Note: Descriptions are shown in the official language in which they were submitted.
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NEW ANTI-ANGIOGENIC EXTRACELLURLAR VESICLES
FIELD OF THE INVENTION
The present invention discloses new anti-angiogenic extracelluiar vesicles
(extracellular vesicles, :EV) formed from stem/stromal mesenchymal cells (also
known as mesenchymal stem cells, MSC), said EVs for the treatment of diseases
characterized by an increased vascularisation, pharmaceutical compositions
comprising the same and methods for the preparation of said EVs.
STATE OF THE ART
Angiogenesis, is the process that leads to the ftrmation of new
blood/lymphatic vessels from a pre-existing vascular system, plays a key role
in
many physiological processes (e.g. during foetal development and in tissue
regeneration) as well as in many pathological conditions such as ischemic,
inflammatory, autoimmune diseases and in cancer.
The process of angiogenesis consists in the succession of finely regulated
different
phases that are characterized by endothelial and extra-cellular matrix
modifications.
At the beginning of the process, an increase in the permeability of the
vessels with
decreased connection between endothelial cells is observed. This is followed
by the
disruption of the capillary basal membrane which is essential to enable the
tissue
invasion by new vessels. At this stage, the endothelial cells organize by
forming new
vascular lumens. Finally, the newly .formed capillary is stabilized with the
construction of the basal membrane and intercellular junctions. This process
can be
altered when vascular insufficiency occurs (such as in stroke) or, on the
opposite, by
excess proliferation, such as in haemangiomas, in tumours and in
retinopathies.
In particular, angiogenesis and inflammation are two closely interconnected
processes. Upon inflammation, if the inflammatory stimulus persists,
angiogenesis is
initiated by the migration of endothelial cells lining the venules into the
tissue. The
generation of new blood vessels is required for the survival of inflammatory
cells
within the tissue, and thus inhibition of factors that promote angiogenesis
may reduce
inflammation and prevent its pathological consequences such as inflammatory
tissue
damage, autoimmunity, -fibrosis or tumour growth.
Consequently, the control of angiogenesis is a very interesting therapeutic
approach
for various pathologies. A positive control is desired when for all the
pathologies
wherein the formation of new vessels has a therapeutic effect, whereas a
negative
control is desired for all the pathologies in which angiogenesis plays a key
role in the
triggering and/or maintenance of the disease such as in inflammatory diseases,
proliferating diseases (retinopathies, cancers, neoplastic diseases),
autoimmune
diseases, fibrosis, transplant rejections and the like.
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Many molecules that interfere with angiogenesis have been developed and tested
both in preclinical and clinical stages, but their effectiveness is generally
limited.
Mesenchymal stem cells (MSCs) are multipotent progenitor cells with self-
renewable capacity and the potential to differentiate into various mesodermal
lineages. MSCs are present in the stromal fraction of many tissues, where they
reside
close to blood vessels, a trait that is shared with pericytes. Indeed, when
analysed in
vitro, MSCs and pericytes display similar morphological and functional
features,
although the two cell types are likely to have different functions in viva
While
pericytes regulate capillary homeostasis and architecture, the in vivo
functional role
of MSC is less clear and it is likely to be tissue-specific. For example, in
the bone
marrow. MSC contribute to the formation of the "niche" for the hematopoietic
stem
cells (HSCs), thus providing an appropriate microenvironment for
haematopoiesis. In
other tissues, MSCs may be involved in homeostatic control and tissue repair.
MSCs have a potent stabilizing effect on the vascular endothelium, having the
5 capacity of inhibiting endothelial permeability after traumatic brain
injury and in
haemorrhagic shock. Therefore, the vascular endothelium seems to be a specific
target of MSC biological activity.
Recently, an anti-angiogenic effect of MSCs was demonstrated by Zanotti et al
(Zanotti et al, Mouse mesenchymal stem cells inhibit high endothelial cell
activation
20 and lymphocyte homing to lymph nodes by releasing TIMP-1, Leukemia
(2016) 30,
1143-.1154) the effect being mediated by soluble factors.
However, although there is a growing interest in using MSCs to treat human
inflammatory diseases, various trials reported non-homogeneous results, with
MSCs
responses varying from 15 to 55% of treated patients.
25 The reasons fur these conflicting results are not clear in the art and,
among others,
may include differences in the number of MSCs that remain viable in patients
overtime, a critical factor that so far is very difficult to control.
Another critical aspect of MSC-based cell therapy is its safety, especially
when
considering long-term complications: MSCs may cause tumour formation or
30 aberrantly differentiate after ectopic engraftment.
Finally, another risk factor is associated with the administration route.
Indeed,
although the number of MSCs required to achieve immunoniodulation in vivo is
basically unknown, injection of large number of cells may he necessary to
obtain
maximal clinical benefit given the low rate of cell retention and survival. In
these
35 conditions, MSCs may form aggregates that could cause pulmonary emboli
or
infarctions in patients.
Encapsulation of MSCs (E-MSCs) has been suggested in order to solve the
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administration problem.
It is clear from the above that, interfering with angiogenesis is a
therapeutic approach
with a broad application spectrum and that the identification of new anti-
angiogenic
tools useful in therapy is of constant interest.
SUMMARY OF THE INVENTION
The authors of the present invention provide a highly effective tool for the
inhibition of angiogenesis that does not present the drawbacks known to be
associated with the use of MSCs.
As already discussed above, it has been reported in the art that MSCs cells
cultured in the presence of certain pro-inflammatory cytokines exhibit an anti-
angiogenic activity (see Zanotti et al 2016). However, the paper disclosing
said
information, also demonstrates that the activity is due to proteins secreted
by the pro-
inflammatory cytokines pre-conditioned MSCs cells.
Surprisingly, the author of the invention have found that EVs MSCs isolated
from MSCs cells cultured in the presence of pro-inflammatory cytokines exhibit
an
anti-angiogenic activity thereby maintaining the anti-angiogenic activity
exhibited by
the MSCs cells due to proteins secreted by said cells.
It is to be noted that, to date, all the EVs MCSs described in the art
exhibited
only pro-angiogenic activity.
The present invention provides, for the first time, Extracellular Vesicles
released by Mesenchymal stemistromal Cells (EVs MSCs) said EVs MSCs
exhibiting angiogenic inhibitory activity.
The authors of the present invention have also demonstrated, with standard
assays for assessing and measuring angiogenic activity, that the new EVs MSCs
of
the invention possess measurable anti-angiogenic activity both in vitro and in
vivo.
EVs have already been used as therapeutic tools in the art, they are complex
biological particles that transmit a series of signals, and may therefore
interfere at
various levels with both the angiogenic,-, process that with that
inflammatory, on the
contrary of the drugs currently in use, which act selectively on a specific
street or
metabolic stage.
Therefore, the new EVs MSCs of the invention provide a new, safe and
powerful tool that can be used for the therapy of diseases in which
angiogenesis
plays a pathogenic role.
Objects of the present inventions are therefore
-Extracellular Vesicles released by Mesenchymal stemistromal Cells (EVs MSCs)
said EVs MSCs exhibiting angiogenic inhibitory activity; said EVs MSCs for use
in
therapy, a pharmaceutical composition comprising said EVs MSCs) wherein said
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EVs MSCs, and at least one pharmaceutically acceptable carrier; said
pharmaceutical
composition for use in therapy; a medical device comprising said EVs MSCs;
process for the preparation of said EVs MSCs, EVs MSCs obtainable or obtained
by
said process, a pharmaceutical composition comprising the same, their use in
therapy
and, the use of the EVs MSCs of the invention, in any embodiment described for
the
preparation of a medicament and a medical treatment comprising the step of
administering to a subject in need thereof a therapeutically effective amount
of the
EVs MSCs of the invention.
DETAILED DESCRIPTION OF 711-IE FIGURES
Figure 1 Ells isolated from primed MSC inhibit angiogenesis in vitro.
The figure shows the results of a comparative test on the effects of MSC EVs
obtained from MSC cells stimulated with inflammatory agents according to the
process of the invention and of MSC EVs obtained from unstimulated MSCs.
The experiment, carried out on a line of murine endothelial cells (SVEC4-10),
is described in detail in the examples section.
The figure reports the quantification of segment length of tubes produced by
SVEC4-10 cells cultured either with EVs obtained from MSC stimulated with
inflammatory agents according to the process of the present invention (EV st
MSC-
CM) or with EVs obtained from MSC that were not stimulated with inflammatory
agents (EV unst MSC-CM). The same analysis was carried out also growing SVEC4-
10 cells with the supernatant of the culture medium of MSC stimulated with
inflammatory agents according to the process of the present invention (St MSC-
CM)
or from MSC that were not stimulated with inflammatory agents (unst MSC-CM).
Figure 2 EVs isolated from primed MSC affect angiogenesis in vivo.
The 'figure shows the results obtained on vascularization model on a matrigel
plug. The experiment is described in detail in the examples section.
Anesthetized 12-,veek-old male C57B116N mice were subcutaneously
injected in the dorsal back either with 5x105 'unst- or st-MSC, or EVs
obtained from
the same cells, mixed with 5000 Matrigel. Matrigel plus 50 riglmL VEGF and
100 ng/ML bEGF was used as positive control. Bare Matrigel was injected as
negative control. The plug was removed after 10 days and the vascularization
of each
plug was evaluated by haemoglobin quantification.
The figure shows that the plugs supplemented with EV at MSC-CM or with at
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MSCs showed a vascularization that was even below the one observed in the
negative control thereby demonstrating the in vivo antiangiogenic effect of
the EVs
MSC of the invention.
Fig. 3: EVs isolated from primed MSC affect angiogenesis in the
5 developing mouse retina.
The figure shows the results obtained using a model of vascularization in the
developing mouse retina upon systemic treatment with EVs obtained from
stimulated
or unstimulated MSC. The experiment is described in detail in the examples
section.
I-day-old C57BILl6N mouse pups were intraperitoneally injected with 50W
EVs from unst or st MSC. Retinas were collected and retina whole mounts were
dissected and stained appropriately before being flat-mounted. Digital images
were
captured using inverted fluorescence corkfocal microscope (A-B). Total retinal
and
vascular areas were measured using Image.f. The relative radial expansion (C)
and
the total retinal branching point (D) were analysed. For the retinal radial
expansion,
is the retinal radius (R, indicated with the black arrow from the optic
nerve to the edge
of the retina) and the vascular radius (v, indicated with the grey arrow from
the optic
nerve to the vascular front) of each petal of the retina were measured. The
retinal
vascular expansion was calculated as the ratio between the vascular radius (v)
and
the retinal radius (R). The data are expressed as means S.E.M. normalized on
control, mice treated with vehicle (n = 4 mice)*P <005, T test.
The figure shows how systemic administration of the EVs of the invention
has a direct effect on the retinal vascularization.
Fig. 4 - EVs from MSC-CM fully mimic the whole conditioned medium
The comparative analysis of the whole medium conditioned by unst- or st-
MSCs (unst MSC-CM or at MSC-CM) and their extracellular vesicles (EV unst or
at
MSC-CM), was assessed by performing the tube formation assay. SVEC4-10 cells
were seeded on the top of a matrigel layer in the presence of appropriate
stimuli.
After 6 hours, images were acquired with a phase contrast inverted microscope
at 4x
objective magnification.A) Representative pictures of the experiment; scale
bar
corresponding to 100um. B) Quantification of the relative tube length
(normalized on
medium), performed with image,1 Angiogenesis Analyzer. 3 independent
experiments, data are expressed as mean -_-Fr SEM (*p b 0.05, **p b 0,01, One-
way
ANOVA).C.) The expression of TIMP-1 on EVs was analysed by Western Blot,
using CD63 an CD9 as EVs-markers. 3 independent experiments; data are
expressed
as mean SEM (*p b 0.05, **p b 0.01, T-test. D) To investigate the role of
TIMP-1
in the angiogenesis inhibition mediated by EV from st MSC-CM, the tube
formation
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assay was performed adding a TIMP-I blocking antibody. Quantification of the
relative tube length (normalized on medium), performed with Image.'
Angiogenesis
Analyzer. 3 independent experiments, data are expressed as mean SEM (*p b
0.05,
**p b 0.01, One-way ANOVA).
Figure 5 - EVs from st- MSC-CM inhibit the migration of endothelial
cells stimulated with VEGF
The evaluation of endothelial cell migration was assessed by the scratch
assay. Wound was made by scratching a line across the bottom of the dish on a
confluent SVEC4-10 monolayer. Cells were treated with extracellular vesicles
isolated from unst- or st- MSC-CM (EV unst or st MSC-CM). 6 hours later, cells
were imaged with a phase contrast inverted microscope at 4x objective
magnifications, A) Representative pictures; scale bar corresponding to 100pm.
B)
quantification of the migration; analysis was performed with Image.1 by
measuring
the initial (time 0) and the final (time 6 hours) length of the scratch. Data
are
expressed as means S.E.M. (n= 3). *P <0.05, One-way ANOVA, C) To investigate
the effect of TIMP-1 in the anti-angiogenic action of EVs from st- MSC-CM, the
same experiment was performed with a TIMP-1 blocking antibody. Here, the
quantification of the migration expressed in kim, is reported. Data are
expressed as
means S.E.M. (n = 3). *P < 0.05, One-way ANOVA.
Figure 6 ¨ Adenosine mediates the second anti-anglogenic effect of the
EVs derived from st- MSC-CM
A) The expression ofectonucleotidases CD39 and CD73 on the EVs surface,
responsible for the ATP hydrolysis, was investigated through the Western Blot
assay,
using CD63 an CD9 as EVs-markers. 3 independent experiments; data are
expressed
as mean SEM (*p b 0.05, **p b 0.01, One-Way ANON/A). The scratch assays were
performed by inhibiting the activity of these enzymes, with ARE: 67156 for
CD39 B),
and AMP-CP for CD73 C) to investigate the implication of the ATP metabolites,
respectively AMP and adenosine, in the suppression of endothelial cell
migration
mediated by EVs derived from st- MSC-CM. Data are expressed as means S.E.M.
(n = 3). *P <0.05. One-way ANOVA.
Figure 7
Adenosine affects migrating endothelial cells, with a
mechanism dependent on ROS accumulation
A) The ability of EVs from st- MSC-CM to induce an increment in the ROS
levels was investigated by using CM-I-12DCFDA, a general oxidative stress
indicator, during scratch migration assays. Here are reported representative
images.
The mean of fluorescence was calculated through IrnageJ and normalized on the
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medium. Data are expressed as means + S.E.M. (n = 3). *P < 0,05, One-way
ANOVA. B) in an effort to evaluate the role of adenosine in this process,
experiments were repeated by blocking the CD73 activity, the final player in
the
ATP hydrolysis. Data are expressed as means S.E.M. (n=2). *P <0.05, One-way
ANOVA. C) The suppression of oxidative stress induced by EVs st- MSC-CMduring
the scratch assay, was assess troughthe treatment with a generic antioxidant N-
Acetyl-L-cysteine (NAG), to confirm the inhibitory effect of ROS. Data are
expressed as means S.E.M. (n = 2.). *P < 0.05, One-way ANOVA.
Figure 8 ¨ In vivo therapeutic potential of !Ns derived from MSCs for
the control of angiogenesis
A) To assess the role of EVs isolated from MSC-CM in the angiogenesis
process of developing mouse retina, 1-day-old C57BL/6N mouse pups were
intraperitoneally injected with 50u1 EVs from unst or st MSC-CM. After 4 days,
mice were sacrificed for retina collection. Retina whole mounts were dissected
and
is stained with isolectin B4 (Green, on the left). -Digital images were
captured using
inverted fluorescence contbcal microscope (on the right). B) Total retinal and
vascular areas were measured using image,' software to calculate the relative
radial
expansion and the relative branching points (both normalized on pups treated
with
the vehicle). Data are expressed as means + S.E.M. (11 =10 pups/group in 3
different
70 experiments). *P < 0,05, One-way ANOVA. C)Anesthetized 12-week-old male
C57BI-16N mice were subcutaneously injected in the dorsal back with EVs from
unst- or st- MSC-CM, mixed with Matrigel plus VEGF. Bare Matrigel was injected
as negative control. After 7 days, mice were sacrificed, and the Matrigel
plugs were
harvested, for the haemoglobin quantification. Plug haemoglobin content was
25 measured using Drabkin's reagent kit 525 (Sigma-Aldrich) and normalized
to the
total protein quantity measured by BCA. Data are expressed as means SEM (n =
8
mice/group). *P < 0.05, T test
DETAILED DESCRIPTION OF THE INVENTION
30 Glossary in the Meaning of the invention:
Mesenehymal stem cells (MSC) are multipotent progenitor cells with self-
renewable capacity and the potential to differentiate into various mesodermal
lineages. MSC according to the invention are present in the stromal fraction
of many
tissues, where they reside close to blood vessels. In the meaning of the
present
35 description MSC can be, depending on the receiver for therapy, of animal
or of
human origin. MSCs are, in the present description, as defined in the art
unless
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differently stated. MSCs are considered adult or somatic stern cells and
remain in a
non-proliferative, quiescent state during most of their lifetime, until
stimulated by the
signals triggered by tissue renewal, damage and remodelling processes, 'When
derived from fetal membranes, such as chorionie and amniotic membranes, MSCs
are considered an intermediate between human embryonic stern cells (hESCs) and
adult stem cells. When the meaning is referred to human MSCs, non-embryonic
MSCs are considered as a possible preferred embodiment of the invention.
In the meaning of the present description Extracellular Vesicles released by
Mesenchymal stemistromal Cells or "EVs MSCs" are Extracellular 'Vesicles
secreted by Mesenchymal stem/stromal Cells. According to the art and to the
present
description EVs MSCs include all kind of EVs secreted/released by l',ASCs.
MSCs
secrete a wide range of extracellular vesicles (EVs) of different size,
morphology,
content and function that interact with target cells and modify their
phenotype and
function. Still according to the art and to the present description EVs can be
Ã5
classified according to their size, origin, and isolation methods, into three
main
classes: (i) Microvesicles or shedding vesicles (size between 50 and 1000 nm,
budding from the plasma membrane, and enriched in CDR)); (ii) Apoptotic bodies
(size between 800 and 5000 mu, derived from fragments of dying cells, and
enriched
in histones and DNA); and (iii) Exosomes, which are small (-30-120 nm)
membrane
vesicles from endoeytie origin (enriched in late endosomal membrane markers,
including Tsg101, CD63, CD9, and CD81). In the meaning of the present
description, in accordance with the state of the art, all the kinds of
vesicles listed
above, released/produced by MSCs are encompassed by the expression
Extracellular
Vesicles released by Mesenchymal stern/stromal Cells or "EVs MSCs".
In the meaning of the present description the term "angiogenesis" may
encompass the formation of new blood vessels and/or lymphatic vessels from pre-
existing vessels (also known in the art, respectively as angiogenesis and
lymphangiogenesis). In the meaning of the present description both meanings
(blood
vessels angiogenesis and lymphatic vessels angiogenesis) are encompassed by
the
more general angiogenesis. Hence, when used herein, the term may refer to the
formation of new blood vessels, to the formation of new lymphatic vessels or
to the
formation of both,
In =the meaning of the present description the term anti-angiogenie or
"exhibiting angiogenesis inhibitory activity" are considered as synonyms and
indicate a compound, a cell, a vesicle, a composition, a molecule, a mixture,
a
moiety, a substance, a product, inhibiting angiogenesis in vitro and/or in
vivo as
defined above. In the meaning of the present description the inhibition may be
at
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least partial i.e. a reduced angiogenesis when compared to a positive control
that is
not treated with the anti-angiogenic compound, cell, vesicle, composition,
molecule,
mixture, moiety or substance tested, or total i.e. no detectable angiogenesis
when
compared to a positive control that is not treated with the anti-angiogenic
compound,
cell, vesicle, composition, molecule, mixture, moiety, product or substance
tested.
in the meaning of the present description, a therapeutically effective
amount is an amount sufficient to exert in a patient or in a disease model
assayed, an
inhibition of the angiogenic activity as defined above in the site of
interest, thereby
provoking at least a reduction of the symptoms of the disease treated. In
other terms,
therapeutically effective amount as used herein, means that amount of active
compound or pharmaceutical agent that elicits the biological or medicinal
response in
a tissue system, animal or human that is being sought by a researcher,
veterinarian,
medical doctor or other clinician, which includes alleviation of the symptoms
of the
disease or disorder being treated.
IS As used herein, the term "composition" is intended to encompass a
product
comprising the specified ingredients in the specified amounts, as well as any
product
which results, directly or indirectly, from combinations of the specified
ingredients in
the specified amounts.
The term "subject" or "patient" as used herein, refers to an animal,
preferably a mammal, most preferably a human, who has been the object of
treatment, observation or experiment.
As already stated above, the present description discloses new Extracellular
Vesicles released by Mesenehymal stem/stromal Cells (EVs MSCs) said EVs MSCs
exhibiting angiogenic inhibitory activity (or anti-angiogenic activity as a
possible
synonym in the whole description as stated above),
According to the present description the EVs MSCs of the present invention
exhibit said angiogenic inhibitory activity is exhibited by said EVs MSCs in
vitro
and/or in vivo. In a preferred embodiment said activity is exhibited both in
vitro and
in vivo.
Hence, according to the invention, the anti-angiogenic EVs MSCs claimed
exhibit a detectable and/or measurable angiogenic inhibitory activity.
According to
an embodiment of the invention, said activity can be assayed in standard in
vitro tube
formation assay. Tube formation assay is a standard test, known in the art and
well
reported in literature, to measure/assess the angiogenic (negative or
positive) activity
of a product.
According to the present invention any tube formation assay disclosed in the
art is suitable for measuring/detecting the anti-angiogenic activity of the
Ely's MSCs
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of the invention.
By way of example, and. not as with a limitative purpose, the tube formation
assay can be canied out as described in Ponce; 2009. Methods Mol Biol.
2009;467:183-8. doi 10.1007/978-1-59745-241-010.Tube formation: an in vitro
5 matrigel angiogenesis assay.
According to another non-limiting example the tube formation assay
according to the present description can be catTied out as detailed in the
experimental
section.
According to another embodiment of the invention, the angiogenic inhibitory
to activity of the EVs MSCs of the invention can be assessed an in vivo
matrigel plug
vascularisation assay.
As the tube formation assay, also the matrigel plug vascularisation assay is a
standard assay to assess the angiogenic activity of a product.
According to the present invention any matrigel plug vascularisation assay
5 disclosed in the art is suitable for measuring/detecting the anti-
angiogenic activity of
the EVs MSCs of the invention.
By way of example, and not as with a limitative purpose, the matrigel plug
vascularisation assay can be carried out as described in Brown et al, 2016.
Methods
Mol Biol. 2016;1430:149-57. doi: 10,1007/978-1-4939-3628-1 9.Tube-Forming
Assays.Brown RM1, Meah 0-2, Heath V12, Styles 1B3, Bicknell R4.
According to another non-limiting example the matrigel plug vascularisation
assay according to the present description can be can ____________________ ied
out as detailed in the
experimental section.
As already defined above, the term angiogenesis according to the present
description, encompasses blood 'vessels angiogenesis and/or lymphatic vessels
angiogenesis, therefore, according to an embodiment of the invention, the EVs
MSCs
described above and in anyone of the following embodiments may exhibit
inhibitory
activity of blood vessels angiogenesis and/or of lymphatic vessels
angiogenesis
As already stated EVs M.SCs of anyone of the embodiments herein disclosed
is suitable for use in therapy. As discussed above, angiogenesis is a feature
of various
diseases. Inflammatory diseases are characterised by angiogenic activity,
proliferative diseases are characterised by angiogenic activity, autoimmune
diseases
are also characterised by angiogenic activity as well as transplant rejection.
Therefore, the Ells MSCs of the invention are particularly useful in the
treatment of diseases, and or condition wherein the inhibition of angiogenesis
results
in a therapeutic effect. Hence, the EVs MSCs of the invention are suitable for
use in
therapies wherein inhibition of angiogenesis is indicated.
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According to an embodiment, the EVs MSCs of the invention are particularly
suitable for use in the treatment of pathologies or conditions characterised
by the
pathological formation of new blood and/or lymphatic vessels by angiogenesis.
According to the present description said pathologies or conditions are
selected from proliferative, inflammatory and autoimmune diseases or
conditions,
ocular and periodontal diseases, obesity- and aging-related conditions.
In an embodiment of the invention said proliferative diseases are selected
from cancers; said inflammatory/autoimmune diseases are selected from
psoriasis,
arthritis, endometriosis, Crohn's disease, inflammatory bowel disease; said
ocular
diseases are selected from diabetic retinopathy, retinopathy of prematurity,
radiation
and solar retinopathies, macular degeneration; said periodontal diseases are
selected
from gingivitis and periodontitis; said Obesity-related conditions are
selected from
obesity-driven neovascularization and obesity-driven inflammation; said aging-
related conditions are selected from wrinkle and cutaneous aging.
Another embodiment of the invention is a pharmaceutical composition
comprising Extracellular Vesicles released by Mesenchy-mal stem/stromal Cells
(EVs
MSCs) wherein said EVs MSCs exhibit angiogenic inhibitory activity and at
least
one pharmaceutically acceptable carrier. Any one of the embodiments described
above for the EVs MSCs apply to the pharmaceutical composition of the
invention.
Therefore, the pharmaceutical composition of claim 11 characterised in that
said
angiogenic inhibitory activity is exhibited by said EVs MSCs both in vitro and
in
vivo and may be assayed via an in vitro tube formation assay and/or an in vivo
matrigel plug vascularisation assay.
According to the present invention, the pharmaceutical composition will exert
anti angiogenic activity over blood vessels angiogenesis and/or a lymphatic
vessels
angiogenesis.
The pharmaceutical composition of the invention will be prepared in a form
suitable for both systemic (enteral and parenteral) and topical
administration.
Non limiting examples of said administration modes are intravenous,
intramuscular, in.tra-organ, transdermal, rectal, eyedrops, intravitreal and
others
commonly used in the art.
Hence, according to the present invention, the pharmaceutical composition of
can be in the form of a solution, a suspension, a cream, a foam, an emulsion,
a paste,
a gel, a lotion, a shake lotion, an ointment, a transdermal patch, a powder, a
sponge,
eye drops, a tape, a suppository, an enema.
Solutions and emulsions can be suitable either for systemic either for topical
administration or both.
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Each form can be prepared by the skilled person according to the best practice
of pharmaceutic preparation, therefore, following the common technical
knowledge,
the skilled person will know how to select the suitable carriers and other
useful
ingredients for the preparation
The EVs MSC's of the invention directly be administered alone and is usually'
preferably made into various pharmaceutical preparations. The pharmaceutical
preparations can be produced by a routine method of pharmaceutics by mixing
the
active ingredient with one or two or more pharmacologically acceptable
carriers.
A carrier may take a wide variety of forms depending on the form of
o preparation desired for administration. These pharmaceutical compositions
are
desirably in unitary dosage form suitable, preferably, for systemic or topical
administration or parenteral injection. For example, in preparing the
compositions in
oral dosage form, any of the usual pharmaceutical media may be employed. For
parenteral compositions, the carrier will usually comprise sterile water, at
least In.
large part, though other ingredients, for example, to aid solubility, may be
included.
Injectable solutions, for example, may be prepared in which the carrier
comprises
saline solution, glucose solution or a mixture of saline and glucose solution.
Injectable suspensions may also be prepared in which case appropriate liquid
carriers
and suspending agents may be employed. In the compositions suitable for
transdennal administration, the carrier optionally comprises a penetration
enhancing
agent and/or a suitable wetting agent, optionally combined with suitable
additives of
any nature in minor proportions, which additives do not cause a significant
deleterious effect to the skin. Such additives may facilitate the
administration to the
skin and/or may be helpful for preparing the desired compositions. These
compositions may be administered in various ways, e.g., as a transdermal
patch, as a
spot-on, as an ointment.
The pharmaceutical composition of the invention will be suitable for use in
therapy. The composition will be useful for the treatment of any of the
diseases or
condition already described above. In particular, the composition is useful
for the
treatment
According to the present description said inflammation-associated
pathologies can be selected from proliferative diseases, inflammatory
diseases,
auto immun e diseases, transplant rejections proliferative, inflammatory and
autoimmune diseases or conditions, ocular and periodontal diseases, obesity-
and
aging-related conditions.
In an embodiment of the invention said proliferative diseases are selected
from cancers; said inftammatory/autoimmune diseases are selected from
psoriasis,
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arthritis, endometriosis, Crohn's disease, inflammatory bowel disease; said
ocular
diseases are selected from diabetic retinopathy, retinopathy of prematurity,
radiation
and solar retinopathies, macular degeneration; said periodontal diseases are
selected
from gingivitis and periodontitis; said obesity-related conditions are
selected from
obesity-driven n.eovascularization and obesity-driven inflammation; said aging
related conditions are selected from wrinkle and cutaneous aging.
In a particular embodiment, the invention relates to a medical device
comprising the EVs MSCs or the pharmaceutical composition as described herein,
in
a particular embodiment said medical device is in the form of a transdermal
patch, in
another embodiment said medical device is in the form of a dispenser for
intranasal
administration.
The present invention also relates to a process for the preparation of MSCs
Pis comprising the following steps:
a. culturing MSCs in a medium complemented with one or more
inflammatory cytokine
b. removing said medium and culturing said MSCs in a medium without said
one or more pro-inflammatory cytokine and collecting EVs from the culture
sumatant.
According to the present description, said one or more pro-inflammatory
cytokine can be selected from IL- 1 0, IL-6, TNFa and chemokines . Therefore,
during
step a) of the process of the invention, one or more of the pro-inflammatory
cytokines listed above can be used to complement the growth medium used,
In a particular embodiment, said pro-inflammatory cytokines are at least two
or at least three.
In a preferred embodiment said pro-inflammatory cytokines are represented
by a mixture of IL-11.1, 11,-6 and TNFct.
According to the invention, a suitable amount of pro-inflammatory cytokines
for carrying out step a. can be represented by a total amount of cytokines of
about
30-70 nglini. When more than one cytokine is used, the concentration can be
concentration between from 15 11g/int to 40 ng/m1 of each c:,'tokine in case
two
cytokines are used or of between from between from 15 ngimi to 30 ng/rid of
each
cytokine in case three cytokines are used.
According to the invention, any known medium commonly used for growing
MSCs is suitable for carrying out steps a. and b. of the invention.
36 Both in steps a. and b. the medium will be further complemented with
standard additional compounds/substances commonly used for the cultivation of
MSCs such as bovine foetal serum (FIBS) in suitable amounts, antibiotics,
suitable
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amino acids and the like.
The concentration of said additional compounds/substances can be readily
assessed by the skilled person following standard protocols.
A non-limiting example of a suitable medium and of suitable additional
compounds/substances is also provided in the experimental section below.
By way of example FBS can be from 5 to 15%, e.g. about 5%, 6%, 7%, 8%,
9%, 10%, 11%, 12%, 13%, 14%, 15%.
Always by way of example, suitable antibiotics can be any antibiotic
commonly used for the culture of MSCs such as streptomycin, penicillin, altri
or a
mixture thereof Commonly used amounts of antibiotics can be used in the
process of
the invention. A non-limiting example is an overall antibiotic amount of 80-
150 U/m1
(eg. About 100 U/m1). In a non-limiting example about 100 Ulm! of
penicillin/streptomycin can be used.
Further additional complementing substances can be one or more amino acid.
in one non limiting example about 2 111M glutamine can be added to the growth
medium.
Suitable media for step a. orb. of the process of the invention are
represented
by any medium commonly used for culturing MSC's. A non-limiting example of
said
media is represented by [MEM low glucose or similar media.
According to the invention, said MSCs can be cultured from 18 to 30 hours
during step a. hence, said MSCs can be cultured about 18, 19, 20, 21, 22, 23,
24, 25,
,27, 28, 29, 30 hours.
In an embodiment of the invention said MSCs are cultured during step a. for
about 22-26 hours, e.g. for about 22, 23, 24, 25 or 26 hours.
25 According to the present description, once the medium of step a. is
removed
and a cytokine-free medium is used in step b. the MSCs cells can be cultured
from 12
to 24 hours during step b.
According to the invention, said MSCs can be cultured from 12 to 24 hours
during step b. hence, said MSCs can be cultured about 12, 13, 1.4, 15, 16, 17,
18, 19,
20, 21, 22, 23, 24 hours.
In an embodiment of the invention said MSCs arc cultured during step b. for
about 16-20 hours, e.g. for about 16, 17, 18, 19 or 20 hours.
The isolation of EVs from culture medium surnatant is known in the art. Any
suitable method can be used for carrying out the process of the invention. In
a non-
limiting embodiment the EVs of the invention can be collected by
ultrafiltration of
the sumatant.
Another object of the invention is represented by the anti-angiogenic EVs
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MSCs obtainable from the process described above.
The anti-angiogenie EVs MSCs obtainable by the process of the invention
(herein referred to also as EVs obtained from stimulated MSC-CM) are
characterised
by the expression of TIMP1, CD73 and CD39.
5 In
particular, the EVs obtainable by the process of the invention, express each
of said markers at least in a two-fold ratio when compared to EVs obtained
from the
same MSC-CM without stimulation during cell culturing as disclosed in the
present
description (herein referred also as EVs obtained from unstimulated MSC-CM).
According to an embodiment, the EVs MSCs of the invention, he. the EVs
io
obtainable from stimulated medium, are characterized by an expression of TEMPI
of
at least two to tenfold higher in comparison to the expression of TIMP1 of the
EVs
MSCs from unstimulated medium as described herein. According to the present
invention, the expression of TIMP1 by the EVs of the invention is at least two-
fold,
three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold,
ten-fold
15 higher
in comparison to the EVs MSCs from unstimulated medium as described
herein.
Further, the EVs MSCs of the invention are characterized by an expression of
CD73 of at least two to three-fold higher in comparison to the expression of
CD73
of EVs MSCs obtainable from unstimulated medium as described herein.
The EVs MSCs of the invention are also characterized by the expression of a
CD39 specific band of molecular mass around 175 kDa in western blots where
proteins extracted therefrom are stained with anti CD39 specific antibodies.
When reference is made to a x-fold expression of a marker in the EVs of the
invention, I fold is the amount of the same marker expressed by the same
amount of
EVs obtained by the same population of cells, in the same culturing conditions
except that the medium is not complemented with one or more inflammatory
cytokine, as disclosed in the present description. In the present description,
the
comparison was carried out on 3lig of total proteins of EVs MSCs extracted
from
each kind of EVs obtained from unstimulated and stimulated medium and
submitted
to western blot for the marker quantification.
The invention also encompasses the use in therapy of said EVs MSCs,
pharmaceutical compositions comprising said -EVs MSCs and uses thereof, and
medical devices comprising said anti-angiogenic EVs MSCs and uses thereof.
All that has been described above with reference to the EVs MSCs of the
invention can be obviously refer to the EVs MSCs obtainable or obtained with
the
process described above, in the experimental section and claimed.
The invention also refers to the medical treatment of all the diseases and
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conditions described above wherein a therapeutically effective amount of the
EVs
MSCs of the invention or of the pharmaceutical composition of the invention
i.s
administered to a subject in need thereof.
EXPERIMENTAL SECTION
Anti-angiogenie EVs NISCs preparation
MSC were plated and let grow until confluence in ventilated cap flask in DMEM
low
glucose supplemented with 20% FBSõ 2mM glutamine, 100 Ulml
penicillin/streptomycin. Culture medium was then substituted with DMEM low
glucose supplemented with 10% FBS, 2mM glutamine, 100 U/m1
penicillin/streptomycin, with or without 25ng/m.1 mIL1 b, 20ng/m1 mIL6,
25nglml
mTNFa for 24 hours. The medium was then changed with DMEM low glucose
supplemented with 2mM glutamine, 100 Ulml penicillin/streptomycin for the
following 18 hours. Thus, conditioned media from unstimulated (unst MSC-CM) or
stimulated MSC (st MSC-CM) were Obtained. .EVs were isolated from unst or st
MSC-CM by ultratiltration using Amicong Ultra 15 mL Filters (Merck Millipore).
Tube formation assay.
The results of this assay are depicted in figure I.
In a flat-bottom 96 well plate, 1,3x104 SVEC4-10 cells (ATCCD CRL_2181TM) were
seeded in a Matrigel coated well (80u.1 Matrigel/well) in 1001.4.1 of either
unst or st
MSC-CM and their EV (EV unst or st MSC-CM). DMEM low glucose with 10%
heat-inactivated FBS was used as positive control. After 4 hours at 37 C 10%
CO2,
cell tubes were imaged with a phase contrast inverted microscope at 4x
objective
magnifications (A). Quantification of segment length was performed using the
Imagej Angiogenesis Analyse plugin (B). Data. are expressed as means S.E.M,
(n= 3). *P <0.05, T test.
Matrigel plug vaseolarization assay
The results of this assay are depicted in figure 2.
EVs isolated from primed MSC according to the invention affect angiogenesis in
vivo, Anesthetized 12-week-old male C5713116N mice were subcutaneously
injected
in the dorsal back either with 5x105 unst- or st-MSC, or EVs obtained from the
same
cells, mixed with 5001,d Matrigel. Matrigel plus 50 ng/iniõ VEGF and 100
nglinL
bFGE was used as positive control. Bare Matrigel was injected as negative
control.
For each group, matrigel was supplemented with Heparin (50 units/m1). After 10
days, mice were sacrificed, and the Matrigel plugs were harvested, weighed and
photographed (A). The haemoglobin quantification in homogenized plugs (B) was
performed using Drabkin's reagent kit 525 (Sigma-Aldrich) Data, normalized to
the
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I. 7
total protein content, are expressed as means S.E.M. (n 4 micelgroup), *P
<0.05,
T test.
Retinal vaseularization inhibition
The results of this test are depicted in figure 3,
EVs isolated from primed MSC according to the invention affect angiogenesis in
the
developing mouse retina.
1-day-aJld C57BL/6N mouse pups were intraperitoneally injected with 50111 EVs
from unst or St MSC, After 4 days, mice were sacrificed for retina collection.
Both
eyes were enueleated and fixed in 4% HA. Retina. whole mounts were dissected
and
stained with biotinylated isoleetin B4 (Vector Laboratories), and stained with
streptavidin¨Alexa 488 (Invitrogen) before being flat-mounted. Digital images
were
captured using inverted fluorescence confocal microscope (A-B). Total retinal
and
vascular areas were measured using Image.!. In details, the relative radial
expansion
(C) and the total retinal branching point (D) were analysed. For the retinal
radial
expansion, the retinal radius (R, indicated with the black arrow from the
optic nerve
to the edge of the retina) and the vascular radius (v, indicated with the grey
arrow
from the optic nerve to the vascular front) of each petal of the retina were
measured.
The retinal vascular expansion was calculated as the ratio between the
vascular
radius (v) and the retinal radius (R). Data are expressed as means S.E.M.
normalized on control, mice treated with vehicle (n = 4 mice)P <0.05, T test,
Extracellular vesicles recapitulate the phenotype of MSC-CM from which they
are isolated
The effect of extracellular vesicles derived from MSC-CM on angiogenesis was
evaluated. EV employment offers several advantages, such as high stability and
wide
dissemination potential, supporting the fascinating possibility of an
industrialized
EVs-based therapy.
EVs were obtained from unstimulated or stimulated murine MSC-CM by
ultrafiltration. Their quality and quantity were validated by Nanosight
analysis (data
not shown). To investigate the effect of EVs on angiogenesis, a tube formation
assay
with SVEC4-10 cells was carried out and the effects of INS isolated from
unstimulated or stimulated MSCs (EVs unst- or st- MSC-CM, respectively) with
whole medium (CM) was compared. As previously demonstrated, stimulated MSC-
CM decreases the tube length, in contrast to control and unstimulated MSC-CM.
Interestingly, the same anti-angiogenic effect was exerted by EVs from st-MSC-
CM,
which strongly inhibit the ability of SVEC4-I.0 to form capillary-like,
tubular
structures (Figure 14 A, B).
As TINE' I has a known prominent role as anti-angiogenic mediator of st-MSCs,
it
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was investigated whether TIMP1 is involved in the anti-angiogenic effect
exerted by
EVs derived from st-MSC-CM. Firstly, the presence of TIMP1 in the E:'µis of
the
invention was verified by Western Blot. TIMP1 is highly enriched in EVs st-MSC-
CM, while the expression of the extracellular vesicles' markers CD63 and CD9
was
unaffected (Figure 4 C). Then, the tube formation assay was repeated using
TIMP1
blocking antibody (Figure 4 D), the result show that the antiangiogenic effect
displayed by EVs isolated from st-MSC-CM is TEMPI -dependent.
Altogether, these results indicate that EV's show the same inhibitory effect
on
angiogenesis of the conditioned media they are isolated from.
The scratch wound healing assay reveals a second anti-angiogenic mechanism of
FiNs sit- MSC-CM
Angiogenie and antiangiogenic activities can be assessed by using several in
vitro
assays, which are typically exploited to investigate different crucial steps
of the in
vivo process124. in particular, through the tube formation assay the matrix
digestion
and the endothelial morphogenesis during the development of the tubular
network
was evaluated. However, the assembly of endothelial cells into vessel tubes in
vivo
also requires the coordinated migration of endothelial cells. To evaluate
whether
-EVs play a regulatory role during this process, the scratch wound healing in
vitro
assay, based on the ability of cells to move in a free space in response to a
pro-
angiogenic factor126 was used.
SVECA4-10 cells were seeded to form a monolayer and, by scratching, a cell-
free
gap in the confluent layer was generated. Stimulation with the pro angiogenie
factor
VEGF induced the subsequent cell migration. EVs derived from unst- or st-MSCs
were added. The distance covered by migrating cells was measured after 6 hours
of
incubation at 37QC 10% CO2 (Figure 5 A). As expected, in response to VEGF, the
migratory ability of SVEC4-10 cells is increased in comparison to =stimulated
cells
(vehicle without VEGF). Treatment with EVs from unst-MSC-CM did not affect the
migration of endothelial cells (Figure 5 B). In contrast, EVs isolated from st-
MSC-
CM completely abolished the SVEC4-10 migratory ability in response to VEGF
(Figure 5 B).
This suggests that ENS isolated from st-MSC-CM negatively control not only the
matrix digestion and endothelial morphogenesis during angiogenesis (Figure 4),
but
also the migratory ability of endothelial cells in response to VEGF.
To evaluate the implication of TIMP1 in this process, the scratch wound
healing
assay in presence of the TIMP1-blocking antibody was carried out (Figure 5 C).
Interestingly, in contrast to what was observed in the tube formation assay,
the
TIMP-1 blocking treatment did not rescue the migratory ability inhibited by
EVs
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from st- MSC-CM (Figure 5 C), suggesting the existence of a TIMPI-indipendent
mechanism -through which EVs derived from st-MSCs exert their anti-angiogenic
effect.
CD39 and C973 confer anti-angiogenic activity to EVs st-MSC-CM
In an effort to characterise the TIMP1-indipendent mechanism of EV-mediated
anti-
angiogenie effects, the possible involvement of adenosine, which has been
reported
to suppress the migration of several cell types was investigated. Adenosine is
a
purine nucleoside that can be generated by the hydrolysis of extracellular
ATP. This
reaction is exerted by two enzymes confined on the cellular plasma membrane,
the
ectonueleotidases CD39 and CD73, catalysing, respectively, the hydrolysis of
ATP
to AMP, and of AMP to adenosine' 25.
To investigate whether EVs from st-MSC-CM induce the accumulation of this
purine, the expression of CD39 and CD73 on the surface of the EVs of the
invention
was analysed by Western Blot (Figure 6 A). The obtained results show an
increased
level of both these proteins in Dis st-MSC-CM in comparison with .vesicles
isolated
from unstimutated MSCs. Additionally, a CD39-specific band of higher molecular
mass (around 175 kDa) only in EVs derived from st- MSC-CM was observed.
To explore whether adenosine could be responsible for the anti-arigiogenic
effect
exerted by EVs isolated from st- MSC-CM, the scratch wound healing assay as
previously described (Figure 5 A) was carried out in the presence of the CD39
or
CD73 inhibitors, frisodium salt hydrate (ARL 67156) and adenosine 5r- (a, [-I-
methylene) diphosphate (AMP-CP), respectively (Figure 6 B, C). Notably, the
inhibition of each enzyme fully recovers the distance covered by SVEC4-10
cells
treated with EV st-MSC-CM in the presence of VEGF.
Thus, these data demonstrate that ATP metabolites, generated by CD39 and CD73
expressed by EVs isolated from st- MSC-CM, inhibit the migration of
endothelial
cells in response to VEGF, with a mechanism completely independent from TIMP I
activity.
EVs st-MSC-CM induce reactive oxygen species (ROS) in migrating endothelial
cells
Recently, it has been extensively reported the involvement of adenosine in the
regulation of the reactive oxygen species (ROS) production, F,xtrac.:ellidar
adenosine
interacts with four subtypes of (ii protein-coupled cell surface receptors
(MR,
A2AR, A2BR, and A3R). Although the mechanism linking the adenosine receptors
with ROS production is far from being fully elucidated, it has been suggested
that
NADPH (nicotinamide adenine dinucleotide phosphate) oxidase (NOX) plays a
crucial role. Indeed, NOX activity, which generates ROS transferring electrons
from
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NADPH to molecular oxygen, has been correlated with the activation of
adenosine
receptors.
Oxidative stress is recognized as a potent inducer of senescence in
endothelial cells
and of dysfunctional cytoskeletal rearrangements two mechanisms potentially
responsible =tbr altered migratory response. Thus, the authors hypothesised
that the
anti-angiogenic EVs (EVs st-MSC-CM) 'hydrolase extracellular ATP and cause
adenosine accumulation on the endothelial cell surface, which in turn induces
oxidative stress in endothelial cells, leading to the inhibition of cell
migration. To
validate this hypothesis, ROS levels in migrating endothelial SVEC4-10 cells
were
10 detected (Figure 7). In particular, a scratch wound healing assay and
assessed ROS
levels by using CM-B2DCFDA, a general oxidative stress indicator was carried
out
Antymycin A (AA), a chemical compound that, by interacting with the
mitochondria'
complex III, inhibits the electron transport causing the mitochondria'
collapse and
the subsequent accumulation of ROS, was used as positive control (Figure 7 A),
15 ROS accumulation was not significantly increased in migrating SVEC4-10
cells
treated with VEGF alone, or in combination with EVs from unst-MSC-CM (Figure 7
A). On the contrary, EVs derived from st- MSC-CM strongly induced ROS
production at the migration front of endothelial cells. These results support
our
hypothesis of an implication of ROS in the inhibition of endothelial cell
migration
20 exerted by EVs st-MSC-CM. Additionally, the block of the adenosine
accumulation,
through the treatment with the AMP-CP (inhibitor of CD73), not only rescued
the
migration of endothelial cells (Figure 6 C), but also reduced ROS levels in
migrating
endothelial cells treated with EVs derived from st-MSC-CM (Figure 7 B). Thus,
these data prove that the local production of adenosine, due to the presence
of
ectonucleotidases on EVs released by st- MSCs, induces ROS production in
endothelial cells, thus affecting their motility.
To finally validate this mechanism, the oxidative stress was reduced with a
generic
antioxidant N-Acetyl-L-cysteine (NAC). Notably, the suppression of oxidative
stress
induced by EVs st- MSC-CM perfectly restores the migratory ability of SVEC4-10
cells (Figure 7 C).
These results highlighted the crucial inhibitory role of EVs st- MSC-CM on
migrating endothelial cells, with a mechanism dependent on ROS accumulation
induced by the local production of adenosine.
inhibition of angiogenesis in vivo through the treatment with EVs derived from
st- MSC-CM
In an effort to develop a new therapeutic approach for pathological
angiogenesis, the
effect of EVs derived from st- MSC-CM in vivo was validated. Thus, the retinal
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21
mouse model, an extensively used approach to study both physiologic and
pathologic
angiogenesis, was used. Indeed, in contrast to humans, mouse pups have an
immature retinal vasculature at the birth; the development completes in some
weeks,
proceeding in a tightly regulated and organized manner, reliable for detection
of any
defect.
Thus, pups were intra-peritoneal injected with unstimulated or stimulated MSCs-
derived EVs, and sacrificed 5 days later to collect retinas. Samples were
dissected
and stained with isolectin-B4 to measure the vasculature formation of
developing
retina by confocal microscopy (Figure 8 A). Remarkably, a decrease in the
retina
to vascular arborisation of pups treated with st- MSCs-derived EVs, but no
effect with
the unstimulated counterpart was observed (Figure 8 B). In accordance with the
in
vitro results, EVs isolated from st-MSC-CM inhibit the angiogenic process also
in
vivo.
This anti-angiogenic effect exerted by :EVs isolated from st- MSC-CM was
further
consolidated with another in vivo approach, the matrigel plug assay. It
consists in the
analysis of the vascularization of a matrigel plug, that was previously
supplemented
with EVs and then implanted in the dorsal back of C57.B116-N mouse (Figure 8
C).
The quantification of the plugs' haemoglobin content reveals a decreased
vasculature
in mice treated exclusively with EVs from st- MSC-CM, thus confirming the
antiangiogenic proprieties of these products.
Together, all these data provide enthusiastic evidence of the therapeutic
potential of
extracellular vesicles derived from MSCs for the control of pathological
angiogenesis.
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BIBLIOGRAPHY
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Brown et al, 2016. Methods Mol Biol. 2016;1430:149-57. doi: 10.1007/978-1-4939-
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Bicknell R4
Ponce, 2009, Methods N/lol Biol. 2009;467;183-8. doi: 10.1007/978-1-59745-241-
0 10.Tuhe formation: an in vitro matrigel angiogenesis assay.
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