Patent Description:
Peripheral arterial disease (PAD) is a widespread condition caused by atherosclerosis of the peripheral arteries. Although surgical or endovascular intervention remains the standard therapy to improve blood flow, even after successful revascularisation, most patients complain of persistent or recurring symptoms. This implies that new therapeutic options for PAD still represent a major unmet need.

The formation of newly born capillary blood vessels, neovascularization, is a crucial event in rescuing tissue after ischemia. A vast array of pre-clinical and clinical data indicates that stem cells can contribute to the healing processes by improving vascularization. Additionally, the transient detection of stem cells into site of tissue damage has suggested that paracrine mechanisms could mainly contribute to stem cell actions. Among such paracrine mechanisms "exosomes", vesicles derived from the endosomal membrane compartment by exocytosis, and "ectosomes/microvesicles", vesicles generated by the budding of cell plasma membranes are included. Due to the overlap in characteristics and biological activities of exosomes and ectosomes/microvesicles, recently the more inclusive term "extracellular vesicles" (EVs) was suggested. EVs re powerful cell-to-cell communication vehicles both in physiological and pathological conditions. Recent studies provide evidences that transfer of EV cargo can dictate recipient cell fate. Indeed, the transfer of EVs cargo, including nucleic acids and proteins, into recipient cells represents the most relevant mechanism of their actions.

So far, the most studied EVs are those released by stem cells, because of their ability to mimic the effects of their cell of origin. Depending on the pathological settings, different phenotypic changes have been described in target cells. In order to try to characterize the mechanism of action of stem cell-derived EVs, transcriptomic and proteomic analysis has been performed. In particular, a robust pro-angiogenic profile has been described in EVs recovered from mesenchymal stem cells derived from bone marrow or adipose tissues. This has suggested the potential impact of these EVs in clinical settings, including ischemic-related diseases.

However, the use of stem cell-derived EVs has the drawback that isolation of such EVs requires in vitro culturing of huge amounts of stem cells in order to obtain a useful number of EVs to be employed.

In order to overcome these and other drawbacks of the prior art, the present inventors studied blood and blood fractions such as serum and plasma as potential sources for EVs. In fact, EVs released within a defined microenvironment can act locally (paracrine action) or they may even act at a distance from the cell of origin (endocrine action). The endocrine action mainly relies on the circulation of EVs in peripheral fluids. This implies that blood could be the natural milieu used by EVs to gain access to their targets. Moreover, EVs can be secreted by all blood cells and retrieved in blood fluids, in which their functions however remain mostly unknown.

As it will be illustrated in detail in the following examples, with their studies the inventors found that EVs derived from a blood component possess a strong proangiogenic activity as measured in a potency test in which vascular endothelial growth factor (VEGF) is used as the reference pro-angiogenic growth factor, whereby EVs derived from a blood component are particularly effective in the therapeutic treatment of ischemic diseases and ischemic injuries or in wound healing by proangiogenic therapy.

In the present description, the extracellular vesicles isolated from a blood component shall be designated in short as "sEVs". sEVs are not the subject matter of the present invention, but the description of their preferred features and properties is useful in order to understand the manufacturing method which forms the subject matter of the invention.

As mentioned, the herein disclosed sEVs are suitable for treating ischemic diseases or ischemic injuries, preferably a vascular disease or injury, such as acute myocardial infarction, acute cerebrovascular disease, acute and chronic peripheral artery disease or acute kidney ischemia.

The herein disclosed sEVs are isolated from a blood component such as blood, serum or plasma, which is preferably prepared from a blood donation of a healthy donor. A composition including the herein disclosed sEVs is preferably a bulk preparation essentially capturing all EVs present in blood, serum, plasma or in the conditioned medium based on size exclusion. A suitable source for sEVs are human cells.

As mentioned above, the inventors found that EVs isolated from a blood component (sEVs) possess a strong pro-angiogenic activity, which makes them particularly effective in the treatment of ischemic diseases and ischemic injuries or in wound healing.

It is envisaged that any composition comprising EVs possessing a pro-angiogenic activity comparable to that of a composition as described above, which comprises EVs isolated from a blood component (sEVs), shall be equally or at least comparably effective in the treatment of ischemic diseases and ischemic injuries and in wound healing, irrespective of the source from which the EVs are prepared.

Accordingly, the present invention provides a method of manufacturing a pharmaceutical preparation of extracellular vesicles (EVs) characterized by a strong proangiogenic activity.

The manufacturing method of the invention comprises the following steps:.

The potency test used in the manufacturing method of the invention to test the pro-angiogenic activity includes testing the EVs activity by means of a BrdU cell proliferation assay, or a tubulogenesis in vitro assay or both a BrdU cell proliferation assay and a tubulogenesis in vitro assay. In all of these embodiments, the BrdU cell proliferation assay and/or the tubulogenesis in vitro assay involve the use of a positive control and a negative control. In both methods, the basic growth medium is preferably endothelial basal medium.

The above-described potency test is suitable to screen for EVs isolated from multiple preparations of body fluid or from the conditioned medium of a cell culture and to identify the pro-angiogenically active preparations for further processing.

In a preferred embodiment of the method of the invention, the potency test comprises the following steps:.

More preferably, the BrdU assay uses HMEC cells seeded in matrigel. In the BrdU cell proliferation assay, serum (preferably <NUM>% serum) is added in the positive control. The negative control is the same medium as the positive control but without the addition of serum. The BrdU assay is preferably based on a concentration of <NUM> EVs per target cell.

In another preferred embodiment of the method of the invention, the potency test comprises the following steps:.

More preferably, the tubulogenesis in vitro assay uses HUVEC cells. In the tubulogenesis in vitro assay, VEGF, preferably <NUM> ng/ml VEGF, is added to the positive control. The negative control is the same medium as the positive control but without VEGF addition. The tubulogenesis in vitro assay is preferably based on a concentration of <NUM> EVs per target cell.

If the EVs measured activity exceeds a predetermined percentage of the positive control measured activity, for example ><NUM>% of the positive control measured activity, the EVs preparation tested is considered as active.

The potency tests to be preferably used in the method of the invention are further detailed in the following description of the experimentations performed by the inventors, in particular in paragraphs <NUM>. <NUM> and <NUM>. <NUM> of the examples herein.

In a further preferred embodiment, the method of the invention comprises both the BrdU cell proliferation assay and the tubulogenesis in vitro assay.

Due to its ability to enhance proliferation of cultured cells, as measured by the BrdU assay, a composition of EVs obtained by the method of the invention is suitable for use as an additive to a culture medium for enhancing proliferation of cultured cells.

Furthermore, a composition of EVs obtained by the method of the invention is suitable for use in the therapeutic treatment of ischemic diseases, ischemic injuries and in wound healing, and it may be provided as a pharmaceutical preparation including a pharmaceutically acceptable carrier, vehicle or diluent. The selection of the carrier, vehicle or diluent as well as of any other excipient required for the preparation of the desired pharmaceutical dosage form falls within the ability of the person skilled in the art.

The person skilled in the art is also able to identify the EVs dose required, which mainly depends on the condition to be treated and the patient's characteristics. In general, a therapeutically effective dose of EVs usually falls within the range of from <NUM>×<NUM><NUM> to <NUM>×<NUM><NUM> EVs/kg of body weight of the subject to be treated. The EVs preparation obtained by the method of the invention may be provided in any suitable pharmaceutical dosage form.

The invention will be better understood from the following examples which are provided by way of illustration only and which make reference to the appended drawings, wherein:.

The invention is further illustrated by the following examples, which are provided by way of illustration only.

Human serum from healthy blood donors (n=<NUM>) was provided by the Blood Bank of Città della Salute e della Scienza di Torino, after informed consent and approval by the internal Review Board of Blood Bank. All human and animal experiments were performed in accordance with the European Guidelines and policies and approved by the Ethical Committee of the University of Turin and by the Italian Institute of Health.

sEVs from each donor were obtained from <NUM> serum bags by centrifuging serum at <NUM> rpm for <NUM> minutes to remove debris. The supernatant was subsequently ultracentrifuged at <NUM> for <NUM> at <NUM>. EVs pellets were then re-suspended in a final volume of <NUM> of RPMI with <NUM>% of DMSO and stored at -<NUM>. sEV were then thawed and used for biological assays and molecular analysis.

sEVs FACS analysis was performed with Guava easyCyte™ Flow Cytometer (Millipore, Germany).

sEVs from healthy donors were incubated with antibodies directed to specific markers for monocyte/macrophages (CD14, CD15), leukocytes (CD45), adhesion molecules (alpha6, CD44, CD29), endothelial cells (CD31, KDR) and platelets (CD42b, CD62P, P SEL). FITC or PE mouse nonimmune isotypic IgG (Beckton Dickinson, Franklin Lakes, NJ) were used as control. sEVs were incubated with specific or control isotype antibody for <NUM> hour at <NUM> and analyzed by FACS.

sEVs were analyzed by the Nanosight LM10 system (Nanosight Ltd. , Amesbury, UK). Briefly, sEVs preparations were diluted (<NUM>:<NUM>) in sterile <NUM>% saline solution and analyzed by NanoSight LM10 equipped with the Nanoparticle Analysis System & NTA <NUM> Analytical Software. The number of total EVs for each patient was obtained by multiplying the value given by the instrument (microparticles/ml) for the dilution made for the analysis and for the number of microliters in which sEVs were resuspended.

In preliminary studies, a dose response curve was performed to evaluate the number of sEVs needed to obtain the best biological response on human microvascular endothelial cells (HMEC). The acronym ECs will be used throughout the study. By using <NUM> different sEV samples, we found that <NUM>×<NUM><NUM> sEVs/target cell was the most effective sEV dose. Thus, <NUM>×<NUM><NUM> EVs/target cell were used throughout the in vitro study.

Therefore, sEVs from single samples were evaluated for their pro-angiogenic activity by either BrdU cell proliferation assay or in vitro tubulogenesis assay or both.

To evaluate sEVs potency (expressed in terms of % activity), a negative and positive control were used. In the BrdU assay, the negative control was medium without serum; the positive control was medium with <NUM>% serum. In the in vitro angiogenesis assay, the positive control was medium with <NUM> ng/ml VEGF, the negative control was medium without added VEGF). The following formula was applied: <MAT>.

Values exceeding <NUM>% of the VEGF pro-angiogenic capability (i.e. the positive control) were considered as efficient sEVs.

Pools of sEVs were obtained by combining <NUM> different sEV samples (<NUM>:<NUM>). Pooled sEVs were also evaluated by BrdU cell proliferation assay and in vitro angiogenesis assays.

A detailed description of the BrdU cell proliferation assay and in vitro angiogenesis assays carried out is provided herein below.

HMEC cells (ATCC CRL <NUM>) were seeded in <NUM> well (<NUM> cell per well) in 300ul of EBM (LONZA) +<NUM>% FBS medium. After <NUM> hours, HMEC cells were starved with DMEM without FBS for <NUM> hours and then stimulated with <NUM>×<NUM><NUM> EVs per well (<NUM> EVs per target cell) resuspended in DMEM without FBS. For negative and positive controls DMEM without FBS and DMEM + <NUM>% FBS were used. After <NUM> hours of EV stimulation, 10ul of BrdU was added to each well (BrdU final concentration <NUM>) and HMEC cells were incubated for <NUM>.

After stimulation and BrdU incubation, BrdU labelling was detected by BrdU labelling and detection kit (ROCHE). Briefly, <NUM> well plate was washed <NUM> times with 250ul RPMI + <NUM>% FBS and cells were fixed by <NUM> ul precooled fixative (Ethanol <NUM>% HCl <NUM>) for <NUM> at-<NUM> and then re-washed <NUM> times with <NUM> ul RPMI + <NUM>% FBS. Plate was incubated with <NUM> ul nuclease solution for <NUM> at <NUM>, washed <NUM> times with <NUM> ul RPMI + <NUM>% FBS and then incubated with <NUM> ul anti-BrdU-POD Fab fragment for <NUM> at <NUM>. Plate was filled with <NUM> ul peroxidase substrate at room temperature for <NUM>-<NUM> and plate was read in a microtiter plate reader at <NUM> with a reference wavelength at <NUM>.

In order to calculate the % of effect, O. derived from EV stimulated samples were elaborate as follow:
The O. of negative control (DMEM without FBS) was set as <NUM>% of effect and the O. of positive control (DMEM + <NUM>% FBS) was set as <NUM>% of effect. of the EV-stimulated samples was converted in % of effect by the following formula: <MAT>.

<NUM> well plate was coated with a thin layer of growth factor reduced matrigel and left <NUM> hour at <NUM>. HUVEC cells (ATCC CRL <NUM>) were seeded in matrigel coated wells (<NUM> cells per well) with <NUM>,<NUM>×<NUM><NUM> EVs per well (<NUM> EVs per target cell) re-suspended in <NUM> ul EBM (endothelial basal medium) (LONZA) without FBS. For negative and positive controls, EBM without FBS and EBM without FBS + <NUM> ng/ml hVEGF (Abcam) were used. After <NUM> hours, <NUM> micrographs for each well were taken, the capillary-like structures were counted by ImageJ software and the mean count of capillary-like structures per field for each EV sample was calculated.

In order to calculate the % of effect, the mean count of capillary-like structures were elaborate as follow:
The capillary-like structure count (CLSC) of negative control (EBM without FBS) was set as <NUM>% and the CLSC of control positive (EBM without FBS + <NUM> ng/ml VEGF) was set as <NUM>%. The CLSC of the samples was converted in % of effect by the following formula: <MAT>.

The sEVs potency was calculated by the arithmetic mean of BrdU and in vitro tubulogenesis test percentage of effect expressed in terms of overall effect percentage.

Animal studies were conducted in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. Angiogenesis was assessed by measuring the growth of blood vessels from subcutaneous tissue into a solid gel of basement membrane, as previously described (<NPL>). First, ECs (<NUM> × <NUM><NUM> cells/injection) were incubated overnight with sEVs (<NUM> × <NUM><NUM> EVs per <NUM> × <NUM><NUM> of ECs). Then, male severe combined immunodeficiency (SCID) mice (<NUM> weeks old) were injected subcutaneously with <NUM> of ice-cold BD Matrigel Matrix Growth Factor Reduced (BD Biosciences, Franklin Lakes, NJ), which had been mixed with pre-stimulated ECs. Equal number of non-stimulated ECs was used as a negative control. The Matrigel plugs were excised <NUM> days after and fixed in <NUM>% paraformaldehyde for <NUM>. Matrigel-containing paraffin sections (<NUM>-<NUM> thick) were stained by trichrome stain method. The vessel lumen area was determined as previously described (<NPL>).

SCID mice (Charles River Laboratories), age <NUM> to <NUM> weeks and weighing <NUM> to <NUM>, were anesthetized with i. injection of zolazepam <NUM>/Kg. Postoperatively, the animals were closely monitored, and analgesia with Ketorolac (<NUM>/Kg) was administered if required.

Under sterile conditions, a small skin incision were made overlying the middle portion of the left hind limb of each mouse. The proximal end of the left femoral artery and the distal portion of the saphenous artery were ligated, dissected and excised. The overlying skin was closed using a sterilized <NUM>-<NUM> silk suture. In preliminary experiments, different sEV number and sEV administration routes were evaluated. The latter included intravenous (iv) or intramuscular (i. ) alone or differently combined. We found that the best administration rout in term of animal distress and response to treatment was: T0 (<NUM>×<NUM>^<NUM> iv), T1 and T2 (<NUM>×<NUM>^<NUM> i. Animals were sacrificed at day <NUM> for histological analysis.

After anesthesia, hair was removed from both legs using a depilatory cream, following which the mice were placed on a heating plate at <NUM> for <NUM> minutes to minimize temperature variations. Hind limb blood flow was measured using a Laser Doppler Blood Flow (LDBF) analyzer (PeriScan PIM <NUM> System, Perimed, Stockholm, Sweden). Immediately before surgery, and at day <NUM>, <NUM>, <NUM> after surgery LDBF analysis was performed on hind limbs and feet. Blood flow was displayed as changes in the laser frequency using different color pixels. After scanning, stored images were analyzed to quantify blood flow. To avoid data variations caused by ambient light and temperature, hind limb blood flow was expressed as the ratio of left (ischemic) to right (non-ischemic) LDBF.

Capillary density within gastrocnemius muscles was quantified by immunofluorescence analysis. Muscle samples were embedded in OCT compound (Miles) and snap-frozen in liquid nitrogen. Tissue slices (<NUM> in thickness) were prepared and capillary endothelial cells were identified by immunofluorescence using a monoclonal antibody against mouse CD31 (Santa Cruz Biotechnology Inc. , Santa Cruz, CA). Fifteen randomly chosen microscopic fields from three different sections in each tissue block were examined for the count of capillary endothelial cells for each mouse specimen. Capillary density was expressed as the number of CD-<NUM>-positive features per high power field (x400).

The gastrocnemius muscle from ischemic and non-ischemic limb was removed at day <NUM> after surgery, immediately fixed with <NUM>% paraformaldehyde for <NUM> hours and then embedded in paraffin. Tissue slices were stained with hematoxylin and eosin. Slides were examined under light microscopy at ×<NUM> magnification. Images were acquired from all the injured area of the ischemic limb sections for the count of the total muscle fibers and of the fibers with small-diameter and with multiple central nuclei that are indicative of the regeneration process. Random images from the total non-ischemic limb section were considered as a control.

Proteins from different sEVs were extracted with NP40 lysis buffer (<NUM> NaCl, <NUM> TRIS-HCl pH <NUM>, <NUM>% NP40). Angiogenic protein profile was performed using Quantibody® Human Angiogenesis array <NUM> (Ray Biotech) which contain a combination of two nonoverlapping array to quantitatively measure the expression of <NUM> human angiogenic proteins. <NUM>µg of protein were loaded for each sample. Data were subjected to background subtraction and protein concentration was obtained from the different standard curve of the array. Functional annotation enrichment analysis was performed using Funrich software and geMANIA plug in on Cytoscape.

sEVs total RNA was isolated using All in One (Norgen, Thorold, ON, Canada) extraction method. RNA concentration and purity were detected by the NanoDrop <NUM> spectrophotometer. The samples of absorbance at <NUM>-<NUM> between <NUM> and <NUM> were adopted.

cDNA was synthesized by RT<NUM> First Strand kit (SABiosciences) following the manufacturer's instructions. Gene expression profiling using the Angiogenesis RT<NUM> Profiler PCR Array (PAHS <NUM>, SA Biosciences) was performed by loading <NUM> ng of cDNA for each sEV sample. The expression profile of <NUM> key genes in angiogenesis was analyzed (list of genes available on website: http://www. sabiosciences. Quantitative reverse transcriptase PCR (RT-PCR) was performed using the StepOne Plus™ System (AB Applied Biosciences). Relative gene expression was determined using the ΔΔCT method. Angiogenesis RT<NUM> Profiler PCR Array was also performed on tubule-like structures formed upon sEV administration. ECs were harvested and purified with the RNeasy Mini kit (Qiagen, GmbH, Germany), then retro transcribed to cDNA and processed using the Angiogenesis RT<NUM> Profiler PCR Array. Five endogenous control genes: beta-<NUM>-microglobulin (B2M), hypoxanthine phosphoribosyl transferase (HPRT1), <NUM> acidic ribosomal protein P0 (RPLP0), glyceraldehyde-<NUM>-phosphate dehydrogenase (GAPDH), and b-actin (ACTB) present on the PCR array were used for normalization. The fold-change for each treated sample relative to the control sample corresponded to <NUM> -ΔΔCt. Changes in gene expression between treated and untreated ECs were illustrated as a fold increase/decrease. Data were further analyzed using the Expression Suite and the Funrich Software.

Surface antigens of different cell populations (platelets, endothelial, monocyte, leukocyte and adhesion molecule markers) were examined in sEVs by Guava FACS analysis. The analysis showed that EVs from serum were a heterogeneous collection coming from different blood cell populations (<FIG>).

By Nanosight analysis, we did not notice significant differences in sEV number and size among different healthy donors. sEV mean particle concentration recovered from a bag of <NUM> was around <NUM>,<NUM>×<NUM><NUM> particles/ml (<FIG>) and sEV diameters was approximately <NUM> with a modal particle size of <NUM> (<FIG>). Starting from <NUM>×<NUM><NUM> of total particles, sEV RNA and protein recovery was similar in all samples analyzed (n= <NUM>) ranging from <NUM> to <NUM> ng (RNA) and about <NUM>µg (proteins) (<FIG>).

To select sEV with proangiogenic activity, a "test of potency" using as internal control VEGF was performed. Efficient sEVs were considered those demonstrating almost <NUM>% of VEGF proangiogenic effects when analyzed for their ability to induce EC proliferation and tube-like structure formation. As shown in <FIG>, <FIG> out of <NUM> sEV samples showed values exceeding the <NUM>% of VEGF activity. To further validate these results selected sEVs sharing comparative pro-angiogenic activities were pooled and analyzed for their biological activities. Of note, we demonstrated that these pooled sEVs showed a comparable biological effects with respect to the distinct sEV preparation (<FIG>). This indicates that sEVs biological activity was not lost even when pooled.

In order to confirm our potency test, in vivo experiments were performed by using an in vivo SCID mouse model. To this purpose ECs were treated with sEVs (<NUM>×<NUM><NUM> sEV/cell), mixed with Matrigel and injected subcutaneously into SCID mice. After one week, wheals were removed and analyzed. As shown in <FIG>, sEVs display proangiogenic activities even when analyzed in vivo. Similar effects were detected when pooled sEVs were analyzed (<FIG>).

The angiogenetic effect of sEVs was evaluated in a murine model of ischemic hind limb that mimics PAD in humans. A total amount of <NUM>×<NUM>^<NUM> sEVs was administrated daily after surgery (<NUM>×<NUM>^<NUM> immediately after intervention (T0), <NUM>×<NUM>^<NUM> at day one (T1) and at day two (T2)). To evaluate whether different ways of administration could impact on the angiogenetic effects, sEVs were administered i) intravenously in the tail vein at T0, T1 and T2 (data not shown); ii) intramuscular directly in the ischemic hind limb at T0, T1 and T2 (data not shown) and iii) intravenously at T0 and intramuscular at T1 and T2. The intravenously administration of sEVs immediately after intervention and the subsequent intramuscular administration proved to lead the best response. Blood perfusion in the ischemic hind limb was evaluated using a Laser Doppler Perfusion imaging. Immediately after surgery, the ratio between the ischemic and non-ischemic hind limb decreased to <NUM>±<NUM> in CTL group and <NUM>±<NUM> in sEVs group (n=<NUM>/group). In the sEVs-treated group hind limb perfusion ratio significantly increased at day <NUM> after surgery compared with the CTL group (<NUM>±<NUM> vs <NUM>±<NUM>, p < <NUM>) (<FIG>). Consistently, capillary density in the ischemic hind limbs was significantly increased in sEVs-treated mice compared to the control group (<NUM>±<NUM> vs <NUM>±<NUM>, p < <NUM>) (<FIG>).

Analysis of tissue damage (gastrocnemius muscles) revealed that muscles from sEVs-treated mice contained a reduced area of muscle damage (<FIG>) and an increased vascular density. Thus, in our model, sEVs appear to protect against ischemia-induced damage.

The above results led us to investigate the contribute of angiogenic-related miRs, mRNAs and proteins carried by sEVs in mediating such functional effects. To this purpose, first, miRs known to be involved in neovascularisation (<NPL>) were evaluated.

Real time PCR analysis reported in <FIG> demonstrates that, compared to ineffective sEVs, effective sEVs showed a significant enrichment of the well-known pro-angiogenic miRs; miR-<NUM>, miR17-5p, miR-92a, miR-<NUM> and miR-<NUM> (effective sEV vs ineffective sEV coefficient of variation CV ranging from <NUM>% for miR-<NUM> to <NUM>% for the miR-<NUM>).

By mRNA and protein microarray analysis we selected the first <NUM> angiogenic mRNA and proteins expressed by sEV (<FIG>). We observed that TGFb1 and Angiostatin were commonly detected in mRNA and proteins (TGFb1: mRNA mean Ct value of <NUM> and protein quantification: <NUM> ng/ml, Angiostatin: mRNA mean Ct value: <NUM> and protein quantification: <NUM> ng/ml).

The cross-match between sEV transcripts and proteins revealed a significant positive correlation with: activation of matrix metalloproteinases and extracellular matrix organization (enrichment GO score: <NUM>,<NUM>; p-value: <NUM>,17E-<NUM> ), cytokine-to-cytokine interaction (enrichment GO score: <NUM>,<NUM>; p-value: <NUM>,67E-<NUM>), endothelial cell migration (enrichment GO score: <NUM>,<NUM>; p-value: <NUM>,03E-<NUM>), positive regulation of blood vessel endothelial migration (enrichment GO score: <NUM>,<NUM>; p-value: <NUM>,49E-<NUM>), VEGF signaling pathway (enrichment GO score: <NUM>,<NUM>; p-value: <NUM>,<NUM> ) (<FIG>).

To validate the biological relevance of sEVs cargo in inducing proangiogenic cues, mRNA-angiogenesis microarrays were analyzed on ECs recovered from capillary-like structures formed in response to sEVs, or VEGF (positive control) and compared to untreated ECs (internal control).

Although the pro-angiogenic effects exerted by sEVs and VEGF was almost comparable, a different pattern of gene expression was detected: upon sEV treatment (<NUM> samples were analyzed) <NUM> angiogenic-related genes were found up-regulated while <NUM> genes down-regulated (<FIG>); upon VEGF treatment <NUM> genes were upregulated and <NUM> genes were down-regulated (<FIG>). For each treatment, the up-regulated transcripts with a fold increase ≥ <NUM> with respect to control samples (untreated ECs) were used for further investigation. On this basis we selected the following <NUM> highest expressed genes. For sEVs treatment: PTGS1, ID1, THBS1, SERPINE1, FN1, SERPINF1, ERBB2, TIE1, IFNG, and CXCL9; for VEGF treatment: IL6, VEGFA, VEGFC, PTGS1, PGF, TIE1, PECAM1, ID1, JAG1, and NOS3 (<FIG>). The Venn diagram reported in <FIG> also shows that PTGS1, ID1 and TIE1 genes were shared by both treatments.

Claim 1:
A method of manufacturing a pharmaceutical preparation of extracellular vesicles (EVs), comprising the steps of:
isolating EVs from multiple preparations of a body fluid or from the conditioned medium of a cell culture;
preparing one or more samples from the isolated EVs at a predetermined concentration of EVs;
testing the activity of each EVs sample in a potency test;
selecting for further processing to a pharmaceutical preparation the samples which exceed a predetermined activity; and
pooling two or more of the active EVs samples.