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    • 专利摘要:
    Procedure for the ex-vivo study of the initial response of different cell types in atherosclerosis. The novelty of this model lies in the incubation of the cells of interest with the complete secretoma of the atheroma plaques to evaluate the cellular response. This incubation allows a more direct study of the cellular response to factors secreted by atheromatous plaques, instead of using isolated factors (vegf, sdf-1). This procedure would allow the evaluation of strategies to modulate the cellular response in response to atherosclerotic damage, for example in the case of endothelial progenitor cells (ecps), to promote vascular remodeling in therapies based on the use of these cells, although it can equally be applied to any cell type associated with atherosclerosis. It also allows to evaluate the effect of drugs in this initial framework of cellular response to atherosclerosis. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2615581A1
    申请号:ES201500877
    申请日:2015-12-07
    公开日:2017-06-07
    发明作者:María Carmen DURÁN RUIZ
    申请人:Universidad de Cadiz；
    IPC主号:
    专利说明:

     PROCEDURE FOR THE EX-VIVO STUDY OF THE INITIAL RESPONSE OF DIFFERENT CELL TYPES IN ATEROSCLEROSIS. SECTOR OF THE TECHNIQUE TO WHICH THE INVENTION IS REFERRED. The present invention consists of a new study model of the initial response of any cell type involved in atherosclerotic damage. Specifically, the model has been applied to evaluate the initial response of progenitor endothelial cells (EPCs) in atherosclerosis. The procedure presented allows the identification of early cellular protein markers in response to the pathology of atherosclerosis, as well as mechanisms of cellular action that can be modulated for use in cell therapy. The present invention is therefore of interest to companies in the pharmaceutical sector focused on the development of treatments against atherosclerosis, including drugs against protein targets as well as cell therapies for the same purpose. 15 STATE OF THE TECHNIQUE. Atherosclerosis is the leading cause of cardiovascular diseases and therefore one of the leading causes of death in developed countries. It is a pathological process that affects blood vessels in response to various cardiovascular risk factors, resulting in the formation of atheroma plaques that are deposited on the endothelium of blood vessels. The rupture of the plaque causes the obstruction of the vessels causing thrombotic or embolic phenomena that usually trigger cerebral or myocardial infarctions (Libby P, Naf Med 2002.8 (11): 1257-1262) 25 In recent years, cell therapy has It has presented as a promising alternative to traditional treatments to combat cardiovascular diseases and the atherosclerotic process itself. Currently, several strategies based on cell therapy are being tested for the treatment of both heart diseases and peripheral vascular diseases (Radrizzani M, J 30 Transl Med 2014, 12: 276). Among the cell types used, progenitor endothelial cells (EPCs) are considered potential candidates in therapeutic applications that pursue revascularization due to the regenerative properties assigned to these cells. EPCs seem to play a fundamental role in the pathology of atherosclerosis DESCRIPTIONand arterial recovery after injury (Hristov M, Curr Opin Lipido / 2008, 19 (5): 491-497), promoting vascular repair, angiogenesis and maintaining a resistant inflammatory surface (Yeh ET et al. Cireulation 2003, 108 ( 17): 2070-2073). In atherosclerosis, a decrease in the number of circulating EPCs is associated with the development of the disease (Chironi G et al. Atheroselerosis 2007, 191 (1): 115-120). Moreover, cells with CD34, KDR and C-kit markers (typical of endothelial cells) have been detected in areas of human atherosclerotic lesions, suggesting their role in the vascular remodeling process (Torsney E, J Mol Ce "Cardio / 2011, 50 (2): 304-311) To date, there are several trials to understand how EPCs behave in response to vascular damage.In vivo studies have shown that EPCs mobilize rapidly after vascular trauma and contribute to revascularization of damaged vessels in response to circulating endothelial vascular growth factor (VEGF) levels induced by pro-inflammatory cytokines (Gil M et al, Cire Res 2001, 88 (2): 167-174; Du F et al, Front Biosei (Landmark Ed) 2012, 17: 2327-2349) In vitro tests have focused more on the response of EPCs against cytokines, pro-inflammatory and pro-atherogenic molecules such as GM-CSF and SDF-1, that increase neovascularization in ischemic tissues (Du F et al, Front Biosei (Landmark Ed) 2012, 17: 2327-2349; Takahashi T et al, Nat Med 1999.5 (4): 434-438). The development of an experimental model to study the molecular and cellular mechanisms by which atheroma plaques interact directly with EPCs is of great interest, and could provide new information on the physiological or pathological relevance of these cells. It is currently unknown if the factors released directly by atheroma plaques have any effect on EPCs or any other circulating cell type. 25 The model presented is a new strategy to determine the initial response of EPCs to atherosclerotic damage, which could allow their modulation to enhance vascular remodeling in therapies based on the use of these cells. However, the procedure presented could also be applied to any cell type associated with atherosclerosis. 30DESCRIPTION OF THE INVENTION The model presented allows to simulate the in vivo situation in which atheroma plaques secrete factors to the circulating medium (blood) and these factors come into contact with the also circulating cells or near the area of injury, 5 triggering an initial response to atherosclerotic damage (figure 1). The novelty of the proposed procedure is mainly to put in direct contact, ex vivo, the secretoma (secreted fraction) of the atheroma plaques of atherosclerotic patients with different cell types involved in this pathology. The term "secretoma" of atheroma plaques refers to the set of 10 molecules, fundamentally and without limitation, protein, glycidic and / or lipid factors, released by the tissue containing atheroma plaques to the medium in which they are being grown. Specifically, the authors have applied the method to study the initial response of EPCs cultured ex vivo with atheroma plaque secrets (Figure 2). To date, it has not been described if the factors directly released by atheroma plaques have any effect on EPCs or any other circulating cell type. The development of this experimental model will allow obtaining new information on the physiological or pathological relevance of these cells in atherosclerosis. The procedure presented allows the identification of molecular markers that are expressed as an early response mechanism against plaque secrecy. The procedure presented allows to detect morphological and / or functional changes in the treated cells as an early response mechanism and activation against the secrecy of the plaques. This procedure therefore allows the evaluation of new strategies to modulate the 25 cells of interest as a previous step to their use in cell therapy applied to atherosclerosis. The novelty of the invention thus lies in incubating the cultured cells ex vivo, take for example the EPCs, with the secretoma of the atheroma plaques. For this, the authors have tested direct incubations, undiluted, or diluting 1: 1 with the culture medium, and different incubation times (1 hour, 24 hours) have also been tested to see the cellular response. To better evaluate the effect produced by the atheroma plaque secretoma on the cells in culture, the authors recommend that another set of cells of interest be incubated in parallel with a control secretoma, obtained from culturing tissue fractions that do not contain atheroma plaque . This secret control can be obtained, without limitation,culturing mammary or radial arteries extracted from individuals during surgical processes, or adjacent, undamaged arterial areas, to complicated areas with atheroma plaques. In order to obtain control secrets, the same procedure is obtained as for obtaining secretion of atheroma plaques, 5 incubating the control tissues 72 hours at 37 ° C in RPM free protein medium. In order to be able to evaluate the effect produced by the contact of the secretoma of the atheroma plaques with the cells of interest, different techniques known from the state of the art can be used, without limitation, including for example techniques for the identification of protein markers whose expression is altered in the initial response of the cells upon contact with the secrecy of the plaques (proteomic analysis, Western blot, ELlSA, cell marking by immunofluorescence, cytometry); or to detect morphological / functional changes in cells (proliferation assays, migration, microscopy analysis of morphological changes, etc.). 15 MODE OF CARRYING OUT THE INVENTION. Performing the procedure presented, consisting of the ex vivo incubation of cultured cells, take for example the EPCs, with the secretoma of atheroma plaques requires: 20 • Obtaining samples of tissue affected by atheroma plaques by endarterectomy ( carotid, femoral or coronary). To obtain the supernatant (the secretoma of atheroma plaques), complicated arteries with atheroma plaques are cultured for 72 hours at 37 ° C in a plastic culture plate that allows a minimum volume of 4 ml of RPMI medium without serum 25 (to ensure that the tissue is covered by said medium), following a previously established protocol (Alvarez-Llamas G et al, Mo / Cel! Proteomics 2007, 6 (4): 589-600 and Duran MC Proteomics 2003, 3 ( 6): 973-978). After 72 hours, said supernatant can be used to incubate directly with the cells or it can be stored aliquoted in microcentrifuge tubes and frozen at -80 ° C for later use. The tissue is discarded after 72 hours. • The type of cell that you want to study. It may be primary cultures or related cell lines. In the model presented, the authors have worked with EPCs isolated from healthy individuals from the peripheral blood mononuclear fraction (PBMCs). There are currently several widely known methods in theprior art, including but not limited to gradient centrifugation methods (for example the gradient with Ficoll, W02008077094) or the use of blood cell separators (Pierelli L. et al, Bone Marrow Transplant, 1991, 7 , 355-361) 5 To obtain EPCs cells, the isolated PBMCs cells are incubated in culture plates that can be of various sizes, consisting of a single surface or divided into several wells. In the presented method, 48-well individual plates have been used, to use a smaller amount of cells (106cells / 0.5 ml per well), a smaller quantity of supernatant (100-200 microliters / well) and to evaluate 10 several conditions in the same plate . Isolated PBMCs cells are incubated in culture plates preferably treated with a polymer that promotes cell adhesion to the culture plate. In the presented method, the plaques have been treated with fibronectin, proceeding according to previously described protocols (Pula, G. et al. Circulation research 104, 32-40, 2009; 15 23 Vasa, M. et al. Circulation research 89, E1-7, 2001), although there are several polymers such as collagen and not limited to, fibronectin, collagen or poly-lysine. Isolated PBMCs cells are incubated for 7 days at 37 ° C and 5% CO2 in a medium enriched in factors that favor the differentiation of PBMCs to EPCs. On day 4 the culture medium is replaced by the same but fresh medium, in order to discard non-adherent cells, and continue to grow until day 7 under the same conditions, 37 ° C and 5% CO2. In the market there are several means currently available for differential growth of EPCs. For example, in the procedure presented, the EBM-2V (Endothelial basal medium 2-vascular medium) supplemented with 5% fetal bovine serum (FBS) has been used and containing growth factors included in the EGM-2 Single Quots kit (Lonza). Once both elements have been obtained (cells of interest and secretoma), the first part of the procedure presented is carried out, consisting of incubating ex vivo cells in culture with the secretoma of atheroma plaques. For this, the medium in which the cells are incubated is removed. EPCs are adherent cells, so the medium is directly discarded. If they were cells in suspension they would have to be previously centrifuged to resuspend them later in the plate supernatant. The supernatant is added to the cells. The authors have tried adding the supernatant directly, undiluted, or making a 1: 1 dilution with the growth medium before adding it to the cells. It has been decided to dilute the secretoma 1: 1 with the cell growth medium to better simulate the situationin which the factors secreted by the plates are released to the circulating medium that contains the cells. The volume that is added to the cells must be proportional to the dimensions of the culture plate. In the case of a 48-well plate, the authors add 200 5 microliters of the 1: 1 mixture for short response tests (1 hour) or 400 microliters when the incubation time is 24 hours. The cells are then incubated in a CO2 incubator, under the optimal conditions of the cells (eg, the EPCs are incubated at 37 ° C at 5% CO2). The incubation time will depend on the type of response to be analyzed (early response, 1 hour, late response, more than 24 hours). The authors have tested responses at 1 hour and 24 hours, observing variations between both times in protein expression. As a control, to another group of the same cultured cells the medium is removed and fresh medium is added in the same amount as what was added to the treated cells (for example 200 or 400 microliters for incubations of 1 hour or 24 hours respectively ) 15 and incubate at 37 ° C and 5% CO2. Additionally, and in parallel, a third group of the cells of interest can be incubated with the secretoma of undamaged arteries with atheroma plaques (control secretoma), to detect the exclusive effect of the plaques. In this case, proceed as before. Both treated and control cells must be incubated at the same time and under the same conditions. 20 The proposed procedure may be applied to evaluate the molecular / cellular changes produced in response to the cells in contact with the supernatant of the arteries complicated by atheroma plaques. In this way, the analysis can focus, as examples on: • The identification / detection of protein markers whose expression is altered in the initial response of the cells to contact with the secrecy of the plaques. For this, various techniques known in the state of the art (proteomic analysis, Western blot, ELlSA, immunofluorescence cell marking, cytometry) may be used, without limitation. For protein identification by proteomic analysis, cells must be pre-lysed with lysis buffer. Lysis can be performed by removing the secretoma mixture: culture medium after the incubation time and adding directly on the lysis buffer wells (about 100 micro liters / well in a 48-well plate), after which the cells are collected in a same microcentrifuge tube (one tube per condition analyzed, that is, cells treated with the secrecy of the plates, 35 control cells). The option selected by the authors is to collect the cells first in a tube, after trypsinization to separate them from the surface of the plate, and after2-3 washes with phosphate buffered saline (PBS), the cells are centrifuged and the cell pellet is resuspended in 100 microliters of lysis buffer. Protein quantification is then performed using methods known in the state of the art, and without limitation, such as Lowry's method or using Bradford's reagent, and proteomic analysis and identification by mass spectrometry are carried out. Alternatively, cell lysate proteins can be analyzed by western blotting to quantify the presence / absence of proteins of interest in response to the incubation of the cells, for example, the EPCs, with the atheroma plate secretoma. • Power / functional tests to determine the effect of the secretoma of the 10 atheroma plaques on the cells of interest. For this, methods known in the state of the art can be carried out, among them, and without limitation, proliferation, migration, angiogenesis, permeability, etc. tests. Among the potency / functional tests, changes in cell mobility can be detected in response to direct contact with the atheroma plaque secretoma. For this, a modification of the procedure is proposed in that the cells are arranged within a Boyden type system (Boyden, Sv J Exp Med 1962. 115 (3): 453-66), consisting of two compartments separated by a porous membrane The cells, in medium containing 0.5-1% FBS, are added to the upper compartment previously treated with an adhesion polymer, among others and without limitation, 20 fibronectin or collagen (in the case of adherent cells such as EPCs) or directly on the upper chamber in the case of suspended cells. In the lower chamber the supernatant / secretoma of atheroma plates diluted 1: 1 in basal medium EBM-2V 5% FBS is added, in sufficient volume so that the lower chamber is covered with said medium (700-800 microliters for plates 24 wells). In parallel another group of cells is added to another boyden chamber but in the lower chamber basal growth medium is added and additionally, a third group of cells can be contacted with the control secretoma of arteries not damaged by atheroma plaques. The cameras are incubated, without limitation, for a minimum of 4 hours, 6 hours, 12 hours, 24 hours or 48 hours. 30 EXAMPLES Example 1. Evaluation of the effect of plaque secrecy in healthy EPCs on the expression of the ICAM-1 protein (intercellular adhesion molecule 1) involved in pro-inflammatory processesTo determine whether direct contact of atheroma plaque secrets affects the protein expression of EPCs, on day 7 of their extraction from the blood of healthy individuals they were incubated for 1 hour and 24 hours with the supernatant of the plaques, as previously described, at 37 ° C and 5% CO2. After this time they were lysed with 5 Laemmli buffer 1x (50 mM Tris pH 6.8, 10% v / v glycerol, 2% w / v SOS, 0.1% w / v bromophenol blue) and separated into 12% polyacrylamide gels, they were transferred to an Immobilon-FL membrane (IPFLoo010 Millipore, MA). The membranes were incubated with specific antibodies against ICAM-1 and a-tubulin as a loading control. As secondary antibodies, IROye 800 CW Goat anti Rabbit and IROye 800 CW 10 Goat anti Mouse (LI-COR) were used. The membranes were analyzed using an Odyssey system (LI-COR). Resu / tado example 1: 15 The ICAM-1 protein is an essential adhesion molecule in cell-cell interactions and also between extracellular cell-matrix, and is involved in the initial steps of the inflammatory response. The data obtained indicate that the ex vivo culture of the EPCs of healthy individuals with the complicated carotid secretoma with atheroma plaques causes an increase in the expression of the ICAM-1 protein, after 1 hour of incubation, and said increase is maintained. after 24 hours of incubation (figure 3). As a load control, a-tubulin protein was used. 25 Example 2. Effect of atheroma plaque secrecy on the migratory capacity of EPCs cells After 7 days culture of EPCs cells isolated from the PBMCs fraction of healthy individuals in fibronectin plates, at 37 ° C and 5% CO2, these were trypsinized to separate them from the plate, centrifuged and resuspended in EBM-2V medium containing 0.5% FBS and counted on a Neubauer plate with Tripan blue. Next, 105 cells / well were added to the top of a Transwell chamber (8 11m pore size, 153422 Corning), previously treated with fibronectin. Subsequently, in the lower chamber, 900 111 of EBM-2V medium containing 5% FBS (EPC cont), 900 111 of a 1: 1 mixture of EBM-2V medium with 5% FBS and secrets of the cells were added in different wells plates (EPC + AP), or 900 111 of a 1: 1 mixture of EBM-2V medium with 35 5% FBS and mammary artery secretoma not damaged by atheroma plates (EPC + MA). After 6 hours of incubation at 37 ° C and 5% CO2, the cells present in the lower side of the membrane of the upper chamber of the transwell were fixed with 4% paraformaldehyde, and the cells in the upper part were discarded. TheExperiments were performed in triplicate for condition and repeated 3 times with plaque secrets and with EPCs cells of different individuals. To quantify cell migration, the nuclei of the cells fixed with DAPI were stained and counted manually in a fluorescence microscope (10 random quadrants per insert). 5 Result example 2: Secrets of atheroma plaques promote a significant increase in the migration of EPCs from healthy individuals (EPC + AP) through the porous membranes of the Transwell system, compared to ECPs in basal conditions (EPC Cont , p <O.01) and also in comparison with the EPC cells incubated with the supernatant / secretoma of mammary arteries without atheroma plaque (EPC + MA, p <O.001), which produced an effect similar to the baseline situation , in which the EPCs were in contact with the basal medium EBM-2V 5% FBS (Figure 4). According to this, only the factors secreted by the arteries complicated with atheroma plaques, and not the mammary arteries, specifically stimulate the mobilization of 15 healthy EPCs cells, presumably by means of a chemo-attractive effect on these cells. DESCRIPTION OF THE FIGURES Figure 1.- Represents the in vivo situation in which atheroma plaques affecting 20 blood vessels secrete proteins and other molecules to the circulating medium (blood). These factors come into contact with circulating cells, activating them and promoting a response to atherosclerotic damage. Figure 2.- Represents the ex-vivo model that seeks to simulate this situation in vivo, 25 directly incubating the factors secreted by atheroma plaques (secrets) with isolated cells (in the case of EPCs) or with cell lines. This simulates contact and the initial response to atherosclerotic damage. Figure 3.-Effect of plaque secrecy on the expression of ICAM-1 30 protein in ECPs. The results obtained by western-blot indicate that in response to the ex vivo incubation of the EPCs of healthy individuals with the supernatant of atheroma plaques for 1 h and 24 h, an increase in the expression of the ICAM-1 protein occurs in treated cells compared to the EPCs without incubating with said supernatant. EPC With !: Control EPCs, untreated, of two healthy individualsdifferent (1, 2). EPC + AP: cells from healthy individuals incubated with the supernatant of atheroma plaques extracted from two different patients (AP1 and AP2) after 1 hour and 24 hours of incubation. 5 Figure 4.-The mobilization of EPCs in response to contact with the supernatant of atheroma plaques was evaluated using Transwell cameras. A) The figure indicates the disposition of the EPCs in the upper chamber and their incubation in the lower part with the basal medium EBM-2V (EPC Cont), the secretion of the mammary arteries (EPC + MA) or the secrecy of the plaques of atheroma (EPC + AP). After 6 hours, the cells that crossed the Transwell's membrane were counted by fluorescence microscopy. B) The data indicate an increase in the migration of EPCs across the membrane when brought into contact with the atheroma plate supernatant (EPC + AP) compared to EPC Cont cells (** p-value <0.01) or cells incubated with the mammary artery supernatant (p-value <O.001), who showed a migration similar to the control EPCs. 20 WAY THE INVENTION IS SUSCEPTIBLE OF INDUSTRIAL APPLICATION. This procedure is applicable to: a) Evaluate, in vitro and ex vivo, the response to the initial contact of the factors secreted by atheroma plaques of any cell type associated with the atherosclerotic process. b) To evaluate ex vivo the initial response of progenitor endothelial cells (EPCs) isolated from healthy individuals against the damage promoted by the secrecy of atheroma plaques extracted from atherosclerotic patients. C) Identify changes in protein expression that result from direct interaction between factors secreted by arteries complicated by atheroma plaques and cell types of interest. The changes in expression will be compared with baseline conditions, in which the cells are not in contact with said plaque secrecy, that is, they are in the basal medium or in contact with control secretoma, obtained from uncomplicated arteries with atheroma plaques. . d) To evaluate the effect of drugs directed towards proteins whose protein expression is affected in the cells in contact with the atheroma plaque secretoma, either to block their expression or promote expression5 10 protein when these favor the process of vascular regeneration in atherosclerosis. e) Evaluate the effect of plaque secrecy on the activity, functions and / or morphology of the cells of interest. This will allow to better understand the initial activation of said cells in response to atherosclerotic damage and at the same time, it will allow to find mechanisms to modulate said cells and use them in cell therapy against atherosclerosis. f) Incorporate this procedure into the stages of validation of a certain drug or cell therapy in atherosclerosis. 
    权利要求:
    Claims (16)
    [1]
    5 10 15 20 25 CLAIMS 1. A procedure, for the ex-vivo study of the initial response of different cell types involved in atherosclerosis, which consists of putting the complete secretome of the atheroma plaques in direct contact with the cell type whose response you want to evaluate.
    [2]
    2. A procedure for the ex-vivo study of the initial response of different cell types involved in atherosclerosis, according to claim 1, where the secretome of the atheroma plaques is obtained from carotid, femoral or coronary endarterectomies applied to atherosclerotic patients.
    [3]
    3. A procedure for the ex-vivo study of the initial response of different cell types involved in atherosclerosis, according to claim 1, where the cell type to be studied consists of primary cultures, obtained by extraction of donor blood, bone marrow cells , umbilical cord, or related cell lines.
    [4]
    4. A procedure for the ex-vivo study of the initial response of different cell types involved in atherosclerosis, according to claims 2 and 3, where the cells to be studied are incubated directly with the supernatant of the atheroma plaques.
    [5]
    5. A procedure for the ex-vivo study of the initial response of different cell types involved in atherosclerosis, according to claim 4, where the supernatant is added to the cell medium (1: 1), in a volume proportional to the type of plaque in the cells are grown, to later incubate the cells in a CO2 incubator, for a time proportional to the type of response to be analyzed. 30
    [6]
    6. A method for the ex vivo study of the initial response of different cell types involved in atherosclerosis, according to claim 5, where the response to contact with the secretome of the plaques is measured in comparison to5 10 15 20 a control situation in which the cells of interest are incubated with the basal growth medium.
    [7]
    7. A method for the ex vivo study of the initial response of different cell types involved in atherosclerosis, according to claim 5, where the response is measured in comparison to a control situation in which the cells of interest are incubated with the secretome of uncomplicated arteries with atheroma plaques, such as, without limitation, mammary or radial arteries obtained in surgical procedures, or adjacent, undamaged areas to arterial tissue complicated with atheroma plaque.
    [8]
    8. Use of the procedure, for the ex vivo study of the initial response of different cell types involved in atherosclerosis, according to claims 6 and 7, in the development of techniques for the identification / detection of protein markers whose expression levels vary in cells of interest after direct contact with the secretomes of arteries damaged by atheroma plaques compared to control conditions.
    [9]
    9. Use of the procedure, for the ex vivo study of the initial response of different cell types involved in atherosclerosis, according to claims 6 and 7, in the development of techniques to determine morphological changes in the cells of interest after direct contact with the secretomes of arteries damaged by atheroma plaques compared to control conditions. 25
    [10]
    10. Use of the procedure, for the ex vivo study of the initial response of different cell types involved in atherosclerosis, according to claims 6 and 7, in the development of techniques to determine functional changes such as, but not limited to, related to migration, permeability, proliferation, of the cells of interest after direct contact with the secretomes of arteries damaged by the atheroma plaques compared to control conditions.
    [11]
    11. Use of the method, according to claims 6 and 7, in any cell type associated with the atherosclerotic process.
    [12]
    12. Use of the method, according to claims 6 and 7, to evaluate the initial response of progenitor endothelial cells (EPCs) isolated from healthy individuals against the damage promoted by the secretome of atheroma plaques extracted from atherosclerotic patients.5 10 15 20
    [13]
    13. A procedure for the ex-vivo study of the initial response of different cell types involved in atherosclerosis, according to claim 12, characterized in that the incubation time to analyze the early response is between 1 and 24 hours, preferably one hour.
    [14]
    14. Use of the method, based on claim 8, to evaluate the activity of a certain drug on protein markers differentially expressed in the cells of interest after direct contact with the secret masses of arteries damaged by atheroma plaques in comparison with control conditions.
    [15]
    15. Use of the procedure, based on claims 9 and 10, to evaluate the activity of a certain drug on the morphological and / or functional changes that occur in the cells of interest after direct contact with the secrets of arteries damaged by atheroma plaques compared to control conditions.
    [16]
    16. Use of the method, according to claims 1 to 15, to demonstrate the validity of a certain drug or cell therapy in atherosclerosis.
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