WO2018109525A1

WO2018109525A1 - New anti-angiogenic extracellurlar vesicles

Info

Publication number: WO2018109525A1
Application number: PCT/IB2016/057608
Authority: WO
Inventors: Antonella Viola; Maurizio Muraca
Original assignee: Università Degli Studi Di Padova; Fondazione Città Della Speranza - Onlus
Priority date: 2016-12-14
Filing date: 2016-12-14
Publication date: 2018-06-21
Also published as: AU2017374947B2; AU2017374947A1; CN110312515A; WO2018109700A1; CN110312515B; EP3554513A1; AU2017374947A8; CA3052739A1

Abstract

The present invention discloses new anti-angiogenic extracellular 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.

Description

NEW ANTI- ANGIOGENIC EXTRACELLURLAR VESICLES

FIELD OF THE INVENTION

The present invention discloses new anti-angiogenic extracellular 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 formation 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. 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 vivo. 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 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 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.

The reasons for 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 aberrantly differentiate after ectopic engraftment.

Finally, another risk factor is associated with the administration route. Indeed, although the number of MSCs required to achieve immunomodulation in vivo is basically unknown, injection of large number of cells may be necessary to obtain maximal clinical benefit given the low rate of cell retention and survival. In these 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 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 proinflammatory 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 stem/ stromal 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 stem/ stromal 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 EVs MSCs, and at least one pharmaceutically acceptable carrier; said pharmaceutical composition for use in therapy; a medical device comprising said EVs MSCs; a 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 THE FIGURES

Figure 1 EVs 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-week-old male C57BL/6N mice were subcutaneously injected in the dorsal back either with 5xl0⁵ unst- or st-MSC, or EVs obtained from the same cells, mixed with 500μ1 Matrigel. Matrigel plus 50 ng/mL VEGF and lOO ng/mL bFGF 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 st MSC-CM or with st 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 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.

1-day-old C57BL/6N mouse pups were intraperitoneally injected with 50μ1 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 confocal microscope (A-B). Total retinal and vascular areas were measured using ImageJ. 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). The data are expressed as means ± S.E.M. normalized on control, mice treated with vehicle (n = 4 mice)*P < 0.05, T test.

The figure shows how systemic administration of the EVs of the invention has a direct effect on the retinal vascularization.

DETAILED DESCRIPTION OF THE INVENTION

Glossary in the meaning of the invention:

Mesenchymal 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 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 differently stated. MSCs are considered adult or somatic stem 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 chorionic and amniotic membranes, MSCs are considered an intermediate between human embryonic stem 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 stem/stromal 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 MSCs. 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 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 CD40); (ii) Apoptotic bodies (size between 800 and 5000 nm, derived from fragments of dying cells, and enriched in histones and DNA); and (iii) Exosomes, which are small (-30-120 nm) membrane vesicles from endocytic origin (enriched in late endosomal membrane markers, including TsglOl, 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 stem/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 preexisting 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-angiogenic 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 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.

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 Mesenchymal 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 EVs MSCs of the invention.

By way of example, and not as with a limitative purpose, the tube formation assay can be carried out as described in Ponce, 2009. Methods Mol Biol. 2009;467: 183-8. doi: 10.1007/978-l-59745-241-0_10.Tube formation: an in vitro matrigel angiogenesis assay. According to another non-limiting example the tube formation assay according to the present description can be carried out as detailed in the experimental section.

According to another embodiment of the invention, the angiogenic inhibitory 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 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-l-4939-3628-l_9.Tube-Forming Assays.Brown RM1, Meah CJ2, Heath VL2, Styles IB3, Bicknell R4.

According to another non-limiting example the matrigel plug vascularisation assay according to the present description can be carried 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 MSCs 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 EVs 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.

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 Mesenchymal 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, intra-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.

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 MSCs 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 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 transdermal 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, autoimmune diseases, transplant rejections proliferative, inflammatory and autoimmune diseases or conditions, ocular and periodontal diseases, obesity- and aging-related conditions.

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 EVs 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 surnatant.

According to the present description, said one or more pro-inflammatory cytokine can be selected from IL-Ιβ, 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-Ιβ, IL-6 and TNFa.

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 ng/ml. When more than one cytokine is used, the concentration can be concentration between from 15 ng/ml to 40 ng/ml of each cytokine in case two cytokines are used or of between from between from 15 ng/ml to 30 ng/ml 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.

Both in steps a. and b. the medium will be further complemented with standard additional compounds/sub stances commonly used for the cultivation of MSCs such as bovine foetal serum (FBS) in suitable amounts, antibiotics, suitable amino acids and the like.

The concentration of said additional compounds/sub stances can be readily assessed by the skilled person following standard protocols.

A non-limiting example of a suitable medium and of suitable additional compounds/sub stances 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/ml (eg. About 100 U/ml). In a non-limiting example about 100 U/ml of penicillin/streptomycin can be used.

Further additional complementing substances can be one or more amino acid. In one non limiting example about 2 mM glutamine can be added to the growth medium.

Suitable media for step a. or b. of the process of the invention are represented by any medium commonly used for culturing MSCs. A non-limiting example of said media is represented by DMEM 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, 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.

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, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours.

In an embodiment of the invention said MSCs are 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 surnatant.

Another object of the invention is represented by the anti-angiogenic EVs MSCs obtainable from the process described above.

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 conditions described above wherein a therapeutically effective amount of the EVs MSCs of the invention or of the pharmaceutical composition of the invention is administered to a subject in need thereof.

EXPERIMENTAL SECTION

Anti-angiogenic EVs MSCs preparation

MSC were plated and let grow until confluence in ventilated cap flask in DMEM low glucose supplemented with 20% FBS, 2mM glutamine, 100 U/ml penicillin/streptomycin. Culture medium was then substituted with DMEM low glucose supplemented with 10% FBS, 2mM glutamine, 100 U/ml penicillin/streptomycin, with or without 25ng/ml mILIb, 20ng/ml mIL6, 25ng/ml mTNFa for 24 hours. The medium was then changed with DMEM low glucose supplemented with 2mM glutamine, 100 U/ml 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 ultrafiltration using Amicon® Ultra 15 mL Filters (Merck Millipore). Tube formation assay.

The results of this assay are depicted in figure 1.

In a flat-bottom 96 well plate, l,3xl0⁴ SVEC4-10 cells (ATCC® CRL-2181™) were seeded in a Matrigel coated well (80μ1 Matrigel/well) in ΙΟΟμΙ 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% C0₂, 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 vascularization 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 C57BL/6N mice were subcutaneously injected in the dorsal back either with 5x105 unst- or st-MSC, or EVs obtained from the same cells, mixed with 500μ1 Matrigel. Matrigel plus 50 ng/mL VEGF and lOO ng/mL bFGF was used as positive control. Bare Matrigel was injected as negative control. For each group, matrigel was supplemented with Heparin (50 units/ml). 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 total protein content, are expressed as means ± S.E.M. (n = 4 mice/group). *P < 0.05, T test.

Retinal vascularization 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-old C57BL/6N mouse pups were intraperitoneally injected with 50μ1 EVs from unst or st MSC. After 4 days, mice were sacrificed for retina collection. Both eyes were enucleated and fixed in 4% PFA. Retina whole mounts were dissected and stained with biotinylated isolectin 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 ImageJ. 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.

BIBLIOGRAPHY

Brown et al, 2016. Methods Mol Biol. 2016; 1430: 149-57. doi: 10.1007/978-1-4939- 3628-l_9.Tube-Forming Assays.Brown RM1, Meah CJ2, Heath VL2, Styles IB3, Bicknell R4

Ponce, 2009. Methods Mol Biol. 2009;467: 183-8. doi: 10.1007/978-1-59745-241- 0 10. Tube formation: an in vitro matrigel angiogenesis assay.

Zanotti et al, Mouse mesenchymal stem cells inhibit high endothelial cell activation and lymphocyte homing to lymph nodes by releasing TIMP-1, Leukemia (2016) 30, 1143-1154

Claims

1. Extracellular Vesicles released by Mesenchymal stem/stromal Cells (EVs MSCs) said EVs MSCs exhibiting angiogenic inhibitory activity.

2. The EVs MSCs of claim 1 wherein said angiogenic inhibitory activity is exhibited by said EVs MSCs both in vitro and in vivo.

3. The EVs MSCs of anyone of claims 1 or 2 wherein said EVs MSCs exhibiti angiogenic inhibitory activity in an in vitro tube formation assay.

4. The EVs MSCs of anyone of claims 1 to 3 wherein said EVs MSCs exhibit angiogenic inhibitory activity in an in vivo matrigel plug vascularisation assay.

5. The EVs MSCs of anyone of claims 1 to 4 wherein said angiogenesis is a blood vessels angiogenesis and/or a lymphatic vessels angiogenesis.

6. The EVs MSCs of anyone of claims 1 to 5 for use in therapy.

7. The EVs MSCs for use according to claim 6 for the treatment of pathologies of conditions wherein a therapeutic inhibition of angiogenesis is required.

8. The EVs MSCs for use according to anyone of claims 6 or 7 for the treatment of pathologies or conditions characterised by the pathological formation of new blood and/or lymphatic vessels by angiogenesis.

9. The EVs MSCs for use according to claim 8 wherein said pathologies or conditions are selected from proliferative, inflammatory and autoimmune diseases or conditions, ocular and periodontal diseases, obesity- and aging-related conditions.

10. The EVs MSCs for use according to claim 9 wherein 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.

11. A pharmaceutical composition comprising Extracellular Vesicles released by Mesenchymal stem/stromal Cells (EVs MSCs) wherein said EVs MSCs exhibit angiogenic inhibitory activity, and at least one pharmaceutically acceptable carrier.

12. The pharmaceutical composition of anyone of claim 1 1 wherein said angiogenesis is a blood vessels angiogenesis and/or a lymphatic vessels angiogenesis.

13. The pharmaceutical composition of anyone of claims 11 or 12 in a form suitable for systemic, enteral or parenteral; or for topical administration.

14. The pharmaceutical composition of claim 13 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.

15. The pharmaceutical composition of anyone of claims 11 to 14 for use in therapy.

16. The pharmaceutical composition for use according to claim 15 for the treatment of pathologies of conditions wherein a therapeutic inhibition of angiogenesis is required.

17. The pharmaceutical composition for use according to anyone of claims 15 or 16 for use in the treatment of pathologies or conditions characterised by the pathological formation of new blood and/or lymphatic vessels by angiogenesis.

18. The pharmaceutical composition for use according to claim 17 wherein said pathologies or conditions are selected from proliferative, inflammatory and autoimmune diseases or conditions, ocular and periodontal diseases, obesity- and aging-related conditions.

19. The pharmaceutical composition for use according to claim 18 wherein 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.

20. A medical device comprising the EVs MSCs of anyone of claims 1 to 5 or the composition of anyone to claims 11 to 14.

21. The medical device of claim 20 in the form of a transdermal patch or in the form of a dispenser for intranasal administration.

22. A process for the preparation of MSCs EVs comprising the following steps:

23. The process of claim 22 wherein said one or more pro-inflammatory cytokine is selected from IL-Ιβ, IL-6, T Fa and chemokines .

24. The process of anyone of claims 22 or 23 wherein said proinflammatory cytokines are at least two or at least three.

25. The process of anyone of claims 22 to 24 wherein said proinflammatory cytokines are IL-Ιβ, IL-6 and TNFa.

26. The process of anyone of claim 25 wherein each of said pro- inflammatory cytokine is at a concentration between from 15 ng/ml to 30 ng/ml in said medium.

27. The process of anyone of claims from 25 to 25 wherein said MSCs are cultured from 18 to 30 hours in step a.

28. The process of anyone of claims from 25 to 27 wherein said MSCs are cultured from 12 to 24 hours in step b.

29. The process of anyone of claims 25 to 28 wherein said EVs are collected by ultrafiltration of said surnatant.

30. Anti angiogenic EVs MSCs obtainable from the process of anyone of claims 25 to 29

31. The anti angiogenic EVs MSCs of claim 30 for use in therapy.

32. A pharmaceutical composition comprising the anti angiogenic EVs MSCs of claim 30 and at least one pharmaceutically acceptable carrier.

33. A medical device comprising the anti angiogenic EVs MSCs of claim

30.