ITMI20060165A1 - TRANSGENIC ANIMALS WITH HYPERTENE HYPERTESUS - Google Patents

Description

Patent application for industrial invention entitled:

Transgenic animals with hypertensive phenotype

FIELD OF THE INVENTION

The invention concerns animal models of hypertensive pathologies, generated by gene modification.

FRONT TECHNIQUE

High blood pressure is an important risk factor in kidney disease, coronary heart disease and acute brain disease. It is quite widespread: it affects about a third of the adult population. Elevated blood pressure can result from either increased cardiac output, and / or increased vascular resistance to blood flow. Recent studies have shown that the elastic component of the extracellular matrix (ECM) plays an important role in blood pressure regulation mechanisms.

The study of transgenic mice in which the elastin gene has been inactivated by gene targeting gives offspring in which homozygotes die from obstruction of the arteries and heterozygotes are stably hypertensive.

These results seem to suggest that components of the ECM in the vascular system are also important in the etiogenesis of hypertension.

Emilins are part of a family of extracellular matrix proteins characterized by a unique arrangement at the structural level, which includes a signal peptide and the EMI domain, consisting of a region of about 80 aa rich in cysteines, at the NH2 end, a region an alpha helix in the central part and a region homologous to the globular C1q domain (gC1q domain) at the carboxy-terminal end. On the basis of the structural homologies in particular in the EMI region, the emilin-1 family, the first isolated (Bressan, G. M. et al., 1993, J Cell Biol 121, 201-12), the emilin -2 (Doliana, R. et al., 2001, J Biol Chem 276, 12003-11), multimerin-1 (Hayward, C. P. et al., 1995, J Biol Chem 270, 18246-51; Braghetta, P et al., 1995, J Biol Chem 270, 18246-51; Braghetta, P et al. al., 2004, Matrix Biol 22, 549-56) a protein secreted by endothelial cells and platelets, and multimerin-2 (Christian, S. et al., 2001, J Biol Chem 276, 48588-95; Braghetta et al., 2004: already cited), also produced by endothelial cells (nomenclature adopted: http://www.aene.ucl.ac.Uk/nomenclature/genefamilv/emilin.html#HGNC ta ble2)

The functional characterization of each of the components of this family has not yet been completed, although recent data have shown that antibodies against emilin inhibit the deposition of elastin by SMC (Smooth Muscle Cells) in vitro. Furthermore, the transgenic mouse in which the emilin 1 locus has been inactivated appears normal, the homozygotes are fertile and have a normal life cycle, but on closer examination, they show alterations in the structure of the elastic fibers and in the cell morphology in the arteries. (Zanetti, M. et al, 2004, Mol Cell Biol 24, 638-50).

SUMMARY

The present invention relates to non-human transgenic animals in which at least one allele of a gene encoding a protein of the EDEN group selected in the group consisting of: emilin-2, emilin-3, multimerin-1 and multimerin-2 is inactivated, interrupted, deleted totally and / or partially with respect to the wild type sequence, animals that are characterized by the fact of being hypertensive, that is to say that they have a significant increase in systemic arterial pressure. This property has made it possible to reveal a totally new mechanism for the etiology of hypertension through an alteration of peripheral vascular resistance.

The use of transgenic animal models developed for the selection of biologically active molecules of utility in the treatment of hypertension and vascular remodeling and of diagnostic and / or prognostic methods in which a decreased or missing expression of a protein of the EDEN family is a sufficient indicator of predisposition to the development of hypertension or the genetic basis of a hypertensive phenotype.

Also part of the invention are cells, isolated from transgenic organisms for the development of assays for the selection of molecules active on the (cardio) vascular system, of antihypertensive drugs, of drugs with activity on vascular remodeling, on atherosclerosis, on aneurysms. and on diabetes-induced vasculopathies, as well as for the study of the etiopathogenesis of hypertension, vascular remodeling, atherosclerosis, aneurysms and diabetes-induced vasculopathies.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Characterization of the vascular phenotype of k.o.

Panel A: Immunofluorescence with anti-emilin-1 antibodies: section of the arterial wall of a normal adult mouse. In green emilin-1, in red the nuclei stained with propidium iodide.

Panel B): mean blood pressure values (systolic and diastolic in mmHg) of wild type and transgenic mice. Light bars: emilin-1 / + mice (wild type); dark bars emilin-1 - / - (knock-out) mice. * P <0.05 vs w.t ..

Panel C): Cardiac output and Total Peripheral Resistance (TPR) of w.t. mice. and transgenic. Light bars: emilina-1 / + (w.t.) mice; dark bars mice emilin-1 - / - (k.o.).

Panel D): variations in the external diameter of the carotid (μm) under conditions of experimentally induced pressure increase: full square: w.t .; empty square: k.o.

Panel E) angiography of the aorta of a w.t. mouse (left) or a knockout mouse. (To the right).

Panel F): mesenteric artery (tract: 50μιτι) of wild type mouse (+ / +) and of k.o mouse (- / -). Under the photograph, the two graphs show MCSA (Media Cross Sectional Area = Area of the media tunic in cross section) and thickness of the media tunic / diameter of the lumen of the artery for wild type mice (+ / +) and for k.o mice. (- / -).

Figure 2. Inhibition of the TGF-β transduction pathway in Xenopus embryos.

Panels A) -E) pattern or pattern of expression by in situ hybridization of the mesodermal marker MyoD in untreated or microinjected Xenopus embryos with the mRNAs for emilin-1, or for emilin-1 without EMI-domain (ΔΕΜΙ), or for the EMI domain only. A) untreated, B) and C) microinjected embryos respectively with mRNA for emilin-1 or for the EMI domain. D) embryos treated with emilin-1 mRNA devoid of EMI-domain (ΔΕΜΙ).

Panels E) -L) RT-PCR of Xenopus embryos co-injected with mRNA for emilin-1, for emilin-1 without EMI domain (ΔΕΜΙ), or only for EMI domain, co-injected into embryos together with different mRNAs, such as shown above the photo; the probes used for PCR detection are shown alongside. Xnr-1: member of the TGF-β family; Xbra, Eomes, Mixer: mesodermal markers induced by Xnr-1; caAlk5: constitutively active type I receptor of TGF-β; Vent-1, Siamois and Xnr-3: mesodermal markers; FGF: Fibroblast Growth Factor. EF1a: check. Figure 3. Increase in TGF-β activity in k.o mice.

Panels A) -B) Immunohistochemical analysis that detects the phosphorylation of phosphorylated Smad-2 (P-Smad2) in the aorta in w.t. mice (A) and k.o (B). Insert: quantization of the number of positive nuclei. The increase of P-Smad-2 in the Vascular Smooth Muscle Cells of k.o. indicates that the lack of emilin-1 leads to an increase in the activation of the TGF-β signaling pathway.

Panels C) -D): Immunohistochemical analysis for GFP (Green Fluorescent Protein) in w.t. A) and k.o. B). GFP is transcribed from a TGF-β responsive transgene (CAGA12-GFP). The lack of emilin-1 determines an activation of the TGF-β transduction pathway with a consequent increase in the expression of GFP (panels C-D).

Panels E) -G) Dependence of the phenotype of k.o. from the gene assay of TGF-β. E): systolic blood pressure of mice generated by crossing between emilin-1 / - and TGF-β / - double heterozygous mice; F) MCSA (Media Cross Sectional Area) of mesenteric arteries of the same mice of panel E). <*> P <0.01 vs emilin-1 / + and TGF-β / +; G) echocardiographic analysis of the ascending aorta; panel H) proliferative capacity of smooth muscle cells. Values: mean ± sd, <*> P <0.05 vs w.t.

Figure 4. Model of TGF-β activation and activity in the vascular system: ECM homeostasis, vascular cell smooth muscle homeostasis and vascular remodeling (Annes et al., 2003, J Celi Sci 116, 217-24 ). TS-1 (Thrombospondin-1), ανβ6 (integrins with alpha chain of type v and β chain of type 6).

Figure 5. Scheme of gene insertion in the emilin-2 locus.

A) murine genomic locus. B) gene targeting vector in which the PGK-neo cassette was inserted in sense orientation in place of the 684 bp Hindlll-Nar I fragment containing exon 2 and part of the first and second introns of emilin-2, in the 7.5 kb genomic clone. A tk cassette was also inserted at one end. The construct contains respectively 3 and 4 kb of homology to the emilin-2 locus. C) Transgenic locus, after vector integration. The target locus lacks exon 2 encoding the signal peptide and the first 15 amino acids of the mature protein. Empty rectangles: untranslated regions; black rectangles: translated (coding) regions exons 1-9; gray rectangles: tk gene and PGK-neo cassette; ATG and TAG codons of start and stop of the emilina-2

Figure 6. Scheme of gene insertion in the multimerin-2 locus. A) murine genomic locus (29 kb and 7 exons); B) gene targeting vector in which the PGK-neo cassette was inserted in the antisense direction in place of the 2772 bp Bgin I fragment (containing part of the region flanking the 5 ', the first exon and part of the first intron), in the clone genomic of 14 kb. A tk cassette was also inserted at one end. The construct contains 4 and 7 kb of homology at the multimerin-2 locus in the left and right arm respectively. C) Transgenic locus, after integration of the vector in B). The target locus lacks exon 1 encoding the signal peptide and the first 32 amino acids of the mature protein. Empty rectangles: untranslated regions; black rectangles: translated (coding) regions exons 1-7; gray rectangles: tk gene and PGK-neo cassette; ATG and TAG start and stop codons of the multimer-2.

Figure 7. Systolic blood pressure of mice w.t. and - / - for emilin-2 and multimerin-2. Blood pressure was measured using a tail sphingomanometer. The reported values represent the mean value of the measurements on 6 animals for each group. The asterisk (*) indicates significantly different values from those of the w.t. mouse

Figure 8. Systolic blood pressure of mice w.t. and null mutants for ultimerina-1. Blood pressure was measured using a tail sphingomanometer. The reported values represent the mean value of the measurements on 6 animals for each group. The asterisk (*) indicates significantly different values from those of the w.t. mouse

Figure 9. Diameter of the ascending aorta in wt mice and in mice - / - for emilin-1 emilin-2 and multimerin-2). The asterisk (*) indicates significantly different values from those of the w.t. mouse

Figure 10. Effect of mRNA for Emilinal on TGF-P3 mRNA activity in Xenopus embryos.

Bottle cells obtained from two-cell Xenopus embryos microinjected with TGF-β 3 mRNA (300 pg) A), in combination with 1 ng of Emilinal B) mRNA; later stages of development of the embryo microinjected with TGF-β3 mRNA (300 pg) C), in combination with 1 ng of Emilinal mRNA, D).

Figure 11. Expression of TGF-β target genes in tissues of Emilinal-deficient mice and controls.

Extracts prepared from aortas of normal (+ / +) and Emilin 1 (- / -) deficient mice were analyzed by western blotting with specific antibodies to PAI-1 (A) and anti c-Myc (B). Control of the quantity of extracts loaded on gel electrophoresis: western-blot with an anti-βactin antibody.

Figure 12. Measurement of the contraction of the aorta in response to a contracting stimulus.

Measurement of aortic vasoconstriction was performed by myograph after stimulation with 80 mM KCI: the contraction of aortas isolated from mice deficient in emilin-1 (- / -) is less than that obtained with control aortas (+ / +) .

Definitions and abbreviations

Transgenic organism: The definition includes multicellular eukaryotic organisms containing a transgene. The organisms according to the invention are, where not specified, non-human mammals.

Null animal or mouse: animal in which mutations (spontaneous or artificially introduced) lead to a complete lack of the gene product (mRNA and / or protein).

Animal or mouse k.o. (knock-out): animal in which a null mutation has been introduced in a certain gene by means of gene targeting techniques.

Family of proteins of the EDEN group (or Emiline): as defined on the basis of structural homologies in particular in the EMI region, it currently includes: emilin-1, the first isolated (Bressan, G. M. et al., 1993, J Celi Biol 121 , 201-12), emilin-2 (Doliana, R. et al., 2001, J Biol Chem 276, 12003-11), emilin-3, multimerin-1 (Hayward, C. P. et al., 1995, J Biol Chem 270, 18246-51; Braghetta, P et al., 2004, Matrix Biol 22, 549-56) a protein secreted by endothelial cells and platelets, multimerin-2 (Christian, S. et al., 2001 , J Biol Chem 276, 48588-95; Braghetta et al., 2004: already cited) also produced by endothelial cells, and also the proteins Emid-1 and Emid-2, according to the nomenclature adopted in

http://www.qene.ucl.ac.Uk/nomenclature/genefamilv/emilin.html#HGNC ta ble2)

DETAILED DESCRIPTION OF THE INVENTION

It has been surprisingly observed that animals transgenic for at least one gene of the EDEN family (EMI Domain Endowed) have a hypertensive phenotype, and in fact reveal a totally unknown pathogenesis mechanism of hypertension, thus representing an important study model for this pathology. in the human field.

In particular, it was observed that the presence of at least one mutated inactivated or silenced allele with respect to the wild type, of a gene selected from: emilin-1; emilin-2, emilin-3, multimerin-1 and multimerin-2, in an animal genome, constitute a sufficient genetic basis for the development of a hypertensive phenotype.

Therefore, the subject of the present invention is transgenic organisms produced by knock-out of one of the genes corresponding to the respective human and murine orthoogues for emilin-2 (human: GenBank GenelD 84034; mouse: GenBank Gene ID 246707), emilin-3 (human: GenBank GenelD 90187; mouse: GenBank GenelD 280635), multimer-1 (human: GenBank GenelD 22915; mouse: GenBank GenelD 70945) and multimer-2, (human: GenBank GenelD 79812; mouse: GenBank GenelD 105450), using for knock out genetic the mouse sequence. The proteins of the EDEN family are characterized by a unique arrangement at the structural level and include a signal peptide, the EMI domain, consisting of a region of about 80 amino acids rich in cysteines at the NH2 end, an alpha helix region in the central part and a region homologous to the globular C1q domain (gC1q domain) at the carboxy-terminal end.

Transgenic animals for emilin-2, emilin-3, multimerin-1 and multimerin-2 are preferably obtained by inactivation (knock out) of the genomic locus. This inactivation is preferably obtained by gene targeting with suitable vectors which insert an expression cassette, such as the pgk-neo cassette, in the respective genomic locus, preferably at the level of the first exon, thus interrupting the translation of the entire gene. Preferably, the animals according to the invention are homozygous null (- / -) that is, the inactivation or modification concerns both alleles.

The transgenic animals are mammals, preferably rodents, and even more preferably mice, of the 129 or CD1 or C57BL6 strain or crosses between these strains, and show, macroscopically, a normal phenotype: the aging process is normal and the adults are fertile. Macroscopically, therefore, they have a normal phenotype, as already observed for mice null for emilin-1: the aging process is normal and the adults are fertile, while alterations in elastogenesis are found (Zanetti, M. et al, 2004, Mol Cell Biol 24, 638-50), attributable to a direct interaction of emilin-1 with elastin.

In transgenic animals according to the invention, a genetically inactivated gene expression level lower than normal (heterozygous / -) or totally absent (homozygous - / -) can be found. This results both by immunohistochemical analysis carried out on organ or tissue sections, and by analysis with molecular methods (Northern blot or RT-PCR) on cells, eg. fibroblasts, isolated from the same transgenic animals. A morphological investigation carried out on the aorta of transgenic mice according to the invention shows that these animals have a reduced vascular diameter. Cardiac output, on the other hand, remains similar to the norm: as a result, total peripheral resistance is significantly increased, with a significant increase in blood pressure in all transgenic animals and particularly in those knock-out for emilin-2.

In particular, in animals according to the invention, the diameter of the aorta, measured by echocardiography, is reduced, as is the thickness of the average wall of the same vessel (indicated by a reduction in the MCSA, average cross-sectional area).

Therefore the invention extends to non-human animal models, preferably small mammals and even more preferably rodents, comprising a gene or a genomic region (coding, non-coding or regulating the expression of a protein of the EDEN group, as identified above, with the exception of emilinal) naturally or artificially inactivated or deleted, where this inactivation and / or deletion determines a decreased or absent expression of a protein of the EDEN group, and in which this genetic anomaly is associated with a hypertensive phenotype, and / or with a altered peripheral vascular resistance, and / or to a decreased proliferative capacity of the peripheral endothelial / vascular system and / or to a reduction in the size of the vessels, and to their use for purposes of research and investigation on the mechanisms of etiogenesis of hypertension.

The Xenopus embryo system was used to investigate through which signal transduction pathway the proteins of the EDEN group act on the vascular system and therefore on the arterial pressure values observed in transgenic mouse models, the Xenopus embryo system was used (Piccolo, S. et al., 1996, Cell 86, 589-98), according to which mRNA coding for different proteins are microinjected into Xenopus embryos and if the protein they express has a biological activity related to some known signal transduction pathway, the development process of the embryo and this alteration is observable and easily traceable to the altered transduction pathway. Accordingly, the expression of target genes of the transduction pathway is also altered, detectable by in situ hybridization on the whole embryo (Sive, H. L. et al., 2000, "Early development of Xenopus laevis. A laboratory manual." Cold Spring Harbor. Laboratory Press, Cold Spring Harbor, New York) or RT-PCR.

Through this technique it was possible to identify that the effect of the whole hemilin is entirely carried out by the EMI domain, which, like the mRNA coding for the whole protein, inhibits the formation of mesodermal tissues, and in particular that the biological effect of EDEN protein alters the signal transduction pathway activated by TGF-β.

Accordingly, the administration of mRNA for factor Xnr-1 (a member of the TGF-β family) induces the formation of mesoderm in the animal caps, as indicated by the presence of the markers Mixer, Eomesodermin and Brachyury, while emilin-1 (or the its EMI-domain), but not the molecule in which the-domain was deleted, block the effect of Xnr-1. Therefore, the biological activity of Emilinal or its EMI domain is similarly expressed on the different TGF-β, with an effect that is preferably highlighted on TGF-βΙ and TGFβ3.

Therefore the phenotype in transgenic animals according to the invention can be determined even only by the lack of an active EMI domain. Further experimental evidence indicates that:

1) the proteins of the EDEN family mentioned above, preferably emilin-1, emilin2, emilin3, multimerinl and multimerin2, act at a stage that precedes the binding of TGF-β to their receptor. In fact, the effect of mRNA coding for a constitutively active form of the receptor is not modified by the simultaneous microinjection of mRNA for emilin-1 or for TEMI domain;

2) the proteins of the EDEN family mentioned above, preferably emilin-1, emilin-2, emilin-3, multimerin-1 and multimerin-2, do not interfere with the signaling of all members of the TGF-β family; eg „the effects of the factors of the BMP (Bone Morphogenetic Protein) subfamily are not modified by emilin-1;

3) The inhibition of signal transduction by EDEN proteins does not affect other growth factors such as eg. FGF (Fibroblast Growth Factor) and Wnt (homologs of the Drosophila Wingless gene).

Therefore in the absence of proteins of the EDEN family, particularly in transgenic organisms deficient in emilin-1, emilin-2, emilin-3, multimerin-1 and multimerin-2, there is an increase in the signaling pathway activated by TGF-β without any alteration of the binding of this growth factor to its receptor.

Epistasis experiments carried out with mice heterozygous for inactivating (k.o.) mutation of TGF-βΙ (Kulkarni, A.B., et al., 1993, Proc Nati Acad Sci U S A 90, 770-4) show that blood pressure increases in null or ko mice according to the invention it disappears when the gene dosage of TGF-β 1 decreases, as in the crossing with the TGF-β1 +/- heterozygous transgenic mouse. This indicates that TGF-β are involved in the determination of the hypertensive phenotype by proteins containing the EMI domain and this represents a totally new pathogenesis mechanism of hypertension.

Similarly, the reduced diameter of the descending aorta and of the mesenteric artery and the reduced thickness of the middle wall, are also to be attributed to an increased signaling of TGF-β, preferably TGF-β1, due to the lack of the emilin product.

A reduced proliferative capacity is also observed in cells isolated from transgenic animals according to the invention, particularly in smooth muscle cells with the Emilin - / - genotype (null for at least one EDEN protein) both in the absence and in the presence of serum. Such isolated cells, preferably smooth muscle or fibroblasts, represent a further object of the invention. They are isolated from the tissues according to standard methods, for example those described in Marigo V. et al. 1992, EurJ Celi Biol 57, 254-264 or in Hogan, B. et al., 1994, "Manipulating the mouse embryo. A laboratory manual." Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. In organisms (animal or isolated cells) according to the invention, in which the expression level of the proteins emilin-2 and / or emilin-3 and / or multimerin-1 and / or multimerin-2, is decreased or absent, there is a greater activation of the TGF-β pathway, evidenced for example through a hyper-phosphorylation of Smad-2 or a modulation of the expression of TGF-β target genes such as PAI-1 (Plasminogen Activator lnhibitor-1) or c-Myc (Figure 11).

A further object of the invention is targeting vectors for the specific targeting of the genomic loci of the genes emilin-2, emilin-3, multimerin-1, or for multimerin-2. The basic vectors are known in the art: their characteristics are described for example in Mansour, S.L, et al., 1988, Nature 336, 348-52. Particularly preferred is the PHM2 vector described in Kaestner K.H. et al. (Kaestner, K. H. et al., 1994, Gene 148, 67-70). Base vectors for targeting must accommodate a genomic region of at least 15 Kb, express at least one marker for positive selection in eukaryotes (e.g. the Neomycin (Neo) gene) and / or negative (e.g., HSV thymidinokinase, or hygromycin or hprt, or diphtheria toxin) and must also be replicable and selectable in bacteria. Within the vectors according to the invention, (structure in the site of interest shown in Figures 6 and 7), murine genomic fragments are cloned, preferably less than 15kb in length isolated by recognition with specific probes from commercially available mouse genomic libraries. The sequence of such inserts is obtainable for specific murine genes from GenBank such as GenelD 246707 (emilin2), GenelD 280635 (emilin3), GenelD 70945 (multimerin1) and GenelD 105450 (multimerin2).

Particularly preferred vectors for gene targeting carry respectively the fragments of genomic sequence corresponding to positions: 4,858,075-4,850,225 (GenBank acc. N.NT_039658.4) for emilin-2 and to positions 7,617,848-7,636,761 (GenBank acc. N. NT_039606.4) for multimerin-2 in which the Hindlll-Narl fragment of 684 bp for the first and the Bglll fragment of 2772 bp for the second, are replaced with the PGKNeo cassette, and which preferably contain the tk gene, under the homologous promoter.

These vectors are used for the preparation of transgenic animals according to a process that preferably includes the linearization of the vector and the electroporation in mouse embryonic stem (ES) cells as described in Mansour et al. (quoted); ES clones with a single integrative event are microinjected into host blastocysts of strain C57BL / 6 (see eg Hogan, B. et al., 1994, "Manipulating the mouse embryo. A laboratory manual." Cold Spring Harbor Laboratory Press , Cold Spring Harbor, N.Y. P.) Heterozygous / - male F1 generation mice are crossed to obtain homozygotes A genomic analysis on ES cells, e.g. by Southern Blot, on caudal tissue and / or cells isolated from animals, can confirm correct insertion of the cassette and verify the actual interruption of the target gene. The invention therefore extends to the process for the preparation of the transgenic animals described using the vectors of the invention.

In transgenic animals according to the invention, in particular in the districts rich in SMC (smooth muscle cells) and / or fibroblasts, an increased activation of the signal transduction pathways activated by TGF-β is observed, (for example an increase in P- Smad2 and PAI-1 and a decrease in c-Myc), which can constitute marker genes of a modulation of activity through the biological mechanism identified by the authors of the present invention.

Transgenic organisms and isolated derived cells therefore become indispensable tools for the selection of molecules and / or drugs active on the (cardio) vascular system, of antihypertensive drugs, of drugs with activity on vascular remodeling, on atherosclerosis, on aneurysms and on vasculopathies induced by diabetes, as well as for the study of the etiopathogenesis of hypertension, vascular remodeling, atherosclerosis, aneurysms and diabetes-induced vasculopathies.

According to a preferred aspect, the invention extends to the use of transgenic animals for emilin 1 (Zanetti, M. et al, 2004, Mol Celi Biol 24, 638-50), for the purposes mentioned above. This use is completely unexpected as it is a consequence of the observation of the hypertensive phenotype in transgenic animals.

Cells derived from transgenic animals with hypertensive phenotype are used, according to requirements, in in vitro systems developed by the skilled in the art: for example, a biological modulation activity of the TGF-β signaling pathway can be highlighted by activation of reporter genes (e.g. luciferase, b-gal, GFP) placed under the control of promoters sensitive to activation / inactivation by TGF-β, preferably TGF-β 1-3, or genes controlled by TGF- β, or by direct detection of genes physologically induced, silenced or modified (eg phosphorylated) by TGF-β (eg P-Smad2, PAI-1 and c-Myc).

The invention therefore extends to a method for the selection of bioactive molecules, in particular of pharmacologically active molecules in hypertension, in vascular remodeling and / or to a method for the development of TGF-β antagonist selection assays. which includes the use of cells derived from the genetically modified organisms according to the invention (or of cells in which the gene for the EDEN protein is inactivated). Cells deficient in at least one protein of the EDEN group are, according to a preferred embodiment, transfected with constructs in which the expression of a reporter gene (such as for example luciferase, etc.) is under the control of a promoter sensitive to activation / inactivation by TGF-β (CAGA12, p15) directly or indirectly.

A further use of transgenic or null animals according to the invention is the establishment of transgenic animal lines, where a further genetic modification is induced. These animals have a null genetic background for at least one protein of the EDEN family as described in the invention, and carry a further transgene, inserted according to known methods in embryonic stem cells or fertilized oocytes isolated from transgenic animals according to the invention. By, but not exclusively, natural methods such as crossing and selection, transgenic animal lines are generated comprising both the described null mutation and a further genetic modification. A specific and exemplary but not limiting realization for the person skilled in the art of this use, is that for which mice with the transgene CAGA12 / GFP (Neptune, E.R. et al., 2003, Nat Genet 33, 407-411) are crossed with mice mutants for emilinal in order to obtain mice with the transgene CAGA12 / GFP in the genetic background emilin1 / + or emilin 1 - / -. The CAGA12 / GFP transgene in which the promoter, the CAGA sequence repeated for 12 times, binds the Smad2-3 / Smad4 complex, is an intracellular effector of the signal activated by TGF-β, activating the transcription of the GFP marker gene. In this model, by highlighting the expression levels of the marker gene with respect to a control in which the marker transgene is in a normal genetic background, target organs or tissues of TGF-β are identified, physiologically or following a pathology and can be recorded increases in the expression or availability of mature TGF- β in mice with null genetic background. In this specific case, an increase in the expression of the GFP marker gene in the sections of the aorta from mice with the two genetic backgrounds, stained eg. by immunoperoxidase with an antibody against GFP, show that at the level of the stained sections there is an increase in the production of TGF-β in mice with a genetic background according to the invention compared to mice with a normal genetic background. having a null genetic background for at least one of the EDEN proteins and a further genetic modification, for example adult embryonic phybroblasts, or stem cells of such animals, are indispensable pharmacological tools for the selection of bioactive molecules useful in the preparation of drugs for prevention and therapy of (cardio) vascular diseases, hypertension, atherosclerosis, aneurysms and vasculopathies induced by diabetes or for vascular remodeling.

According to this aspect, the cells, derived from transgenic mice carrying a construct sensitive to the activity of TGF-β, such as p15-lux or CAGA12-lux in a genetic background for wild type or null emilinal, are used according to methods known to the technician. of the branch to test the activity of TGF-β antagonist drugs by comparing the activity of luciferase produced by treated and untreated cells with the molecules or collections of molecules and / or compounds (libraries) whose biological activity is to be tested , comparing the response of cells with wild type or mutant genotype. In this case the effect of a drug is revealed by a decrease in the activity of the marker gene, e.g. luciferase or GFP.

As a consequence of the identification of the phenotype and of the mechanisms inhibited by the lack of proteins of the EDEN family in the transgenic animals of the invention, the invention also extends to methods of selection of molecules or bioactive drugs in which a genetically modified animal according to the invention ( bearing at least one allele of the gene coding for emilin-1, emilin-2, emilin-3, multimerin-1 and multimerin-2 in an inactivated or interrupted form, or totally and / or partially deleted, thanks to a natural event or specific targeting of the genomic sequence that reduces or cancels the expression of the corresponding protein), and phenotypically hypertensive, is treated with such molecules or collections of molecules or drugs and in which the restoration to normal values (significantly not different from the wild type) of one of the following indices : arterial pressure, vessel diameter, peripheral vascular resistance, represents an indication of pharmacological activity of tera analysis of the compound or libraries of compounds tested.

Furthermore, the invention extends to in vitro assays for the diagnosis of a genetic predisposition to the development of hypertension and / or a genetic basis to hypertension or to an altered peripheral vascular resistance which includes the determination of at least one of the following parameters: sequence nucleotide or expression level of at least one protein of the EDEN group, preferably of: emilin-1, emilin-2, emilin-3, multimerin-1 and multimerin-2 and in which an alteration with respect to the wild type of one or the other This parameter indicates an alteration in the mechanisms of blood pressure regulation or a genetic predisposition to the development of (cardio) vascular diseases or hypertension, abnormal vascular remodeling, atherosclerosis, aneurysms and diabetes-induced vasculopathies. This genetic determination is preferably performed on samples of nucleic acids isolated from mammals, including humans.

According to a preferred aspect and in accordance with the experimental observations, the induced alteration is inversely correlated to the expression of the protein with the EMI domain, therefore a decreased expression of these proteins corresponds to the induction of that set of alterations that lead to a hypertensive phenotype or predisposing to cardiovascular alterations.

EXPERIMENTAL PART

Example 1. Preparation of knock-out transgenic animals for emilin-2 and multimerin-2.

The emilin-2 and multimerin2 genes were obtained from phage clones λΕΜΒί3 derived from a mouse genomic library of strain 129 / SvJ (Stratagene), by selection with the respective cDNAs. The clone containing emilin-2 is a 7.5 kb clone.

The genomic inserts, containing contiguous exonic-intronic regions, were subcloned in the pHM2 vector for gene-targeting (Kaestner, K. H. et al., 1994, Gene 148, 67-70) in which the tk (thymidino-kinase) gene was also inserted ) for negative selection. For the emilin-2 gene, a 684 bp Hindlll-Narl region, comprising the second exon and part of the first and third introns, was replaced with the pgk-neo cassette of the pHM2 vector in the forward orientation (see Figure 5). In the final vector, the homology region for the gene is 3 Kb to the right and 4 Kb to the left. For the multimerin2 gene, a region of 2772 bp, comprising part of the 5'-flanking region, the first exon and part of the first intron, was deleted and replaced with the pgk-neo cassette of the pHM2 vector in reverse orientation (see figure 6) .

The vectors were linearized and used for the electroporation of mouse embryonic stem (ES) cells (Nagy, A. et al., 1993, Proc NatlAcad Sci U S A 90, 8424-8), selected and grown according to standard methods as described in: Hogan, B. et al., 1994, "Manipulating the mouse embryo. A laboratory manual." Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. ES cell clones with a single integrative event were microinjected into host blastocysts of strain C57BL / 6. Heterozygous male mice <+/-> of F1 generation were crossed to obtain homozygous <- / ->. Primary fibroblasts were also isolated and cultured. Genomic analysis was carried out by Southern blot on ES cells and on caudal tissue and cells isolated from mice, confirming the correct insertion of the cassette in the genes that were therefore interrupted.

For the emilin-2 gene, 7 chimeric mice derived from an ES cell clone with a correct gene targeting event were obtained. These mice were crossed with C57BL / 6 mice to obtain heterozygous and homozygous mice for the mutation.

The genomic locus of emilin-2 after homologous recombination with the knock-out vector lacked exon 2, coding for the signal peptide and for the first 15 amino acids of the mature protein.

For the multimerin-2 gene, 23 different chimeric mice derived from 4 independent ES cell clones with a correct gene targeting event were obtained.

The genomic locus of multimerin-2 after homologous recombination with the knock-out vector lacked exon 1, coding for the signal peptide and the first 32 amino acids of the mature protein.

Example 2. Phenotypic characterization of emilinal (emilin 1 - / -) deficient transgenic mice.

The protein emilin-1 is strongly expressed in the cardiovascular system, as shown in Figure 1A, in which the distribution of the protein in an aorta of a normal adult mouse was visualized by the immunofluorescence technique following the method previously described (Zanetti et al . 2004: already mentioned). Consequently, the cardiovascular system of mutant animals was studied by various methods.

An important parameter is systemic blood pressure, which was measured by the telemetry method (described in Vecchione et al 2002, Circulation 105, 1700-7). The results show that blood pressure levels of emilin-1 - / - mice significantly increased (Figure 1B).

Cardiac output and peripheral resistance were measured in emilin-1 - / - mice. The method used is that described in Brancaccio, M. et al., 2003, Nat Med 9, 68-75, which is based on the use of a four-pole catheter for pressure / volume measurements (Millar 1.4 F product, SPR 671, Millar Instruments, Ine., USA), which is inserted into the left ventricle via the right common carotid artery; total peripheral resistance is calculated by dividing the mean arterial pressure, measured at the same time through a catheter introduced into the femoral artery, by the cardiac output. As shown in Figure 1C, while cardiac output was similar in mutant and wild type mice, total peripheral resistance was significantly reduced, statistically significantly. At the same time, the elastic property of the vessels was also evaluated. For this, parts of the artery were mounted in a pressure myograph on an inverted microscope connected to a camera-computer system that allows the continuous detection of internal and external vascular diameters. Internal pressure was increased from 20 to 200 mmHg in small increments and the extensibility calculated using the formula: defined pressure extensibility (p) = [(diameter at p 25 mmHg -diameter at p - 25 mmHg) / (diameter at p - 25 mmHg)] x 100 (Li, D. Y. Et al., 1998, J din Invest 102, 1783-7). The pressure-diameter graph of Figure 1D, recorded for the common carotid, indicates that the extensibility of the vessel is similar for mutant and control mice, since the two curves are practically parallel.

The conclusion of the experiments described above is that the cause of the hypertension found in the emilin mutant mice is due to an altered peripheral resistance.

The morphology of the great vessels in vivo was studied by echocadiography and angiography. The method used for the first is that described by De Acetis et al. (De Acetis, M. et al., 2005, drc Res 96, 1087-94), while for angiography the mice were anesthetized, the contrast agent lohexol injected into the left ventricle, while the images were acquired by means of a digital angiograph (OEC 9800, General Electric) and an image capture system. The data obtained show a reduction in the diameter of the aorta (figure 9) and of the whole vascular system (figure 1E and F) in emilin 1 - / - mice. It has also been shown that, at physiological pressure levels, the arteries of - / - mice, albeit of smaller caliber, show a normal elastic response (Figure 1D).

Subsequently, the remodeling of the small vessels (resistance vessels) induced by the deficiency of emilin-1 was evaluated according to the methods described (Vecchione et al, 2002, already mentioned). The results are shown in Figure 1F and show that the small vessels (mesenteric arteriole in the figure) of emilinal - / - mice also have a reduced caliber.

Taken as a whole, the results described so far suggest that the main cause of the increase in blood pressure in mice deficient in emilin is the increase in peripheral resistance resulting from the reduction in the diameter of the resistance vessels.

Example 3. Characterization of the alterations induced by the expression of mRNA coding for emilin in Xenopus embryos.

The hemi-domain of emilin-1 (and also of the other emilins) is a region of the molecule rich in cysteines, a characteristic that unites it to various types of secreted protein domains whose ability to modulate the activity of signal transduction pathways. The Xenopus embryo system was used to verify whether-domain plays a regulatory role in signaling pathways. In this procedure, mRNAs transcribed and capped in vitro (Piccolo, S. et al., 1996, Cell 86, 589-98) are microinjected into embryos of 2-4 cells. If the protein they express has a biological activity related to some signal transduction pathway, it can alter the development process of the embryo. Furthermore, the expression of target genes of the signaling pathway is altered and can be detected by in situ hybridization on the whole embryo (Sive, H. L. et al., 2000, "Early development of Xenopus laevis. A laboratory manual." Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York) or RT-PCR (Ausubel, F. M. et al., 1993, "Current protocols in molecular biology." John Wiley & Sons, Ine., New York, N.Y.) on so-called animai caps . The latter constitute the so-called animal portion of the early embryo, which is excised immediately after microinjection and maintained in vitro until the stage corresponding to blastula or gastrula (Sive, 2000).

Microinjection of mRNA encoding emilin-1 (Figure 2B) inhibits the formation of mesodermal tissues, as indicated by the loss of hybridization for MyoD, a muscle tissue marker (Figure 2A). This biological activity of emilin-1 is attributable to the EMI-domain, since the microinjection of mRNA for this region alone has the same effect (figure 2C), while the remaining part of the molecule has no consequence on development (figure 2D ). Accordingly, the administration of mRNA for factor Xnr-1 (a member of the TGF-β family) induces the formation of mesoderm in the animal caps, as indicated by the presence of the markers Mixer, Eomesodermin and Brachyury, while emilin-1 (or the its EMI-domain), but not the molecule in which the-domain was deleted, block the effect of Xnr-1 (see Figures 2E and 2F).

Further experiments indicated that:

1) emilin-1 acts at a stage that precedes the binding of TGF-β to their receptor. In fact, the effect of mRNA coding for a constitutively active form of the receptor (type I, caAlk5) is not modified by the simultaneous microinjection of mRNA for emilin-1 (figure 2G); 2) Emilin-1 does not interfere with the signaling of all members of the TGF-β family; eg, the effects of the factors of the BMP subfamily are not modified by emilin-1 (Figure 2H);

3) The inhibition of signaling by emilin-1 does not affect other growth factors such as eg. FGF and Wnt (Figure 21 and 2L).

Example 4. Recovery (Rescue) of the hypertensive phenotype by crossing with other transgenic mice.

As foreseen by the hypothesis suggested by the experiments described in Examples 2 and 3, that is, that emilin-1 inhibits the signaling of some factors of the TGF-β family, in the tissues of mice deficient in emilin-1 it is shown an increase in TGF-β activity. This is documented in Figure 3 with two different methods. In the first, aortas of control mice (Figure 3A) and emilinal - / - (Figure 3B) were stained by immunoperoxidase with antibodies against phosphorylated Smad2 (P-Smad2) using the Vectastain ABC kit (Vector) and the procedure indicated therein. Smad2 is an intracellular component of the TGF- β transduction pathway that is phosphorylated when TGF- β binds to its receptor. The results show a significant increase in the number of Smad2-positive phosphorylated nuclei in the mutant mice. In the second method, mice with the CAGA12 / GFP transgene (a construct, described in Neptune, E.R. et al., 2003, Nat Genet 33, 407-411 whose promoter, the CAGA sequence repeated 12 times, binds the Smad2-3 complex / Smad4, intracellular effector of the signal activated by TGF-β, activating the transcription of the marker gene GFP) were crossed with mice mutants for emilin 1 in order to obtain mice with the transgene CAGA12 / GFP in the background emilin 1 / + or emilin 1 - / -. Aortas from mice with the two genotypes were stained by immunoperoxidase with an antibody against GFP. The results show an increase in GFP expression in emilin-1 deficient mice (compare Figure 3C and 3D).

To verify whether the increased TGF-β signaling found in mice mutants for emilin-1 is implicated as a cause of the phenotypic alterations that they manifest (arterial hypertension and decrease in the diameter of the arteries), epistasis experiments concerning emilin-1 were carried out. and TGF- βΙ. These experiments allow to verify if there is an interaction between genes in the determination of a certain phenotypic character; if the two genes act on the same path, the phenotype determined by the mutation of one of them will be modified by mutations of the other gene; vice versa in the opposite case. Therefore, double heterozygous mice (emilinal / -; TGF-βΙ / -) were crossed with each other to obtain the following genotypes: emilin1 + / +; TGF β1 + / +, emilinal - / -; TGF-βΙ 1 + / +, emilinal / +; TGF-βΙ +/- and emilinal - / -; TGF-βΙ +/-. As shown in Figure 3E, the blood pressure increase induced by emilin-1 deficiency (genotype emilinal - / -; TGF-βΙ + / +) disappears when the dosage of TGF-βΙ is reduced by the inactivation of an allele (genotype emilin 1 - / -; TGF-β 1 +/-): this indicates that TGF- β1 is involved in the determination of the hypertensive phenotype in mice deficient in emilin-1. Similarly, Figures 3G and 3F demonstrate that the reduced diameter of the ascending aorta, as measured by echocardiography, and the reduced thickness of the same vessel mean (indicated by a reduction in MCSA, mean cross-sectional area), are also from attributed to increased TGF-β1 signaling.

Consistent with a role of TGF-β in the determination of the phenotype of emilin 1 - / - is also the observation reported in figure 3H, in which the proliferative capacity of smooth muscle cells of aorta cultured in vitro and derived from emilinal mice is compared / + and emilina 1 - / -. Cells were isolated from tissues as described in Marigo, V. et al., 1992, Eur J Cell Biol 57, 254-64) and cultured in vitro in DMEM (Dulbecco modified Eagle's medium) in the presence of 20% FCS (fetal calf serum). For the experiment the cells were plated at a density of 700 cells / well of 96-well microplates and cultured for 12 hours in DMEM + 20% FCS to adhere to the plastic. They were then grown for 24 in DMEM without FCS to inhibit growth and then in DMEM supplemented with 5 μg / ml of insulin and 100 ng / ml PDGF-BB. At different times (12-36 hours), the samples were subjected to cell growth tests with the Cell Titer 96 Aqueous One solution (Promega) kit according to the manufacturer's instructions. The proliferation of smooth muscle cells from animals with different genotypes was compared by assigning a value of 1 to cells cultured simultaneously without serum. The results show that hemilin 1 - / - cells grow slower than hemilin 1 / + cells.

Example 5. Characterization of TGF- β signaling food effect in emilin 1 - / - mice on the vascular system.

Deficiency of emilin-1 has a dual effect on blood vessels. On the one hand, the increased signaling of TGF-β causes inhibition of cell proliferation and reduction of the diameter and thickness of the media of the arteries and arterioles with a consequent increase in peripheral resistance and hypertension. On the other hand, it modifies the response of the smooth muscle cells of the arteries to contractile stimuli. This is demonstrated in Figure 12. In it, the aorta of emilinal / + and emilinal - / - mice was fitted into a myograph and the contraction of smooth muscle cells stimulated with 80 mM KCI as described (Lembo G. et al. , 2000, Diabetes, 49: 293-297): the contraction of emilin-1 deficient aortas is less than that obtained with control aortas.

Example 6. Characterization of the phenotype of mice with targeted or spontaneous gene inactivation of emilin-2, multimerin-1 and multimerin-2.

A characterization of the cardiovascular system similar to that described for emilin-1 was also performed on mice deficient in each of the other emilins. Emilin-2 (emilin 2 - / -) and multimerin2 (multimerin 2 - / -) knockout mice were used in this study. Mice of the C57BI / 60laHsd strain, (Harland, Indiana-USA), showing in homozygosity a spontaneous deletion of 365 Kb between positions 60.976 and 61.431 Mb of chromosome 6, comprising the six exons of the Snca gene and the eight exons of the multimerinal gene (Spechi, C. G., and Schoepfer, R., 2004, Genomics 83, 1176-8), lacking all the transcribed portion of the multimerin-1 gene, therefore represent a complete natural knock-out of multimerin-1. Characterization of mutant mice demonstrated increased blood pressure in emilin2 - / - and multimerin2 - / - null mice (Figure 7) and in C57BI / 60laHsd mice (Figure 8). Furthermore, a reduction in the diameter of the aorta was also found in the same mutant mice (Figure 9).

Example 7. Effect of Emilinal on TGF- β3 activity.

To verify that emilin activity extended to other members of the TGF-β family, two-cell Xenopus embryos were microinjected at the animal pole with TGF-β3 mRNA (300 pg) alone or in combination with 1 ng of Emilinal mRNA, as described in Example 3.

As shown in Figure 10, overexpression of TGF-β 3 in ectodermal cells induces the formation of bottle cells (A) and, at later stages of development, secondary trunk / tail structures (C). These effects are inhibited by Emilinal (B and D). These experiments show that Emilinal antagonizes the biological effects of TGF-β 3 as well.

Example 8. Expression of TGF-β target genes in tissues of Emilinal and control deficient mice.

To verify that emilin deficiency had the same regulating effect on TGF-β-controlled genes also in vivo in the tissues of k.o. mice, extracts from aortas of normal and Emilinal-deficient mice were prepared and analyzed by western blotting with specific anti- PAI-1 and c-Myc. As a check that the amount of extract was the same for the different samples, a western-blot with an anti-b-actin antibody was performed on the same samples.

As shown in Figure 11 in the aortas of knock out mice, the absence of Emilinal induces an increase in PAI-1 (A) and a decrease in c-Myc (B), where PAI-1 and c-Myc are genes known to be induced and inhibited by TGF- β, respectively (Siegei, P.M., and Massague, J., 2003, Nat Rev Cancer 3, 807-21). Therefore, the experiment in Figure 11 demonstrates that the expression of TGF-β target genes is modified in the tissues of Emilinal-deficient mice due to an increase in the activity of this growth factor.

Claims (44)

CLAIMS 1. Non-human transgenic animal where at least one allele of a gene encoding a protein of the EDEN family selected in the group consisting of: emilin-2, emilin-3, multimerin-1 and multimerin-2 is inactivated, interrupted, totally deleted and / or partially with respect to the wild type sequence and characterized by the fact of being phenotypically hypertensive. 2. Transgenic animal according to claim 1 wherein such animal is a mammal. 3. A transgenic animal according to claim 2 wherein such mammal is a rodent. 4. Transgenic animal according to claim 3 wherein such rodent is a mouse. 5. Transgenic animal according to claims 1-4 in which the allele encodes emilin -2. 6. Transgenic animal according to claims 1-4 in which the allele encodes the multimerin-1. 7. Transgenic animal according to claims 1-4 in which the allele encodes the multimerin-2. 8. Cell isolated from a transgenic animal according to claims 1-7 An isolated cell according to claim 8 comprising a further transgene and / or further genetically transformed in vitro. 10. Animal according to claims 1-7 or cell according to claims 8-9 in which the expression level of the proteins emilin-2 and / or emilin-3 and / or multimerin-1 and / or multimerin-2 is decreased or absent. 11. Vector for the specific targeting of genes for emilin-2, for multimerin-1, or for multimerin-2, comprising at least one fragment of 100 nucleotides of the genomic sequence of the corresponding gene. Vector according to claim 10 wherein said genomic sequence fragment of the gene for emilin-2, for multimerin-1, or for multimerin-2 is interrupted by a cassette for positive selection in eukaryotes. 13. Vector according to claim 12 where this cassette is pgk-Neo for the expression of the gene for resistance to neomycin. Vector according to claims 12-13 where such fragment has nucleotide sequence as deposited in GenBank as GenelD 246707 (emilin2), GenelD 280635 (emilin3), GenelD 70945 (multimerin1) and GenelD 105450 (multimerin2) respectively. 15. Vector according to claim 14 where such fragment corresponds to the nucleotide positions 4,858,075-4,850,225 for emilin-2, to positions 7,617,848-7,636,761 for multimerin-2, according to the numbering of the mouse sequences known in GenBank with access number NT_039658.4 and NT_039606.4, respectively. 16. Process for the preparation of a phenotypically hypertensive transgenic animal according to claims 1-7 and 10 which comprises the inactivation of a gene or a genomic region coding for a protein of the EDEN family and selected from the group consisting of: emilin-2 , emilin-3 multimer-1 and multimerin-2. A method according to claim 16 wherein such inactivation is carried out by introducing a vector for gene targeting according to claims 11-15 into a non-human embryonic stem cell or a non-human fertilized oocyte. 18. Process according to claims 16-17 wherein said embryonic stem cell or said oocyte are of mouse origin. 19. Use of a non-human animal in which a gene or a genomic region coding for a protein of the EDEN group is naturally or artificially inactivated or deleted and where such inactivation and / or deletion results in a decreased or absent expression of the corresponding protein, and having a hypertensive phenotype, for the selection of antihypertensive drugs, and / or active on the (cardio) vascular system, in vascular remodeling, in atherosclerosis, in aneurysms and in diabetes-induced vasculopathies. Use according to claim 19 wherein said inactivation relates to a gene or a genomic region selected from the genes coding for emilin-1, emilin-2, emilin-3, multimerin-1 or multimerin-2. 21. Use according to claim 20 wherein such gene or genomic region is emilin-1 (GenBank GenelD 100952). Use according to claim 19-21 wherein such gene or genomic region is inactivated in the first exon by interruption with a LacZpgk-neo expression cassette. 23. Use according to claim 19 where such non-human animal is the transgenic animal according to each of claims 1-7 and 10. 24. Use according to claim 23 where the animal is k.o. for emilin-2 according to claim 5. 25. Use according to claim 23 where the animal is k.o. for multimer-1 according to claim 6. 26. Use according to claim 23 where the animal is k.o. for multimer-2 according to claim 7. 27. Use according to claim 19 where the animal carries a spontaneous deletion of chromosome 6 in the genomic region comprising the gene coding for multimerin-1 for the selection of antihypertensive molecules or drugs, for the selection of molecules or drugs active on the cardiovascular system and / or in vascular remodeling and / or in atherosclerosis and / or in aneurysms and / or in vasculopathies induced by diabetes and / or for the study of the mechanisms of aetiogenesis of hypertension, aetiogenesis of cardiovascular diseases, aetiogenesis of atherosclerotic diseases , the etiogenesis of aneurysms, the etiogenesis of diabetes-induced vasculopathies. Use according to claim 27 where such animal is a mouse. 29. Use of a cell according to claims 8-9 for the selection of antihypertensive molecules or drugs, for the selection of molecules or drugs active on the cardiovascular system and / or in vascular remodeling and / or in atherosclerosis and / or aneurysms and / or in diabetes-induced vasculopathies and / or for the study of the mechanisms of aetiogenesis of hypertension, the etiogenesis of cardiovascular diseases, the etiogenesis of atherosclerotic diseases, the etiogenesis of aneurysms, the etiogenesis of diabetes-induced vasculopathies. 30. Use of a cell according to claim 29 for the selection of TGF-β antagonist molecules. 31. Use of a cell according to claim 29 for the selection of emilin agonist molecules. 32. Use of a cell according to claim 29 for the selection of inhibitory molecules of the furin-convertase enzymatic activity for the processing and / or activation of pro TGF-β to mature TGF-β. 33. Use of a transgenic animal according to each of claims 1-7 and 10, for the preparation of transgenic animals comprising a further genetic modification other than that concerning one of the following genes or genomic regions coding for: emilin-1, emilin-2, emilin-3, multimerin-1, multimerin-2, Emid-1, Emid-2 (filing n ° in GenBank respectively: GenelD 100952 (emilinal) GenelD 246707 (emilina2), GenelD 280635 (emilina3), GenelD 70945 (multimerinal) and GenelD 105450 (multimer2)). 34. Use according to claim 33 where such genetic modification consists in the introduction of a transgene comprising a marker gene. Use according to claim 34 wherein such marker gene encodes a fluorescent protein. 36. Process for the preparation of transgenic animals comprising a further genetic modification different from that concerning one of the following genes or genomic regions coding for: emilin-1, emilin-2, emilin-3, multimerin-1, multimerin-2, Emid -1, Emid-2 having a deposit number in genBank respectively GenelD 100952 (emilinal) GenelD 246707 (emilina2), GenelD 280635 (emilina3), GenelD 70945 {multimerinal) and GenelD 105450 (multimerinal2), NM_080595 and NM_024474, where this modification Genetics is carried out by introducing a vector for gene targeting into an embryonic stem cell or fertilized oocyte isolated from a non-human transgenic animal according to claims 1-7 and 10. 37. A process according to claim 36 wherein said embryonic stem cell or oocyte is of mouse origin. 38. Process according to claims 36-37 wherein said vector for gene targeting comprises a coding sequence for a marker or reporter protein. 39. Method for the selection of antihypertensive drugs and / or active on the cardiovascular system, characterized by the fact of using a genetically modified animal bearing at least one allele of the gene coding for emilin-1, emilin-2, emilin-3, multimerin-1 and multimerin-2 in inactivated or interrupted form, or totally and / or partially deleted and phenotypically hypertensive, in which the restoration to normal values (significantly not different from the wild type) of one of the following indices: arterial pressure and / or diameter of the vessels and / or peripheral vascular resistance, represents an indication of pharmacological activity of therapeutic interest. 40. Method according to claim 39 where the genetically modified animal is the animal according to claims 1-7 and 10. 41. Method for the selection of drugs or antihypertensive molecules and / or drugs or molecules active on the cardiovascular system and / or in vascular remodeling, in atherosclerosis, in aneurysms, in vasculopathies induced by diabetes and for the study of the mechanisms of the etiogenesis of hypertension, the etiogenesis of cardiovascular diseases, the etiogenesis of atherosclerotic diseases, the etiogenesis of aneurysms, the etiogenesis of vasculopathies induced by diabetes, characterized by using a cell according to claims 8-9. 42. In vitro assay for the diagnosis of a genetic predisposition to the development of hypertension and / or the genetic component to hypertension in a mammal which includes the in vitro determination of the nucleotide sequence or expression level of at least one selected protein from the group consisting of: emilin-1, emilin-2, emilin-3, multimerin-1 and multimerin-2 and in which an alteration with respect to the wild type of one or the other parameter is indicative of an alteration of the blood pressure regulation mechanisms or it indicates a genetic predisposition to the development of cardiovascular diseases or hypertension, atherosclerotic diseases, aneurysms, diabetes-induced vasculopathies. 43. Assay according to claim 42 where this mammal is man. 44. Kit for the determination of a genetic predisposition to the development of hypertension and / or for the identification of the genetic component to hypertension in a mammal which includes the in vitro determination of the nucleotide sequence or the expression level of at least one protein selected in the group consisting of: emilin-1, emilin2, emilin-3, multimerin 1 and multimerin 2, where this kit includes suitable oligonucleotide primers or specific antibodies.