Although the molecular content and functional activities of MVs,
produced by E-AdMSCs, remain to be characterised, the results of
the present study have indicated that MSCs from equine adipose
tissue constitutively produce MVs that may be partly responsible
for their paracrine activity. Since MSCs undoubtedly induce angiogenesis through a complex set of mechanisms, involving both soluble
factors and MVs, further research is required, comparing the effects
of whole conditioned medium, MV-deprived conditioned medium
and conditioned medium obtained after MV inhibition, to clarify
the contribution of each pathway in modulating angiogenesis. Our
results support the concept of using E-AdMSC-MVs in veterinary
regenerative medicine, either alone or in combination with MSCs,
to enhance therapeutic efficacy.
Conflict of interest statement
None of the authors of this paper has a financial or personal relationship with other people or organisations that could
inappropriately influence or bias the content of the paper.
Acknowledgement
This work was partly supported by Fondazione Cassa di Risparmio
di Perugia. Grant 2013.0240.021. The authors are grateful to Mrs.
Maria Gabriella Mancini and Mrs. Claudia Pellegrini for their helpful
suggestions and for technical support.
Appendix: Supplementary material
Supplementary data to this article can be found online at
doi:10.1016/j.tvjl.2014.08.021.
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Please cite this article in press as: Luisa Pascucci, et al., Membrane vesicles mediate pro-angiogenic activity of equine adipose-derived mesenchymal stromal cells, The Veterinary
Journal (2014), doi: 10.1016/j.tvjl.2014.08.021