CN113574179A

CN113574179A - Method for predicting the pro-angiogenic potential of Extracellular Vesicles (EV)

Info

Publication number: CN113574179A
Application number: CN202080021024.9A
Authority: CN
Inventors: 马里亚·费利切·布里齐; 乔瓦尼·卡穆西; 克劳迪亚·卡瓦拉里
Original assignee: Unicyte EV AG
Current assignee: Unicyte EV AG
Priority date: 2019-03-15
Filing date: 2020-03-13
Publication date: 2021-10-29

Abstract

The present invention relates to an in vitro method for predicting the pro-angiogenic activity of a preparation of Extracellular Vesicles (EVs), preferably blood-derived EVs, wherein said method is based on a combined determination of the transforming growth factor beta (TGF β) and micro RNA-130a content. Also disclosed are methods of making Extracellular Vesicle (EV) preparations predicted to have potent angiogenic activity and EV preparations thereof, which are effective for the therapeutic treatment of ischemic diseases, ischemic injuries and pathological conditions associated with cardiovascular disease risk, or for wound healing.

Description

Method for predicting the pro-angiogenic potential of Extracellular Vesicles (EV)

The present invention relates to methods of predicting the pro-angiogenic activity of Extracellular Vesicle (EV) preparations, EV preparations thereof and therapeutic applications thereof.

Extracellular Vesicles (EVs) are vesicles that shed from almost all cell types in both normal and pathological conditions. They mainly comprise microvesicles produced by budding of the cytoplasmic membrane and exosomes derived from endosomal membrane compartments by exocytosis. Recent evidence suggests that EV may serve as a mediator of a variety of pathophysiological processes. Increased levels of circulating EV are associated with vascular injury and hypercoagulability (hypercoagulability), particularly in patients with diabetes and acute coronary syndrome, suggesting a role in driving cardiovascular disease. Furthermore, elevated circulating EV levels, primarily derived from platelets and endothelial cells, are considered to be a hallmark of cellular dysfunction. EV has been widely reported to act as a bioactive carrier and participate in the exchange of information between circulating cells and many cell types, including endothelial cells. Indeed, it has also been suggested that platelet-derived EV plays a role in the pathogenesis of atherosclerosis.

EV acts as a biological mediator primarily by delivering proteins, active lipids and extracellular RNA, commonly referred to as EV cargo (cargo) (Pathan m., et al. visiclepedia 2019: opportunistic of RNA, proteins, lipids and metabolites in extracellular vectors (2019) Nucleic Acids res.47: D516-D519) however, the most studied EV-mediated biological processes rely on miR transfer. mirs are a class of small, non-coding RNAs that regulate gene expression following transcription. mirs are stably expressed in serum/plasma, and their unique expression patterns have been proposed for use as disease fingerprints in many clinical settings. Furthermore, it has been shown that activated platelets can use EV to transfer functional mirs into vascular cells. This in turn modulates ICAM-1 expression and the vascular inflammatory response. Indeed, changes in circulating EV cargo have been shown to be associated with endothelial cell and smooth muscle cell dysfunction in diabetes.

There is increasing evidence that EVs can serve as potential therapeutic, diagnostic and prognostic tools.

Increased risk of cardiovascular events is a common feature of patients with diabetes and obesity. In these clinical settings, impaired angiogenesis is still considered to be a relevant mechanism leading to abnormal vascular remodeling. Thus, promoting neovascularization of damaged tissue remains necessary to improve patient outcome. Different treatments have been proposed to improve vascular remodeling in patients with cardiovascular risk factors. However, they fail to provide real benefits, indicating that new treatment options are still needed.

WO2018069408 discloses compositions of blood-derived EVs characterized by strong pro-angiogenic activity. Furthermore, WO2018069408 teaches an assay capable of measuring the pro-angiogenic potential of EV comprising determining the ability of EV to induce cell proliferation and/or tubular structure formation in vitro.

The present invention is based on the following findings: the composition of the EV cargo represents a reliable predictor of whether these vesicles exhibit pro-angiogenic activity. Unexpectedly, experimental studies carried out by the inventors (detailed in the experimental section below) revealed that EV with pro-angiogenic activity can be distinguished from inactive vesicles based on the concentration of transforming growth factor beta (TGF β) used in combination with the measurements of miR-130 a. In fact, as deduced from ROC analysis, tests based on a combination of the above measurements show a strong predictive power of EV pro-angiogenic activity, both in terms of sensitivity and specificity, enabling an accurate selection of EVs effective in the therapeutic treatment of diseases or lesions positively affected by pro-angiogenic therapy.

Accordingly, a first aspect of the invention is a method of predicting whether an Extracellular Vesicle (EV) composition has pro-angiogenic activity, comprising the steps of:

(a) quantifying miR-130a microRNA content in an EV composition, an

(b) Quantifying transforming growth factor beta (TGF β) content in the EV composition;

(c) determining whether the miR-130a content is higher than a first predetermined value and whether the TGF beta content is higher than a second predetermined value,

wherein:

predicting the EV composition to have pro-angiogenic activity when the miR-130a content is above the first predetermined value and the TGF β content is above the second predetermined value.

As used herein, the term "pro-angiogenic activity" refers to stimulation or enhancement of angiogenesis and/or endothelial cell proliferation.

As explained in further detail below, the inventors performed microarray-based expression profiling on micrornas (mirnas) in pro-angiogenic potent and ineffective EVs isolated from the blood of healthy donors and patients with cardiovascular risk factors and unexpectedly found a significant correlation between the pro-angiogenic activity of these vesicles and miR-130a content. Further studies have shown that EVs with pro-angiogenic activity contain higher amounts of TGF β protein than non-active EVs.

Without wishing to be bound by any theory, the inventors believe that the role that miR-130a plays in promoting the angiogenic process can be explained by the interaction of this molecule with several genes involved in the angiogenic process (e.g., KDR, HOXA5, ROCK1, and EPHB6), as revealed by IPA informality bioinformatics analysis performed by the inventors. In addition, as a result of this analysis, TGF β and TGFBR1 were also found in genes under the control of miR-130a, which further confirms the cooperation of miR-130a and TGF β in driving the pro-angiogenic activity of biologically active EV.

According to the invention, the miR-130a nucleotide sequence comprises or consists of the following nucleotide sequence: 5'-CAGUGCAAUGUUAAAAGGGCAU-3' (SEQ ID NO. 1).

In the method according to the invention, the quantification of miR-130a in the EV composition is preferably performed by nucleic acid-based amplification techniques, more preferably by real-time PCR.

In a preferred embodiment, the miR-130 content assessed in the EV composition by real-time PCR is measured as a threshold cycle (Ct) value.

Generally, nucleic acid quantification by real-time PCR relies on plotting amplification signal (e.g., fluorescence) against cycle number on a logarithmic scale. As used herein, the term "Ct value" refers to the number of PCR cycles required for the amplification signal to reach an intensity above background level during the exponential phase of a nucleic acid amplification reaction. Thus, the Ct value is inversely proportional to the amount of target nucleic acid initially present in the sample, i.e., the greater the abundance of the target nucleic acid, the smaller the Ct value. Methods for determining background levels in real-time PCR reactions are well established and known to those skilled in the art.

More specifically, the inventors observed that a miR130a content with a Ct >30, measured as Ct value in both serum EV (serum EV, sov) from healthy subjects and patients, is predictive of pro-angiogenic null vesicles (< 50% as measured in an in vitro potency test).

Thus, the EV compositions in the methods of the invention are determined to have miR130a content above a predetermined value. Preferably, the EV composition in the method of the invention is determined to have a miR130a content measured as Ct value with a Ct of less than or equal to 35, more preferably with a Ct of less than or equal to 33, more preferably with a Ct of less than or equal to 30, even more preferably with a Ct comprised in the range of 10 to 29, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29.

In a preferred embodiment, the EV composition in the method of the invention is determined to have a miR130a content measured as Ct value of Ct < 30.

In a preferred embodiment, the amount of miR-130a content in the EV is determined by applying 2^-(ΔCt)The method is determined as Ct value based on a formula that allows the calculation of the relative fold-gene expression of the sample, as described in Livak KJ and Schmittgen TD, "Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta Delta C (T)) method" (2001) Methods 25: 402-. As will be explained in detail in the examples below, the present inventors have considered Ct values measured for all examined genes and housekeeping genes in order to use the delta-delta Ct method. Since the entire analysis was based on different housekeeping genes, Ct values were previously averaged for all housekeeping genes (Human U)&snRNA,RNU43snoRNA,Hm/Ms/RT U1 snRNA)。

In another preferred embodiment, quantification of miR-130a content in an EV is achieved by using a standard curve of known amounts of miR-130a and inserting, with the aid of the standard curve, a Ct value determined by real-time PCR in an unknown sample.

Although reference is made to real-time PCR, it is to be understood that other nucleic acid amplification methods known in the art may be used within the scope of the present invention. Such methods include, but are not limited to, nucleic acid sequence-based amplification (NASBA) and digital PCR.

By the present inventorsAs a result of investigation, the present inventors found that the TGF-beta content is less than a predetermined value, preferably less than 23pg/10¹⁰Both sEV from healthy subjects and patients of EV are predicted to have no pro-angiogenic activity.

Thus, the EV composition in the method of the invention is determined to have a TGF content above a predetermined value, preferably above that contained in 20pg/10¹⁰EV to 50pg/10¹⁰Values in the EV range, more preferably higher than 23pg/10¹⁰EV to 40pg/10¹⁰Values in the EV range, even more preferably higher than 25pg/10¹⁰EV to 35pg/10¹⁰Values in the EV range, for example 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35pg/10¹⁰EV。

In a preferred embodiment, the EV composition in the method of the invention is determined to have at least 23pg/10¹⁰TGF β content of EV.

Quantification of TGF-beta protein in EV according to the methods of the invention may be performed in any suitable manner, such as those known in the protein art.

Preferably, the TGF β content of the EV is measured by immunoassay. Any suitable immunoassay may be used, for example, enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay (CLIA), Fluorescent Immunoassay (FIA), Radioimmunoassay (RIA), precipitated immunoassay, particle immunoassay, competitive binding assay, and the like. More preferably, the immunoassay employed in the method of the invention is an ELISA assay. It is clear that the use of any type of immunoassay format, the choice of which falls within the skill of the person skilled in the art, is within the scope of the present invention.

In all of the above embodiments, it is also preferred that the EV is derived from human cells.

According to one embodiment of the method of the invention, the pro-angiogenic activity of the EV composition is quantified by an in vitro potency assay involving testing the EV by a BrdU cell proliferation assay or a tube generation (tubulogenesis) in vitro assay, or by both a BrdU cell proliferation assay or a tube generation in vitro assay.

In a preferred embodiment, the efficacy test comprises the steps of:

-measuring the activity of the EV composition by a BrdU cell proliferation assay;

-measuring the activity of the negative control by a BrdU cell proliferation assay;

-measuring the activity of the positive control by a BrdU cell proliferation assay;

-calculating the% activity of the composition in the BrdU cell proliferation assay by applying the following formula:

according to this embodiment, the method of the invention further comprises a step (d) of quantifying the pro-angiogenic activity of said EV composition by a potency test comprising the following steps:

-testing the EV composition by a BrdU cell proliferation assay to obtain a composition value;

-testing a negative control by a BrdU cell proliferation assay to obtain a negative control value;

-testing a positive control by a BrdU cell proliferation assay to obtain a positive control value;

-calculating the% pro-angiogenic activity of the EV composition in the BrdU cell proliferation assay by applying the following formula:

the BrdU assay preferably uses HMEC cells seeded in matrigel (matrigel). In the BrdU cell proliferation assay, serum (preferably 10% serum) is added to the positive control. The negative control was the same medium as the positive control, but no serum was added. The BrdU assay is preferably based on 10000 EV/concentration of target cells.

In another preferred embodiment, the efficacy test comprises the steps of:

-measuring the activity of the EV composition by a tube generation assay;

-measuring the activity of the negative control by a tube generation assay;

-measuring the activity of the positive control by a tube-generating assay;

-calculating the% activity of the composition in the tube production assay by applying the following formula:

-testing the EV composition by a tube generation assay to obtain a composition value;

-generating a test negative control by the tube to obtain a negative control value;

-testing a positive control by a tube generation assay to obtain a positive control value;

-calculating the% pro-angiogenic activity of the EV composition in the tube production assay by applying the following formula:

in vitro tube production assays HUVEC cells are preferably used. In a tube-generated in vitro assay, VEGF, preferably 10ng/ml VEGF, is added to a positive control. The negative control was the same medium as the positive control, but no VEGF was added. The in vitro tube-generating assay is preferably based on a concentration of 50000 EV/target cells.

In a more preferred embodiment, the activity of a given EV composition is tested relative to the positive control activity using both a BrdU cell proliferation assay and a tube production in vitro assay, in which case the average% activity values from the BrdU cell proliferation assay and the tube production assay are compared.

According to this embodiment, the method of the invention further comprises a step (d) of quantifying the pro-angiogenic activity of the EV composition by a potency assay comprising both the BrdU cell proliferation assay and the in vitro angiogenic assay described above.

A test EV preparation is considered active if the EV measured activity exceeds a predetermined percentage of the positive control measured activity, e.g., > 50% of the positive control measured activity.

Thus, the EV compositions in the methods of the invention are preferably identified as having at least 50% pro-angiogenic activity.

Furthermore, the predictive test described above is suitable for screening EVs isolated from various preparations of body fluids or from conditioned medium of cell cultures and identifying pro-angiogenic active preparations for further processing.

Accordingly, a second aspect of the invention is a process for preparing a preparation of pro-angiogenic Extracellular Vesicles (EV), comprising the steps of:

-isolating EV from various preparations of body fluids or from conditioned medium of cell cultures;

-preparing one or more samples from the isolated EV at a predetermined EV concentration;

-predicting the pro-angiogenic activity of each EV sample using the method described above;

-selecting a sample wherein the miR-130a content is above said first predetermined value and TGF β is above said second predetermined value; and optionally

-combining two or more active EV samples,

thereby obtaining the preparation of the angiogenesis promoting EV.

EV from serum or other blood components of healthy donors or patients with cardiovascular risk factors can induce pro-angiogenic signals in vitro and in vivo, and this effect does not disappear even when combined sEV.

As described above, through the studies of the present inventors, the present inventors found that EV compositions determined to have miR-130a and TGF β contents higher than the first and second predetermined values, respectively, are predicted to have strong angiogenesis promoting properties. These characteristics make them particularly suitable for the treatment of ischemic diseases, ischemic injuries and pathological conditions associated with the risk of cardiovascular diseases, or for wound healing.

Thus, a third aspect of the invention is a preparation of pro-angiogenic Extracellular Vesicles (EV), wherein the preparation has a miR-130a content as measured by real-time PCR as a Ct value that is Ct less than or equal to 35 and a TGF beta content that is higher than that contained in 20pg/10¹⁰EV to 50pg/10¹⁰A value in the EV range, for use in the therapeutic treatment of a disease or injury positively affected by a pro-angiogenic treatment, or for use in wound healing.

Preferably, the TGF beta content of the pro-angiogenic EV preparations of the invention is higher than that contained in 23pg/10¹⁰EV to 40pg/10¹⁰Within the EV range, it is more preferably 25pg/10¹⁰EV to 35pg/10¹⁰Values in the EV range, for example 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35pg/10¹⁰EV。

In a preferred embodiment, the TGF β content of the pro-angiogenic EV preparations of the invention is>23pg/10¹⁰EV。

Preferably, the miR-130a content in the pro-angiogenic EV preparations of the invention, measured as Ct value by real-time PCR, is a Ct of less than or equal to 33, more preferably a Ct of less than or equal to 30, even more preferably a Ct comprised in the range of 10 to 29, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29.

In a preferred embodiment, the miR-130a content in the pro-angiogenic EV preparations of the invention, measured as Ct value by real-time PCR, is Ct < 30.

In a more preferred embodiment, in the pro-angiogenic EV preparations according to the invention, the miR-130a content, measured as Ct value by real-time PCR, is Ct<30 and a TGF beta content of>23pg/10¹⁰EV。

A fourth aspect of the invention is a preparation of pro-angiogenic Extracellular Vesicles (EV) for use according to the invention as defined above, which is obtainable by the above-mentioned preparation process.

The disorders associated with the risk of cardiovascular diseases treated with the EV preparations according to the invention are preferably characterized by impaired vascular remodeling, more preferably obesity, diabetes, dyslipidemia or hypertension.

In a preferred embodiment, the EV preparation according to the invention is suitable for the treatment of a disease selected from: acute myocardial infarction, acute cerebrovascular disease, acute and chronic peripheral arterial disease, acute renal ischemia, obesity and diabetes.

Extracellular vesicles are produced by many different cell types (so-called donor cells) and are ubiquitous in biological fluids as well as cell or tissue cultures. Thus, according to the present invention, EV compositions may be obtained from any suitable cell type, preferably nucleated mammalian cells, more preferably stem cells, even more preferably adult stem cells.

In the context of the present specification, the expression "adult stem cell" is intended to mean a stem cell isolated from adult tissue, as opposed to an "embryonic stem cell" isolated from the inner cell population of a blastocyst. Adult stem cells are also referred to as "somatic stem cells".

According to an alternative embodiment, the EV preparation used according to the invention is isolated from a biological fluid or from conditioned cells or tissue culture medium.

Preferably, the EV preparation used according to the invention is separated from blood components, more preferably from whole blood, plasma or serum (sEV).

In one embodiment, the pro-angiogenic EV is prepared from a blood donation from a healthy donor.

In another embodiment, the pro-angiogenic EV is prepared from a blood donation from a patient, more preferably from a patient with cardiovascular risk factors.

In a more preferred embodiment, pro-angiogenic EVs prepared from the blood of a patient with cardiovascular risk factors are suitable for use as a medicament for treating the same patient.

Furthermore, in all the above embodiments, the preparation of the invention may be a pharmaceutical preparation comprising a pro-angiogenic EV as defined above and a pharmaceutically acceptable excipient and/or carrier and/or diluent. The choice of carrier, carrier or diluent, and any other excipients, falls within the skill of the art, especially considering the selected pharmaceutical dosage form, route of administration, and administration regimen, as well as the characteristics of the patient and the disease to be treated.

The invention will be better understood from the following examples, which are provided by way of illustration only and with reference to the accompanying drawings, in which:

fig. 1 shows the results of sEV characterization by Nanosight. (A) Representative images of NTA analysis, related to individual patient groups. (B) Dot-plot diagram representing NTA size distribution with average size values per individual subject (healthy donor, obese, diabetic/obese and ischemic). (C) Histograms of the number of EVs recovered from sera from individual patient groups are reported. D ═ diabetes; o ═ obesity; OD ═ obesity/diabetes; IC ═ ischemic patients. Obesity and ischemic patients vs healthy subjects p < 0.05; (one-way ANOVA followed by multiple comparison test) (n-9 patients/group).

Figure 2 shows the in vitro and in vivo pro-angiogenic activity of serum EV from healthy donors and patients. (A) Representative micrographs of angiogenesis in response to potent and ineffective sEV are shown. Each number refers to sEV (upper panel: invalid sEV; lower panel: valid sEV) prepared from individual subjects (each group n: 3, except OD, same sample was used in 3 independent experiments). (B) Results of in vivo quantitative analysis of angiogenesis. For each experimental condition, blood vessels were counted in 10 sections of matrigel. The data show the average number of blood vessels counted in untreated mice (C) (n ═ 3) or mice treated with the following EV preparations: angiogenic ineffectiveness sEV (i-sEV) from healthy donors, angiogenic effectivity sEV (e-sEV) from healthy donors; angiogenic inactiveness sEV from diabetic patients (D i-sEV), angiogenic inactiveness sEV from diabetic patients (D e-sEV); proangiogenic ineffectiveness sEV from obese patients (Oi-sEV), proangiogenic effectivity sEV from obese patients (O e-sEV); angiogenesis-ineffective sEV from diabetic/obese patients (OD i-sEV), angiogenesis-effective sEV from diabetic/obese patients (OD e-sEV); angiogenesis was ineffective sEV from ischemic patients (IC i-sEV) and effective angiogenesis was effective sEV from ischemic patients (IC e-sEV). Healthy e-sevv vs healthy i-sEV, < 0.05; diabetes e-sEV vs diabetes i-sEV, § p < 0.05; obesity e-sEV vs obesity i-sEV, # p < 0.05; diabetes/obesity e-sEV vs diabetes/obesity i-sEV, p.degree.p < 0.05; ischemia e-sEV vs ischemia i-sEV ischemia, + p < 0.05; (one-way ANOVA followed by multiple comparison test). (each group n-3, except OD, same sample was used in 3 independent experiments). (original magnification:. times.200; scale bar: 12 μm).

Figure 3 shows that the pro-angiogenic activity of sEV correlates with its TGF β content. The figure reports data obtained from serum EV samples prepared from individual subjects of each group (healthy donors, diabetic, obese and ischemic patients). For each group of patients, the upper curve refers to the curve measured in pg/10 at sEV¹⁰TGF β content by EV, while the lower curve refers to% pro-angiogenic activity measured in an in vitro potency assay. Dotted lines indicate the pro-angiogenic effective and ineffective TGF beta of sEV>23pg/10¹⁰Cutoff value for EV (cut-off). Each number corresponds to an individual patient (n-3 per group).

Figure 4 shows the results of the miRNA expression profiling analysis in sEV. (A) Distribution of Ct values for miR-130a measured in proangiogenic active (black circles) and inactive (white circles) sEV from individual patients and healthy subjects. The results are reported as 40-Ct. (B) Network analysis of pathways positively correlated with miR-130 a. Data were obtained by DIANAmiRpath analysis. Only pathways containing at least 15 genes were selected.

FIG. 5(A) analysis of the network between miR-130a and mRNA targets. The line represents the interaction between the gene predicted by IPA software and miR-130 a: indirect interaction (dotted line), direct interaction (continuous line). Squares include TGF β and TGFBR. The circles include genes involved in angiogenesis (KDR, EPHB6, ROCK1, HOXA 5). (B) Receiver Operating Characteristic (ROC) curves and corresponding areas under the curves (AUC) indicate that miR-130a and TGF β have predictive abilities to distinguish proangiogenic potent sEV from non-potent vesicles. For ROC analysis, the results obtained for sEV from all patients and healthy subjects were considered. AUC values are reported in the table below the ROC curve, along with standard error, p-value, and threshold.

Examples

1. Method of producing a composite material

1.1 patients

In the studies conducted by the present inventors, thirty-six patients with cardiovascular risk factors and nine sex-matched healthy volunteers were included. Specifically, 9 diabetic patients (D: n ═ 9), 9 obese patients (O: n ═ 9), 9 diabetic and obese patients (OD: n ═ 9), and 9 ischemic patients (patients who underwent surgical treatment for lower limb ischemia) (IC: n ═ 9) were examined. All diabetic patients were not treated with insulin. All experiments were conducted according to European guidelines and policies and were approved by the Ethical Committee of the University of Turin, Italy. Serum from all patients was obtained after admission (D, O, OD) and for ischemic patients (IC), before surgery. Informed consent was obtained for all patients. Human serum from healthy donors (n-9) was provided by the urban public health sciences city Blood Bank (Blood Bank of "Citt jade della salve della Scienza di Torino") after informed consent and approval from the internal Review Board of the Blood Bank.

1.2. Study approval

Animal studies were conducted according to the Italian National institutes of Health Guide for the Care and Use of Laboratory Animals (protocol no: 490/2105-PR). Mice were bred according to the European Association of Experimental Animal sciences Guidelines (Federation of European Laboratory Animal Science Association Guidelines) and the university of City ethical Committee. All experiments were performed according to relevant guidelines and regulations.

1.3. Serum EV separation

Human blood was obtained from healthy and patient donors by venipuncture. A total of 9ml serum/donor was recovered from each donor and stored at-80 ℃. After thawing, all EVs were isolated and purified by ultracentrifugation at 100,000 × g for 2 hours, followed by centrifugation at 3000g to remove debris. The precipitate was washed once with PBS and centrifuged at 100.000 Xg for 1 hour at 4 ℃. Samples were used either fresh or thawed after storage at-80 °.

1.4 nanoparticle tracking analysis

sEV was analyzed by Nanoparticle Tracking Analysis (NTA) using a NanoSight LM10 system (NanoSight ltd., Amesbury, UK) equipped with a 405nm laser and NTA2.3 analysis software to determine its size and characteristics. All acquisitions were completed with the camera level set to 14 and for each sample, three videos of 30 second duration were recorded. sEV was diluted (1:1000) in 1ml of a vesicle-free physiological solution (Fresenius Kabi, Runcorn, UK). Post-acquisition NTA settings were optimized and held constant between samples, and each video was then analyzed to measure EV size, distribution and concentration.

1.5 sEV angiogenesis assay

Primary macroendothelial cells (EC) and microvascular endothelial cells (HMEC) were purchased from Lonza (Basel, Switzerland) and cultured as described by the manufacturer's instructions. In vitro and in vivo angiogenic potency tests were performed as previously described (Cavallari C.et al, "Serum-derived Extracellular Vessels (EVs) impact on vascular modification and present multiple damage in acid project iso 2017 (1): 8180). Briefly, 5X 10 was administered throughout the in vitro study⁴sEV/target cell. The pro-angiogenic activity of sEV from a single sample was evaluated using BrdU and in vitro tube production assays. EV of all analysis groups were classified as pro-angiogenic or inactive EV according to a 50% cut-off value.

In vivo angiogenesis was assessed by measuring the growth of blood vessels as previously described (Lopatina T.et al, "Platlet-derivGrowth factor specifications of the series of additional services by addition of sensitive cells and enhancement of the same genetic potential (2014) Cell Commun Signal.12: 26). Briefly, EC (1X 10)⁶Single cell/injection) and sEV (5X 10)¹⁰ EV 1X 10 per unit⁶EC) were incubated together overnight. Male Severe Combined Immunodeficiency (SCID) mice (6 weeks old) were then injected subcutaneously. The same number of unstimulated ECs was used as a negative control. Matrigel plugs were recovered and fixed on day 7 and stained using a trichrome staining procedure. The vascular lumen area was determined as previously described (Lopatina T.et al, "plate-derived growth factor definitions the section of extracellular vessels by adsorption media cells and processes the same (2014) Cell communication Signal.12: 26).

1.6 TGF-beta ELISA assay

The TGF β content in EV isolated from serum samples of healthy subjects and patients was measured using a solid phase sandwich enzyme-linked immunosorbent assay (ELISA, Invitrogen Multispecies TGF- β 1 kit, Germany) according to the manufacturer's instructions. For the system containing 1 × 10¹¹Samples of EV were run in triplicate. The intensity of the coloured product obtained in the assay was determined using an ELISA iMarkTM microplate absorbance reader (Bio Rad, Switzerland) which had an absorbance at 450 nm. TGF-beta concentrations present in EV preparations in pg/10¹⁰And EV means.

1.7 miRNA expression profiling

The expression profile of the miRNA (so-called miRNome) contained in sEV was assessed by real-time PCR on 1140 micrornas according to the manufacturer's recommended protocol using miRNome micrornafilters QuantiMir (SBI, System Biosciences). The kit comprises an assay for human microRNAs in pre-formatted plates with three endogenous reference RNAs on each plate as normalization signals (human U6 snRNA, nucleolar small RNARNU43 and Hm/Ms/Rt U1 snRNA).

Briefly, a high capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, California, U) was usedSA) reverse transcribed 100ng RNA. All qRT-PCR reactions were performed in StepOneplus^TMThe real-time PCR system was performed under the following conditions: 15' at 95 ℃ (PCR initial activation step), followed by 3 cycles (15 at 94 ℃, 30 at 55 ℃, 30 at 70 ℃), for 40 cycles. In screening, miRNome performed profiling against sEV collected from sera of healthy subjects, these sEV had been assessed as having pro-angiogenic activity (n-3) and pro-angiogenic inactivity (n-3) by the efficacy test described above. For each sEV sample analyzed, the Ct value of the miRNA was extrapolated. Ct representing the mean Ct values of different samples from both the valid and invalid sEV populations (n-3) were normalized to endogenous reference RNA and converted to 2^-(ΔCt)Values (Livak KJ and Schmittgen TD, "Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta Delta C (T)) method" (2001) Methods 25: 402-.

sEV from healthy donors and patients were subjected to miRNA validation using the MiScript SYBRGreen PCR kit (Qiagen, Valencia, Calif., USA). Briefly, 100ng of input RNA isolated from sEV samples was reverse transcribed using the mixcript reverse transcription kit, and the cDNA thus obtained was used for detection and quantification of the miRNA of interest. Experiments were run in triplicate using 3ng cDNA per reaction as described in the manufacturer's protocol (Qiagen). The following mirnas were screened in sEV from all patients: miR-126(SEQ ID NO.2), miR-21(SEQ ID NO.3), miR-296-3p (SEQ ID NO.4), miR-210(SEQ ID NO.5), miR-130a (SEQ ID NO.1), miR-27a (SEQ ID NO.6), miR-29a (SEQ ID NO.7) and miR-191(SEQ ID NO. 8). Amplification data obtained by qRT-PCR were normalized using RNU6B and RNU43 reference genes as internal controls. It was shown that the amplification efficiency of the target sequence and endogenous control was approximately equal.

1.8 approach and target predictive analysis of miRNA EV content

For EV miRNA target prediction and biological pathway enrichment analysis, the web-based program DIANAmirPath (Collino F. et al, "Exosome and Micronescale-engineered Fractions Isolated from Mesench culture Cells by Gradient Separation) was used (2017) culture Cell Rev. (2): 226-43). The algorithm microT-CDS was chosen to predict EV-derived miRNA targets using a default threshold (microT 0.8). Only biological pathways that show a P value <0.01 for all known Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways are considered significantly enriched. Ingenity IPA pathway analysis was used to predict miR-130a target genes. The inventors set up a miRNA target filter tool on IPA (Qiagen: http:// www.qiagen-bioinformatics. com/products/ingnenuity-pathway-analysis /) to associate miR-130a with predicted mRNA targets.

1.9 ROC analysis

The main data for the two study groups "true pro-angiogenic activity sEV"/"true pro-angiogenic inactivity sEV" considered as Reference Standard (RS) are expressed as mean, Standard Deviation (SD), median and 95% confidence interval. To evaluate the predictability of miR-130a and TGF β, the realization of RS was evaluated using the ROC curve (Grund B and Sabin C. "Analysis of biorarer data: logs, odds rates, and receiver operating characteristics curves" (2010) Curr Opin HIV AIDS 5(6): 473-9). Based on miR-130a content measured as Ct value and in pg/10¹⁰The EV meter measured TGF β content, classifying sEV compositions into the following categories:

1. sEV showing a Ct value of miR-130a of >30 is considered to be pro-angiogenic ineffective sEV;

2. showing TGF beta content<23pg/10¹⁰sEV for EV is considered to be pro-angiogenic ineffective sEV.

To evaluate the "goodness" of the cut-off score based on ROC curve analysis of the above measurements of "true pro-angiogenic inactivity sEV" defined in the prediction RS, the predictive power of each of the two measurements was evaluated separately, as well as by combining the two measurements in a "tandem" approach (sEV, which is pro-angiogenic ineffective for both measurements, was considered "pro-angiogenic ineffective sEV", and vesicles, which are "non-pro-angiogenic ineffective sEV" for at least one of the two measurements, were considered "non-pro-angiogenic ineffective sEV").

The analysis is based on sensitivity (Se), specificity (Sp), and positive likelihood ratio (LH +) [ "the probability that a true pro-angiogenic effect sEV" is identified as a "pro-angiogenic effect sEV" compared to a "true pro-angiogenic effect sEV"), and relative 95% confidence interval values.

1.10 statistical analysis

Data were analyzed using GraphPad Prism 6.0Demo program. Results are expressed as mean ± SD or ± SEM unless otherwise reported. Statistical analysis was performed using one-way ANOVA followed by Tukey's post hoc or multiple comparisons, student's t-test (for 2 group comparisons) and Newman-Keuls multiple comparison test (where appropriate). The cutoff for statistical significance was set at p <0.05 (. p <0.05,. p <0.01,. p < 0.001).

2. Results

2.1 characterization of serum EV

In the studies conducted by the present inventors, the number and size of sEV samples derived from healthy individuals and sEV samples derived from a patient cohort were examined. sEV did not show any significant difference in size between healthy individuals and patients (FIGS. 1A and B). The average particle size observed was about 138 nm. sEV were present in higher numbers in patients than in healthy subjects (FIG. 1C). Significantly higher sEV levels were detected in obese and ischemic patients (fig. 1C).

2.2 proangiogenic Activity of patient-derived serum EV

To evaluate the angiogenic activity of sEV derived from different patient groups in vitro, efficacy tests were performed as described in example 1.3 above. sEV compositions showing an average of over 50% were considered to be pro-angiogenic.

The results of the angiogenic efficacy test were validated in vivo using proangiogenic potent and ineffective sEV from different patient groups (fig. 2A-B).

2.3 TGF-beta content in 2.3 sEV and its angiogenic potential

To investigate whether TGF β content in sEV could lead to its angiogenic potential, the inventors performed ELISA assays on EV isolated from serum samples of healthy subjects and patients (diabetic, obese, diabetic/obese and ischemic). As shown in figure 3, the TGF β content measured in the sEV composition was significantly correlated with the pro-angiogenic potential of these vesicles in the patient cohort as well as in healthy donors. Based on the following observations: TGF beta content<23pg/10¹⁰EV's of EV's are more likely to be proangiogenic inactive, and a cut-off value was determined that distinguishes between proangiogenically effective EV's and non-effective vesicles, which corresponds to 23pg/10¹⁰TGF β concentration of EV.

2.4 miRNome Spectroscopy of 2.4 sEV

miRNome analysis by the present inventors of potent and potent proangiogenic sEV from healthy donors (3 samples per donor) led to the identification of the 8 vas-mirnas with the greatest differences in expression: miR-126, miR-21, miR-296-3p, miR-210, miR-130a, miR-27a, miR-29a and miR-191. In particular, in sEV with pro-angiogenic ability, miR-126, miR-130a, miR-27a and 296-3p is up-regulated, while miR-21, miR-29a, miR-191 and miR-210 are down-regulated.

To investigate whether the observed differences in miRNA expression levels in EVs correlate with their functional activity, the inventors conducted studies by comparing the expression of selected mirnas in sEV derived from individual healthy donors and patients with the levels of pro-angiogenic activity of these vesicles measured by in vitro potency assays. Expression analysis was performed by real-time PCR (cut-off Ct value 30). As shown in fig. 4A, the Ct value distribution of miR-130a measured in EVs from individual subjects (healthy donors and patients) was significantly correlated with the results of the angiogenic efficacy test performed on these EV samples. In particular, it was observed that EVs with miR130a content Ct >30, measured as Ct values, have a higher probability of being pro-angiogenic ineffective.

Interestingly, the inventors also found that miR-210 was enriched in patient-derived sEV, as previously described in Shalaby SM.et al, "Serum miRNA-499and miRNA-210, Apotensal role in early diagnosis of acid coronary syndrome," IUBMB Life.2016; 68(8) 673-82. However, no significant correlation was detected between miR-210 content of sEV and the pro-angiogenic activity of these vesicles.

By selecting pathways involving at least 15 genes, the DIANAmirpath assay was interrogated using miR-130 a. Again, a significant enrichment of genes involved in the TGF β pathway was detected in particular (fig. 4B).

Several genes closely related to the angiogenic process were identified by IPA prediction of the network of miR-130a target genes, such as KDR, HOXA5, ROCK1, EPHB6 (fig. 5A). In addition, the TGF β and TGFBR1 genes were found in the miR-130a interactor. Collectively, the above results also support the contribution of the TGF signaling pathway in sEV-mediated mechanisms of action.

2.5 miR-130a and TGF-beta content in 2.5 sEV represents a means to identify a "truly pro-angiogenic ineffective" sEV Predictive markers of value.

The inventors performed a Receiver Operating Characteristic (ROC) analysis to assess whether miR-130a and TGF β content in sEV have predictive power to distinguish between sEV and null vesicles that exhibit pro-angiogenic ability. From the ROC curve shown in fig. 5B, it was concluded that both miR-130a and TGF β are good predictive measures of "true proangiogenic null sEV" identified by RS, which showed statistically significant AUC values.

Both measurements showed good sensitivity to identify the "true pro-angiogenic inefficiency sEV" identified by RS as "pro-angiogenic ineffectiveness". This is particularly evident and further emphasized by: for miR-130a (Se ═ 0.94, IC 95%, 0.73 to 0.99) and for TGF β (Se ═ 0.88, IC 95%, 0.66 to 0.97), LH + ═ 1.88, IC 95%, 1.49 to 2.27. However, two measured low specificity values were detected (miR-130 a: Sp ═ 0.50; TGF β Sp ═ 0.64).

By combining the two measurements "in series", i.e. considering those sEV defined as "proangiogenic ineffective" in both measurements as "proangiogenic ineffective", a good level of sensitivity and an increased specificity value (Sp ═ 0.75; Se ═ 0.82) were detected. These results are further supported by the LH + values reported in table 1 below.

TABLE 1

The tests of miR-130a and TGF beta 1 are combined "in tandem". The list of values obtained for both measurements was combined "in tandem" (sEV defined as "pro-angiogenic ineffective" in both miR-130a and TGF β 1 measurements was considered "pro-angiogenic ineffective").

Claims

1. A method of predicting whether an Extracellular Vesicle (EV) composition has pro-angiogenic activity, comprising the steps of:

(a) quantifying miR-130a microRNA content in the EV composition, an

wherein:

2. The method of claim 1, wherein the miR-130a content is quantified as a Ct value by real-time polymerase chain reaction (real-time PCR), and wherein there is a negative correlation between the miR-130a content and the Ct value.

3. The method of claim 2, wherein the first predetermined value is a Ct value < 30.

4. The method according to any one of claims 1 to 3, wherein the TGF β content is measured by an immunoassay, preferably by an enzyme-linked immunosorbent assay (ELISA).

5. The method of any one of claims 1-4, wherein the second predetermined value is 23pg/10¹⁰Amount of TGF β of EV.

6. The method according to any one of claims 1-5, further comprising a step (d) of quantifying the pro-angiogenic activity of the EV composition by a potency test, comprising the steps of:

7. the method according to any one of claims 1-5, further comprising a step (d) of quantifying the pro-angiogenic activity of the EV composition by a potency test, comprising the steps of:

8. the method of any one of claims 1-5, further comprising step (d) of quantifying the pro-angiogenic activity of the EV composition by a potency assay comprising both a BrdU cell proliferation assay of claim 6 and an in vitro tube production assay of claim 7.

9. The method of any one of claims 1-8, wherein the EV composition is determined to have at least 50% pro-angiogenic activity.

10. The method of any one of claims 1-9, wherein the EV is from a human cell.

11. A process for preparing a preparation of pro-angiogenic Extracellular Vesicles (EV), comprising the steps of:

-predicting the pro-angiogenic activity of each EV sample with the method according to any one of claims 1 to 10;

-combining two or more active EV samples,

thereby obtaining the preparation of the angiogenesis promoting EV.

12. The method of claim 11, wherein the miR-130a content is quantified by real-time PCR as a Ct value and the first predetermined value is a Ct value of < 30.

13. The method of claim 11 or 12, wherein the second predetermined value is 23pg/10¹⁰Amount of TGF β of EV.

14. The method of any one of claims 11-13, wherein the EV preparation is determined to have at least 50% pro-angiogenic activity.

15. The method of any one of claims 11-14, wherein the EV is from a human cell.

16. A preparation of pro-angiogenic Extracellular Vesicles (EV) obtainable by the method according to any one of claims 11 to 15, wherein the miR-130a content in the preparation, measured as Ct value by real-time PCR, is Ct <30, and the TGF β content in the preparation is>23pg/10¹⁰EV for use in the therapeutic treatment of a disease or injury positively affected by a pro-angiogenic treatment, or for use in wound healing.

17. Preparation of pro-angiogenic Extracellular Vesicles (EV) with miR-130a content of Ct value measured as Ct value by real-time PCR<30 and/or a TGF beta content of>23pg/10¹⁰EV for use in the therapeutic treatment of a disease or injury positively affected by a pro-angiogenic treatment, or for use in wound healing.

18. The preparation of claim 16 or 17, wherein the EV preparation has at least 50% pro-angiogenic activity.

19. A preparation for use according to any one of claims 16 to 18, wherein the EV is derived from a biological fluid or a conditioned cell or tissue culture medium.

20. A preparation for use according to claim 19, wherein the biological fluid is whole blood, plasma or serum.

21. The preparation for use according to claim 20, wherein the EV is prepared from serum of a healthy donor or serum of a patient with cardiovascular risk factors.

22. The preparation for use according to any one of claims 16 to 21, wherein the disease or injury is a vascular disease or injury, or a condition associated with risk of cardiovascular disease.

23. The preparation for use according to any one of claims 16 to 22, wherein the disease or injury is selected from the group consisting of acute myocardial infarction, acute cerebrovascular disease, acute and chronic peripheral arterial disease, acute renal ischemia, obesity, and diabetes.