AU2017374947B2 - New anti-angiogenic extracellular vesicles - Google Patents

Abstract

The present invention discloses new aiiti-angiogenic extracellular vesicles (extracellular vesicles, EV) formed from stent/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 extracelliular 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. STATEOF THE ART Angiogenesis, is the process that leads to the formation of new blood/lyuphatic 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 venues into the tissue. The generation of new blood vessels is required fbr 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 stein 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 ofmany 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 mesenchynal 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. Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of the common general knowledge in the field. Unless the context clearly requires otherwise, throughout the description and the claims, the words "comprise", "comprising", and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to".

SUMMARY OF THE INVENTION

According to a first aspect, the present invention provides a process for the preparation of MSCs EVs comprising the following steps: a. culturing MSCs in a medium complemented with at least three pro-inflammatory cytokines, wherein said pro-inflammatory cytokines are IL- , IL-6 and TNFa, b. removing said medium and culturing said MSCs in a medium without said three or more pro-inflammatory cytokines and collecting EVs from the culture supernatant, wherein the MSCs EVs thus obtained exhibit angiogenic inhibitory activity, characterised by the expression of TIMPI, CD73 and CD39. According to a second aspect, the present invention provides anti-angiogenic EVs MSCs when obtained by the process of the first aspect. According to a third aspect, the present invention provides the anti-angiogenic EVs MSCs of the second aspect, when used for therapy. According to a fourth aspect, the present invention provides a pharmaceutical composition comprising anti-angiogenic MSCs EVs when obtained by the process of the first aspect, and at least one pharmaceutically acceptable carrier.

3a

According to a fifth aspect, the present invention provides a medical device comprising anti-angiogenic MSCs EVs when obtained by the process of the first aspect. According to a sixth aspect, the present invention provides a method of treatment of a pathology or condition wherein a therapeutic inhibition of angiogenesis is required comprising the step of administering a therapeutically effective amount of the anti-angiogenic EVs MSCs according to the second aspect or of the pharmaceutical composition according to the fourth aspect to a subject in need thereof. According to a seventh aspect, the present invention provides use of the anti angiogenic EVs MSCs according to the second aspect or of the pharmaceutical composition according to the fourth aspect in the manufacture of a medicament for treating a pathology or condition wherein a therapeutic inhibition of angiogenesis is required. 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 MS Cs. As already discussed above, it has been reported in the art that MSC 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 has 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

3b

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. In some aspects, the present inventions relate to Extracellular Vesicles released by Mesenchymal stern/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 notstimulated 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 amatrigel 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 5x105 unst- or st-MSC, or EVs obtained from 500 1 Matrigel. Matrigel plus 50ng/mL VEGF and the same cells, mixed with 100 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 t

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. I-day-old C57BL/6N mouse pups were intraperitoneally injected with 50pl 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. Fig. 4 - EVs from MSC-CM fully mimic the whole conditioned medium The comparative analysis of the whole medium conditioned by unst- or st M/ISCs (unst MSC-CM or st MSC-CM) and their extracellular vesicles (EV unst or st 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 100pm. B) Quantification of the relative tube length (normalized on medium), performed with ImageJ Angiogenesis Analyzer. 3 independent experiments, data are expressed as mean + SEM (*p b 0.05, **p b 0.01, One-way ANOVA).C) The expression of TIMP-l 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-i in the angiogenesis inhibition mediated by EV from st MSC-CM, the tube formation assay was performed adding a TIMP-1 blocking antibody. Quantification of the relative tube length (normalized on medium), performed with ImageJ 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 ImageJ 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 im, 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-angiogenic 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 ANOVA). The scratch assays were performed by inhibiting the activity of these enzymes, with ARL 67156 for CD39 B), and ANP-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.EM (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 by0usingC-H2DCFDA, a general oxidative stress indicator, during scratch migration assays. Here are reported representative images. The mean of fluorescence was calculated through InageJ and normalized on the medium. Data are expressed as meansiS.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 t 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 (NAC), 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 EVs 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 50ul EVs from unst or st MSC-CM. After 4 days, mice were sacrificed for retina collection. Retina whole mounts were dissected and stained with isolectin B4 (Green, on the left). Digital images were captured using inverted fluorescence confocal microscope (on the right). B) Total retinal and vascular areas were measured using imageJ 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. (n=10 pups/group in 3 different experiments). *P<0.05, One-way ANOVA. C)Anesthetized 12-week-old male C57BL/6N mice were subcutaneously injected in the dorsal back with EVs from unst- or st- MSC-CM, mixed with Matrigel plus VEGP. 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 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 OPTHE 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 3 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 -5 are considered an intermediate between human embryonic stern cells (hESCs) and adult stern 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 Vesieles 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 nn) membrane vesicles from endocytic origin (enriched in late endosomal membrane markers, including Tsgl0l, 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 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-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 Mesenchyrnal 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 viv. 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 aimitative purpose, the tube formation assay can be carried out as described in Ponce, 2009. Methods Mol Biol. 2009;467:183-8. doi: 10.1007/978-1-59745-241-0 10.Tube formation: an in vitro Tmatrigel 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 1o 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: 10,1007/978-1-4939-3628-1_9.Tube-Forming Assavs.Brown RMI 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 angiogenie 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 inflrmatory/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 phannaceutically 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 II characterized 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 phannaceutic 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, fbr 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 an embodiment of the invention said proliferative diseases are selected from cancers; said inflammatory/autoimmune diseases are selected from psoriasis, arthritis, endometriosis, Crolm'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. 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 forintranasal 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-13, 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-13, IL-6 and TNFu. 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 15ng/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/substances 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/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/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-fee mediumis 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 anti-angiogenic 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. 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, i.e. the EVs obtainable from stimulated medium, are characterized by an expression of TIMP Iof at least two to tenfold higher in comparison to the expression of TIMP Iof the EVs MSCs from unstimulated medium as described herein. According to the present invention, the expression of TIMPI 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 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 3ug 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 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/streptomnycin, with or without 25ng/ml mILlb, 20ng/ml mIL6, 25ng/ml mTNFa for 24 hours. The medium was then changed with DMEM low glucose supplemented with 2mM glutamine, 100 U/mI 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, ,3x10 SVEC4-10 cells (ATCC CRL-2181Tm) were seeded in a Matrigel coated well (80pl Matrigel/well) in 100p] 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 lmageJ 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 500pl Matrigel. Matrigel plus 50 ng/mL VEGF and 100 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/mi). 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.EM. (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 50pl 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. nonnalized 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 urine 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 ENs 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 SVEC4-0 to forn capillary-like, tubular structures (Figure 14 A, B). As TIMP1 has a known prominent role as anti-angiogenic mediator of st-MSCs, it was investigatedwhether TIMPi is involved in the anti-angiogenic effect exerted by EVs derived from st-MSC-CM. Firstly, the presence of TIMPI in the EVs of the invention was verified by Western Blot. TIMPI 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 usingTIMP1 blocking antibody (Figure 4 D), the result show that the antiangiogenic effect displayed by EVs isolated from st-MSC-CM is TIMPI-dependent. Altogether, these results indicate that EVs 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 ENs st- MSC-CM Angiogenic 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 fee space in response to a pro angiogenic factorl26 was used. SVECA4-10 cells were seeded to forn a monolayer and, by scratching, a cell-free gap in the confluent layer was generated. Stimulation with the pro anlgiogenic 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 37C 10% C02 (Figure 5 A). As expected, in response to VEGF, the migratory ability of SVEC4-10 cells is increased in comparison to unstimulated 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 EVs 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 TIMPI in this process, the scratch wound healing assay in presence of the TIMPI-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 from st- MSC-CM (Figure 5 C), suggesting the existence of a TIMP-indipendent mechanism through which EVs derived from st-MSCs exert their anti-angiogenic effect. CD39 and CD73 confer anti-angiogenic activity to EVs st-MSC-CM In an effort to characterise the TIMPI-indipendent mechanism of EV-mediated anti angiogenic 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 ectonucleotidases CD39 and CD73, catalysing, respectively, the hydrolysis of ATP to AMP, and of AMP to adenosinel25. 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 EVs st-MSC-CM in comparison with vesicles isolated from unstimulated 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-angiogenic 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, trisodium salt hydrate (ARL 67156) and adenosine 5'- (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 TIMP1 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. Extracellular adenosine interacts with four subtypes of G protein-coupled cell surface receptors (AIR, 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

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 for 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 detected (Figure 7). In particular, a scratch wound healing assay and assessed ROS levels by using CM--2DCFDA, a general oxidative stress indicator was carried out. Antymycin A (AA), a chemical compound that, by interacting with the mitochondrial complex III, inhibits the electron transport causing the mitochondrial collapse and the subsequent accumulation of ROS, was used as positive control (Figure 7 A). ROS accumulation was not significantly increased in migrating SVEC4-10 cells treated with VEGF alone, or in combination with EVs fom unst-ISC-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 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 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

[0 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 C57BL6-N mouse (Figure 8 C). The quantification of the plugs' haernoglobin 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.

BIBLIOGRAPHY Brown et al, 2016. Methods Mol Biol. 2016;1430:149-57. doi: 10.007/978-1-4939 3628-1_9.Tube-Forming Assays.Brown RMI, 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 mesenchyral stem cells inhibit high endothelial cell activation and lymphocyte homing to lymph nodes by releasing TIMP-1, Leukemia (2016) 30, 1143-1154

Claims (11)

1. A process for the preparation of MSCs EVs comprising the following steps: a. culturing MSCs in a medium complemented with at least three pro

inflammatory cytokines, wherein said pro-inflammatory cytokines are IL- I, IL-6

and TNFa, b. removing said medium and culturing said MSCs in a medium without said

three or more pro-inflammatory cytokines and collecting EVs from the culture

supernatant, wherein the MSCs EVs thus obtained exhibit angiogenic inhibitory activity, characterised by the expression of TIMP1, CD73 and CD39.

2. The process of claim 1, wherein each of said pro-inflammatory cytokines is at a

concentration between from 15 ng/ml to 30 ng/ml in said medium.

3. The process of claim 1 or 2, wherein said MSCs are cultured from 18 to 30 hours

in step a.

4. The process of any one of claims I to 3, wherein said MSCs are cultured from 12 to 24 hours in step b.

5. The process of any one of claim 1 to4, wherein said EVs are collected by

ultrafiltration of said supernatant.

6. Anti-angiogenic EVs MSCs when obtained by the process of any one of claims 1

to 5.

7. The anti-angiogenic EVs MSCs of claim 6, when used for therapy.

8. A pharmaceutical composition comprising anti-angiogenic MSCs EVs when

obtained by the process of any one of claims I to 5, and at least one pharmaceutically acceptable

carrier.

9. A medical device comprising anti-angiogenic MSCs EVs when obtained by the

process of any one of claims I to 5.

10. A method of treatment of a pathology or condition wherein a therapeutic inhibition of angiogenesis is required comprising the step of administering a therapeutically effective

amount of the anti-angiogenic EVs MSCs according to claim 6 or of the pharmaceutical

composition according to claim 8 to a subject in need thereof.

11. Use of the anti-angiogenic EVs MSCs according to claim 6 or of the

pharmaceutical composition according to claim 8 in the manufacture of a medicament for

treating a pathology or condition wherein a therapeutic inhibition of angiogenesis is required.

UniversitA degli Studi di Padova

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SPRUSON & FERGUSON