Method for detecting endothelial extracellular vesicles

Abstract

The present invention notably relates to a method for detecting endothelial extracellular vesicles (EEVs) in a biological sample of a subject, and to an in vitro method for capturing endothelial extracellular vesicles (EEVs) in a biological sample of a subject, that are based on the detection of the expressions of CD62E and EPhA2 on said EEVs, or capture of extracellular vesicles expressing CD62E and EPhA2 on said EEVs.

Description

[0001]METHOD FOR DETECTING ENDOTHELIAL EXTRACELLULAR VESICLES FIELD OF THE INVENTION The present invention notably relates to a method for detecting endothelial extracellular vesicles (EEVs) in a biological sample of a subject, and to an in vitro method for capturing endothelial extracellular vesicles (EEVs) in a biological sample of a subject, that are based on the detection of CD62E and EPhA2 on said EEVs, or captures of EEVs expressing at least CD62E and EEVs expressing at least EPhA2. BACKGROUND OF THE INVENTION Extracellular vesicles (EVs) constitute all of the vesicles emitted by the cells into the extracellular environment. Some of these vesicles convey a large amount of information due to their content of lipids, proteins and nucleic acids. In addition, they can be considered as potential biomarkers, for example of certain cardiovascular, immuno- inflammatory diseases and cancer and are found in many biological fluids such as blood, pleural fluid or urine. In particular, endothelial extracellular vesicles (EEVs) released by activated or injured vessel-wall endothelium can be detected in peripheral blood. Currently available methods for detecting EEVs have limitations in terms of specificity and sensitivity, the challenge being to distinguish these EVs from those released by circulating blood cells such as leukocytes and platelets. The use of a strategy based on several markers, either present or necessarily absent for EVs to be considered as EEVs, are frequently used in flow cytometry; typically EEVs are often defined as CD31+ / CD41- or CD146+ / CD45-. This strategy consists of using two different fluorochromes to exclude in particular CD31+ / CD41+ EVs (platelet EVs) or CD146+ / CD45+ EVs (leukocyte EVs) respectively, in order to compensate for the lack of strict endothelial specificity of the markers CD31 or CD146 used alone with respect to EEVs. Indeed, CD31 is a marker of EVs but also of platelets; thus, it does not specifically target EEVs. On the other hand, CD146 is not specific enough of endothelial cells, since it can be detected on leukocytes, more precisely on a subclass of T lymphocytes, albeit at low level of expression. Furthermore, the patent document US11231422 discloses that the levels of microvesicles of endothelial origin (CD62E+) could predict the occurrence of HCC in patients with cirrhosis. Therefore, it relates to a method for determining whether a patient suffering from cirrhosis is at risk of having or developing hepatocellular carcinoma comprising determining the level of endothelial-derived microvesicles (e.g. by flow cytometry) in a blood sample obtained from the patient (by detection of CD62E on said EEVs in said biological sample). PROBLEM TO BE SOLVED It is therefore necessary to identify a combination of markers that allows a more specific and more sensitive detection of EEVs. Especially, there is a need for a reliable and robust method for detecting and / or capturing EEVs, said method being specific, sensitive and easy to perform. The present invention solves these needs. SUMMARY OF THE INVENTION The inventors have now identified that the cell-surface antigens CD62E and EPhA2, and optionally CDH13, are detectable on endothelial extracellular vesicles (EEVs). As shown in the examples, the inventors observed no expression of these markers on extracellular vesicles derived from platelets, neutrophils and monocytes; this shows that these markers are specific to the endothelium-derived EEVs. The inventors demonstrated that the simultaneous detection of CD62E and EPhA2 antigens at the membrane of EVs specifically identifies EEV among EV from other sources, and increases the sensitivity of EEV detection. Thus, the present invention relates to a method for detecting endothelial extracellular vesicles (EEVs) in a biological sample of a subject, which comprises the detection of CD62E and EPhA2 on said EEVs in said biological sample, wherein said detection is indicative of the presence of EEVs. Said method is an in vitro method. Preferably, CD62E and EPhA2 are detected simultaneously. The present invention also relates to an in vitro method for capturing endothelial extracellular vesicles (EEVs) in a biological sample of a subject (also called the “in vitro EEV capture method of the invention”), which comprises: - capturing EVs expressing at least CD62E and EVs expressing at least EPhA2 in said biological sample, - and optionally collecting said EVs. Optionally, the method comprises further determining biological characteristics of the collected EVs, including biological activities or molecular features. The present invention also relates to a method for studying a cell sample or a tissue sample or an organ sample, which comprises an in vitro method as described above. The present invention also relates to a method for diagnosing a subject with a disease involving an endothelial alteration, for example a myocardial infarction or a stroke or a cerebrovascular accident or a heart failure or a ventricular fibrillation, said method comprising: i. performing an in vitro EEV detection method according to the invention or an in vitro EEV capture method according to the invention in a biological sample of the subject; ii. comparing the concentration, amount or activity level or molecular expression of the EEVs with its respective predetermined reference value; and iii. determining that when said concentration, amount or activity level or molecular expression of the EEVs is higher than its respective predetermined reference value, then the subject is very likely to be afflicted with a disease involving an endothelial alteration, for example a myocardial infarction or a stroke or a cerebrovascular accident or a heart failure or a ventricular fibrillation. The present invention also relates to a method for predicting a complication on a subject with a disease involving an endothelial alteration, for example an inflammatory disease or a cardiovascular disease or a thrombosis, said method comprising: i. performing an in vitro EEV detection method according to the invention or an in vitro EEV capture method according to the invention in a biological sample of the subject; ii. comparing the concentration, amount or activity level or molecular expression of the EEVs with its respective predetermined reference value; and iii. determining that when said concentration, amount or activity level or molecular expression of the EEVs is higher than its respective predetermined reference value, then the subject is very likely to have a complication due to the disease involving an endothelial alteration. The present invention also relates to a method for predicting the efficacy of a treatment on a subject with a disease involving an endothelial alteration, for example an inflammatory disease or a cardiovascular disease or a thrombosis, said method comprising: i. performing an in vitro EEV detection method according to the invention or an in vitro EEV capture method according to the invention in a biological sample of the subject; ii. comparing the concentration, amount or activity level of the EEVs with its respective predetermined reference value; and iii. determining that when said concentration, amount or activity level of the EEVs is higher than its respective predetermined reference value, then the subject is expected to have a favorable response to a treatment against the disease involving an endothelial alteration, for example an anti-inflammatory treatment or an anti-thrombotic treatment. The present invention also relates to an in vitro method for monitoring the efficacy of a treatment on a subject with a disease involving an endothelial alteration, for example an inflammatory disease or a cardiovascular disease or a thrombosis, comprising: (i) performing an in vitro EEV detection method according to the invention or an in vitro EEV capture method according to the invention from a first biological sample obtained from said subject at a first time point to obtain first collected EEVs from the subject, (ii) determining the concentration, the amount or the activity level or molecular expression of the first collected EEVs, (iii) performing an in vitro EEV detection method according to the invention or an in vitro EEV capture method according to the invention from a second biological sample obtained from said subject at a second time point to obtain second collected EEVs from the subject, (iv) determining the concentration, the amount or the activity level or molecular expression of the second collected EEVs, (v) comparing the concentration, the amount or the activity level or molecular expression of first collected EEVs and second collected EEVs, and (vi) concluding about the treatment efficacy, depending on the concentration, the amount or the activity level or molecular expression of the second collected EEVs as compared to the concentration, the amount or the activity level or molecular expression of the first collected EEVs. The present invention also relates to an in vitro method for eliminating EEVs in a biological sample of a subject, which comprises: - capturing or filtrating-out EVs expressing at least CD62E and EVs expressing at least EPhA2 in said biological sample. The methods of the invention are in vitro methods. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for detecting EEVs in a biological sample of a subject (also called the “EEVs detection method of the invention”), which comprises the detection of CD62E and EPhA2 on said EEVs in said biological sample, wherein said detection is indicative of the presence of EEVs. Preferably, CD62E and EPhA2 are detected simultaneously. Preferably, said method further comprises the detection of CDH-13 on said EEVs in said biological sample, wherein the detection of CD62E, EPhA2 and CDH-13 is indicative of the presence of EEVs. Indeed, CD62E, EPhA2 and optionally CDH-13 are expressed on said EEVs (i.e. at their surface). They are different from their possible soluble forms (i.e. free forms). CD62E (CD62 antigen-like family member E), also known as E-selectin, is a cell adhesion molecule belonging to the selectin family of proteins, expressed only on endothelial cells activated by cytokines. It is encoded in humans by the SELE gene. The human protein sequence can be retrieved in UniProt under accession number P16581. EPhA2 (EPH receptor A2), also called ephrin type-A receptor 2, is a protein that binds ephrin-A ligands. It is encoded in humans by the EPHA2 gene. The human protein sequence can be retrieved in UniProt under accession number P29317. CDH-13 (cadherin-13), also called T-cadherin, is a unique member of the cadherin superfamily of proteins, because it lacks the transmembrane and cytoplasmic domains common to all other cadherins and is instead anchored to the cell’s plasma membrane by the GPI anchor. It is encoded in humans by the CDH13 gene. The human protein sequence can be retrieved in UniProt under accession number P55290. By “endothelial extracellular vesicles” or “EEVs”, it is meant any extracellular vesicle that can be released or secreted by an endothelial cell in its environment. The definition of said extracellular vesicles is well-established, notably using Thery et al., Journal of Extracellular Vesicles of 2023 (MISEV 2023). This includes microparticles, microvesicles, ectosomes, apoptotic vesicles and exosomes released or secreted by an endothelial cell in its environment. As used herein, the term “subject” or “patient” means a mammal, preferably a human. Typically the subject presents an endothelial alteration responsible for the generation of endothelial extracellular EEVs. Preferably, the subject is a patient, preferably human, with a disease involving an endothelial alteration. The disease involving an endothelial alteration is preferably an inflammatory disease or a cardiovascular disease or a thrombosis, more preferably a myocardial infarction or a stroke or a cerebrovascular accident or a heart failure or a ventricular fibrillation. Preferably, the subject is a patient, preferably human, with an immunoinflammatory disease or a cardiovascular disease, or at risk of thrombosis, or at risk of haemorrhagic or ischemic disease or the subject is a patient with cancer at risk of thrombosis. Preferably the subject is a patient with an immuno-inflammatory disease, such as an autoimmune or infectious disease, and at risk of thrombosis or haemorrhagic or ischemic disease or the subject is a patient with cancer at risk of thrombosis. As used herein, the term “biological sample” is any biological sample of said subject. Preferably, the biological sample of the invention is a sample of blood, urine, pleural fluid, cerebrospinal fluid, lymph, saliva, tears or vitreous humor, preferably a blood sample. As used herein, the term "blood sample" refers to a whole blood, serum or plasma sample. Preferably, the biological sample of the invention is a blood sample, more preferably is a plasma sample. Especially, the methods of the invention allow detecting the presence and / or measuring the amount and / or biological activities of EEVs that are released by “endothelial” cells which are hardly accessible in the blood, especially the plasma, or in other biological fluids. For example said cells may be cells forming the vessel wall / vascular endothelium or from or araising from atherosclerotic plaques or any other vascularized tissue. Thus the methods of the invention based on EEV determination in liquid samples to explore cells, tissues or organs in a non-invasive way, enter in the field of methods applicable onto a “liquid biopsy”. By “molecular expression” of EEVs, it is meant the expression of a protein, a carbohydrate, a lipid or a nucleic acid such as RNA or miRNA, at the surface of said EEV or inside said EEVs. Preferably, the detection of CD62E is performed using at least one anti-CD62E antibody or one of its fragments, and / or the detection of EPhA2 is performed using at least one anti-EPhA2 antibody or one of its fragments. Preferably, the detection of CDH-13 is performed using at least one anti-CDH13 antibody or one of its fragments. The antibody may be polyclonal or monoclonal, preferably monoclonal. Polyclonal antibodies directed against CD62E or EPhA2, or optionally CDH-13, can be raised according to known methods by administering the appropriate antigen or epitope to a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others. Various adjuvants known in the art can be used to enhance antibody production. Although antibodies useful in practicing the invention can be polyclonal, monoclonal antibodies are preferred. Monoclonal antibodies against CD62E or EPhA2, or optionally CDH-13, can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for production and isolation include but are not limited to the hybridoma technique originally described by Kohler and Milstein (1975); the human B-cell hybridoma technique (Cote et al., 1983); and the EBV-hybridoma technique (Cole et al. 1985). Alternatively, techniques described for the production of single chain antibodies (see e.g. U.S. Pat. No. 4,946,778) can be adapted to produce anti-CD62E single chain antibodies, or anti-EPhA2 single chain antibodies, or optionally anti-CDH-13 single chain antibodies. Antibodies useful in practicing the present invention also include anti-CD62E (or EPhA2 or optionally CDH-13) fragments including but not limited to F(ab')2 fragments, which can be generated by pepsin digestion of an intact antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab and / or scFv expression libraries can be constructed to allow rapid identification of fragments having the desired specificity to CD62E or EPhA2, or optionally CDH-13. For example, phage display of antibodies may be used. In such a method, single-chain Fv (scFv) or Fab fragments are expressed on the surface of a suitable bacteriophage, e.g., M13. Briefly, spleen cells of a suitable host, e.g., mouse, that has been immunized with a protein are removed. The coding regions of the VL and VH chains are obtained from those cells that are producing the desired antibody against the protein. These coding regions are then fused to a terminus of a phage sequence. Once the phage is inserted into a suitable carrier, e.g., bacteria, the phage displays the antibody fragment. Phage display of antibodies may also be provided by combinatorial methods known to those skilled in the art. Antibody fragments displayed by a phage may then be used as part of an immunoassay. Preferably, the anti-CD62E antibody, the anti-EPhA2 antibody, the anti-CDH-13 antibody, or one of their fragments, are chosen from polyclonal antibodies, monoclonal antibodies, single chain antibodies (such as single-chain Fv or scFv), F(ab')2 fragments and Fab fragments. Preferably, the detection of CD62E and EPhA2 on said EEVs, and optionally the detection of CDH-13 on said EEVs, is performed with a technique chosen from immunodiagnostic techniques and cell analysis techniques; preferably chosen from flow cytometry (associated or not with size exclusion chromatography), immunoassays including fluorescence-based or chemiluminescence-based immunoassays, enzyme- mediated immunoassays such as ELISAs, radioimmunoassays, biotin / avidin type assays, , immunoelectrophoresis, immunoprecipitation, solid-phase affinity chromatography, Western Blots, agglutination tests, super resolution microscopy, fluorescence microscopy, electron microscopy with nanoparticle staining. More preferably, the detection of CD62E and EPhA2 on said EEVs, and optionally the detection of CDH-13 on said EEVs, is performed with flow cytometry. The antibody may be labelled with a detectable molecule or substance, such as a fluorescent molecule, a radioactive molecule or any others labels known in the art. Labels are known in the art that generally provide (either directly or indirectly) a signal. As used herein, the term "labeled", with regard to the antibody, is intended to encompass direct labeling of the antibody by coupling (i.e., physically linking) a detectable substance, such as a radioactive agent, a chemiluminescent agent or a fluorophore (e.g. fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or Indocyanine (Cy5)) to the antibody, as well as indirect labeling of the antibody by reactivity with a detectable substance. An antibody may be labeled with a radioactive molecule by any method known in the art. The aforementioned assays may involve the bounding of one or several antibody(ies) to a solid support. Solid supports which can be used in the practice of the invention include substrates such as nitrocellulose (e. g., in membrane or microtiter well form); polyvinylchloride (e. g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, and the like. More particularly, an ELISA method can be used, wherein the wells of a microtiter plate are coated with a set of antibodies against CD62E or EPhA2 or optionally CDH-13. A blood sample containing or suspected of containing EVs exposing CD62E or EPhA2 or optionally CDH-13 is then added to the coated wells. After a period of incubation sufficient to allow the formation of antibody-antigen complexes, the plate(s) can be washed to remove unbound moieties and a detectably labeled secondary binding molecule added. The secondary binding molecule is allowed to react with any appropriate marker protein present on captured sample EEVs, the plate washed and the presence of the secondary binding molecule detected using methods well known in the art. As an example, marker proteins likely present on captured sample EEVs may include, without being limiting, CD55, CD59, CD29, HLA class I, as well as CD31 and CD146, that are present on EEVs without being specific for them. Alternatively, capturing antibodies may include those reacting with EVs in general (e.g. CD55, CD59, anti-HLA Class I…), preferably those reacting not only with EEVs (such as CD31, CD9 or CD146…), and the detection method is rendered specific for EEVs by using reporter antibodies such as the ones chosen from the group of anti-CD62E, anti- EPhA2 and optionally anti-CDH-13. Preferably, the detection is performed by flow cytometry. Preferably flow cytometry involves the use of labeled antibodies against CD62E and of labeled antibodies against EPhA2, wherein all antibodies are coupled to a fluorophore, preferably are coupled to the same fluorophore or to fluorophores emitting photons in the same wavelength range. In the EEVs detection method of the invention, the detection of CD62E and EPhA2 is indicative of the presence of EEVs. Preferably, the detection of CDH-13, CD62E and EPhA2 is indicative of the presence of EEVs. Thus, the EEVs detection method of the invention, when performed in a given sample, can easily bring a response about the presence or absence of EEVs in the sample. Preferably, the detection of CD62E and EPhA2, and optionally of CDH-13, on the EEVs, comprises determining a concentration or an amount of EVs staining positive for CD62E and EPhA2 and optionally CDH-13 present in the sample. More preferably, the detection of CD62E and EPhA2, and optionally of CDH-13, on the EEVs e.g. by Flow cytometry, comprises determining a concentration or an amount of EVs staining positive for CD62E and EPhA2 and optionally CDH-13 present in the sample. This allows a more specific result of the method: the method of the invention allows determining the concentration and / or amount of EVs staining positive for CD62E and / or EPhA2 (and optionally for CDH-13) present in the sample, and thus allows measuring the concentration and / or amount of EEVs in the sample. For example, the concentrations or amounts of CD62E positive and / or EPhA2 positive EVs and optionally CDH-13 EVs may be determined by flow cytometry. The present invention also relates to an in vitro method for capturing EEVs in a biological sample of a subject (also called the “in vitro EEV capture method of the invention”), which comprises: - capturing EVs expressing at least CD62E and EVs expressing at least EPhA2 in said biological sample, - and optionally collecting said EVs. Optionally the method comprises measuring additional features of said EVs, including genetic material, expression of proteins e.g. membrane antigens and biological activities. By “collecting”, it is meant that the EVs are gathered together among the biological sample and / or separated from the biological sample. The collecting step can be a separation step targeting EVs expressing at least CD62E and at least EPhA2. The collecting step is typically a step of isolating EVs expressing at least CD62E and at least EPhA2. The collecting step of said EVs is very useful as a preliminary step for further investigations, such as quantifying the captured EVs, measuring molecular expression or measuring biological activities of the captured EVs and / or characterizing / detecting genetic features of said EVs such as RNA, DNA or miRNA. Optionally the in vitro EEV capture method of the invention further comprises capturing EVs expressing at least CDH-13 in said biological sample, and EVs expressing at least CD62E, at least EPhA2 and at least CDH-13 are EEVs. Preferably, the capture is performed with a capturing technique using a substrate onto which at least one anti-CD62E antibody or one of its fragments and / or at least one anti-EPhA2 antibody or one of its fragments, and optionally at least one anti-CDH-13 antibody or one of its fragments, is(are) grafted. By “substrate”, it is meant any support that can be used for antibody grafting. The substrate is preferably a solid support, which is preferably chosen from nitrocellulose (e.g., in membrane or microtiter well form); polyvinylchloride (e.g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, and the like. Notably said substrate is a bead. Typically, according to an embodiment, the substrate can be divided into a first set of substrate onto which only at least one anti-CD62E antibody or one of its fragments is grafted, and a second set of substrate onto which only at least one anti-EPhA2 antibody or one of its fragments is grafted. It may comprise a third set of substrate onto which only at least one anti-CDH-13 antibody or one of its fragments is grafted. According to an other embodiment, at least one anti-CD62E antibody or one of its fragments, at least one anti-EPhA2 antibody or one of its fragments, and optionally at least one anti-CDH-13 antibody or one of its fragments, are grafted onto each substrate. Typically, the substrate is a bead. Thus, preferably, according to an embodiment, the beads are divided into a first set of beads onto which only at least one anti-CD62E antibody or one of its fragments is grafted, and a second set of beads onto which only at least one anti-EPhA2 antibody or one of its fragments is grafted. It may comprise a third set of beads onto which only at least one anti-CDH-13 antibody or one of its fragments is grafted. According to an other embodiment, at least one anti-CD62E antibody or one of its fragments, at least one anti-EPhA2 antibody or one of its fragments, and optionally at least one anti-CDH-13 antibody or one of its fragments, are grafted onto each bead. Preferably, as shown in the example, the capture is performed by immunomagnetic separation (IMS). IMS follows the following principle: antibodies-coated paramagnetic beads bind to CD62E, EPhA2 and optionally CDH-13 present on the surface of the EEVs, thus ensure EEVs capture, and facilitate the concentration of these bead-attached EEVs. Concentration of the EEVs is created by a magnet placed on the side of the recipient bringing the beads onto it. Preferably, the in vitro EEV capture method of the invention comprises collecting said EVs. Preferably, the in vitro EEV capture method of the invention comprises determining a concentration, an amount, an activity level or molecular expression of the EEVs. By “activity level”, it is meant the biological activity of EEVs. Typically, the activity level is the procoagulant activity and / or profibrinolytic activity of EEVs. The protocol that can be followed to measure said activity level is detailed in the example, i.e. the procoagulant functional assay. Molecules such as proteins classically present on all EVs may have interest for quantitating the amount of EEVs such as CD55, CD59 or HLA-Class I. Typically, the activity level is the one of a protein with a procoagulant activity and / or profibrinolytic activity, such as TF (Tissue Factor) or tPA (Tissue-type Plasminogen Activator) expression, on the surface of EEVs. The protocol that can be followed to measure said protein expression can be flow cytometry or sandwich-type immunoassays wherein EEVs have been collected by the capturing method of the invention, as alternative of a procoagulant functional assay. The present invention also relates to a method for studying a cell sample or a tissue sample or an organ sample, which comprises an in vitro EEVs detection method of the invention or an in vitro EEV capture method of the invention. Notably, said method is useful for analyzing EEVs biological functions and / or to provide information about the structure and / or role of these EEVs. Enzymatic tests may be used in said methods. An example could be the detection of Tissue Factor (TF) dependent procoagulant activity of EEV captured by IMS (based on CD62E / EPhrA2), using a chromogenic enzymatic assay generating activated Factor X. An alternative is to use a colorimetric test for measuring EV activities by functional assay. Detectable substances such as chromogenic or fluorogenic substrates, or fluorescent, luminescent or radioactive agents or substances detectable by molecular hybridation, or nanoparticles based tags, may be used in said methods. Preferably, the fluorophore may be chosen from fluorescein isothiocyanate (FITC), phycoerythrin (PE) and Indocyanine (Cy5). The present invention also relates to a method for diagnosing a patient with a disease involving an endothelial alteration, preferably a myocardial infarction or a stroke or a cerebrovascular accident or a heart failure or a ventricular fibrillation (herein “diagnostic method”), said method comprising: i. performing an in vitro EEV detection method according to the invention or an in vitro EEV capture method according to the invention, in a biological sample of the patient; ii. comparing the concentration, amount or activity level or molecular expression of the EEVs with its respective predetermined reference value; and iii. determining that when said concentration, amount or activity level or molecular expression of the EEVs is higher than its respective predetermined reference value, then the patient is very likely to be afflicted with a disease involving an endothelial alteration. Other biomarkers may be used in addition to step i. Notably, a method for diagnosing a patient with a myocardial infarction may comprise detecting circulating endothelial cells (CEC) and / or detecting troponin. The detection of CEC and / or of troponin may comprise determining the concentration, amount or activity level of CEC or troponin. The predetermined reference value used in the diagnostic method can be any EEVs concentration, amount or activity level value or molecular expression value that is observed in average in healthy patients, i.e. patients who are not afflicted with a myocardial infarction, a stroke, a cerebrovascular accident, a heart failure or a ventricular fibrillation. The present invention also relates to a method for predicting a complication on a patient with a disease involving an endothelial alteration, for example an inflammatory disease or a cardiovascular disease or a thrombosis, said method comprising the following steps of: i. performing an in vitro EEV detection method according to the invention or an in vitro EEV capture method according to the invention, in a biological sample of the patient; ii. comparing the concentration, amount or activity level or molecular expression of the collected EEVs with its respective predetermined reference value; and iii. determining that when said concentration, amount or activity level or molecular expression of the collected EEVs is higher than its respective predetermined reference value, then the patient is very likely to have a complication due to the disease involving an endothelial alteration, for example the inflammatory disease or the cardiovascular disease or the thrombosis. The predetermined reference value used in the method for predicting a complication can be any EEVs concentration, amount or activity level or molecular expression value correlated with at least one complication due to diseases involving an endothelial alteration, for example inflammatory diseases, cardiovascular diseases or thrombosis. The above diagnostic method or the above method for predicting a complication optionally comprises an in vitro EEV capture method of the invention, which further comprises capturing EVs expressing at least CDH-13 in the biological sample, and EVs expressing CD62E, EPhA2 and CDH-13 are EEVs. The present invention also relates to a method for predicting the efficacy of a treatment on a patient with a disease involving an endothelial alteration, for example an inflammatory disease or a cardiovascular disease or a thrombosis (herein “prediction method”), said method comprising: i. performing an in vitro EEV detection method according to the invention or an in vitro EEV capture method according to the invention in a biological sample of the patient; ii. comparing the concentration, amount or activity level of the EEVs with its respective predetermined reference value; and iii. determining that when said concentration, amount or activity level of the EEVs is higher than its respective predetermined reference value, then the patient is expected to have a favorable response to a treatment against the disease involving an endothelial alteration, for example an anti-inflammatory treatment or an anti-thrombotic treatment. Such a prediction method may allow patient stratification according to cardiovascular or immuno-inflammatory risk. Preferably, the step of performing an in vitro EEV detection or capture in a biological sample of the patient (step i) is performed in the prediction method before any treatment of the patient. The predetermined reference value used in the prediction method can be any EEVs concentration, amount or activity level value correlated with efficacy of a given treatment, preferably an anti-inflammatory treatment or an anti-thrombotic treatment. The comparison of step ii. is indicative whether the patient is expected to have a favorable response to an anti-inflammatory treatment or an anti-thrombotic treatment. For example, each predetermined reference value is determined in a statistical analysis. The above method for predicting the efficacy of a treatment preferably comprises an in vitro EEV detection or capture method of the invention, which further comprises detecting or capturing EVs expressing at least CDH-13, and EVs expressing at least CD62E, at least EPhA2 and at least CDH-13 are EEVs in the biological sample. The present invention also relates to an in vitro method for monitoring the efficacy of a treatment on a patient with a disease involving an endothelial alteration, for example an inflammatory disease or a cardiovascular disease or a thrombosis (herein “monitoring method”), comprising: (i) performing an in vitro EEV detection method according to the invention or an in vitro EEV capture method according to the invention from a first sample obtained from said patient at a first time point to obtain first collected EEVs from the patient, (ii) determining the concentration, the amount or the activity level or molecular expression of the first collected EEVs, (iii) performing an in vitro EEV detection method according to the invention or an in vitro EEV capture method according to the invention from a second sample obtained from said patient at a second time point to obtain second collected EEVs from the patient, (iv) determining the concentration, the amount or the activity level or molecular expression of the second collected EEVs, (v) comparing the concentration, the amount or the activity level or molecular expression of first collected EEVs and second collected EEVs, and (vi) concluding about the treatment efficacy, depending on the concentration, the amount or the activity level or molecular expression of the second collected EEVs as compared to the concentration, the amount or the activity level or molecular expression of the first collected EEVs. Step (vi) especially comprises determining if the EEVs have procoagulant or profibrinolytic function. Regarding step (vi), concluding on the effect of the treatment depends on the biological significance of the measured EEV-associated biomarker: For a debilitating characteristics of endothelium such as procoagulant function (e.g. TF-associated generation of FXa), if the concentration, the amount or the activity level or molecular expression of the second collected EEVs is lower than the concentration, the amount or the activity level or molecular expression of the first collected EEVs, then the treatment is efficient. If the concentration, the amount or the activity level or molecular expression of the second collected EEVs is higher than or equal to the concentration, the amount or the activity level or molecular expression of the first collected EEVs, then the treatment is not efficient. For a favorable characteristics of endothelium such as profibrinolytic function (e.g. uPA- or tPA-associated generation of plasmin), if the concentration, the amount or the activity level or molecular expression of the second collected EEVs is higher than the concentration, the amount or the activity level or molecular expression of the first collected EEVs, then the treatment is efficient. If the concentration, the amount or the activity level or molecular expression of the second collected EEVs is lower than or equal to the concentration, the amount or the activity level or molecular expression of the first collected EEVs, then the treatment is not efficient. Preferably, the first time point is anterior to the second time point. Preferably, the treatment evaluated in the monitoring method is chosen in the group consisting in anticoagulant drugs, fibrinolytic agents and antiplatelet drugs. Anticoagulant drugs inhibit thrombus formation. Fibrinolytic agents degrade fibrin. Antiplatelet drugs inhibit platelet activation or aggregation. Preferably, the anticoagulant drug is chosen from non-fractionated heparin (NFH), low-molecular-weight heparin (LMWH) and direct oral anticoagulant (DOA). DOA may be apixaban, rivaroxaban or dabigatran. Preferably, the fibrinolytic agent is tranexamic acid. Preferably, the antiplatelet drug is chosen from aspirin, clopidogrel and ticagrelor. Preferably, in the monitoring method, the patient starts his treatment between the collection of the two samples (i.e. between steps (i) and (iii)). Preferably, in the monitoring method, the patient starts his treatment before the collection of any sample (i.e. before steps (i) and (iii)). Preferably, the time interval between the first and second time points is of a few days, preferably 2 to 7 days, or of a few months, preferably two to six months. The present invention also relates to an in vitro method for eliminating endothelial extracellular vesicles (EEVs) in a biological sample of a subject, which comprises: Capturing or filtrating-out EVs expressing at least CD62E and EVs expressing at least EPhA2 in said biological sample. The present invention also relates to a method for treating a subject, which comprises: Capturing or filtrating-out EVs expressing at least CD62E and EVs expressing at least EPhA2 in said subject, notably in the blood of said subject, and collecting said EVs. The collected EVs are not to be re-administered to the subject. Said method may be useful in the case of pathogenic deleterious EEVs. The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1: Production method and characterization of EEVs Methods of primary endothelial culture to obtain Not Stimulated (NS) and TNF-α Stimulated EEVs containing supernatants and concentrated EEVs. These supernatants were produced from three primary cell types: HCAEC, HCMEC and HRGEC and were used for the large screening of endothelial antigenic specificities by flow cytometry. Figure 2: EndoEV contained in culture supernatant were phenotyped with a panel of 43 selected markers. The results were generated from three different endothelial primary cells derived Evs (HCAECs, HCMEC and HRGEC). Data are presented as the mean value of triplicates with standard deviation (SD). PC : Polyclonal antibodies; Purifié = purified. Figure 3: Validation of the antibody specificity on monocytes, neutrophils and platelets Monocytes, Neutrophils and Platelets derived EVs were phenotyped with the eight selected markers divided in classical targets (left) and new targets (right). Data are presented as the mean value of triplicates with standard deviation (SD). Figure 4: PE-labelled antibodies combined strategy improve EEV detection by flow cytometry Mean percentage of double-positive EEVs detected among Annexin V+FITC events between the different specific antibodies tested and their combination on not stimulated derived EV (A) and TNF-α stimulated derived EEV (B) in washed condition. All the experiments were performed at least five independent times (n=5). Mann-Whitney t test. Figure 5: Combined strategy is useful to capture efficiently EEVs by IMS (A,B) Mean percentages of double-positive (AnnV-FITC + / CD59-PE+) EEVs depleted by the different beads on not stimulated derived EV (A) and TNF-α stimulated derived EEV in PBS BA conditions (B). (C,D) Mean percentages of double-positive (AnnV-FITC+ / CD59-PE+) EEVs depleted by the different beads on not stimulated derived EVs (C) and TNF-α stimulated derived EEVs in spiked MVFP (D). The percentages of depletion were calculated using the number of remaining EEV in the post-IMS supernatant in comparison to the number of EEV in the without beads condition (WO Beads). All the experiments were performed at least three independent times (n=3) on the three types of concentrated EVs (HCAEC, HCMEC, HRGEC) at 100, 1000 and 5000 EEVs / µL. Mann-Whitney t test. 1 Endothelial reference.2Panvesicular positive control Figure 6: The new endothelial combined strategy does not capture the other subsets of plasmatic EVs Mean percentage of depletion ± SD of Neutrophils (NdEV), Platelets (PdEV), Monocytes (ModMV), Erythrocytes (ErydEV), Lymphocytes T4, T8, B (LT4dEV, LT8dEV, LBdEV) and NK (NKdEV) derived EVs by the four differents beads in spiked PBS BA. EVs were spiked at 2000 Evs / µL All the experiments were done at least three independent times (n=3). Figure 7: TF activity assay with the new endothelial combined IMS strategy in spiked model and patients (A) IMS steps diagram, (B) Principle diagram of TF activity assay, (C) Mean percentage of TF activity normalized on the PanEV positive control beads CD29 / 59 TF activity. TF activity was measured on the IMS beads with three concentrations of HCMEC derived TNF-α stimulated EEV spiked in MVFP. (D) Mean TF activity expressed in fM on the different plasmatic derived EV spiked in MVFP at 1000 EV / µL. All the experiments were done at least three independent times. Mann-Whitney test. EXAMPLE(S) This example illustrates the sensitivity and specificity of the invention to detect and quantify EEV by flow cytometry, to capture EEV by immunomagnetic separation and to measure the TF-dependent procoagulant activity on the captured EEV. Materials & Methods Antibodies and reagents: All the antibodies used for the large screening are listed in Figure 2. Cell culture and production of endothelial extracellular vesicles (EEVs): Three types of primary endothelial cells were used: the Human Cardiac Arteries Endothelial Cells (HCAEC), the Human Cardiac Microvascular Endothelial Cells (HCMEC) were purchased from Promocell and the Human Renal Glomerular Endothelial Cells (HRGEC) were from Cliniscience. The three types of primary endothelial cells were grown in EGMV2 medium from Promocell supplemented with 5% Free EV FCS, EGF (5ng / ml), bFGF (10 ng / mL), IGF (20 ng / mL), VEGF (0.5 ng / mL), ascorbic acid (1 µg / mL) and hydrocortisone (0.2 µg / mL) at 37°C in a humidified atmosphere with 5% CO2. The fetal calf serum (FCS) of the EMGV2 kit was replace by free EV FCS obtained by ultracentrifugation of FCS at 4°C during 18h at 100000g. When cells reached 90 % of confluency, the medium supernatants containing extracellular vesicles (EVs) were collected after 24 hours. In some case, the cells were activated by the pro-inflammatory cytokine TNF-α (10 ng / mL - Miltenyi) during 18 hours. After collection, the medium supernatants were centrifugated twice at 400g during 5 minutes to remove cell debris and once at 1400g during 10 minutes to remove apoptotic bodies. Finally, the endothelial vesicles (EEV) were concentrated by ultracentrifugation at 20000g during 180 minutes at 4°C. The pellets were resuspended in PBS, aliquoted and stored at -80°C. Production of EV Low Plasma (EVLP): Blood bags were divided in few tubes and centrifugated twice at 2500g during 15 minutes at room temperature and with a minimal break. The Platelet Free Plasma (PFP) obtained was then ultracentrifugated at 100000g during 3 hours at 4°C. The plasma was finally filtrated with 0.1 µm filter in order to obtain EV Low Plasma (EVLP). Production of plasmatic EVs: The sorting of the various blood cell types were realized on Astrios EQ Instrument (Beckman Coulter) from blood bags. First, PMNs and mononuclear cells were separated using gradient density separation (1.077 g / ml, lymphocyte separation medium, Eurobio, Courtaboeuf, France) and centrifuged at 500g for 20 min. The upper part of the gradient was discarded, and peripheral blood mononuclear cells (PBMCs) were collected independently from the neutrophil-red blood cell fraction. Red blood cells were lysed using 50-mM ammonium chloride buffer for 10min. PMNs were washed with Ca / Mg-free phosphate-buffered saline (PBS), bovine serum albumin (BSA) 0.1%, centrifuged for 5 min at 300 g and resuspended in PBS and BSA 0.1% buffer. For the cell sorting, a multicolor panel was performed on cells using CD3- PB (Biolegend), CD4-PE (Beckman), CD8-APC-Cy5 (Biolegend), CD19-AF700 (Biolegend), CD56-PC7 (Beckman), CD45-ECD (Beckman), CD14-KrO (Beckman) and CD66b-APC (Beckman) antibodies. The purity of sorted cells was greater than 95 %. The T4, T8, B- lymphocytes, NK cells, monocytes and neutrophils were added in RPMI1640 medium (Thermofisher) supplemented with 10 % free EV FCS and with pro-inflammatory cytokines during 24 hours to induce EV production at 37°C in a humidified atmosphere with 5% CO2. Monocytes were stimulated with LPS (20µg / mL), neutrophils with TNF-α (10 ng / mL), and the other cells with PMA (50 mM). After collection, the medium supernatants were centrifugated twice at 400g during 5 minutes to remove cell debris and once at 1400g during 10 minutes in order to remove apoptotic bodies. Finally, the EVs were concentrated by ultracentrifugation at 20000g during 180 minutes at 4°C. The pellets were resuspended in PBS, aliquoted and stored at -80°C. Production of platelets derived EVs (PdEVs): Blood was collected from healthy volunteers in accordance with the Helsinky declaration of 1964. Blood samples were immediately treated by 200g centrifugation at 37°C during 13 minutes to obtain PRP (Plasma Rich Platelets). PRP was then centrifuge at 900g during 13 minutes at 37°C to obtain platelets pellet. The pellet was carefully resuspended in Tyrode / BSA buffer (138mM NaCl, 2,9M KCl, 12 mM NaHCO3, 0,36mM NaH2PO4, 5,5 mM Glucose, 10 mM Hepes, 2% BSA, pH 7,4) supplemented with apyrase (0,02U / mL – sigma Aldrich) and prostacyclin (0,1µM PGI2). Platelets were washed twice in Tyrode / BSA buffer, finally washed platelets were resuspended in Tyrode / BSA buffer without apyrase nor prostacyclin. Washed platelets were kept at 37°C during 30 minutes before stimulation (resting time). Washed platelets were count by flow cytometry using Cytoflex LX (Beckman Coulter). The concentration was adjusted to 250.106platelets / mL using Tyrode buffer. To induce vesiculation, platelets were stimulated with thrombone (1U / mL) and collagen (10µg / mL) during 1h with a gentle agitation. The activated platelet suspension was then centrifugated at 900g during 13 minutes. The supernatant was collected and centrifuged twice at 400g during five minutes, and once at 1400g during 10 minutes. Finally, the PdEVs were concentrated by ultracentrifugation at 20000g during 180 minutes at 4°C. The pellets were resuspended in PBS, aliquoted and stored at -80°C. Production of erythrocytes derived EVs (ErydEVs): Blood was collected from healthy volunteers in accordance with the Helsinky declaration of 1964. Blood samples were immediately treated by 200g centrifugation during 13 minutes to remove PRP (Plasma Rich Platelets) and leucocytes ring. The erythrocyte phase was washed twice using PBS and two successive centrifugations at 300g during 10 minutes. The vesiculation of erythrocytes was allowed during 10 days at room temperature. Once aged, erythrocytes were centrifugated at 300g during 10 minutes. After collection, the supernatant was centrifugated twice at 400g during 5 minutes to remove cell debris. Then, they were centrifugated at 1400g during 10 minutes in order to remove apoptotic bodies. Finally, the ErydEVs were concentrated by ultracentrifugation at 20000g during 180 minutes at 4°C. The pellets were resuspended in PBS, aliquoted and stored at -80°C. Saturating curves: The affinities of the selected antibodies were determined on HCAEC cells. At 90 % of confluency, the cells were detached and counted. In total, 2.105 cells were incubated with eight increasing concentrations of antibodies (from 0.1 to 10µg / mL). The expression of the target and the median fluorescence intensity (MFI) were evaluated by flow cytometry using Cytoflex LX. Large screening of antibodies by flow cytometry: First, saturating concentrations of each antibody were determined on EEVs derived from HCAEC in stimulated conditions using five final concentrations (from 0.1µg / mL to 10µg / mL). All antibodies were centrifugated for 2 min at 13,000g to remove aggregates prior to use. In total, 0.5 million of EEVs were incubated with 10µL of antibodies and with Annexin-V FITC (2,5 µg / mL - Tau Technology) during 20 minutes at room temperature. To stabilize the binding of Annexin V to phosphatidyl-serine on EVs, a volume of AnnV binding buffer was added at the end of incubation. Briefly, EVs analysis by FCM was performed using a 5-laser (Near UV-Violet– Blue–Yellow–Red) CytoFLEX LX cytometer (Beckman Coulter) provided with a plate reader. Instrument performances were checked daily using CytoFLEX Daily QC fluorosphere beads and SPHERO Rainbow 8-peak (Spherotech, Lake Forest, USA). The stability of the large EVs scatter gate was monitored by the use of Megamix-Plus Forward Scatter (FSC) and Side Scatter (SSC) purchased from BioCytex (Marseille, France). Once selected, the expressions of some targets were evaluated after a washing step using size exclusion chromatography (Izon) according to the manufacturer’s instructions in order to decrease the free antibody dependent background noise. Biotinylation of antibodies and coating with beads: Antibodies were biotinylated using Sulfo-NHS-LC-Biotine EZ-Link™ kit (Thermo Scientific). Briefly, 1 mg of Sulfo-NHS-LC- Biotin was solubilized in 180 µL of distilled water. After few minutes of stabilization, 72µg of Sulfo-NHS-LC-Biotine were added to 1 mg of purified antibodies and incubated during 30minutes at room temperature. After biotinylation, free molecules of biotin were removed using PD-10 columns according to the manufacturer’s instructions. Regarding Sera-Mag™SpeedBeads Blocked Streptavidin particles (Cytiva) coating also called SM1, biotinylated antibodies were used at the rate of 60µg to 1mg of SM1 beads. Immunomagnetic Separations: Es were spiked in PBS BA or EVLP at various concentrations. SM1 coated beads were added at the final concentration of 1µg / µL and incubated with spiked EEV during 1h in rotation at room temperature. Supernatants were collected and the capture efficiencies were evaluated by flow cytometry using cytoflex LX after Annexin V-FITC and antibodies labelling. Beads were washed twice with PBS BA and used for functional TF activity assays. Procoagulant functional assay: A modified version of Cy-Quant TF activity assay (Biocytex) was used to evaluate pro-coagulant EVs activity. EVs bearing SM1 beads were washed once with R1-1X and put in a final volume of 125µL of R1-1X. The TF activity assay was performed according to the manufacturer’s instructions. Data analysis: All FCM data were analysed with Kaluza analysis 2.1 software. Data were analysed for their statistical relevance with GraphPadPrism 8 software (GraphPad Software). Statistical tests used were Mann–Whitney test. The results are presented as the mean ± standard deviation. Results Characterization of EEVs EEVs from three primary endothelial cells (HCAEC, HCMEC and HRGEC) were produced and for some experiments concentrated according to the Minimal Information for Studies Extracellular Vesicles 2018 (MISEV 2018) as illustrated in Figure 1. They were characterized as EVs structure according to several methods (CMF, WB, TRPS). Large screening of antibodies by sensitive flow cytometry (Figure 2) The different specificities tested during the screening were selected by an extensive bibliography on the endothelium and the EEVs and with the team expertise. The goal was to find new antigenic specificities that sufficiently and specifically recognize EEVs. The large screening antibody was performed on EEVs contained in the supernatants of three primary endothelial cells from cardiac and kidney origins (HCAEC, HCMEC and HRGEC) in basal conditions (Not Stimulated endothelial supernatant - NS) and in inflammatory conditions (TNF-α stimulated endothelial supernatant – STIM). In total, 22 different molecules were selected for the screening. According to the availability of the antibodies, one, two or three different monoclonal antibodies (MAbs) were chosen for each antigenic specificity with various conjugates (PE, Biotin, Purified). For biotin- conjugated and purified antibody, the saturating concentrations of secondary reagents (both Streptavidin-PE and PE-conjugated secondary antibody were determined using as a model of EEV-bound MAbBiotin-conjugated and purified forms of CD59 antibodies by flow cytometry (data not shown). In the same manner, the saturating concentration of each MAb was determined on TNF-α Stimulated EEVs from HCAEC before testing on all other EVs. To take into account the endothelial heterogeneity found in vivo, the inventors decided to combine the results obtained with the different cell types. According to the mean percentage of double positive (AnnV FITC+ / Target PE+) EEVs detected by flow cytometry, among the 22 antigenic specificities tested, the inventors found 8 interesting targets with more than 5% of double positivity (considered as positive expression) : CD105, CD146, CD54, CD62E, CDH13, EPhA2, ESAM and MADCAM-1. For CD105, two of the three MAbs tested were interesting with an EEV positive expression percentage of 38.7 ± 6.1 (NS) and 36.4 ± 10.4 (STIM) for REA 794 MAb and of 32 ± 6.2 (NS) and 26.6 ± 9.2 (STIM) for TEA3 / 17.1.1 MAb. Only one representative Mab was tested for CD146 (S-Endo1) without significant difference between both types of conjugates with 40,7 ± 5.6 (NS) and 39,7 ± 12.6 (STIM) for PE-conjugated antibody and 34,8 ± 7.0 (NS) and 33,1 ± 8.2 (STIM) for Biotin- conjugated antibody. The percentages of double positive EEVs were 15,7 ± 1.1 (NS) and 33,3 ± 7.3 (STIM) for CD54 (PE-conjugated MAb 84H10), 0,9 ± 0.8 (NS) and 18,3 ± 15.2 (STIM) for CD62E (Biotin conjugated MAb REA280), 3,0 ± 4.1 (NS) and 8,4 ± 7.3 (STIM) for CD62E (PE-conjugated MAb HAE-1f), 6,2 ± 4.4 (NS) and 8,1 ± 4.0 (STIM) for CDH13 (Biotin- conjugated MAb REA 752), 9,2 ± 4.4 (NS) and 9,2 ± 4.0 (STIM) for CDH13 (PE-conjugated MAb392411), 11,9 ± 2.2 (NS) and 11,5 ± 1.9 (STIM) for EPhA2 (PE-conjugated MAb SHM- 16), 7,2 ± 2.7 (NS) and 5,5 ± 1.5 (STIM) for ESAM (Biotin-conjugated MAb REA901), 8,9 ± 2.0 (NS) and 5,2 ± 3.0 (STIM) for ESAM (PE-conjugated MAb 408519) and 45,8 ± 8.5 (NS) and 38,9 ± 4.1 (STIM) for MADCAM-1 (Purified MAb 683715). For all these specificities no significant differences were found in terms of expression levels between EEVs derived from not stimulated and TNF-α stimulated cells. Reactivity of the selected antibodies on the other main plasmatic EVs In order to determine the endothelial specific characteristics of CD105, CD146, CD54, CD62E, EPhA2, CDH13, ESAM and MADCAM-1 antibodies the inventors evaluated their reactivity on the other main plasmatic EVs such as platelets-, neutrophils- and monocytes-derived EVs (PdEVs, NdEVs and ModEVs, respectively). The expression of these targets on PdEVs, NdEVs and ModEVs were assessed using the same saturating concentration as the one for EEVs with one antibody per candidate specificity: MAbs REA794 (CD105), S-Endo1 (CD146), 84H10 (CD54), HAE-1f (CD62E), 392411 (CDH13), SHM- 16 (EPhA2), REA901 (ESAM), 683715 (MADCAM-1). As mentioned previously, the positive expression of the targets was considered from 5% to 100%. Flow cytometry analysis showed that CD105, CD54, ESAM and MADCAM-1 are not specific of the endothelium. Indeed, as illustrated in Figure 3, ModEVs are positive for CD105, CD54 and MADCAM-1 with a percentage of 7, 6 and 21 % respectively (AnnV-FITC + / Target +). ESAM and MADCAM-1 positive expressions were also found on PdEVs with 11 and 29% of double positive EVs respectively. Moreover, 14% of NdEVs were positive for AnnV and MADCAM- 1. In contrast, the inventors observed no expression of CD62E, EPhA2 and CDH13 on PdEVs, NdEVS and ModEVs showing indirectly their endothelial specificity. Selection of the best monoclonal antibodies for EEV detection by flow cytometry The goals of this study were to improve the specificity and the detection of EEVs by flow cytometry in comparison to the gold standard reference CD146 (S-Endo1 MAb). In this way, it was necessary to select the MAbs with the best affinity. For this purpose, the inventors compared the affinity profiles of four different representative MAbs for each specificity (CD62E, EPhA2 and CDH13) on HCAEC cells using flow cytometry. Unfortunately, because of a production issue at the manufacturer’s site with the anti- CDH13MAb 392411 by, only three MAbs were tested for CDH13 (Purified OTI3H6, purified E-9 and Biotin-conjugated REA752). For EPhA2, the affinity profiles of PE-conjugated SHM- 16, purified 371805, purified 1C1 and purified 1C11A12 MAbs were assessed. For CD62E, the comparisons were between PE-conjugated HAE-1f, purified P2H3, purified BBIG-E1 and purified BBIG-E4. To study the affinity profiles of each clone, a concentration range from 0.01 to 10 µg / mL was applied on not stimulated (NS) or 4-hour TNF-α stimulated (STIM) HCAEC endothelial cells. All the purified or Biotin-conjugated antibodies were revealed with a saturating concentration of PE-conjugated secondary anti-mouse Ig antibodies or PE-Streptavidin. The MAbs which had the smallest saturating concentration were considered as the MAbs with the best affinity. SHM-16 for EPhA2 and REA752 for CDH13 were the MAbs with the best affinity with a saturating concentration of 0.05 µg / mL and 5 µg / mL respectively (data not shown). Concerning CD62E, the P2H3, BBIG-E1 and BBIG-E4 MAbs had a stronger affinity than HAE-1f clone with a saturating concentration of 5 µg / mL versus 10 µg / mL respectively. Here, the MFI PE value cannot be compared between MAbs because of the different revealing process. From these results, the inventors selected the SHM-16, REA-752 and P2H3 MAbs to study their ability to detect EEVs and to improve their detection by flow cytometry. PE-labelled antibodies combined strategy improve EEV detection by flow cytometry A strategy to improve EEV detection is to increase the absolute fluorescent signal of EEVs by combining several antibodies conjugated with the fluorochrome (PE). For this purpose, the inventors combined two or three of the selected antibodies (CD62E, EPhA2 and / or CDH13) and they compared EEV staining after labelling with the gold standard CD146. These experiments were done on not stimulated and on TNF-α stimulated derived EEVs. For not stimulated EEVs (Figure 4A), in term of double positive events detected, the inventors show that CD146 allows the significantly highest detection with an average of 66.8 ± 4.9 % in comparison to CD62E, EPhA2, CDH13 or CD62E / EPhA2 combinations. The use of the three combined antibodies CD62E, EPhA2 and CDH13 rose the percentage of detection to 56.7 ± 15.1 % and was not significantly different from CD146. By contrast, for TNF-α stimulated derived EEVs (Figure 4B), the highest percentage of detected double positive events was obtained for the three combined antibodies CD62E / EPhA2 / CDH13 with 81 ± 7.1 % followed by the combination of CD62E / EPhA2 with 79.4 ± 6.6 % and CD146 with 72 ± 8.4 %. Used alone, the average percentage of double positive events was 60.3 ± 9.6 % for CD62E, 56.5 ± 8.6 % for EPhA2 and 45.9 ± 23.3 % for CDH13; that was significantly lower than CD146 and the two other combinations. These results show that an antibody combined strategy improves the detection of EEVs in comparison to the gold standard CD146. Development of a new tool to capture EEVs by ImmunoMagnetic Separation (IMS) To go further in the evaluation of this new combined antibody strategy, the inventors assessed the ability of these combinations to efficiently capture by IMS EEVs that were spiked either in buffered saline (noted PBS BA) and in plasmatic conditions. The capture efficiency was compared to the gold standard CD146 and to a PanEV IMS tool, i.e. CD29 / 59 beads, which is known to detect and to capture all plasmatic EVs (ref. Franco et al.). Briefly, the streptavidin-coated IMS beads were incubated with one or several (combined at equivalent dose) biotinylated antibodies and washed extensively. The efficiency of antibody coating was checked by flow cytometry using secondary antibodies. EEVs of the three territories were spiked in buffer (PBS BA) at a final concentration of 5000, 1000 or 100 / µL and then incubated with the different IMS beads during 60 minutes under agitation. Then the supernatants were collected and the non- depleted EEVs were analyzed by flow cytometry using CD59-PE labelling. The percentages of depletion were calculated using a control without beads (WO beads). The isotypic control beads Mouse IgG1 and Mouse IgG2b capture less than 5% of the spiked EEVs. For unstimulated derived EEVs (Figure 5A), the mean percentages of depletion obtained were 11.1 ± 11.4 % for CD62E beads, 55.1 ± 7.4 % for EPhA2 beads, 45.1 ± 10.8 % for CDH13 beads and were significantly lower than CD146 and CD29 / 59 with 70.6 ± 10.7 % and 83.1 ± 8.3 % ± respectively. The mix of the CD62E beads with EPhA2 beads (CD62E + EPhA2) failed to improve the depletion rate in comparison to CD146 with 48.9 ± 6.6 % and 47.9 ± 7.6 % respectively. Surprisingly, certainly due to steric hindrance, the mix of the CD62E beads with EPhA2 beads and CDH13 beads has a low depletion percentage with only 24.4%. However, when the IMS was performed sequentially with CD62E / EPhA2 beads and then CDH13 beads after removing of CD62E / EPhA2 beads, this percentage rose se to 63.5 % (CD62E / EPhA2 + CDH13 (seq)). Due to the higher expression of CD62E in inflammatory conditions, the CD62E beads were more efficient on TNF-α stimulated derived EEVs with a mean depletion percentage of 75 ± 8.7 % (Figure 5B). In the same manner, all the combinations using CD62E were more efficient on TNF-α stimulated derived EEVs than unstimulated cell- derived EEVs. Indeed, the mean depletion percentages were 81.6 ± 5.6 % for CD62E / EPhA2 beads, 82.4 ± 5.3 % with CD62E + EPhA2 beads that was as effective as CD146 and CD29 / 59 beads which had a depletion rate of 78.2 ± 7.1 % and 84.5 ± 5.7 % respectively. As observed in unstimulated cell-derived EEVs, the use of the three beads CD62E, EPhA2 and CDH13 simultaneously was less efficient with only 52.4 ± 8.6 % of depletion. When used sequentially, these beads allowed a capture of 86.3 ± 4.2 % of TNF- α EEVs which is not significantly different from CD146 beads. Taken together, these results indicate that the combined strategy allows a capture efficiency of TNF-α EEVs equivalent to the gold standard CD146 in PBS BA conditions. As demonstrated, the use of two different beads (CD62E + EPhA2) had the same efficiency as the combination of the two antibodies on the same bead (CD62E / EPhA2). Finally, the inventors could observe that CDH13 did not provide additional efficacy in the capture of TNF-α EEVs and they decided to continue the study only on CD62E / EPhA2. As plasmatic environment is richer and more viscous than simple buffer (PBS BA), they decided to evaluate the impact of plasma on the efficiency of IMS using four beads: Isotypic control beads (Mouse IgG1 / IgG2b), CD62E / EPhA2, CD146 and CD29 / 59 beads. Briefly, EEVs of the three territories were spiked in EVLP at 2000, 1000 and 200 / µL and then incubated with IMS beads. The supernatant post-IMS were collected and analyzed by flow cytometry using CD59-PE labelling. The percentages of depletion were calculated using a control without beads (WO beads). As illustrated in figure 5C and D, the isotypic control beads showed a higher unspecific depletion compared with non-plasmatic condition with 17 ± 8.9 and 16.6 ± 9.3 % for unstimulated and TNF-α stimulated EEV respectively. Concerning specific depletion on unstimulated EEVs, CD62E / EphA2 beads were as effective as CD146 beads with 64.4 ± 12.7 and 65.2 ± 12.4 % of capture, respectively. In that case, the depletion obtained with CD29 / 59 Pan-MV beads was significantly higher with 73.4 ± 8.0%. By contrast, on TNF-α stimulated EEVs, the mean depletion percentage of CD62E / EPhA2 beads was significantly higher than CD146 and CD29 / 59 beads, with 85.1 ± 9.9% against 66.2 ± 11.5 and 75.7 ± 10.2%, respectively. These results show that CD62E / EPhA2 beads are as effective as CD146 beads in the capture of unstimulated EEVs and more effective in the capture of TNF-α EEVs than CD146 beads in plasmatic conditions. Validation of the specificity of the CD62E / EPhA2 beads As the inventors showed that CD62E / EPhA2 beads are a good tool to capture EEVs, they wanted to check the capture specificity of these beads. To that aim, they produced vesicles from several blood cells under stimulation: Neutrophil-derived EVs (NdEV), Plateletderived EVs (PdEV), Monocyte-derived EVs (ModEV), Eythrocyte-derived EVs (ErydEV), T4, T8 and B lymphocytes-derived EVs (LT4dEV, LT8dEV, LBdEV) and Natural Killer-derived EVs (NKdEV). Briefly, EVs were spiked is PBS BA at 2000 EV / µL and incubated as below with IMS Beads. The supernatant post-IMS were collected and analyzed by flow cytometry using CD41-PE for PdEVs, CD15-PE for NdEVs, HLA-DR-PE for ModEVs and CD59- PE for others EVs. As shown in Figure 6, CD29 / 59 positive control beads capture all the plasmatic EVs tested with different rates depending on the EVs subtypes. As expected, CD62E / EPhA2 as well as CD146 beads do not capture the EVs subtypes tested. These results indicate that CD62E / EPhA2 beads are specific of EEVs and could be used in plasma without any specific capture of the other subset(s) of plasmatic EVs. TF activity assay using CD62E / EPhA2 beads in spiked model and in patients The main goal of this study was the development of a new tool to capture circulating EEVs in the plasma of patients and evaluate their procoagulant behavior by measuring TF activity. To that aim, the inventors performed TF activity assays on the bead- captured EV as illustrated in figure 7A and 7B. Briefly, EEVs were spiked in EVFP at 200, 1000 and 2000 EVs / µL and incubated with IgG Beads, CD29 / 59 beads, CD62E / EPhA2 beads or CD146 beads. The post-IMS beads were washed and TF-activity assays were performed on them. For EEVs, the results are expressed in percentage of TF activity normalized on TF activity present on the positive control, CD29 / 59 beads. As shown in figure 7C and 7D, TF activity generated from EEVs on CD62E / EPhA2 beads was higher than EEVs on CD146 beads for all the tested concentrations and higher than that for CD29 / 59 beads except for 200 EEVs / µL; that is consistent with their better ability to capture EEVs even if this increased activity was not significant. As expected, when other plasmatic EVs were spiked in plasma, no TF activity was detected using CD62E / EPhA2 and CD146 beads for IMS. By contrast, the CD29 / 59 beads (positive control) allowed the detection of TF activity provided by Monocyt-derived EVs. This result show that CD62E / EPhA2 beads could be used in a sample of patient’s plasma to evaluate the specific procoagulant activity of circulating EEVs.

Claims

CLAIMS 1. In vitro method for detecting endothelial extracellular vesicles (EEVs) in a biological sample of a subject, which comprises the detection of CD62E and EPhA2 on said EEVs in said biological sample, wherein said detection is indicative of the presence of EEVs.

2. Method according to claim 1, wherein the detection of CD62E is performed using at least one anti-CD62E antibody or one of its fragments, and / or the detection of EPhA2 is performed using at least one anti-EPhA2 antibody or one of its fragments.

3. Method according to claim 1 or 2, wherein the detection of CD62E and EPhA2 on said EEVs is performed with a technique chosen from immunodiagnostic techniques and cell analysis techniques; preferably chosen from flow cytometry (associated or not to size exclusion chromatography), Western Blots, agglutination tests, enzyme-labeled and mediated immunoassays, such as ELISAs, biotin / avidin type assays, radioimmunoassays, immunoelectrophoresis, immunoprecipitation, solid-phase affinity chromatography, super resolution microscopy, fluorescence microscopy, electron microscopy with nanoparticle staining and interferometry; and more preferably is flow cytometry.

4. Method according to any one of the preceding claims, wherein the detection of CD62E and EPhA2 on said EEVs comprises determining a concentration or an amount of extracellular vesicles (EVs) staining positive for CD62E and EPhA2 present in the sample.

5. Method according to any one of the preceding claims, which further comprises the detection of CDH-13 on said EEVs in said biological sample, wherein the detection of CD62E, EPhA2 and CDH-13 is indicative of the presence of EEVs.

6. In vitro method for capturing endothelial extracellular vesicles (EEVs) in a biological sample of a subject, which comprises: - capturing EVs expressing at least CD62E and EVs expressing at least EPhA2 in said biological sample, - and optionally collecting said EVs.

7. Method according to claim 6, wherein the capture is performed with a capturing technique using a substrate onto which at least one anti-CD62E antibody or one of its fragments and / or at least one anti-EPhA2 antibody or one of its fragments is(are) grafted, preferably by immunomagnetic separation.

8. Method according to any one of claims 6 or 7, which further comprises capturing EVs expressing at least CDH-13 in said biological sample, and EVs expressing CD62E, EPhA2 or CDH-13 are EEVs.

9. Method according to any one of claims 6 to 8, which comprises determining a concentration, an amount or an activity level or molecular expression of the EEVs.

10. Method according to any one of the preceding claims, wherein the subject is a patient with an immuno-inflammatory disease or a cardiovascular disease, or is at risk of thrombosis, or is at risk of haemorrhagic or ischemic disease, preferably the subject is a patient with an immuno-inflammatory disease such as an autoimmune or infectious disease and at risk of thrombosis or haemorrhagic or ischemic disease or the subject is a patient with cancer at risk of thrombosis.

11. Method according to any one of the preceding claims, wherein the biological sample is a blood sample, a urine sample, a pleural fluid sample, a lymph sample or a saliva sample, preferably a blood sample.

12. Method for studying a cell sample or a tissue sample or an organ sample, which comprises a method according to any one of the preceding claims.

13. Method for diagnosing a patient with a disease involving an endothelial alteration, for example a myocardial infarction or a stroke or a cerebrovascular accident or a heart failure or a ventricular fibrillation, or method for predicting a complication on a patient with a disease involving an endothelial alteration, for example an inflammatory disease or a cardiovascular disease or a thrombosis, said method comprising: i. performing an in vitro EEV detection method according to claim 4 or an in vitro EEV capture method according to claim 9 in a biological sample of the patient; ii. comparing the concentration, amount or activity level or molecular expression of the EEVs with its respective predetermined reference value; and iii. determining that when said concentration, amount or activity level or molecular expression of the EEVs is higher than its respective predetermined reference value, then the patient is very likely to be afflicted with a disease involving an endothelial alteration, for example a myocardial infarction or a stroke or a cerebrovascular accident or a heart failure or a ventricular fibrillation, or the patient is very likely to have a complication due to the disease involving an endothelial alteration.

14. Method for predicting the efficacy of a treatment on a patient with a disease involving an endothelial alteration, for example an inflammatory disease or a cardiovascular disease or a thrombosis, said method comprising: i. performing an in vitro EEV detection method according to claim 4 or an in vitro EEV capture method according to claim 9 in a biological sample of the patient; ii. comparing the concentration, amount or activity level of the EEVs with its respective predetermined reference value; and iii. determining that when said concentration, amount or activity level of the EEVs is higher than its respective predetermined reference value, then the patient is expected to have a favorable response to a treatment against the disease involving an endothelial alteration, for example an anti-inflammatory treatment or an anti-thrombotic treatment.

15. Method according to any one of claims 13 to 14, wherein the in vitro EEV capture method further comprises capturing EV expressing at least CDH-13 in the biological sample, and EVs expressing CD62E, EPhA2 and CDH-13 are EEVs.

16. In vitro method for monitoring the efficacy of a treatment on a patient with a disease involving an endothelial alteration, for example an inflammatory disease or a cardiovascular disease or a thrombosis, comprising: (i) performing an in vitro EEV detection method according to any one of claims 1 to 5 or an in vitro EEV capture method according to any one of claims 6 to 9 from a first sample obtained from said patient at a first time point to obtain first collected EEVs from the patient, (ii) determining the concentration, the amount or the activity level or molecular expression of the first collected EEVs, (iii) performing an in vitro EEV detection method according to any one of claims 1 to 5 or an in vitro EEV capture method according to any one of claims 6 to 9 from a second sample obtained from said patient at a second time point to obtain second collected EEVs from the patient, (iv) determining the concentration, the amount or the activity level or molecular expression of the second collected EEVs, (v) comparing the concentration, the amount or the activity level or molecular expression of first collected EEVs and second collected EEVs, and (vi) concluding about the treatment efficacy, depending on the concentration, the amount or the activity level or molecular expression of the second collected EEVs as compared to the concentration, the amount or the activity level or molecular expression of the first collected EEVs.

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