Method for preventing and / or treating angiogenesis

Abstract

The present invention relates to an in vitro method for selecting mesenchymal stem cells (MSC) for use in treating and / or preventing pathological angiogenesis. The invention further relates to the mesenchymal stem cells for use in treating and / or preventing pathological angiogenesis, as well as diseases related to same.

Description

[0001] METHOD FOR THE PREVENTION AND / OR TREATMENT OF PATHOLOGICAL ANGIOGENESIS

[0002] FIELD OF INVENTION

[0003] The present invention relates to the medical field. In particular, the present invention relates to an in vitro method for selecting mesenchymal stem cells (MSCs) for use in the treatment and / or prevention of pathological angiogenesis; or to the mesenchymal stem cells themselves for use in the treatment and / or prevention of pathological angiogenesis, as well as related diseases.

[0004] STATE OF THE ART

[0005] Pathological angiogenesis is the process by which new blood vessels form abnormally or uncontrollably, which can contribute to various diseases. In the context of arteriovenous malformations (AVMs), pathological angiogenesis is particularly relevant.

[0006] Arterial malformations (AVMs) are developmental abnormalities of blood vessels involving direct connections between arteries and veins, bypassing normal capillaries. This disrupts blood flow and can lead to a range of problems. Pathological angiogenesis in this context contributes to the formation and growth of these malformations, as it generates abnormal vascular tissue that fails to perform its normal function of oxygen and nutrient exchange.

[0007] This process of abnormal vessel formation can be mediated by imbalances in angiogenic growth factors, such as vascular endothelial growth factor (VEGF), which stimulates the proliferation and migration of endothelial cells. Under pathological conditions, an excess of these factors or defective regulation can contribute to disorganized vessel growth and the formation of abnormal connections.

[0008] Arterial malformations (AVMs) can occur in different parts of the body, but when they are located in the brain, for example, they can increase the risk of bleeding, seizures, and other neurological symptoms. The exact pathogenesis of AVMs is not yet fully understood, but it is known that genetic and molecular factors play a crucial role in regulating angiogenesis, which leads to these malformations. In short, pathological angiogenesis is fundamental to the formation and development of AVMs, where blood vessels form abnormally, contributing to a defective vascular architecture that affects blood flow functionality and can have significant clinical consequences.

[0009] There is an unmet medical need for therapeutic strategies to treat pathological angiogenesis and related diseases such as arteriovenous malformations (AVMs). The present invention relates to a novel therapeutic strategy for treating these diseases.

[0010] DESCRIPTION OF THE INVENTION

[0011] Brief description of the invention

[0012] The inventors of the present invention demonstrate that treatment with MSC reduces the occurrence and severity of arteriovenous malformations (AVMs) in an animal model of Hereditary Hemorrhagic Telangiectasia (HHT). This disease, characterized by the appearance of AVMs that seem to result from alterations in inflammation and angiogenesis, is caused by mutations in various genes, the most important of which are ACVRL1 (ALK1) and ENG (endoglin).

[0013] Specifically, the inventors of the present invention demonstrate that endoglin plays a role in modulating the biological properties of MSCs and that, therefore, changes in the expression of this protein alter MSC function. Consequently, endoglin deficiency affects the functionality of these cells, and HHT patients with mutations in the endoglin gene may present with non-functional MSCs. This finding explains pathophysiological aspects of the disease and represents a significant step toward the development of a new therapy.

[0014] As shown by the results presented in the Examples of the present invention, endoglin deficiency alters the primary culture of MSCs, the immunomodulatory capacity of MSCs is modified by endoglin expression, endoglin expression modifies the ability of MSCs to modulate angiogenesis, and overexpression of endoglin produces a pro-angiogenic effect, while silencing endoglin promotes an anti-angiogenic effect.Furthermore, the results shown in the Examples of the present invention show that the creation of skin wounds, which causes an angiogenic / inflammatory stimulus, in mice with an absence of endoglin expression is a reproducible and medium model of AVM generation; treatment with human MSCs in the murine model of AVM reduces the frequency of occurrence and severity of vascular malformations; treatment with human MSCs results in a decrease in the muscular and fibrotic layer of the larger vessels that are generated in the murine model of AVM; and treatment with human MSCs causes changes in the immune environment of the area where the vascular malformations are developing, which is evidenced by a significant increase in total perivascular macrophages.

[0015] Therefore, the first aspect of the present invention relates to an in vitro method for selecting MSCs for use in the treatment and / or prevention of pathological angiogenesis, characterized in that the method comprises evaluating the expression level of the endoglin protein, or the ENG gene, in the mesenchymal stem cell.

[0016] The second aspect of the present invention relates to the MSCs themselves (MSCs of the invention), or to a pharmaceutical composition comprising them, characterized by the expression of endoglin, or of the ENG gene, for use in a method for the prevention and / or treatment of pathological angiogenesis. Or, alternatively, to a method for the prevention and / or treatment of pathological angiogenesis comprising administering to the patient a therapeutically effective dose of the MSC of the invention, or to a pharmaceutical composition comprising them.

[0017] In a preferred aspect, the method for the prevention and / or treatment of angiogenesis comprises administering MSCs with an increased expression level of endoglin, or of the ENG gene, compared to a pre-established threshold value.

[0018] In a preferred aspect, an increased expression level of the endoglin protein, or the ENG gene, in the mesenchymal stem cell, compared to a pre-established threshold value, is indicative that the mesenchymal cell can be used in the treatment of pathological angiogenesis.

[0019] In a preferred aspect, the MSCs of the invention can be used in the treatment and / or prevention of pathologies with excessive angiogenesis, preferably selected from hemangiomas, psoriasis, Kaposi's sarcoma, ocular neovascularization, retinopathy of prematurity, rheumatoid arthritis, endometriosis and atherosclerosis; or pathologies with deficient angiogenesis, preferably selected from myocardial ischemia, peripheral limb ischemia, cerebral ischemia, delayed wound healing and impaired ulcer healing; or pathologies with defective angiogenesis, preferably selected from AVMs and telangiectasias.

[0020] In a preferred aspect, the MSCs of the invention can be used specifically in the treatment and / or prevention of hereditary hemorrhagic telangiectasia.

[0021] In a preferred aspect, the expression level of endoglin, or the ENG gene, has been altered by genetic modification or pharmacological treatment of mesenchymal stem cells.

[0022] The third aspect of the present invention relates to the in vitro use of endoglin, or transcripts of the ENG gene, (from a kit comprising reagents for assessing the expression level of the endoglin protein, or of the ENG gene) to select MSCs for use in promoting or inhibiting angiogenesis or to identify mesenchymal stem cells that promote or inhibit angiogenesis.

[0023] The present invention further relates to an in vitro method for selecting mesenchymal stem cells for use in the treatment and / or prevention of pathological angiogenesis, characterized in that the method comprises evaluating the expression level of the endoglin protein, or the ENG gene, in the mesenchymal stem cell, characterized in that an increased expression level of the endoglin protein, or the ENG gene, in the mesenchymal stem cell, compared to a pre-established threshold value based on the expression of endoglin, or the ENG gene, measured in wild-type mesenchymal stem cells, is indicative that the mesenchymal cell can be used in the treatment of pathological angiogenesis.

[0024] In vitro selection of mesenchymal stem cells can be performed using any of the commonly employed techniques for quantifying gene or protein expression, including, but not limited to, qPCR, Western blot, or flow cytometry. Selection is based on comparing endoglin expression with a predefined threshold value, obtainable from reference populations (e.g., wild-type MSCs). Cells with an expression level above the threshold are considered selected for use in the prevention and / or treatment of pathological angiogenesis.The present invention also relates to a mesenchymal stem cell, or pharmaceutical composition comprising it, characterized by having an increased expression level of the endoglin protein, or the ENG gene, compared to a pre-established threshold value based on the expression of endoglin, or the ENG gene, measured in wild-type mesenchymal stem cells, for use in a method for the prevention and / or treatment of pathological angiogenesis.

[0025] In a preferred aspect, the method of the invention is directed to selecting mesenchymal stem cells for use in the treatment and / or prevention of pathologies with excessive angiogenesis, preferably selected from hemangiomas, psoriasis, Kaposi's sarcoma, ocular neovascularization, retinopathy of prematurity, rheumatoid arthritis, endometriosis and atherosclerosis; or pathologies with deficient angiogenesis, preferably selected from myocardial ischemia, peripheral limb ischemia, cerebral ischemia, delayed wound healing and impaired ulcer healing; or pathologies with defective angiogenesis, preferably selected from vascular or arteriovenous malformations (AVMs) and telangiectasias.

[0026] In a preferred aspect, the method of the invention is directed to selecting mesenchymal stem cells for use in the treatment and / or prevention of hereditary hemorrhagic telangiectasia.

[0027] In a preferred embodiment, the present invention relates to the stem cell of the invention for use in the treatment and / or prevention of pathologies with excessive angiogenesis, preferably selected from hemangiomas, psoriasis, Kaposi's sarcoma, ocular neovascularization, retinopathy of prematurity, rheumatoid arthritis, endometriosis, and atherosclerosis; or pathologies with deficient angiogenesis, preferably selected from myocardial ischemia, peripheral limb ischemia, cerebral ischemia, delayed wound healing, and impaired ulcer healing; or pathologies with defective angiogenesis, preferably selected from vascular or arteriovenous malformations (AVMs) and telangiectasias.

[0028] In a preferred aspect, the present invention relates to the stem cell of the invention to be used in the treatment and / or prevention of Hereditary Hemorrhagic Telangiectasia.

[0029] In a preferred embodiment, the expression level of Endoglin, or the ENG gene, has been increased by genetic modification or pharmacological treatment of mesenchymal stem cells. For the purposes of this invention, the following terms and abbreviations are defined:

[0030] • The term “comprising” includes, but is not limited to, what follows the word “comprising”. Thus, the use of the term “comprising” indicates that the listed elements are mandatory, but that other elements are optional and may or may not be present.

[0031] • The term “consisting” means that it includes, and is limited to, what follows the phrase “consisting of”. Thus, the expression “consisting of” indicates that the listed elements are mandatory, and that no other elements may be present.

[0032] • The term “pathological angiogenesis” refers to a pattern of vascularization associated with a pathological condition. Pathological angiogenesis, which can also be called inadequate angiogenesis, is responsible for several types of diseases:

[0033] (1) pathologies with an excess of angiogenesis / vascular remodeling, such as hemangiomas, psoriasis, Kaposi's sarcoma, ocular neovascularization, retinopathy of prematurity, rheumatoid arthritis, endometriosis and atherosclerosis;

[0034] (2) pathologies with impaired angiogenesis / vascular remodeling, including myocardial ischemia, peripheral limb ischemia, cerebral ischemia, delayed wound healing and impaired ulcer healing; and

[0035] (3) pathologies with defective angiogenesis / vascular remodeling, in which the number of vessels formed is normal, but their structure and cellular composition are abnormal, such as arteriovenous malformations and telangiectasias [Núñez-Gómez, Elena et al. “The role of endoglin in post-ischemic revascularization.” Angiogenesis vol. 20,1 (2017): 1-24. doi:10.1007 / sl0456-016-9535-4],

[0036] • 4-OHT: 4-Hydroxytamoxifen.

[0037] • DNA: Deoxyribonucleic acid.

[0038] • cDNA: Complementary deoxyribonucleic acid.

[0039] • ALK1 : Activin receptor-like kinase type I (Activin receptor-like kinase / ).

[0040] • RNA: Ribonucleic acid.

[0041] • mRNA: Messenger ribonucleic acid.

[0042] • ATCC: American Type Culture Collection.

[0043] • BMP9: Bone Morphogenetic Protein 9 (Bone Morphogenetic Protein 9).

[0044] • BrdU: 5-bromo-2-deoxyuridine.

[0045] • BSA: Bovine Serum Albumin.

[0046] • CD: Cluster of Differentiation. • CD: Dendritic cells.

[0047] • CVM: Cauliflower Mosaic Virus.

[0048] • DMEM: Dulbecco's Modified Eagle Medium culture medium.

[0049] • DMSO: Dimethyl Sulfoxide.

[0050] • dNTP: Deoxyribonucleotide triphosphates.

[0051] • ECFC: Endothelial Colony-Forming Cells.

[0052] • EDTA: Ethylenediaminetetraacetic acid disodium salt dihydrate.

[0053] • SEM: Standard error of the mean.

[0054] • EGM-2: Endothelial Growth Medium.

[0055] • ELISA: Enzyme-Linked Immunosorbent Assay.

[0056] • ENG: Human endoglin.

[0057] • Eng: Mouse endoglin.

[0058] • EtOH: Ethanol.

[0059] • FBS: Fetal Bovine Serum.

[0060] • FGF: Fibroblast Growth Factor.

[0061] • FITC: Fluorescein Isothiocyanate (Fluorescein Isothiocyanate).

[0062] • GDF2: Growth / Differentiation Factor 2.

[0063] • GI: Gastrointestinal.

[0064] • HHT: Hereditary Haemorrhagic Telangiectasia.

[0065] • HTJP: Combined HHT and juvenile polyposis syndrome (Juvenile polyposis / Hereditary haemorrhagic telangiectasia syndrome).

[0066] • HGF: Hepatocyte Growth Factor.

[0067] • hMSC: Human Mesenchymal Stem Cells.

[0068] • HUVEC: Human Umbilical Vein Endothelial Cells.

[0069] • IFN-y: Interferon y.

[0070] • KO: Mouse in which the expression of both copies of the gene has been eliminated (Knockout mouse). • LPS: Lipopolysaccharide.

[0071] • AVM: Arteriovenous Malformations.

[0072] • ECM: Extracellular matrix.

[0073] • MHC: Major histocompatibility complex.

[0074] • MMP: Matrix Metalloproteinase.

[0075] • MSC: Mesenchymal Stem Cells.

[0076] • NK: Natural Killers.

[0077] • PBS: Phosphate Buffered Saline.

[0078] • PCR: Polymerase Chain Reaction.

[0079] • PDGF: Platelet-derived growth factor.

[0080] • PFA: Paraformaldehyde.

[0081] • PVDF: Polyvinylidene Fluoride (PolyVinylDene Fluoride).

[0082] • qPCR: Quantitative PCR.

[0083] • RPS 13: Ribosomal Protein S 13 (Ribosomal Protein SI 3).

[0084] • SDS-PAGE: Sodium Dodecyl Sulphate-Poly Acrylamide Gel Electrophoresis.

[0085] • SEA: Animal Experimentation Service.

[0086] • sEng: Soluble endoglin.

[0087] • siENG: human endoglin siRNA.

[0088] • siRNA: Small interfering RNA.

[0089] • Smad : Small protein Mothers Against Decapentaplegic.

[0090] • TGF-p Transforming Growth Factor beta (TGF-β).

[0091] • TNF-α: Tumor necrosis factor α (Tumor Necrosis Factor d).

[0092] • Treg: Regulatory T cells or regulatory T lymphocytes.

[0093] • VEGF: Vascular Endothelial Growth Factor.

[0094] • WT: Wild Type.

[0095] • ZP: Zona Pellucida Domain.

[0096] Description of the figures

[0097] Figure 1. Murine models used in the study. Transgenic mouse lines used in this work to obtain mesenchymal stem cells (MSCs), labeled with the color code employed. WT as controls, ENG mice overexpressing human endoglin, iKOEng endoglin conditional knockout mice, and Eng 1 like an endoglin knockout.

[0098] Figure 2. Two approaches to obtaining MSC Eng' / _ . (A) Generate endoglin knockout mice (Eng' / _ ) from iKOEng mice by intraperitoneal injection of tamoxifen for five consecutive days and subsequently, isolate the MSC Eng' / _ (B) Isolate iKOEng MSCs from endoglin conditional knockout (iKOEng) mice and subsequently treat with 4-OHT in vitro for four consecutive days to inactivate the endoglin gene and obtain Eng MSCs. / _ .

[0099] Figure 3. Gene and protein expression of endoglin after tamoxifen administration. (A) Schematic representation of tamoxifen administration in the murine model. (B) Gene expression of endoglin in iKOEng Cre mice + and iKOEng Cre' mice analyzed by conventional PCR. (C) Expression of the endoglin protein in different tissues, specifically liver (H), kidney (R), lung (P) and heart (C) of iKOEng Cre mice + and iKOEng Cre' mice, analyzed by Western blot, using the Stain Free technique as a load control. Adapted from the thesis of Laura Silva Sousa.

[0100] Figure 4. Relative frequency of success / failure in obtaining primary MSC cultures. Graphical representation of the relative frequency of success / failure in obtaining mesenchymal stem cells (MSCs) from adipose tissue of transgenic murine models with different levels of endoglin expression. Fisher's contingency test: *p<0.05.

[0101] Figure 5. Gene and protein expression of endoglin in iKOEng MSCs after treatment with 4-OHT. (A) Photomicrographs of iKOEng MSCs after treatment with 4-OHT. The white lines represent the scale corresponding to 100 pM. (B) Murine endoglin gene expression of MSCs analyzed by conventional PCR. (C) Analysis of endoglin mRNA expression by qPCR. (D) Analysis of endoglin protein levels by Western blot; iKOEng mouse MSCs treated with ethanol are used as a control; Stain-free technology is used as a loading control.

[0102] Figure 6. Human endoglin expression in MSC ENG + (A) Analysis of human endoglin expression in MSC WT and MSC ENG + (B) Analysis of human endoglin protein levels in MSC WT and MSC ENG using the qPCR technique. + , analyzed by Western blot; Stain-free technology was used as a load control. Figure 7. Mesenchymal stem cells (MSCs) with different levels of endoglin in culture. Photomicrographs of MSCs isolated from mice with different levels of endoglin expression growing in DMEM 10% FBS + P / S medium. MSCs obtained from WT mice (MSC WT), MSCs obtained from mice with endoglin overexpression (MSC ENG). + ), MSCs obtained from tamoxifen-induced endoglin conditional knockout mice (MSC iKOEng) and MSCs obtained from 4-OHT-treated iKOEng mice (MSC Eng' / _ The white lines represent the scale corresponding to 100 pm.

[0103] Figure 8. Trilineage differentiation of MSCs with different levels of endoglin. Photomicrographs of adipogenesis, osteogenesis, and chondrogenesis produced in WT and ENG MSCs + and MSC Eng' / _ Adipogenic differentiation is visualized by Oil Red staining, osteogenic differentiation by NBT / BCIP, and chondrogenic differentiation by Alzian Blue staining of sulfated proteoglycans. The black lines represent the scale corresponding to 100 pM.

[0104] Figure 9. Characterization of the immunophenotype of WT MSCs. (A) MSC-specific immunophenotype panel used. (B) Expression levels of the markers CD29, (C) Seal, (D) CD105, (E) CD31 and (F) CD45 in WT MSCs measured by flow cytometry.

[0105] Figure 10. BrdU incorporation by cultured T lymphocytes. Quantification of T lymphocyte proliferation by BrdU incorporation in co-culture with MSC WT, MSC ENG + and MSC Eng' / _ Each bar represents the average of four independent experiments. ANOVA statistical test: *p<0.05, ****p<0.0001.

[0106] Figure 11. Transendothelial migration of T lymphocytes attracted by MSCs. (A) Transendothelial migration of T lymphocytes over time attracted by MSCs WT, ENG + and Eng' / _ (B) T lymphocyte migration to the lower compartment at 24 hours. (C) T lymphocyte migration to the lower compartment at 120 hours. #: There is interaction in the two-way ANOVA statistical test. Each bar represents the average of three independent experiments. ANOVA statistical test: *p<0.05, ***p<0.001, ****p<0.0001.

[0107] Figure 12. BrdU incorporation by regulatory T lymphocytes in culture. Quantification of Treg proliferation by BrdU incorporation in co-culture with WT MSCs and ENG MSCs + and MSC Eng' / _ Each bar represents the average of three independent experiments. ANOVA statistical test: **p<0.01, ****p<0.0001. Figure 13. Macrophage polarization analysis by qPCR. (A) Schematic representation of the experimental design carried out in the macrophage polarization assay. (B) Analysis of IL-1RA expression levels in macrophages by qPCR. (C) Analysis of CD206 expression levels in macrophages by qPCR. Without MSC (n=6) and with MSC (n=6). Student's t-test statistical test.

[0108] Figure 14. Analysis of marker expression in MI and M2 phenotype macrophages. (A) Analysis of IL-1RA expression levels by qPCR in macrophages co-cultured with the three MSC types. (B) Analysis of CD206 expression levels by qPCR in macrophages co-cultured with the three MSC types. MSC WT (n=6), MSC ENG + (n=6) and MSC Eng® (n=6). Statistical test of ANOVA.

[0109] Figure 15. Skeleton of the structure generated by the “Skeleton” tool 44 from Fiji. (A) The analyzed and quantified parameters are indicated in the image by black arrows. (B) Representation of the longest shortest paths in pink.

[0110] Figure 16. Comparative analysis of pseudocapillaries formed in Matrigel® by HUVECs and MSCs with different levels of endoglin expression. (A) Representative photographs of the pseudocapillaries created. Scale bars: 200 pM. (BF) Quantification of the structures created by HUVECs and MSCs WT, ENG + or Eng' / _ (B) number of independent structures, (C) number of branches, (D) number of joining points, (E) average branch length, and (F) longest shortest path. With MSC WT (n=42), MSC ENG + (n=42) and MSC Eng' (n=43); each bar represents the mean of three experiments ± SEM; ANOVA statistical test: **p<0.01, ***p<0.001, ****p<0.0001.

[0111] Figure 17. Sprouting of aortic rings in Matrigel® with conditioned medium of the MSC WT, ENG + and Eng / _3 days after seeding. (A) Quantification of the number of tip cells, (B) sprouts (C) and sprouting rate (tip cells +1, sprouts +2) 3 days after seeding of the aortic rings. Each bar represents the average of four independent experiments. ANOVA statistical test: *p<0.05, **p<0.01.

[0112] Figure 18. Sprouting of aortic rings in Matrigel® with conditioned medium of the MSC WT, ENG + and Eng / _ 6 days after sowing. (A) Representative photomicrographs of aortic rings in Matrigel® with conditioned medium of MSCs with different levels of endoglin expression. The white lines represent the scale corresponding to 200 pM. (B) Quantification of the area occupied by the sprouts and (C) the maximum distance in the presence of conditioned medium of the different MSCs. Each bar represents the average of four independent experiments. ANOVA statistical test: *p<0.05, ***p<0.001.

[0113] Figure 19. Sprouting of aortic rings with MSC WT, ENG + and Eng / _ embedded in Matrigel® 3 days after seeding. (A) Quantification of the number of tip cells, (B) sprouts (C) and sprouting rate (tip cells +1, sprouts +2) 3 days after seeding the aortic rings. Each bar represents the average of four independent experiments. ANOVA statistical test: *p<0.05, **p<0.01, ***p<0.001.

[0114] Figure 20. Sprouting of aortic rings with MSC WT, ENG + and Eng / _Embedded in Matrigel® 6 days after sowing. (A) Representative photomicrographs of aortic rings in Matrigel® with MSCs exhibiting different levels of endoglin expression. White lines represent the scale corresponding to 500 pM. (B) Quantification of the area occupied by the sprouts and (C) the maximum distance in the presence of the different MSCs. Each bar represents the average of four independent experiments. ANOVA statistical test: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0115] Figure 21. Sprouting of aortic rings in Matrigel® with the MSC WT, ENG + and Eng / _ 3 days after seeding. (A) Quantification of the number of tip cells, (B) sprouts, (C) and sprouting rate (tip cells +1, sprouts +2) 3 days after seeding of the aortic rings. Each bar represents the average of four independent experiments. ANOVA statistical test: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0116] Figure 22. Sprouting of aortic rings in Matrigel® with the MSC WT, ENG + and Eng / _ 6 days after sowing. (A) Representative photomicrographs of aortic rings in Matrigel® with MSCs exhibiting different levels of endoglin expression. White lines represent the scale corresponding to 200 pM. (B) Quantification of the area occupied by the sprouts and (C) the maximum distance in the presence of the different MSCs. Each bar represents the average of four independent experiments. ANOVA statistical test: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0117] Figure 23. Distance migrated by HUVEC in culture with conditioned medium of MSC WT, ENG + and Eng / _in the Wound Healing assay. (A) Representative photomicrographs of the migration assay at 0 and 6 hours for each condition. The white lines represent the scale corresponding to 200 pM. (B) Quantification of the distance migrated by HUVECs in the presence of conditioned medium of MSCs with different endoglin expression. Each bar represents the average of three experiments. Kruskal-Wallis statistical test: *p<0.05, ***p<0.001, ****p<0.0001.

[0118] Figure 24. Distance migrated by HUVEC in co-culture with MSC WT, ENG +and Fng in the Wound Healing assay. (A) Micrographs representative of the migration assay at 0 and 6 hours for each condition. The white lines represent the scale corresponding to 200 pM. (B) Quantification of the distance migrated by HUVECs in the presence of MSCs with different endoglin expression. Each bar represents the average of three experiments. Kruskal-Wallis statistical test: **p<0.01, ****p<0.0001.

[0119] Figure 25. BrdU incorporation by HUVECs in culture. (A) Quantification of endothelial cell proliferation by BrdU incorporation with conditioned medium from MSCs with different endoglin expression. (B) Quantification of endothelial cell proliferation by BrdU incorporation in co-culture with WT and ENG MSCs + and Eng' / _ Each bar represents the average of three experiments ± SEM. ANOVA statistical test: **p<0.01, ***p<0.001, ****p<0.0001.

[0120] Figure 26. Human endoglin gene silencing in hMSCs. (A) Percentage of human endoglin gene silencing in hMSC-TA and hMSC-MO analyzed at different time points by flow cytometry. (B) Human endoglin protein levels in hMSC-AT, measured by flow cytometry 39 h after siRNA transfection. (C) Human endoglin protein levels in hMSC-MO, measured by flow cytometry 72 h after siRNA transfection.

[0121] Figure 27. Distance migrated by ECFCs in co-culture with hMSC-TA control and siENG in the Wound Healing assay. (A) Micrographs representative of the migration assay at 0 and 6 hours for each condition. (B) Quantification of the distance migrated by ECFCs in the presence of hMSCs with different endoglin expression. With hMSC-TA Ctrl (n=190), with hMSC-TA siENG (n=195). Student's t-test: ****p<0.0001.

[0122] Figure 28. Distance migrated by ECFCs in co-culture with hMSC-MO control and siENG in the Wound Healing assay. (A) Micrographs representative of the migration assay at 0 and 6 hours for each condition. (B) Quantification of the distance migrated by ECFCs in the presence of hMSCs with different endoglin expression. With hMSC-MO control (n=110), with hMSC-MO siENG (n=110). Student's t-test: **p<0.01.

[0123] Figure 29. Sprouting in the day 1 cytodex assay with hMSC-TA as feeders. (A) Representative photomicrographs of the day 1 cytodex assay under the two different conditions. (B) Quantification of the number of tip cells, (C) number of sprouts, (D) and sprouting rate (tip cells +1, sprouts +2). Each bar represents the mean of three experiments ± SEM. Mann-Whitney U test: ****p<0.0001. Black arrows indicate examples of tip cells and sprouts that were manually quantified.

[0124] Figure 30. Sprouting in the day 1 cytodex assay with hMSC-MO as feeders. (A) Representative photomicrographs of the day 1 cytodex assay under the two different conditions. (B) Quantification of the number of tip cells, (C) number of sprouts, (D) and sprouting rate (tip cells +1, sprouts +2). Each bar represents the mean of three experiments ± SEM. Mann-Whitney U test: ***p<0.001, ****p<0.0001. Black arrows indicate examples of tip cells and sprouts that were quantified manually.

[0125] Figure 31. Cytodex assay on day 3 with hMSC-TA as feeders. (A) Immunofluorescence micrographs of the Cytodex assay on day 3, in which actin filaments (phalloidin) are labeled green and cell nuclei (TO-PRO) are labeled red. (B) Quantification of the number of sprouts. Each bar represents the mean of three experiments ± SEM. Mann-Whitney U test: ****p<0.0001.

[0126] Figure 32. Cytodex assay on day 3 with hMSC-MO as feeders. (A) Photomicrographs of the immunofluorescence of the Cytodex assay on day 3, in which actin filaments are labeled green (phalloidin) and cell nuclei red (TO-PRO). (B) Quantification of the number of sprouts. Each bar represents the mean of three experiments ± SEM. Mann-Whitney U statistical test: ****p<0.0001.

[0127] Figure 33. Analysis of AVM occurrence. (A) Representative photomicrographs of vessels perfused with latex, showing the absence or presence of AVMs. The white lines represent the scale corresponding to 5 mm. (B) Percentage of AVM occurrence in untreated mice (without MSC), mice treated with MSC WT, and mice treated with MSC Eng' / _ . Wounds of mice without MSC (n=32), wounds of mice treated with MSC WT (n=34) and wounds of mice treated with MSC Eng' / _(n=32). Fisher's contingency test: **p<0.01. Figure 34. Analysis of AVM severity grades. (A) Micrographs representative of AVM grades in vessels perfused with latex. White lines represent the scale corresponding to 5 mm. (B) Quantification of the percentage of different AVM grades in untreated mice (without MSC), mice treated with MSC WT, and mice treated with MSC Eng' / _ . Wounds of mice without MSC (n=32), wounds of mice treated with MSC WT (n=34) and wounds of mice treated with MSC Eng' / _ (n=32). Fisher's contingency test: *p<0.05.

[0128] Figure 35. Gene and protein expression of endoglin after tamoxifen administration. A) Schematic representation of the murine model after tamoxifen administration; B) Gene expression of endoglin in the tails of iKO-Eng Cre mice +and iKO-Eng Cre' analyzed by conventional PCR; C) Levels of the endoglin protein in different tissues, specifically liver (H), kidney (R), lung (P) and heart (C) of iKO-Eng Cre mice + and iKO-Eng Cre', analyzed by Western Blot, using the stain-free technique as a load control.

[0129] Figure 36. Photomicrographs of vessels perfused with latex from a control mouse (A) and an Eng'' mouse (B). A) Representative photomicrograph of the vasculature surrounding the wound in a control mouse; B) Representative photomicrograph of the vasculature surrounding the wound in an Eng' mouse / _ .

[0130] Figure 37. Quantification of the vascular area occupied by latex and vessel caliber in control and Eng'' mice. A) Percentage of AVM occurrence; ****p<0.0001; contingency test; B) Quantification of the number of blue pixels **p<0.01; C) Quantification of the diameter of vessels perfused with latex ***p<0.001. Each bar represents the mean ± SEM, Mann-Whitney U test (B and C). Wounds in control mice (n=39) and wounds in Eng' mice / _ (n=44).

[0131] Figure 38. Analysis of AVM grades and severity in control and Eng' mice / _ A) Representative photomicrographs of the AVM grades of vessels perfused with latex; B) Quantification of the percentage of the different AVM grades in control mice and Eng' mice / _; C) Quantification of the percentage of severity of AVMs in control and Eng' mice / -, 0.0001; Contingency test, wounds of control mice (n=39) and wounds of Eng' mice / _ (n=44).

[0132] Figure 39. Analysis of the degrees of MAV tortuosity in control and Eng' mice / _ .

[0133] A) Representative photomicrographs of the tortuosity degrees of vessels perfused with latex; B) Quantification of the percentage of the different degrees of tortuosity of the AVMs in control and Eng' mice / _ ; C) Quantification of the percentage of severity of AVMs according to tortuosity in control and Eng' mice / _ , 0.0001; Contingency test, wounds of control mice (n=39) and wounds of Eng' mice / _ (n=44).

[0134] Figure 40. Analysis of various complexity parameters in the different degrees of AVM.

[0135] A) Quantification of the perimeter / area ratio in control and Eng mice _ / Mann-Whitney U statistical test, wounds of control mice (n=39) and wounds of Eng' mice / _ (n=44);

[0136] B) Quantification of the number of branches in the different degrees of MAV ****p<0.0001; C) Quantification of the number of triple points in the different degrees of MAV ***p<0.001 and *p<0.05; D) Quantification of the average length of the branches in the different degrees of MAV **p<0.01 and *p<0.05; Kruskal Wallis statistical test, No MAV ( n= 8), MAV 1-2 ( n= 9), MAV 3-4 ( n= 7), (B, C and D).

[0137] Figure 41. Representative photomicrographs of Hematoxylin-Eosin staining in control mice (left) and Eng / _(right). The pound sign (#) indicates the thickened wall of the blood vessel, and the asterisk (*) shows the tortuous appearance of the vessel. The black line represents the scale and corresponds to 50 pm in the photos in the top row and 20 pm in the photos in the bottom row. Wounds in control mice (n=24) and wounds in Eng' mice / _ (n=38).

[0138] Figure 42. Representative photomicrographs of Masson's Trichrome staining (top row), a-SMA immunohistochemical staining (middle row) and Weigert-Van Gieson staining (bottom row) in control mice (left) and Eng / _ (right). The black line represents the scale and corresponds to 20 pm in all photos. Wounds of control mice (n=24) and wounds of Eng' mice / _ (n=38).

[0139] Figure 43. Representative photomicrographs of the immunohistochemical staining of hCD105 in Eng mice / _treated with hMSC. The black line represents the scale, wounds of the Eng' mice / _ treated with hMSC (n =26).

[0140] Figure 44. Quantification of the vascular area occupied by latex and vessel caliber in Img mice and Img mice treated with hMSC. A) Percentage of AVM occurrence; **p<0.01; contingency test; B) Quantification of the number of blue pixels; C) Quantification of the diameter of vessels perfused with latex, **p<0.01; Each bar represents the mean ± SEM, Mann-Whitney U test (B and C). Wounds of Eng' mice / _ (n=44) and wounds of the Eng' mice / _ treated with hMSC (n=29). Figure 45. Analysis of AVM severity in Eng mice and in Eng mice treated with hMSC. A) Quantification of the percentage of AVM severity in Eng mice' / _ and in the Eng' mice / _treated with hMSC, *p<0.05; B) Quantification of the percentage of severity of AVMs according to tortuosity in Eng' mice / _ and in the Eng' mice / _ treated with hMSC, ***p<0.001; Contingency test, wounds of Eng' mice / _ (n=44) and wounds of the Eng' mice / _ treated with hMSC (n=29).

[0141] Figure 46. Analysis of the effect of hMSC treatment on various AVM complexity parameters. A) Quantification of the perimeter / area ratio in Eng' mice / _ with MAV and in Eng' mice / _ treated with hMSC, wounds of Eng mice / _ (n=44) and wounds of the Eng' mice / _ treated with hMSC (n=33); B) Quantification of the number of branches in Eng' mice / _ with MAV and in Eng' mice / _ treated with hMSC, **p<0.01; C) Quantification of the number of triple points in Eng' mice / _ with MAV and in Eng' mice / _treated with hMSC, **p<0.01; D) Quantification of the mean length of the branches in Eng' mice / _ with MAV and in Eng' mice / _ treated with hMSC, ***p<0.001; wounds of Eng' mice / _ (n=47) and wounds of the Eng' mice / _ treated with hMSC (n=42), (B, C and D); Mann-Whitney U statistical test.

[0142] Figure 47. Representative photomicrographs of Hematoxylin-Eosin staining (top row) and Weigert-Van Gieson staining (bottom row) in Eng (left) and Eng mice treated with hMSC (right), the black line represents the scale.

[0143] Wounds of the Eng mice / _ (n=38) and wounds of the Eng' mice / _ treated with hMSC (n=26) Figure 48. Structural analysis of the wall of the largest vessels of Eng mice / _ and Eng / _treated with hMSC. A) Representative photomicrographs of Masson's Trichrome staining (top row) and a-SMA immunohistochemical staining (bottom row) in Eng' mice / _ (left) and Eng' / _ treated with hMSC (right); The black line represents the scale and corresponds to 20 pm in all photos; B) Quantification of the thickness of the fibrotic layer of the vessels by analysis of Masson's Trichrome staining, ****p< 0.0001, Mann-Whitney U statistical test, Eng' vessels / _ (n=136) and Eng' vessels / _ treated with hMSC (n=435); C) Quantification of the thickness of the muscular layer of the vessels by analysis of a-SMA staining, 0.0001, Mann-U statistical test

[0144] Whitney, Eng glasses / _ (n=259) and Eng' vessels / _ treated with hMSC (n=391).

[0145] Figure 49. Immunohistochemical analysis of the cytokines TNF-α and HGF involved in the inflammatory process of wound healing. A) Representative photomicrographs of the immunohistochemical staining of TNF-α (top row) and of the immunohistochemical staining of HGF (bottom row) in Eng mice / _ (left) and Eng' / _ treated with hMSC (right); The black line represents the scale and corresponds to 20 pm in all photos; B) Quantification of TNF-α expression in percentage of pixels, Mann-Whitney U statistical test, Eng' wound photos / _ (n=20) and photos of wounds Eng' / _ treated with hMSC (n=15); C) Quantification of HGF expression in percentage of pixels, Mann-Whitney U statistical test, Eng' wound photos / _ (n=27) and wounded photos Eng' / _ treaties (n=22)

[0146] Figure 50. Immunohistochemical analysis of the number of macrophages present in the wound environment. A) Representative photomicrographs of immunohistochemical staining with F4 / 80, marker of total perivascular macrophages (top row) and of immunohistochemical staining of CD206, marker of M2 perivascular macrophages (bottom row) in Eng mice / _ (left) and Eng' / _ treated with hMSC (right); The black line represents the scale and corresponds to 2 mm in all photos; B) Quantification of the total number of macrophages in the wound environment of the two groups of mice; C) Quantification of the number of perivascular macrophages in the wounds of the two groups of mice; D) Quantification of the number of perivascular macrophages with M2 profile in the wounds of the two groups of mice; Eng' wounds / _ (n=38) and wounds Eng' / _ treated with hMSC (n=26), Mann-Whitney U statistical test (B, C and D).

[0147] Detailed description of the invention

[0148] The present invention is illustrated by the following Examples, which are set forth without the intention of limiting its scope.

[0149] Example 1. ANALYSIS OF ENDOGLIN LEVELS AND THEIR IMPLICATIONS IN MSC FUNCTIONALITY

[0150] Example 1.1. Obtaining primary MSC cultures from transgenic mice with different endoglin expression

[0151] To study the role of endoglin in modulating the biological and therapeutic properties of mesenchymal stem cells (MSCs), it is necessary to use primary MSC cultures with different endoglin expression levels. For this purpose, the transgenic murine models used in this work were wild-type (WT) C57BL / 6J mice to isolate cells with basal endoglin expression, and mice that overexpress the human endoglin gene (ENG). +) and, to obtain MSCs without endoglin expression, endoglin conditional knockout mice (iKO / ú / ) and endoglin knockout mice (Eng~ / That is, mice that have already been treated with tamoxifen to induce endoglin silencing (Figure 1). Two different approaches were developed to obtain Eng~ ~ MSCs. The first was based on generating the Eng~ ~ mouse by intraperitoneal administration of tamoxifen and, after achieving endoglin gene inactivation, obtaining the primary culture of Eng~ ~ MSCs. Since endoglin (CD 105) is a selection marker for MSCs, we did not know if it would be possible to isolate Eng~ ~ MSCs from the Eng~ ~ mouse. For this reason, the second approach consisted of isolating cells directly from the KOEng mouse, and subsequently inducing the loss of the endoglin gene by in vitro treatment with 4-OHT (Figure 2).

[0152] In our laboratory, we had previously optimized the generation of the endoglin knockout mouse (Eng^') from the KOEng mouse (Figure 3). This involved intraperitoneal injection of tamoxifen for five consecutive days, which induced Cre recombinase activity. Endoglin gene inactivation was confirmed by conventional PCR using genomic DNA extracted from tissue fragments of the KOEng Cre mice. + 21 days after the start of treatment (Figure 3B). Litter-sibling mice that do not express the Cre recombinase (KOEng Cre') were used as controls. After complete gene inactivation, residual protein expression could still remain; therefore, the loss of endoglin protein expression was analyzed by Western blot. For this purpose, lysates from the liver, kidney, lung, and heart of mice (KOEng Cre) were used. +and mice (KOEng Cre'. Complete loss of protein expression was observed 35 days after the first tamoxifen injection (Figure 3). Therefore, we established this time both for performing primary cultures of Eng~ ~ mice and for performing in vivo assays with these mice.

[0153] Primary MSC cultures were obtained from adipose tissue of the aforementioned transgenic murine models. First, we analyzed the relative success / failure rates in obtaining these primary MSC cultures. Figure 4 shows significant differences between the Eng~ ~ mouse and the other murine models, as it was not possible to isolate MSCs from Eng^ endoglin knockout mice. Regarding the other groups, the highest success rate was observed in obtaining WT MSCs (75%), followed by ENG MSCs. +with approximately half of the primary cultures and, finally, the MSC KOEng with a 40% success rate. Although the statistical analysis of the frequencies did not determine that there were significant differences. As a consequence of the above, the obtaining of the MSC Eng~ ~ was developed from the second approach proposed, that is, from the in vitro treatment with 4-OHT of the isolated MSC iKOEng (Figure 2B).

[0154] To induce endoglin gene loss in iKOEng MSCs, Cre recombinase was activated by treatment with the 4-OHT metabolite for four consecutive days; the doses used were 2 pM and 2.5 pM. As a control, we used iKOEng MSCs treated with ethanol (EtOH), the solvent used to dilute the 4-OHT. Since the potential residual expression of the protein depends on its half-life, we performed several passages at confluence to ensure that the cells divided numerous times before analyzing endoglin expression and conducting the experiments.

[0155] As shown in Figure 5A, after in vitro treatment, no appreciable morphological differences were observed between the different iKOEng MSCs. To verify that the treated MSCs were indeed Eng^', genomic DNA extracted from iKOEng MSCs treated with 4-OHT and EtOH was analyzed by conventional PCR after two and three cell passages (P2TT and P3TT, respectively). In addition, mRNA expression levels were evaluated by qPCR and protein expression by Western blot. Figure 5B-D shows that the MSCs are indeed endoglin knockouts (Eng^') because they do not express endoglin. Therefore, we established the lower concentration, 2 pM, as the one used for the experiments.

[0156] Next, we confirm that the MSC ENG +We isolated human endoglin expresses MSCs using qPCR and, in addition, we confirmed the expression of the protein using the Western blot technique. We used WT MSCs isolated from C57BL / 6J mice as a control (Figure 6).

[0157] Before beginning the experiments, we checked whether the different MSCs used in this work had similar morphological characteristics. As shown in Figure 7, no significant morphological differences were observed with the naked eye between the different cell types used in the study.

[0158] The identification and characterization of mesenchymal stem cells is defined by three basic minimum criteria: adherence to a plastic surface under standard culture conditions, the capacity for trilineage differentiation in vitro, and the expression of a specific immunophenotype. For this reason, we wanted to characterize the different cell types based on these three criteria. As we can see in Figure 8, the three types of MSCs (WT, ENG) + and Eng~ ~ grow attached to a plastic surface under standard growing conditions.

[0159] To test the ability of MSCs to differentiate into adipocytes, osteocytes, and chondrocytes, we performed cell differentiation experiments by incorporating adipogenic, osteogenic, and chondrogenic supplements into MSCs cultured in vitro. Subsequently, we carried out various staining techniques that allowed us to visualize the different cell types induced by the supplements.

[0160] Oil Red staining revealed the presence of adipocytes by staining lipids and vacuoles in orange-red in MSC WT, ENG + and Eng~ ~. The presence of osteocytes was evidenced by the alkaline phosphatase activity, which produced a purple stain in all three cell types. Finally, Alcian Blue staining marked the chondrocytes blue in the different MSCs. Therefore, the results show that both WT MSCs and ENG MSCs +and MSC Eng~ ~ are able to differentiate in vitro into adipocytes, osteocytes and chondrocytes (Figure 9).

[0161] The final criterion for identifying MSCs, the expression of a specific immunophenotype, was analyzed using flow cytometry. For this purpose, different markers were studied in WT and ENG MSC cells. + and MSC Eng~ ~.

[0162] First, we characterized the WT MSCs that are our control cells and verified that they meet the specific immunophenotype (Figure 10).

[0163] Subsequently, we analyzed the different markers in the MSC ENG. + and MSC Eng~ ~ and we verified that they retain the immunophenotype of the MSC WT except in the case of endoglin that the MSC Eng~ ~ lack expression of the protein (data not shown).

[0164] In short, the MSC WT, the MSC ENG +and the MSC Eng~ ~ meet the minimum requirements for identification and characterization of mesenchymal stem cells.

[0165] Example 1.2. Effect of endoglin expression on the immunomodulatory capacity of MSCs

[0166] One of the most important biological properties of mesenchymal stem cells (MSCs) is their immunomodulatory capacity. MSCs can modulate tissue inflammation by secreting trophic factors, promoting an anti-inflammatory immune response by exerting an immunosuppressive function. In fact, MSCs can modulate both innate and adaptive immune responses by acting on various types of immune cells, such as T cells, B cells, NK cells, dendritic cells, and macrophages, among others.

[0167] Example 1.3. Effect of MSCs with different endoglin expression on T lymphocyte function

[0168] As a first approach to the effect of modifying endoglin levels on the immunomodulatory capacity of MSCs, we used T lymphocytes and regulatory T lymphocytes to carry out the experiments.

[0169] MSCs inhibit T cell proliferation, reduce the secretion of pro-inflammatory cytokines, limit the differentiation of pro-inflammatory Th subtypes, and promote the differentiation and activation of regulatory T cells (Tregs).

[0170] Study of T lymphocyte proliferation

[0171] To evaluate the effect of endoglin modification on T lymphocyte proliferation, we analyzed the incorporation of BrdU by T lymphocytes in co-culture with WT MSCs and ENG MSCs. + and MSC EngG

[0172] According to the results, WT MSCs significantly reduce T lymphocyte proliferation, consistent with findings described in the literature. ENG MSCs +They reduce proliferation more than WT. However, MSC Eng~ ~ do not reduce T lymphocyte proliferation, as no differences are observed between the group co-cultured with MSC Eng~ ~ and those grown in the absence of MSC (Figure 11).

[0173] Therefore, modifying the expression of endoglin in MSCs alters their ability to inhibit T lymphocyte proliferation.

[0174] Analysis of transendothelial migration of T lymphocytes

[0175] T lymphocytes migrate across the endothelial barrier, attracted by an inflammatory focus, to perform their function during an immune response. This occurs through chemoattraction, which draws T lymphocytes toward a chemokine gradient. MSCs may stimulate this process by synthesizing chemoattractant molecules.

[0176] To evaluate the effect of modifying MSC endoglin levels on T lymphocyte chemoattraction across the endothelial barrier, a cell migration assay was performed using a transwell with a pore size sufficient to allow cell passage. Different MSCs were seeded in the lower compartment to act as chemoattractants for T lymphocytes placed in the upper compartment. As shown in Figure 11, the regression curve obtained represents the transendothelial migration of T lymphocytes attracted by each MSC (WT, ENG). + and Eng~ ~ over time and a two-way ANOVA statistical test was performed. The statistical analysis reveals interaction (#), that is, the behavior of each of the curves over time depends on the type of MSC; for this reason, it is not possible to analyze the result jointly, but rather an independent analysis is necessary.

[0177] Consequently, we independently compared the transendothelial migration of T lymphocytes over a short time frame (24 hours) and a longer time frame (120 hours). At 24 hours, a significant increase in the migration of T lymphocytes attracted by wild-type MSCs and MSCs overexpressing endoglin was observed compared to the control without MSCs. However, endoglin knockout MSCs reduced T lymphocyte migration compared to other MSC types (Figure 11B).

[0178] When analyzing the transmigration of T lymphocytes at 120 hours, a very significant increase in the migration of lymphocytes attracted by MSC ENG is observed + with respect to the other experimental conditions (Figure 11C).

[0179] With the results obtained from the independent analyses along with the regression curves, we can conclude that during the first 24 hours the MSC WT and the ENG +They stimulate the migration of T lymphocytes, but over time the curve of WT MSCs stabilizes and the MSCs with overexpression of endoglin continue to further stimulate the migration of T lymphocytes.

[0180] Example 1.4. Effect of MSCs with different endoglin expression on the proliferation of regulatory T lymphocytes

[0181] To study the effect of different levels of endoglin on the proliferation of regulatory T lymphocytes, we evaluated the incorporation of BrdU by regulatory T cells in co-culture with WT MSCs and ENG MSCs. + and MSC EngG

[0182] As shown in Figure 12, all three types of MSCs significantly reduce the proliferation of regulatory T lymphocytes. However, Eng~~ MSCs reduce proliferation even more than WT MSCs. On the other hand, we found no differences between the behavior of WT MSCs and MSCs with endoglin overexpression.

[0183] Consequently, endoglin silencing modifies the effect of MSCs on regulatory T lymphocyte proliferation. Example 1.5. Effect of MSCs with different endoglin expression on macrophage polarization

[0184] Another approach to studying the effect of MSCs on immunomodulation was to evaluate their effect on macrophage polarization, since MSCs have been described as favoring the polarization of macrophages from a pro-inflammatory phenotype (MI) to an anti-inflammatory phenotype (M2).

[0185] To study this immunomodulatory capacity, macrophages were co-cultured with WT, ENG MSCs +and Eng~ ~ for 24 hours. Previously, we preconditioned the MSCs with LPS for three days and polarized the M0 macrophages to an MI phenotype with IFN-γ and LPS (Figure 13A). Subsequently, the expression of the M1 phenotype marker, IL-1RA, and the M2 phenotype marker, CD206, was analyzed by qPCR in order to evaluate the polarization of the macrophages to an M2 phenotype.

[0186] First, macrophage polarization to an M2 phenotype was assessed in the presence of control MSCs. A decrease in MI marker expression and an increase in M2 marker expression were observed after co-culture with control MSCs (Figure 13B-C).

[0187] Subsequently, we compared the effect of different endoglin expression on this polarization. As shown in Figure 14, the MSC ENG +Eng~ and Eng~ ~ stimulate the polarization of macrophages from a pro-inflammatory to an anti-inflammatory phenotype in a slightly different way. However, we did not observe any statistically significant difference.

[0188] Example 1.6. Study of the effect of endoglin expression on the ability of MSCs to modulate angiogenesis

[0189] As previously mentioned, another essential biological property of MSCs is their ability to modulate angiogenesis. Several studies demonstrate the capacity of these cells to promote the formation of new blood vessels through various mechanisms, such as the secretion of pro-angiogenic molecules, direct cell interaction, modulation of the microenvironment, and transdifferentiation into endothelial cells or mural cells.

[0190] To analyze the effect of modifying endoglin levels on the ability of MSCs to modulate angiogenesis, we used different in vitro angiogenesis models that allowed us to understand the possible role that endoglin plays in this biological property of MSCs and to understand the mechanism by which MSCs act in the angiogenic process.

[0191] Example 1.7. Effect of MSCs with different endoglin expression on the pseudocapillary formation assay

[0192] Endothelial cells have the ability to organize themselves into capillary-like structures (pseudocapillaries) when seeded onto a three-dimensional matrix, such as Matrigel®, fibrin, collagen, or other polymeric components. Therefore, our initial approach to studying the effect of modifying endoglin expression on this MSC capacity was the pseudocapillary formation assay on Matrigel®.

[0193] For this purpose, human umbilical vein endothelial cells (HUVECs) were seeded onto Matrigel® in the presence of different types of MSCs on Matrigel®. To subsequently differentiate between the two cell types, the HUVECs were pre-labeled with calcein-AM (which fluoresces green) and the MSCs were labeled with CellTracker™ (which fluoresces red). Three hours after seeding, photographs were taken of the resulting structures.

[0194] As shown in Figure 16A, HUVECs, together with the various MSCs, form the characteristic pseudocapillaries of endothelial cells seeded on a three-dimensional matrix. However, regarding the complexity of the resulting structure, a simple visual analysis of the images suggests that the structure formed by HUVECs with MSCs is ENG + And with MSC Engf it is more disorganized and less complex than with MSC WT.

[0195] To quantify the structures created in Matrigel®, a digital skeleton of the formed structures was created using Fiji software. Using the same software, we analyzed the number of independent structures, number of branches, number of joining points, average branch length, and the longest shortest path parameter on this skeleton (Figure 15).

[0196] The resulting analysis shows a significant increase in independent structures in HUVECs seeded with MSC Eng~ ~ compared to MSC WT and MSC ENG + (Figure 16B), indicating that the overall structure is less complete and less complex. A significant decrease in the number of branches (Figure 16C) and junction points (Figure 16D) is also observed in the structures formed by HUVECs with MSC ENGs. +and the MSC Eng~ ~ with respect to those formed with the MSC WT, resulting in a more linear and less branched two-dimensional clustering. Likewise, the structures formed by the HUVEC with the MSC ENG + They show a tendency to have a greater length, compared to the pseudocapillaries formed by HUVECs and MSC Eng~ ~ (Figure 16F).

[0197] These results indicate that modifying endoglin levels alters the ability of MSCs to modulate angiogenesis in the pseudocapillary formation assay because it modifies the organization of endothelial cells.

[0198] Example 1.8. Effect of MSCs with different endoglin expression on the sprout assay from aortic rings

[0199] To complement the results obtained in the in vitro pseudocapillary angiogenesis model, a sprouting assay was performed using aortic rings. This model allows us to study angiogenesis in an environment closer to in vivo physiological conditions because it uses ex vivo tissue.

[0200] Mouse aortic rings are seeded in Matrigel®, allowing for the evaluation of initial vessel sprouting, followed by matrix remodeling and vessel luminization. It is noteworthy that the sprouts arising from the ring anatomically mimic in vivo microvessels, making this assay suitable for analyzing whether different microvascular structures (MVCs) promote or inhibit angiogenesis.

[0201] The aortic ring assay was performed using three different approaches that addressed different questions. The first involved adding conditioned medium containing the various MSCs after polymerization of Matrigel® with the aortic ring; the second involved resuspending the MSCs in Matrigel® before polymerization with the aortic ring; and the last involved adding the MSCs to the medium after polymerization of Matrigel® with the aortic ring, so they adhered to the plaque outside of Matrigel®.

[0202] Sprout growth begins on day two. For this reason, three days after sowing the rings, photographs were taken and the onset of sprouting was studied by quantifying the number of tip cells and sprouts. In addition, the sprouting rate parameter was calculated by assigning a value of 1 to each tip and a value of 2 to each sprout.

[0203] To analyze the peak of sprouting, the area occupied by the sprouts and the maximum distance a sprout could travel were calculated on day six after ring seeding. Effect of the MSC secretome on sprouting from aortic rings

[0204] To evaluate the effect of the secretome secreted by the different MSCs on sprouting, aortic rings were seeded in the presence of conditioned medium from WT and ENG MSCs. + and MSC Eng ~.

[0205] Initially, it is observed that the secretome of MSC Eng~ ~ has a different effect than that of MSC WT and MSC ENG + at the beginning of sprouting. An anti-angiogenic trend is observed in MSCs without endoglin; however, the reduction is only significant compared to the conditioned medium of control MSCs and MSCs with overexpression of endoglin (Figure 17).

[0206] The results obtained on day six are consistent with those previously observed. In an initial visual analysis (Figure 18A), sprouting is very poor in the ring sown in the presence of the conditioned medium of the MSCs without endoglin. When analyzing the different parameters, it is observed that the rings cultivated with the secretome of the MSCs Eng~' show a significant reduction in the area occupied and the maximum distance compared to those cultivated with the secretome of the MSCs WT. Furthermore, these rings show a significant reduction in the maximum distance compared to the control (without MSCs), which means that the secretome of the MSCs Eng^ somehow reduces the length of the sprouts (Figure 18B-C).

[0207] In summary, the secretome of endoglin-deficient MSCs affects sprouting, significantly reducing the various parameters studied compared to control MSCs and MSCs with endoglin overexpression. Therefore, we may be observing an anti-angiogenic effect of Endoglin-deficient MSCs.

[0208] Direct effect of MSCs on sprouting from aortic rings

[0209] In light of the results obtained with conditioned medium, we considered the possibility that MSCs exert an effect through direct cell-cell contact and that, in some way, different MSCs may integrate into sprouting formation by exerting their pro-angiogenic or anti-angiogenic action. This idea arises from the findings described in the literature regarding the possible pro-angiogenic mechanisms of MSCs. Several studies suggest a role in blood vessel formation through direct transdifferentiation into endothelial cells and / or mural cells. Other studies demonstrate that they exert their function through paracrine action by secreting pro-angiogenic factors that stimulate this process. Therefore, to evaluate this direct effect of different MSCs on sprouting, WT MSCs and ENG MSCs were resuspended. + and MSC Eng~ ~ in the Matrigel® and were allowed to polymerize together with the aortic ring.

[0210] At the beginning of sprouting (Figure 19), the MSC ENG + They stimulate this process, as evidenced by a significant increase in tip cells and sprouting rate compared to the control situation (without MSCs). Again, Eng~ MSCs appear to inhibit this process, as a significant reduction in all three parameters is observed compared to ENG MSCs. + .

[0211] When observing the sprouts generated from the aortic rings at the time of maximum sprouting (Figure 20A), it appears that the Eng~ ~ MSCs exhibit less growth. However, this is difficult to analyze visually because the MSCs are located within the Matrigel®, albeit on a different plane. Objective analysis shows that the results are consistent, as MSCs with endoglin overexpression stimulate sprout length, while endoglin knockout MSCs significantly reduce the area occupied and the maximum sprout distance compared to the ENGs. + (Figure 20B-C)

[0212] According to the results, MSCs with endoglin overexpression produce a pro-angiogenic effect on sprouting formation, as evidenced by a significant increase in the various parameters studied compared to the control (without MSCs). On the other hand, Eng~~ MSCs show a tendency to inhibit this process; however, no significant differences were observed compared to the control or WT MSCs.

[0213] Effect of MSCs on sprouting from aortic rings

[0214] Based on the results obtained with conditioned medium and direct cell contact, we considered the possibility of observing a more potent effect from the different MSCs. For this reason, we studied the approach of adding the MSCs to the medium after the polymerization of Matrigel® with the aortic rings. In this way, the MSCs could exert their function through paracrine cell signaling. This type of signaling occurs when a cell secretes a signaling molecule that induces changes in nearby cells, altering their behavior or differentiation.

[0215] First, it is observed that the different MSCs react differently to the onset of sprouting. The WT MSCs show a significant increase in the various parameters, but the ENG MSCs +They show even more significant differences. However, the Eng~ ~ exhibit a significant reduction compared to the other MSCs. Thus, the WT MSCs stimulate this process, the ENG + They stimulate it even more, while the Eng~ ~ inhibit it (Figure 21).

[0216] When the aortic rings were observed on day six, those cultured in the presence of MSCs without endoglin showed inhibition of sprout growth compared to the other conditions (Figure 22A). Quantifying the area occupied by the sprouts and the maximum distance between them showed results consistent with those observed in previous days. The MSCs ENG + They show a significant increase in the length of the sprouts while the Eng~ ~ show a reduction of the parameter compared to the other experimental conditions, coinciding with what we had perceived at first glance (Figure 22B-C).

[0217] Analyzing all the results obtained from studying the effect of endoglin expression on the ability of MSCs to modulate angiogenesis, it is observed that under normal conditions, MSCs appear to stimulate angiogenesis in vitro. However, while endoglin overexpression seems to increase this pro-angiogenic effect, endoglin silencing completely alters this effect, eliminating it or even making it anti-angiogenic. Specifically, sprout growth is practically halted.

[0218] Regarding the different approaches studied, it is observed that they are complementary; however, the most potent effect occurs when MSCs are added to the medium after polymerizing Matrigel® with the aortic ring. Therefore, it can be deduced that paracrine cell signaling is crucial for the performance of the biological properties of MSCs.

[0219] Example 1.9. Effect of MSC endoglin expression on endothelial functions

[0220] Sprouting is one of the phases of angiogenesis, encompassing essential endothelial cell processes such as migration and proliferation. After observing that altered endoglin levels affect the ability of MSCs to modulate angiogenesis in various in vitro angiogenesis assays, and specifically in the sprouting process, we investigated whether endothelial migration and proliferation functions might be altered by varying endoglin expression.

[0221] Analysis of endothelial cell migration

[0222] As mentioned, one of the endothelial functions involved in sprouting is the migration of tip cells. To study the effect of different endoglin expression on this migration process, the Wound Healing assay was performed. In this assay, a wound is created on a monolayer of endothelial cells, which then migrate to "close the wound," occupying the free space. Therefore, the endothelial migration capacity is evaluated by measuring the distance migrated. In our case, two approaches were used: the first consisted of incubating the cells with conditioned medium containing the three types of MSCs, and the second consisted of co-culturing the endothelial cells with different types of MSCs on the same surface. The distance migrated by HUVEC endothelial cells was measured over 6 hours.

[0223] The results obtained with conditioned medium indicate that the secretome of MSC Eng~ does not stimulate endothelial cell migration, but rather inhibits this process. Regarding MSC ENG + , its secretome appears to stimulate HUVEC migration as they travel a greater distance than HUVEC in the control condition (without MSC) (Figure 23).

[0224] When we analyzed the same process using co-culture with different MSCs, we observed the same trend. Co-culture of HUVECs with endoglin-overexpressing MSCs induced significantly faster migration than when co-cultured with control MSCs. However, there was a very significant decrease in endothelial migration when HUVECs were co-cultured with Englin-overexpressing MSCs (Figure 24).

[0225] In conclusion, we can say that MSC Eng~ ~ inhibits the endothelial cell migration process in the Wound Healing assay. Regarding MSC ENG, we can state that their secretome stimulates endothelial cell migration in this assay.

[0226] Study of endothelial cell proliferation

[0227] Another endothelial function involved in sprouting is stalk cell proliferation. To analyze the effect of different endoglin expression on this endothelial proliferation process, we evaluated BrdU uptake by cultured endothelial cells using two different procedures: 1) incubating HUVECs with conditioned medium from different MSCs and 2) co-culturing HUVECs with WT MSCs and ENG MSCs + and MSC Eng' / '.

[0228] The results obtained in the conditioned medium approach indicate that endoglin overexpression in MSCs stimulates endothelial cell proliferation; however, wild-type MSCs and eng-type MSCs do not appear to stimulate or inhibit proliferation (Figure 25A). In the co-culture case, both wild-type MSCs and eng-type MSCs + They significantly increase the proliferation of endothelial cells; however, MSC Eng~ ~ does not stimulate HUVEC proliferation (Figure 26B).

[0229] Once again, it is observed that both the secretome assay and the co-culture assay follow the same trend; however, the latter provides more significant differences since the MSCs are present interacting with the other cell types.

[0230] In summary, these results suggest that the effect of endoglin expression modification observed in in vitro angiogenesis assays may be partly due to its effect on endothelial functions essential for sprouting in the angiogenic process. Therefore, the observed anti-angiogenic action of Eng~~ MSCs could be a consequence of their not stimulating either endothelial process, but rather inhibiting endothelial cell migration.

[0231] Example 1.10. Effect of endoglin expression and human MSC origin on their ability to modulate angiogenesis

[0232] After confirming that modifying endoglin expression in MSCs alters their ability to modulate angiogenesis, we wanted to evaluate whether these results obtained in murine cells were reproducible in human cells and, furthermore, whether the origin of the hMSCs could influence the effect of modifying endoglin levels. To this end, we used two types of hMSCs: 1) hMSCs derived from adipose tissue (hMSC-TA) and 2) hMSCs derived from bone marrow (hMSC-MO).

[0233] Endoglin gene silencing in human MSCs

[0234] To analyze the effect of modifying endoglin expression in human MSCs on their ability to modulate angiogenesis, it was necessary to inhibit endoglin gene expression in hMSC-TA and hMSC-MO and determine the time of maximum gene silencing to perform the different experiments. Silencing was achieved by transfection with siRNA or interfering RNA and analyzed at different time points using flow cytometry.

[0235] As shown in Figure 26, flow cytometry results confirm that hMSC-TA siENG cells reach their maximum endoglin gene silencing (65%) 39 hours after transfection with endoglin siRNA. In the case of hMSC-MO siENG cells, 70% endoglin gene silencing is achieved 72 hours after transfection. Based on these results, we performed experiments with hMSC-TA cells (control and siENG) 39 hours after transfection and with hMSC-MO cells (control and siENG) 72 hours after transfection.

[0236] Effect of endoglin expression and hMSC origin on endothelial cell migration

[0237] As a first step, we wanted to evaluate the effect of endoglin silencing in hMSCs on endothelial cell migration using the Wound Healing assay. In this case, the endothelial cells used were endothelial cell fibroblasts (ECFCs), and the approach involved co-culturing ECFCs with different types of hMSCs. Subsequently, the distance migrated by the endothelial cells over 6 hours was calculated.

[0238] The results obtained with adipose tissue hMSCs show, similarly to what was observed previously with murine MSCs, a very significant reduction in the distance migrated by endothelial cells cultured with endoglin-silenced hMSC-TA compared to those cultured with control hMSC-TA (Figure 27).

[0239] On the other hand, the same trend is observed in the results obtained with bone marrow hMSCs, since endothelial cells co-cultured with siENG hMSC-MO travel a shorter distance than those cultured with control hMSC-MO (Figure 28). These differences are less significant than in TA hMSCs.

[0240] These results indicate that gene silencing of endoglin in hMSCs alters their ability to stimulate endothelial cell migration in the Wound Healing assay. Furthermore, we can specify that the effect of gene silencing is independent of the hMSC origin, as it has been observed in both cell types.

[0241] Effect of endoglin expression and hMSC origin on the cytodex assay

[0242] To study the effect of endoglin silencing in hMSCs on their ability to modulate angiogenesis, the Cytodex fibrin gel assay was performed in the presence of different hMSC types. This assay is a more sophisticated model that allows for a more detailed analysis of the cellular parameters that affect angiogenesis. The model consists of collagen-coated dextran beads to which endothelial cells adhere, forming a monolayer, similar to the arrangement of endothelial cells in blood vessels. Therefore, this technique allows observation of sprouting from the time the tip cell differentiates and begins to migrate. The hMSCs act as feeders, providing necessary factors through paracrine action. We monitored the experiment and took photographs on day 1 to study the onset of sprouting and on day 3 to assess tube formation.On day 1, the beginning of sprouting is observed, marked by the migration of the tip cell, and with a first visual analysis, it appears that the number of tip cells and sprouts per spheroid is greater in the presence of control hMSCs than in the presence of siENG hMSCs, both in hMSCs from adipose tissue (Figure 29A) and in hMSCs from bone marrow (Figure 30A).

[0243] To perform an objective analysis, we manually quantified the tip cells and sprouts generated from the beads under different conditions. We also quantified the sprouting rate parameter, defined by assigning a value of 1 to tip cells and a value of 2 to sprouts. The results obtained reveal a highly significant reduction in the number of tip cells, sprouts, and the sprouting rate parameter when endoglin-silenced hMSCs were used as feeders, as we had previously observed visually. This decrease occurred in both hMSC-TA (Figure 29B-D) and hMSC-MO (Figure 30B-D).

[0244] Therefore, hMSC siENG appear to act as antiangiogenic feeders in the sprouting initiation process.

[0245] To analyze the tube formation that occurs in the following days, immunofluorescence of the day 3 assay was performed, staining actin filaments green with phalloidin and nuclei red with TO-PRO. In an initial visual analysis, as before, a decrease in the number of sprouts as well as their length was observed in the cytodex assays with hMSC siENG as feeders, regardless of the origin of the hMSCs: hMSC-TA (Figure 31A) or hMSC-MO (Figure 32A).

[0246] To analyze the results objectively, we manually quantified the sprouts generated from the beads under different conditions; in this case, we did so using photographs taken in visible light before immunofluorescence. The results show a highly significant decrease in the number of sprouts in beads seeded using hMSC-TA with silenced endoglin as feeders compared to control hMSC-TA (Figure 31B). Similarly, this significant reduction was observed in the case of hMSCs derived from bone marrow (Figure 32B).

[0247] Based on the results, hMSC siENG appear to act as anti-angiogenic feeders, not only during the sprouting initiation process but also in later stages of angiogenesis, such as tube and anastomosis formation. This effect seems to be independent of the hMSC cell origin, as the results are consistent for both cell types, hMSC-TA and hMSC-MO.

[0248] In conclusion, the effect of altered endoglin expression in MSCs on their ability to modulate angiogenesis, as observed in human MSCs, is consistent with the results obtained in murine MSCs. Therefore, we can determine that decreased endoglin levels in MSCs impair their ability to modulate angiogenesis, shifting them from a pro-angiogenic to an anti-angiogenic effect.

[0249] Example 1.11. Effect of treatment with MSCs with different endoglin expression on the occurrence and severity of AVMs

[0250] Recently, in our laboratory, we have demonstrated that treatment with human MSCs in a murine model of arteriovenous malformation (AVM) development, which attempts to replicate what occurs in HHT, reduces the frequency and severity of AVMs. The hypothesis is that this result could be due to the immunomodulatory and angiogenic effects of the MSCs.

[0251] In view of the in vitro results obtained in this work, which demonstrate that changes in the expression of endoglin in MSCs modify their immunomodulatory capacity and modulation of angiogenesis, we consider the possibility that treatment with MSCs with different expression of endoglin may modify the effect on AVMs.

[0252] To this end, the previously developed murine model of AVM generation was used. The model consists of creating skin wounds on the backs of Eng~ ~ mice generated after intraperitoneal administration of tamoxifen (Figure 3). During wound healing, angiogenesis is stimulated, along with a significant inflammatory response, leading to the development of arteriovenous malformations in the wound environment of the endoglin knockout mice. Ten days after wound creation, the mouse blood vessels are perfused with a blue latex solution, allowing visualization of the vasculature and, in particular, the AVMs that have developed in the tissue near the wound. In the previous study, hMSC-TA resuspended in Matrigel® were administered at the time of wound generation.In this case, murine-derived wild-type (WT) and endoglin-derived MSCs were administered in Matrigel® because we wanted to study the effect of endoglin expression on the ability of MSCs to limit the development of arteriovenous malformations (AVMs). WT and endoglin-derived MSCs were administered in Matrigel® at the time of wound formation. Ten days later, the vessels were perfused with a blue latex solution, and the vasculature formed near the wound was analyzed.

[0253] First, the presence of AVMs was analyzed in the different experimental groups (no MSCs, MSC WT, and MSC Eng). The absence of AVMs was identified with a vasculature organized in a monolayer with non-tortuous, thin, and linear vessels. Conversely, the presence of AVMs was associated with a disorganized and overlapping vasculature with dilated, tortuous, and branching vessels (Figure 33A). When evaluating the relative frequencies of this subjective analysis, it was observed that the group treated with MSC WT showed a significant reduction in the occurrence of AVMs compared to the untreated group (no MSCs) and the group treated with MSC Eng, as they appeared in 50% of the mice versus 80% and 90%, respectively (Figure 33B).

[0254] To complete the study, the severity grades of the arteriovenous malformations (AVMs) generated in the different experimental groups were subjectively characterized. For this purpose, the Yakes classification, widely used in clinical practice, was adapted (Figure 34A). Grade 1 is associated with a direct connection between an artery and a vein; grade 2 is characterized by multiple inflow arteries in a nidus with outflow venules; and finally, grade 3 is related to the presence of multiple arterioles connected to a highly dilated vein. In our study, no grade 4 AVMs were observed.

[0255] As shown in Figure 34B, there is a statistically significant difference between the group treated with MSC WT and the other two groups, untreated and treated with MSC Eng~'. All three groups have a similar percentage of grade 1 AVMs, around 40%. Differences begin to appear in grade 2 AVMs, as the group treated with MSC WT has approximately half the number of grade 2 AVMs compared to the other two groups of mice. However, the greatest variation between groups is seen in grade 3 AVMs, since the control MSC group does not have any AVMs of this severity.

[0256] Overall, the results show that MSC WT reduces the generation of AVMs, as well as their severity. However, MSC Eng~ ~ shows no effect on this process.

[0257] Therefore, silencing endoglin in MSCs alters the treatment effect on the occurrence and severity of AVMs in the endoglin knockout mouse model of AVM generation. Example 2. TREATMENT OF HEREDITARY HEMORRHAGIC TELANGIECTASIA

[0258] Example 2.1. Development of an endoglin-dependent MAV model

[0259] To study the appearance and development of arteriovenous malformations, as well as a therapeutic approach to these lesions, we first focused on obtaining and verifying the murine model of this process.

[0260] Some authors previously found that in mice heterozygous for endoglin (or ALK1, which are the genetic model for HHT), AVMs appeared infrequently and depended on the genetic background. These malformations only appeared in knockout models that were also undergoing an angiogenic or inflammatory process. Therefore, we set out to determine whether AVMs formed in Eng~ mice ! ~ that were undergoing a skin wound healing process. Since endoglin deficiency is lethal during embryonic development, we used a tamoxifen-inducible knockout mouse model.

[0261] To get mice Eng~ ! Cre recombinase activity was induced in Eng-floxecL mice by intraperitoneal administration of tamoxifen for 5 consecutive days (Figure 35A). To ensure that the tamoxifen had taken effect and the mice were fully Eng-floxecL !~, we performed conventional PCR with genomic DNA extracted from the terminal portion of the tail of KO-Eng Cre' and KO-Eng Cre mice + Complete loss of the endoglin gene was observed to occur 21 days after the first tamoxifen injection (Figure 35B), since in the preceding days partial presence of the gene was still observed.

[0262] However, even if the endoglin gene is lost from the genomic DNA, residual protein expression may still occur, as it depends on the protein's half-life. Therefore, we used Western blot to study when complete loss of the endoglin protein occurred. To do this, we isolated the protein from different tissues (liver, kidney, lung, and heart) of Eng-C Q mice. +and Eng-C Q treated with tamoxifen and compared endoglin expression. Our results show that protein expression is no longer detectable 35 days after the first tamoxifen injection (Figure 35C). For this reason, this was the time we determined to begin the wound healing assays. Example 2.2. Macroscopic analysis under stereoscopic microscope of latex-perfused vessels in skin wounds

[0263] To try to develop the MAV model, we made wounds in the backs of animals that had lost endoglin expression, i.e., iKO-Cre mice. + and we analyzed the vasculature formed during that process. We compared it with iKO-Cre' mice as controls. Ten days after the wounds were generated, we perfused the tissues with a blue latex that allowed us to analyze the vasculature formed.

[0264] The tissues perfused with latex were analyzed and photographed under a stereoscopic microscope, which allowed us to obtain a complete picture of these samples.

[0265] Even at first glance, in the control samples of the back wound, it can be seen that the vasculature is organized and arranged in a monolayer, and at the same time, the vessels are characterized as being thin and linear (Figure 36A).

[0266] However, in Eng~ ~ mice, the vasculature is disorganized, and overlapping vessels are observed. Furthermore, these vessels appear more dilated, tortuous, and branched (Figure 36B). Since latex barely penetrates the capillaries, the blue-stained vessels will be large vessels that supply or surround the wound area.

[0267] As a first step to quantify the observed differences, we assessed the presence of arteriovenous malformations (AVMs), that is, a tangle of more or less tortuous vessels, generally with an increased diameter, around previously generated skin wounds. This allowed us to calculate the frequency of AVMs in the two study groups. It was observed that the control mice presented almost no AVMs; that is, only 5% of the mice had developed arteriovenous malformations, while the majority of the Eng~ mice did. ! ~, 57% of the mice do generate them (Figure 37A). These differences are statistically significant.

[0268] Our goal is to obtain a model in which we can take quantitative and not just qualitative measurements, so we wanted to quantify the differences observed with respect to the structure and organization of the vessels and for this we used two objective parameters.

[0269] First, thanks to the blue color of the latex used, we analyzed the area occupied by the blood vessels by estimating it from the number of blue pixels surrounding the wound area. A cropping template generated in Adobe Photoshop was applied to all the photos taken, ensuring they had the same size and number of pixels and were representative of the skin wounds.

[0270] The analysis showed that the group of mice Eng~ !~ exhibits a significantly larger vascular area than the control mouse group (Figure 37B). On the other hand, we studied the caliber of the vessels by taking five random measurements of the diameter of the largest vessels surrounding the wound. It was observed that the Eng~ mice ! ~ present vessels of significantly larger caliber compared to the control group (Figure 37C).

[0271] Next, we set out to establish a grading system for the observed AVMs, since in the clinic this is a way to assess their severity and predict their behavior.

[0272] To analyze the grades of AVMs, we relied on the Yakes clinical classification (Neto CASF and Durans M., 2019), in which grade 1 is characterized by a direct arteriovenous fistula, that is, a direct artery-to-vein connection. Grade 2 is an AVM generally characterized by multiple incoming arteries in a "nidus" pattern with direct arteriole-to-vein structures or a nidus of malformations that then drain into a single vein. Grade 3 consists of multiple arterioles connecting within the wall of an enlarged vein. And grade 4 AVMs are microfistulas of countless arteriole-like structures that branch off into numerous venular connections that diffusely infiltrate tissue (Figure 38A).

[0273] Following this assessment and according to our subjective analysis, we observed that the control group only has some vessels that we could identify as grade 1 AVMs, which would correspond to the mildest grade, while in the Eng~ mice! ~, arteriovenous malformations present a grade that ranges between grade 1 and grade 4 (Figure 38B).

[0274] If we consider only severe AVMs, that is, AVMs with a grade higher than grade 1, to avoid possible misclassification of normal vessels as AVMs, we observe that the control mice do not have severe AVMs, while 32% of the Eng~ mice ! ~ present grade 2, 3, and 4 AVMs. Upon closer examination, these mice present 44% grade 1 AVMs, 36% grade 2 AVMs, 8% grade 3 AVMs, and 12% grade 4 AVMs, so the majority of the AVMs generated are grade AVMs higher than 1 (Figure 38C). According to our analysis, these results are statistically significant.

[0275] Another relevant parameter in the characterization of arteriovenous malformations is their degree of tortuosity, that is, the degree of curves or twists that the blood vessels present in their course.

[0276] Vessel tortuosity was classified using grades 1 through 3, where grade 1 corresponds to slightly tortuous vessels, grade 2 to vessels with some tortuosity, and grade 3 to highly tortuous vessels (Figure 39A). After assigning these values, it was observed that the control group exclusively presented vessels with little tortuosity, that is, all were grade 1 (Figure 39B), while the Eng~ mice ! The different degrees of tortuosity were observed, with 56% of AVMs exhibiting grade 1 tortuosity, 28% grade 2, and 16% grade 3 (Figure 39C). According to our analysis, these results are statistically significant. After verifying the visually observed differences, we felt it was relevant to study the complexity of the vascular structures.

[0277] Within this study, we analyzed the ratio of perimeter to area occupied by vessels perfused with latex in the two genotypes under study.

[0278] The ratio of circumference to area occupied by a blood vessel is a measure that helps us understand the vessel's shape and structure. An increase in this ratio could correlate with a greater degree of vessel tortuosity.

[0279] When we analyzed the perimeter / area ratio of the vessels surrounding the wound, we found that the Eng~ ~ mice had a higher ratio compared to the control group, which could mean that these vessels are more complex and tortuous, that is, they have more twists in their course while maintaining the area of ​​their lumen (Figure 40A).

[0280] On the other hand, we used Fiji software and its Skeletonize tool, which allows us to corroborate the subjective analysis previously carried out, since, by converting the vessels into digital skeletons, it provides several parameters for analyzing the structure of the vasculature.

[0281] To characterize the different grades of AVMs observed and study their structural differences, we have grouped the malformations according to their degree of complexity as Grade 1-2 or Grade 3-4 and compared them with the absence of AVMs.

[0282] The analysis identifies different complexity parameters of the digital skeleton generated by the Fiji software, after which we selected the skeleton parameters that we considered could be correlated with the different degrees of AVM.

[0283] Specifically, we selected the number of branches, the average length of the branches, and the triple points, that is, the points that are joined to three different branches, and compared them with the MAV grade classification that we had carried out.

[0284] As a result, we observed that more severe AVMs, specifically grades 3 and 4, exhibit more branched vessels (Figure 40B) with shorter branches compared to the absence of AVMs (Figure 40D). In contrast, grade 1 and 2 AVMs show an increased number of branches (Figure 40B) and a decrease in the average branch length compared to vessels in the absence of AVMs (Figure 40D). All differences are statistically significant.

[0285] Regarding triple points, we observed that both grades 1 and 2 and grades 3 and 4 showed an increase in triple points when compared to vessels without AVMs. In the case of more severe AVMs, this difference was statistically significant (Figure 40C). Vessels without arteriovenous malformations were characterized by being longer (Figure 40D), having fewer branches (Figure 40B), and fewer triple points (Figure 40C). Therefore, thanks to this analysis, we could objectively differentiate the severity grades of AVMs and study whether the subsequently administered treatment could act on any of these parameters, which would mean that the severity of the AVMs was being reduced.

[0286] Example 2.3. Microscopic analysis and histological characterization of the vessels surrounding the skin wound

[0287] Next, we set out to microscopically analyze the organization and structure of the skin vessels on the backs of mice 10 days after the creation of skin wounds. Histologically, an arteriovenous malformation can be identified by the presence of dilated and abnormal blood vessels, as well as a decrease in the number of vessel layers or an inability to define and distinguish them. Simultaneously, abnormal proliferation of endothelial cells in the vessel walls can be observed, which could lead to increased wall thickness. Furthermore, degenerative changes may be present, including fibrosis, accumulation of connective tissue, and deposition of abnormal substances such as calcium. In addition, in some cases, direct communication between arteries and veins without intervening capillaries may be identified.

[0288] To carry out a structural study of the vessels of the mice under study and to be able to investigate the identifying criteria of arteriovenous malformations, we first performed the routine Hematoxylin-Eosin staining, which allows us to have a global view of the tissue, as well as to study the arrangement and organization of the vessels in the tissue.

[0289] In a first analysis, we observed the presence of large vessels, mostly with a thickened wall (#) and in some cases it is possible to distinguish the tortuous appearance (*) of these when compared with the control samples (Figure 41).

[0290] Furthermore, considering the alterations of the vascular wall that occur in the remodeling of vessels when they are subjected to a wound, we wanted to study the composition of fibers generated in the wound regeneration process, as well as the structure of its basement membrane or the matrix formed around the AVM.

[0291] To this end, we used the Weigert-Van Gieson and Masson's Trichrome techniques, where we analyzed the components of the basal lamina and the extracellular matrix, and on the other hand, the immunohistochemical staining of a-SMA, which allows us to analyze the muscular layer of the vessels (Figure 42). Since arteries and veins are composed of three layers—the adventitia, the media, and the intima—made up of different cells, a staining technique is necessary to identify and study the different components.

[0292] The intima layer consists of endothelial cells distributed continuously and supported by a subendothelial layer of connective tissue and supporting cells. The media layer is composed of muscle cells, elastic tissue, and connective tissue, while the externa layer is the outermost and thickest layer, composed entirely of connective tissue fibers and surrounded by an elastic lamina.

[0293] Specifically, the Masson Trichrome and Weigert-Van Gieson techniques allow the examination of the quantity and distribution of elastic and collagen fibers and the evaluation of the degree of fibrosis, which helps to understand the progression of vascular malformations and their structural changes.

[0294] Using WVG staining, we observed that the vessels contain few elastic fibers, stained purple, at this stage of tissue remodeling, which is characteristic 10 days after the healing of a skin wound (Figure 42).

[0295] On the other hand, although it has been described that some arteries of arteriovenous malformations could show rupture of the internal elastic lamina highlighted by WVG staining, we have not been able to observe it at this stage of wound healing.

[0296] Furthermore, using Masson's trichrome staining in the AVM model, we observed an increase in collagen fibers, stained green, consistent with an increased fibrotic layer typically seen in arteriovenous malformations compared to control tissues (Figure 42). The same occurs with the muscular layer, highlighted by immunohistochemical staining that detects the a-SMA protein and marks the vascular smooth muscle cells in the medial layer of the vessel wall in brown (Figure 42).

[0297] Example 2.4. Effect of human mesenchymal stem cell treatment on the occurrence and severity of AVMs

[0298] Verification of the presence and location of human mesenchymal cells at the microscopic level

[0299] Once the model for the development of arteriovenous malformations in Eng' mice was fine-tuned / _We then moved on to the next objective of this study: to verify the effect of hMSC treatment on this model. First, we wanted to determine if stromal mesenchymal cells were present in the skin sample containing the wound and, furthermore, to ascertain their location and arrangement within the skin tissue.

[0300] To do this, we performed an immunohistochemical staining that detected the human CD105 (endoglin) marker, one of the identifying markers of mesenchymal cells.

[0301] Since the mouse does not express endoglin, there is no risk of nonspecificity due to cross-linking of the antibody with mouse endoglin, making this an ideal antibody for detecting human MSC cells.

[0302] We have observed that a large number of MSC cells persist after ten days and, moreover, have migrated to the wound environment, as we observed many cells dispersed throughout the tissue. A large number of cells are also detected around the vessels (#) and even integrated into their walls (*) (Figure 43).

[0303] Macroscopic analysis of latex-perfused vessels in skin wounds in mice Eng / _ and mice Eng / _ treated with hMSC

[0304] Next, we analyzed the same parameters quantified previously in samples from vessels in the wound environment perfused with latex, but this time in the presence of treatment with human mesenchymal stromal cells. First, we studied the occurrence of arteriovenous malformations (AVMs) in Eng' mice and in Eng' mice treated with hMSCs. We observed that in the presence of hMSCs, only 37% of the Eng' mice / _They generate MAVs, while this occurs in 57% of Eng' mice / _ without treatment (Figure 44A).

[0305] Regarding the vascular area and diameter of the vessels in the study groups, we observed that in the mice treated with hMSC there was a slight decrease in the number of blue pixels, corresponding to the area occupied by the vessels (Figure 44B) and a significant decrease in their caliber (Figure 44C).

[0306] On the other hand, when we analyzed the severity of the AVMs generated in the Eng~ ~ mice, we observed that only 16% of the treated mice generated severe AVMs compared to 38% of the untreated Eng~ ~ mice (Figure 45A).

[0307] Regarding the tortuosity of the vessels, the same pattern is observed; that is, only 21% of the treated mice exhibit tortuosity, while this occurs in 44% of the untreated mice (Figure 45B). These differences are statistically significant.

[0308] Regarding the complexity parameters analyzed previously, we considered whether treatment with human MSC cells could alter any parameters related to the severity of AVMs. As we saw in the previous section, AVM severity was associated with a significant increase in the number of branches and triple points, and, conversely, with a significant decrease in the mean length of the branches. Furthermore, a non-significant increase in the perimeter-to-area ratio was observed.

[0309] Regarding the perimeter / area ratio, our results show a slight decrease in the ratio in Eng~ ~ mice in the presence of hMSC compared to Eng~ ~ mice (Figure 46A)

[0310] On the other hand, we observed that when treated with human MSCs, the Eng~ ~ mice exhibited significantly longer branches (Figure 46D), fewer branches (Figure 46B), and fewer triple points (Figure 46C). In other words, all the skeletal analysis parameters confirm that these malformations are less severe in the presence of the treatment.

[0311] Example 2.3. Histological analysis of the effect of hMSC cell treatment in the environment of skin wounds

[0312] Structural analysis of AVMs

[0313] In view of the results obtained, where it has been seen that treatment with human mesenchymal cells produces changes in different parameters at a macroscopic level, we considered studying whether this effect was reflected in tissue changes that imply a lower severity of AVMs.

[0314] As a first approximation to the general architecture of the tissue, we observed by Hematoxylin-Eosin staining that in Eng~ ~ mice treated with hMSC cells, the vessels are apparently smaller and thinner (Figure 47).

[0315] Secondly, we studied the composition of the vascular wall and the structure of its basal lamina and the matrix formed around the AVM.

[0316] With respect to the vessel wall, it is observed that with the treatment there appears to be a decrease in the amount of collagen fibers, evidenced by Masson's Trichrome staining (Figure 48A) and that the elastic fibers remain intact and continuous, as highlighted by WVG staining (Figure 47).

[0317] In order to analyze whether the differences observed in the vessel walls were significant, we carried out, on the one hand, Masson's Trichrome staining which highlights the fibrotic layer of the vessels and, on the other hand, the immunohistochemical staining of a-SMA which highlights the muscular layer (Figure 48A).

[0318] Our results demonstrate that the administration of hMSCs at the time of skin wound formation significantly induces a reduction in both the fibrotic layer (Figure 48B) and the muscular layer (Figure 48C) of blood vessels. Effect of hMSC treatment on the immune microenvironment in the area of ​​AVM development

[0319] As described in the literature, treatment with mesenchymal cells in the context of a skin wound improves the survival and migration of fibroblasts and increases the deposition of extracellular matrix by this cell type, enhancing the healing effects in the proliferative phase.

[0320] During the proliferative phase, fibroblasts are the main key factors, but macrophages or T cells can also modulate their activation. MSCs secrete various growth factors and cytokines that can regulate the response of neutrophils, macrophages, and lymphocytes.

[0321] During the maturation and repair phase, the number of fibroblasts must decrease, and collagen fibers must be properly reorganized for correct tissue remodeling. It is widely documented that MSC cells release numerous cytokines and growth factors with anti-fibrotic properties, such as HGF (liver growth factor), interleukin 10 (IL-10), and adrenomedullin. Furthermore, MSC cell signaling activates neighboring cells to produce the extracellular matrix correctly and also secrete various factors that promote vascular stability and vasoprotection, thus regulating the components involved in wound healing. In addition, MSC cells have been shown to suppress pro-inflammatory TNF-α released by MI macrophages and increase myofibroblast activity.These cells migrate to the wound and secrete HGF and PGE2, and both factors are able to inhibit myofibroblast differentiation and prevent epithelial-mesenchymal transition (Guillamat-Prats, 2021; Kimura and Tsuji, 2021).

[0322] Our hypothesis is that these same inflammatory factors that regulate healing could affect the development of AVMs and could be indicative of the effect of MSCs on tissue.

[0323] Therefore, we decided to study the expression of the markers TNF-α and HGF, two of the cytokines involved in the inflammatory process of skin wound healing, using immunohistochemistry. Our results show a tendency for TNF-α expression to be lower in treated mice than in untreated Eng~ ~ mice (Figure 49A and Figure 49B). Consistent with this result, higher HGF expression was observed in the mice treated with hMSC (Figure 49A and Figure 49C), although the results were not statistically significant.

[0324] Macrophages play a crucial role in regulating angiogenesis, suggesting they may be involved in the development of arteriovenous malformations (AVMs). In this regard, some studies have demonstrated an abnormally high number of macrophages around the vessels that make up AVMs, associating this with a potentially more active role in the pathogenic process and an increase in the severity of these malformations, although there is no evidence that this is actually the effect.

[0325] On the other hand, MSCs can modulate the chemoattraction, proliferation, or differentiation of macrophages, since they can polarize macrophages from a pro-inflammatory MI profile to a reparative / anti-inflammatory M2 activation.

[0326] This activation change of macrophages from an inflammatory MI phenotype to a reparative / anti-inflammatory M2 phenotype is a key step for wound healing and for controlling inflammation.

[0327] Therefore, we decided to study the presence of macrophages in skin wounds using immunohistochemistry of the F4 / 80 marker, which detects total macrophages, whether MI or M2 profile. First, we quantified the total number of macrophages in skin samples from the wound area (Figure 50A). After manually counting the total number of macrophages in the stained skin samples, we observed no significant differences between the treated and untreated Eng~ ~ mice (Figure 50B) for this marker.

[0328] However, during quantification, we noticed an apparent accumulation of macrophages around the blood vessels. Therefore, we decided to analyze whether there were differences in the number of macrophages recruited to the vicinity of the blood vessels. To do this, we quantified perivascular macrophages and observed a significant increase in total macrophages in Eng~ ~ mice treated with hMSC compared to untreated mice (Figure 50C). We analyzed whether the macrophages recruited to the perivascular area were of MI or M2 profile using CD206 immunohistochemical staining, which exclusively marks M2 profile macrophages. Thus, we compared the number of perivascular M2 profile macrophages in Eng' mice / _We compared mice treated with hMSCs and untreated mice, and observed that in the presence of hMSCs there was a non-significant increase in the number of perivascular M2 macrophages (Figure 50D). These results suggest that in the presence of hMSCs, perivascular macrophage recruitment is increased without a clearly defined M2 profile.

Claims

1. CLAIMS 1. An in vitro method for selecting mesenchymal stem cells for use in the treatment and / or prevention of pathological angiogenesis, characterized in that the method comprises evaluating the expression level of the endoglin protein, or the ENG gene, in the mesenchymal stem cell, characterized in that an increased expression level of the endoglin protein, or the ENG gene, in the mesenchymal stem cell, compared to a pre-established threshold value based on the expression of endoglin, or the ENG gene, measured in wild-type mesenchymal stem cells, is indicative that the mesenchymal cell can be used in the treatment of pathological angiogenesis.

2. Mesenchymal stem cell, or pharmaceutical composition comprising it, characterized by having an increased level of expression of the endoglin protein, or of the ENG gene, compared to a pre-established threshold value based on the expression of endoglin, or of the ENG gene, measured in wild-type mesenchymal stem cells, to be used in a method for the prevention and / or treatment of pathological angiogenesis.

3. An in vitro method, according to claim 1, for selecting mesenchymal stem cells for use in the treatment and / or prevention of pathologies with excessive angiogenesis, selected from hemangiomas, psoriasis, Kaposi's sarcoma, ocular neovascularization, retinopathy of prematurity, rheumatoid arthritis, endometriosis and atherosclerosis; or pathologies with deficient angiogenesis, selected from myocardial ischemia, peripheral limb ischemia, cerebral ischemia, delayed wound healing and impaired ulcer healing; or pathologies with defective angiogenesis, selected from vascular or arteriovenous malformations (AVMs) and telangiectasias.

4. In vitro method, according to claim 1 or 3, for selecting mesenchymal stem cells for use in the treatment and / or prevention of hereditary hemorrhagic telangiectasia.

5. Mesenchymal stem cell to be used, according to claim 2, in a method for the treatment and / or prevention of pathologies with excessive angiogenesis, selected from hemangiomas, psoriasis, Kaposi's sarcoma, ocular neovascularization, retinopathy of prematurity, rheumatoid arthritis, endometriosis and atherosclerosis; or pathologies with deficient angiogenesis, selected from myocardial ischemia, peripheral limb ischemia, cerebral ischemia, delayed wound healing and impaired ulcer healing; or pathologies with defective angiogenesis, selected from vascular or arteriovenous malformations (AVMs) and telangiectasias.

6. Mesenchymal stem cell to be used, according to claims 2 or 5, in a method for the treatment and / or prevention of Hereditary Hemorrhagic Telangiectasia.

7. Mesenchymal stem cell to be used according to claims 2, 5 or 6, wherein the expression level of Endoglin, or of the ENG gene, has been increased by genetic modification or pharmacological treatment of the mesenchymal stem cells.

8. In vitro use of Endoglin, or of ENG gene transcripts, to select mesenchymal stem cells for use in promoting or inhibiting angiogenesis or to identify mesenchymal stem cells that promote or inhibit angiogenesis.

9. In vitro use of a kit comprising reagents for assessing the expression level of the Endoglin protein, or the ENG gene, for selecting mesenchymal stem cells for use in promoting or inhibiting angiogenesis or for identifying mesenchymal stem cells that promote or inhibit angiogenesis.

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